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Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides.
Lohse, Jonas; Swier, Lotteke J Y M; Oudshoorn, Ruben C; Médard, Guillaume; Kuster, Bernhard; Slotboom, Dirk-Jan; Witte, Martin D
In chemical biology, azides are used to chemically manipulate target structures in a bioorthogonal manner for a plethora of applications ranging from target identification to the synthesis of homogeneously modified protein conjugates. While a variety of methods have been established to introduce the azido group into recombinant proteins, a method that directly converts specific amino groups in endogenous proteins is lacking. Here, we report the first biotin-tethered diazotransfer reagent DtBio and demonstrate that it selectively modifies the model proteins streptavidin and avidin and the membrane protein BioY on cell surface. The reagent converts amines in the proximity of the binding pocket to azides and leaves the remaining amino groups in streptavidin untouched. Reagents of this novel class will find use in target identification as well as the selective functionalization and bioorthogonal protection of proteins.
Selective Flotation of Calcite from Fluorite: A Novel Reagent Schedule
Directory of Open Access Journals (Sweden)
Zhiyong Gao
Full Text Available Fluorite is an important strategic mineral. In general, fluorite ores will contain a certain amount of calcite gangue mineral. Thus, they need to be separated from each other. For an economic separation, a reverse flotation process is used to float calcite gangue from fluorite. However, little information on the separation is available. In this study, a novel reagent schedule using citric acid (CA as the depressant, sodium fluoride (NaF as the regulator and sulfoleic acid (SOA as the collector, was developed to separate calcite from fluorite. The results demonstrated a high selectivity for the flotation of calcite from fluorite using this new reagent schedule. The best selective separation for a single mineral and mixed binary minerals was obtained when 200 mg/L of NaF, 50 mg/L of CA, and 6 mg/L of SOA were used at pH 9. In addition, a batch flotation experiment was carried out using a run-of-mine feed material. Selective separation was achieved with 85.18% calcite removal while only 11.2% of fluorite was lost. An attempt was made to understand the effect of the new reagent schedule on the flotation of calcite. The results from both microflotation and bench scale flotation demonstrated a great potential for industrial application using this novel reagent schedule to upgrade fluorite ore.
Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.
Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T
A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides
Directory of Open Access Journals (Sweden)
Michael E. Østergaard
Full Text Available Antisense oligonucleotides (ASOs have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT and wild-type HTT (wtHTT mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.
Recyclable bio-reagent for rapid and selective extraction of contaminants from soil
International Nuclear Information System (INIS)
Lomasney, H.L.
This Phase I Small Business Innovation Research program is confirming the effectiveness of a bio-reagent to cost-effectively and selectively extract a wide range of heavy metals and radionuclide contaminants from soil. This bioreagent solution, developed by ISOTRON reg-sign Corporation (New Orleans, LA), is flushed through the soil and recycled after flowing through an electrokinetic separation module, also developed by ISOTRON reg-sign. The process is ex situ, and the soil remains in its transport container through the decontamination process. The transport container can be a fiberglass box, or a bulk bag or open-quotes super sack.close quotes Rocks, vegetation, roots, etc. need not be removed. High clay content soils are accommodated. The process provides rapid injection of reagent solution, and when needed, sand is introduced to speed up the heap leach step. The concentrated waste form is eventually solidified. The bio-reagent is essentially a natural product, therefore any solubizer residual in soil is not expected to cause regulatory concern. The Phase I work will confirm the effectiveness of this bio-reagent on a wide range of contaminants, and the engineering parameters that are needed to carry out a full-scale demonstration of the process. ISOTRON reg-sign scientists will work with contaminated soil from Los Alamos National Laboratory. LANL is in the process of decontaminating and decommissioning more than 300 sites within its complex, many of which contain heavy metals or radionuclides; some are mixed wastes containing TCE, PCB, and metals
Recyclable bio-reagent for rapid and selective extraction of contaminants from soil
Energy Technology Data Exchange (ETDEWEB)
Lomasney, H.L. [ISOTRON Corp., New Orleans, LA (United States)
This Phase I Small Business Innovation Research program is confirming the effectiveness of a bio-reagent to cost-effectively and selectively extract a wide range of heavy metals and radionuclide contaminants from soil. This bioreagent solution, developed by ISOTRON{reg_sign} Corporation (New Orleans, LA), is flushed through the soil and recycled after flowing through an electrokinetic separation module, also developed by ISOTRON{reg_sign}. The process is ex situ, and the soil remains in its transport container through the decontamination process. The transport container can be a fiberglass box, or a bulk bag or {open_quotes}super sack.{close_quotes} Rocks, vegetation, roots, etc. need not be removed. High clay content soils are accommodated. The process provides rapid injection of reagent solution, and when needed, sand is introduced to speed up the heap leach step. The concentrated waste form is eventually solidified. The bio-reagent is essentially a natural product, therefore any solubizer residual in soil is not expected to cause regulatory concern. The Phase I work will confirm the effectiveness of this bio-reagent on a wide range of contaminants, and the engineering parameters that are needed to carry out a full-scale demonstration of the process. ISOTRON{reg_sign} scientists will work with contaminated soil from Los Alamos National Laboratory. LANL is in the process of decontaminating and decommissioning more than 300 sites within its complex, many of which contain heavy metals or radionuclides; some are mixed wastes containing TCE, PCB, and metals.
Trans-Selective Rhodium Catalysed Conjugate Addition of Organoboron Reagents to Dihydropyranones
Directory of Open Access Journals (Sweden)
Hannah J. Edwards
Full Text Available The selective synthesis of 2,6-trans-tetrahydropyran derivatives employing the rhodium catalysed addition of organoboron reagents to dihydropyranone templates, derived from a zinc-catalysed hetero-Diels-Alder reaction, is reported. The addition of both arylboronic acids and potassium alkenyltrifluoroborates have been accomplished in high yields using commercially-available [Rh(cod(OH]2 catalyst. The selective formation of the 2,6-trans-tetrahydropyran stereoisomer is consistent with a mechanism involving alkene association and carbometalation on the less hindered face of the dihydropyranone.
Phenylmercuric hydroxide. A highly selective reagent for the hydration of nonconjugated terminal alkynes
International Nuclear Information System (INIS)
Janout, V.; Regen, S.L.
This article describes an unusual and highly selective method for hydrating nonconjugated terminal alkynes based on the use of phenylmercuric hydroxide as a reagent. Unlike classical mercury catalyzed procedures, sigma-bonded mercury acetylides are formed initially as stable intermediates and subsequently reacted with water under neutral pH to form the corresponding methyl ketone. Isolated yields which have been obtained by using this approach lie in the range of 49-65%. The high selectivity toward nonconjugated terminal alkynes which characterizes the procedure described herein should make it a useful supplement to existing hydration methods
Factors involved in the selection of limestone reagents for use in wet FGD systems
International Nuclear Information System (INIS)
Jarvis, J.B.; Roothaan, E.S.; Meserole, F.B.; Owens, D.R.
With recent activity in the design and construction of retrofit flue gas desulfurization (FGD) systems, many utilities are faced with the task of selecting limestones which will allow FGD systems to function as designed, and at the same time, provide cost-effective operation. The Electric Power Research Institute (EPRI) has sponsored research to identify factors which should be considered in the reagent selection process. A set of capabilities has been developed which is currently being employed to assist six utilities in selecting cost-effective reagent sources. The major elements in the selection package consist of an analytical characterization of candidate limestones; grindability, reactivity, and magnesium availability testing; and performance modeling utilizing EPRI's FGD PRocess Integration and Simulation Model (FGDPRISM). The results from these measurements are used to perform a site-specific economic analysis which can be used to rank the candidate limestones and quantify the impact of various limestone properties on plant operating costs. This paper includes a description of each element in the selection package along with a review of current research activities aimed at improving predictions of limestone reactivity and magnesium availability. An example is presented which illustrates how reactivity and magnesium availability affect both the performance of an FGD system and plant operating costs
Selective-Reagent-Ionization Mass Spectrometry: New Prospects for Atmospheric Research
Sulzer, Philipp; Jordan, Alfons; Hartungen, Eugen; Hanel, Gernot; Jürschik, Simone; Herbig, Jens; Märk, Lukas; Märk, Tilmann D.
Proton-Transfer-Reaction Mass Spectrometry (PTR-MS), which was introduced to the scientific community in the 1990's, has quickly evolved into a well-established technology for atmospheric research and environmental chemistry [1]. Advantages of PTR-MS are i) high sensitivities of several hundred cps/ppbv, ii) detection limits at or below the pptv level, iii) direct injection sampling (i.e. no sample preparation), iv) response times in the 100 ms regime and v) online quantification. However, one drawback is a somehow limited selectivity, as in case of quadrupole mass filter based instruments only information about nominal m/z are available. In Time-Of-Flight (TOF) mass analyzer based instruments selectivity is drastically increased by a high mass resolution of up to 8000 m/Δm, but e.g. isomers still cannot be separated. In 2009 we introduced an advanced version of PTR-MS, which permits switching the reagent ions from H3O+ to NO+ and O2+, respectively [2]. This novel type of instrumentation was called Selective-Reagent-Ionization Mass Spectrometry (SRI-MS) and has been successfully used to separate isomers, e.g. the biogenic compounds isoprene and 2-methyl-3-buten-2-ol as shown by Karl et al. [3]. Switching the reagent ions dramatically increases selectivity and thus applicability of SRI-MS in atmospheric research. Here we report on the latest results utilizing an even more advanced embodiment of SRI-MS enabling the use of the additional reagent ions Kr+ and Xe+ [4]. With this technology important atmospheric compounds, such as CO2, CO, CH4, O2, etc. can be quantified and selectivity is increased even further. We present comparison data between diesel and gasoline car exhaust gases and quantitative data on indoor air for these compounds, which are not detectable with classical PTR-MS. Additionally, we show very recent examples of isomers which cannot be separated with PTR-MS but can clearly be distinguished with SRI-MS. Finally, we give an overview of ongoing SRI
Optimization of Cu-Zn Massive Sulphide Flotation by Selective Reagents
Soltani, F.; Koleini, S. M. J.; Abdollahy, M.
Selective floatation of base metal sulphide minerals can be achieved by using selective reagents. Sequential floatation of chalcopyrite-sphalerite from Taknar (Iran) massive sulphide ore with 3.5 % Zn and 1.26 % Cu was studied. D-optimal design of response surface methodology was used. Four mixed collector types (Aer238 + SIPX, Aero3477 + SIPX, TC1000 + SIPX and X231 + SIPX), two depressant systems (CuCN-ZnSO4 and dextrin-ZnSO4), pH and ZnSO4 dosage were considered as operational factors in the first stage of flotation. Different conditions of pH, CuSO4 dosage and SIPX dosage were studied for sphalerite flotation from first stage tailings. Aero238 + SIPX induced better selectivity for chalcopyrite against pyrite and sphalerite. Dextrin-ZnSO4 was as effective as CuCN-ZnSO4 in sphalerite-pyrite depression. Under optimum conditions, Cu recovery, Zn recovery and pyrite content in Cu concentrate were 88.99, 33.49 and 1.34 % by using Aero238 + SIPX as mixed collector, CuCN-ZnSO4 as depressant system, at ZnSO4 dosage of 200 g/t and pH 10.54. When CuCN was used at the first stage, CuSO4 consumption increased and Zn recovery decreased during the second stage. Maximum Zn recovery was 72.19 % by using 343.66 g/t of CuSO4, 22.22 g/t of SIPX and pH 9.99 at the second stage.
Recovery of molybdenum using alumina microspheres and precipitation with selective organic reagents
International Nuclear Information System (INIS)
Carvalho, Fatima Maria Sequeira de; Abrao, Alcidio
In this paper is presented a study for the optimization of dissolution of the UAL x plates used for irradiation and production of radiomolybdenum. The alloy is dissolved in nitric acid with mercury as catalyst. The separation and concentration of the molybdenum was achieved using a chromatographic grade alumina microspheres column. the purified eluted molybdenum is finally precipitated using one of the selective reagents: alizarine blue, α,α'- bipyridine and 1,10-phenanthroline. Any one of the obtained precipitate can be fired to the molybdenum trioxide. The interference of the following elements was studied: Re(VII), U(VI), Cr(VI), W(VI), V(V), Te(IV), Ti(IV), Zr(IV), Th(IV), Fe(III), Au(III), Ru(III), Al(III), Bi(III), Sb(III), Ce(IV), Pr(III), Sc(III), Y(III), Sm(III), Ba(II), Sr(II), Ni(II), Co(II), Cs(I). The molybdenum precipitates were characterized by gravimetric, CHN, TG, DTG, IR and X-ray diffraction analyses. (author)
Microfluidics in the selection of affinity reagents for the detection of cancer: paving a way towards future diagnostics.
Hung, Lien-Yu; Wang, Chih-Hung; Fu, Chien-Yu; Gopinathan, Priya; Lee, Gwo-Bin
Microfluidic technologies have miniaturized a variety of biomedical applications, and these chip-based systems have several significant advantages over their large-scale counterparts. Recently, this technology has been used for automating labor-intensive and time-consuming screening processes, whereby affinity reagents, including aptamers, peptides, antibodies, polysaccharides, glycoproteins, and a variety of small molecules, are used to probe for molecular biomarkers. When compared to conventional methods, the microfluidic approaches are faster, more compact, require considerably smaller quantities of samples and reagents, and can be automated. Furthermore, they allow for more precise control of reaction conditions (e.g., pH, temperature, and shearing forces) such that more efficient screening can be performed. A variety of affinity reagents for targeting cancer cells or cancer biomarkers are now available and will likely replace conventional antibodies. In this review article, the selection of affinity reagents for cancer cells or cancer biomarkers on microfluidic platforms is reviewed with the aim of highlighting the utility of such approaches in cancer diagnostics.
Fluorimetric determination of copper(II) in aqueous solution using lucifer yellow CH as selective metal reagent
Energy Technology Data Exchange (ETDEWEB)
Mayr, T.; Wencel, D.; Werner, T. [Univ. of Regensburg (Germany) Inst. of Analytical Chemistry, Chemo- and Biosensors
Lucifer yellow CH is shown to be a highly selective fluorescent reagent for the determination of Cu(II) in the {mu}g L{sup -1} concentration range. The fluorophore is statically quenched by Cu(II); the carbohydrazide group was assigned as the complexing part of the dye molecule. A total range of Cu(II) determination from 0.06 mg L{sup -1} (1 {mu}mol L{sup -1}) to 6.3 mg L{sup -1} (100 {mu}mol L{sup -1}) with a limit of detection of 0.019 mg L{sup -1} (0.3 {mu}mol L{sup -1}) was obtained, along with surprisingly high selectivity. There was no interference from alkaline and earth alkaline metal ions. The cross sensitivity to heavy metal ions was evaluated by the separate solution method and by competitive binding experiments. Calibration plots are shown for Cu(II) determination at different pH and the dissociation constant was determined. The application of the reagent was demonstrated by the determination of the Cu(II) content of tap water samples. (orig.)
A selective and rapid microdetermination of molybdenum(VI) using 2`, 4`-dihydroxyacetophenone benzoylhydrazone as a new reagent
Energy Technology Data Exchange (ETDEWEB)
Dass, Rameshwar; Mehta, J R [Kurukshetra Univ. (India). Dept. of Chemistry
A simple, rapid and selective spectrophotometric method for the microdetermination of molybdenum(VI) using 2`,4`-dihydroxyacetophone benzoylhydrazone (DHABH) as a new reagent has been developed. The greenish-yellow Mo(VI)-DHABH complex with {lambda}sub(max) at 402 nm, is extractable into benzene from 0.01-0.10 M HCl medium containing 0.5-1.4 ml of 0.2% solution of the reagent in acetone. The 1:1 species in benzene obeys Beer`s law in the range 0-17.5 {mu}g Mo ml{sup -1} and is stable for more than one week. The molar absorptivity, Sandell`s sensitivity and standard deviation are 0.940 x 10{sup 4} mol{sup -1} cm{sup -1}, 0.0102 {mu}gMo cm{sup -2} and {+-} 0.0055 respectively. The method has been used for the determination of molybdenum(VI) in various alloy steels and a wide variety of synthetic samples with satisfactory results. (author). 8 refs., 1 fig.
Poly(4-vinylpyridine) as a reagent with silanol-masking effect for silica and its specific selectivity for PAHs and dinitropyrenes in a reversed phase
International Nuclear Information System (INIS)
Ihara, Hirotaka; Fukui, Megumi; Mimaki, Takamasa; Shundo, Atsuomi; Dong, Wei; Derakhshan, Mahnaz; Sakurai, Toshihiko; Takafuji, Makoto; Nagaoka, Shoji
This paper demonstrates that poly(4-vinylpyridine) is applicable as an effective masking reagent for silica to reduce undesirable side effects due to silanol groups. It also shows that this chemical modification brings about unique retention behaviors absolutely different from conventional ODS, which appear in molecular-shape selectivity for polycyclic aromatic hydrocarbons and in selectivity for position isomerism, especially for electron-withdrawing substitution compounds. Separation of 1,6- and 1,8-dinirtopyrenes as carcinogens is also described
Antisense Treatments for Biothreat Agents
National Research Council Canada - National Science Library
Warfield, Kelly L; Panchal, Rekha G; Aman, M J; Bavari, Sina
... a variety of pathogens in cell culture studies and nonhuman primate models of infection. For these reasons, antisense technologies are being pursued as treatments against biothreat agents such as Ebola virus, dengue virus and Bacillus anthracis...
Selection of organic acid leaching reagent for recovery of zinc and manganese from zinc-carbon and alkaline spent batteries
Yuliusman; Amiliana, R. A.; Wulandari, P. T.; Ramadhan, I. T.; Kusumadewi, F. A.
Zinc-carbon and alkaline batteries are often used in electronic equipment that requires small quantities of power. The waste from these batteries contains valuable metals, such as zinc and manganese, that are needed in many industries and can pollute the environment if not treated properly. This paper concerns the recovery of zinc and manganese metals from zinc-carbon and alkaline spent batteries with leaching method and using organic acid as the environmental friendly leaching reagent. Three different organic acids, namely citric acid, malic acid and aspartic acid, were used as leaching reagents and compared with sulfuric acid as non-organic acid reagents that often used for leaching. The presence of hydrogen peroxide as manganese reducers was investigated for both organic and non-organic leaching reagents. The result showed that citric acid can recover 64.37% Zinc and 51.32% Manganese, while malic acid and aspartic acid could recover less than these. Hydrogen peroxide gave the significant effect for leaching manganese with non-organic acid, but not with organic acid.
E- or Z-Selective synthesis of 4-fluorovinyl-1,2,3-triazoles with fluorinated second-generation Julia-Kocienski reagents.
Kumar, Rakesh; Singh, Govindra; Todaro, Louis J; Yang, Lijia; Zajc, Barbara
A highly modular approach to N-substituted 4-(1-fluorovinyl)triazoles is described. In situ desilylation and Cu-catalyzed ligation reaction of TMS-protected α-fluoropropargyl benzothiazole sulfone with aryl, alkyl, and metallocenyl azides furnished second-generation Julia-Kocienski reagents in good to excellent yields. Condensation reactions of these reagents with aldehydes can be tuned to yield E or Z-alkenes selectively. Under mild conditions with DBU as the base, reactions of aldehydes furnished E-alkenes as the major isomer. On the other hand, in condensation reactions with LHMDS as the base and in appropriate solvents, both aldehydes and ketones reacted to yield fluoroalkenes with Z-selectivity. Stereochemical assignment of E/Z olefins obtained in the reaction of a ketone with two Julia reagents was performed via X-ray crystallographic analysis and comparisons of NMR data. The method allows efficient and ready diversification of the N1-substituent and substituents at the double bond.
Dyslipidemia, sense, antisense or nonsense?
Visser, M.E.
Maartje Visser onderzocht het remmen van de synthese van apoB met behulp van antisense - een nieuwe farmacologische techniek. Dit blijkt het slechte LDL-cholesterol op een effectieve manier te verlagen. Bij sommige proefpersonen resulteerde dit in leververvetting. Of dit op de lange termijn
Selective optical sensing of biothiols with Ellman's reagent: 5,5'-Dithio-bis(2-nitrobenzoic acid)-modified gold nanoparticles.
Güçlü, Kubilay; Ozyürek, Mustafa; Güngör, Nilay; Baki, Sefa; Apak, Reşat
Development of sensitive and selective methods of determination for biothiols is important because of their significant roles in biological systems. We present a new optical sensor using Ellman's reagent (DTNB)-adsorbed gold nanoparticles (Au-NPs) (DTNB-Au-NP) in a colloidal solution devised to selectively determine biologically important thiols (biothiols) from biological samples and pharmaceuticals. 5,5'-Dithio-bis(2-nitrobenzoic acid) (DTNB), a versatile water-soluble compound for quantitating free sulfhydryl groups in solution, was adsorbed through non-covalent interaction onto Au-NPs, and the absorbance changes associated with the formation of the yellow-colored 5-thio-2-nitrobenzoate (TNB(2-)) anion as a result of reaction with biothiols was measured at 410nm. The sensor gave a linear response over a wide concentration range of standard biothiols comprising cysteine, glutathione, homocysteine, cysteamine, dihydrolipoic acid and 1,4-dithioerythritol. The calibration curves of individual biothiols were constructed, and their molar absorptivities and linear concentration ranges determined. The cysteine equivalent thiol content (CETC) values of various biothiols using the DTNB-Au-NP assay were comparable to those of the conventional DTNB assay, showing that the immobilized DTNB reagent retained its reactivity toward thiols. Common biological sample ingredients like amino acids, flavonoids, vitamins, and plasma antioxidants did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. DTNB-adsorbed Au-NPs probes provided higher sensitivity (i.e., lower detection limits) in biothiol determination than conventional DTNB reagent. Under optimized conditions, cysteine (Cys) was quantified by the proposed assay, with a detection limit (LOD) of 0.57μM and acceptable linearity ranging from 0.4 to 29.0μM (r=0.998). Copyright © 2013 Elsevier B.V. All
Mono(pyridine-N-oxide) analog of DOTA as a suitable organic reagent for a sensitive and selective fluorimetric determination of Ln(III) ions
Energy Technology Data Exchange (ETDEWEB)
Vanek, Jakub [Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno (Czech Republic); Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno (Czech Republic); Lubal, Premysl, E-mail: firstname.lastname@example.com [Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno (Czech Republic); Central European Institute of Technology (CEITEC), Masaryk University, Kamenice 5, 625 00 Brno (Czech Republic); Sevcikova, Romana [Department of Chemistry, Faculty of Science, Masaryk University, Kotlarska 2, 611 37 Brno (Czech Republic); Polasek, Miloslav; Hermann, Petr [Department of Inorganic Chemistry, Faculty of Science, Charles University, Hlavova 2030, 128 40, Prague (Czech Republic)
The mono(pyridine-N-oxide) analog of the H{sub 4}dota macrocylic ligand, H{sub 3}do3a-py{sup NO}, is capable of forming thermodynamically stable and kinetically inert Ln(III) complexes. Its Eu(III) and Tb(III) complexes display a strong long-lived fluorescence as a result of the antenna effect of the pyridine-N-oxide fluorophore in the reagent. It is shown that H{sub 3}do3a-py{sup NO} can be used as a fluorogenic reagent for the determination of Eu(III) and Tb(III) at pH 6.5 and c{sub L}=1 mM. At an excitation wavelength of 286 nm, the emission maxima are 615 nm (Eu(III)-complex), and 547 nm (Tb(III)complex). Detection limits are at concentrations around 1.0 {mu}M and linearity of the method spans over 2 orders of magnitude. The method was applied to artificial and real samples (spiked mineral waters, extracts from cathode ray tube luminophore dust) and gave satisfactory results. The method is simple, rapid, and hardly interfered by other metal ions. - Graphical Abstract: A DOTA-like ligand with pyridine-N-oxide pendant arm is used for a quick, selective and sensitive determination of Eu{sup 3+} and Tb{sup 3+} ions through sensitized emission with excitation at 286 nm. The presented fluorimetric method is not interfered by transition metal or other lanthanide(III) ions and has a high dynamic range. Highlights: Black-Right-Pointing-Pointer Quick, selective and sensitive determination of Eu{sup 3+}/Tb{sup 3+} ions was developed. Black-Right-Pointing-Pointer Sensitized emission with excitation at 286 nm through pyridine-N-oxide pendant arm. Black-Right-Pointing-Pointer No interference of transition metal or other Ln(III) ions within high dynamic range.
Energy Technology Data Exchange (ETDEWEB)
Alnajjar, Mikhail S.; Haynie, Todd O.
Pyrophoric reagents are extremely hazardous. Special handling techniques are required to prevent contact with air and the resulting fire. This document provides several methods for working with pyrophoric reagents outside of an inert atmosphere.
Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde.
Gavrilyuk, Julia; Ban, Hitoshi; Nagano, Masanobu; Hakamata, Wataru; Barbas, Carlos F
4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene glycol) chains (PEGylation), and functional small molecules onto model proteins and to label the surface of living cells.
Formylbenzene diazonium hexafluorophosphate reagent for tyrosine-selective modification of proteins and the introduction of a bioorthogonal aldehyde
Gavrilyuk, Julia; Ban, Hitoshi; Nagano, Masanobu; Hakamata, Wataru; Barbas, Carlos F.
4-Formylbenzene diazonium hexafluorophosphate (FBDP) is a novel bench-stable crystalline diazonium salt that reacts selectively with tyrosine to install a bioorthogonal aldehyde functionality. Model studies with N-acyl-tyrosine methylamide allowed us to identify conditions optimal for tyrosine ligation reactions with small peptides and proteins. FBDP-based conjugation was used for the facile introduction of small molecule tags, poly(ethylene) glycol chains (PEGylation), and functional small m...
On extraction reagents for hydrometallurgy
International Nuclear Information System (INIS)
Zolotov, Yu.A.
Fundamental requirements to the extractants are considered. Ways of obtaining selective extractants are discussed in particular on the basis of coordination chemistry achivements. Attention is drawn to expediency of study (as extractants) of flotation reagents, additions to the oil, pesticides, accelerators of caoutchouc vulcanization
A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin
Directory of Open Access Journals (Sweden)
Nikola Štambuk
Full Text Available Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.
Compilation of minimum and maximum isotope ratios of selected elements in naturally occurring terrestrial materials and reagents
Coplen, T.B.; Hopple, J.A.; Böhlke, J.K.; Peiser, H.S.; Rieder, S.E.; Krouse, H.R.; Rosman, K.J.R.; Ding, T.; Vocke, R.D.; Revesz, K.M.; Lamberty, A.; Taylor, P.; De Bievre, P.
Documented variations in the isotopic compositions of some chemical elements are responsible for expanded uncertainties in the standard atomic weights published by the Commission on Atomic Weights and Isotopic Abundances of the International Union of Pure and Applied Chemistry. This report summarizes reported variations in the isotopic compositions of 20 elements that are due to physical and chemical fractionation processes (not due to radioactive decay) and their effects on the standard atomic weight uncertainties. For 11 of those elements (hydrogen, lithium, boron, carbon, nitrogen, oxygen, silicon, sulfur, chlorine, copper, and selenium), standard atomic weight uncertainties have been assigned values that are substantially larger than analytical uncertainties because of common isotope abundance variations in materials of natural terrestrial origin. For 2 elements (chromium and thallium), recently reported isotope abundance variations potentially are large enough to result in future expansion of their atomic weight uncertainties. For 7 elements (magnesium, calcium, iron, zinc, molybdenum, palladium, and tellurium), documented isotope-abundance variations in materials of natural terrestrial origin are too small to have a significant effect on their standard atomic weight uncertainties. This compilation indicates the extent to which the atomic weight of an element in a given material may differ from the standard atomic weight of the element. For most elements given above, data are graphically illustrated by a diagram in which the materials are specified in the ordinate and the compositional ranges are plotted along the abscissa in scales of (1) atomic weight, (2) mole fraction of a selected isotope, and (3) delta value of a selected isotope ratio. There are no internationally distributed isotopic reference materials for the elements zinc, selenium, molybdenum, palladium, and tellurium. Preparation of such materials will help to make isotope ratio measurements among
Biotin-tagged proteins: Reagents for efficient ELISA-based serodiagnosis and phage display-based affinity selection.
Verma, Vaishali; Kaur, Charanpreet; Grover, Payal; Gupta, Amita; Chaudhary, Vijay K
The high-affinity interaction between biotin and streptavidin has opened avenues for using recombinant proteins with site-specific biotinylation to achieve efficient and directional immobilization. The site-specific biotinylation of proteins carrying a 15 amino acid long Biotin Acceptor Peptide tag (BAP; also known as AviTag) is effected on a specific lysine either by co-expressing the E. coli BirA enzyme in vivo or by using purified recombinant E. coli BirA enzyme in the presence of ATP and biotin in vitro. In this paper, we have designed a T7 promoter-lac operator-based expression vector for rapid and efficient cloning, and high-level cytosolic expression of proteins carrying a C-terminal BAP tag in E. coli with TEV protease cleavable N-terminal deca-histidine tag, useful for initial purification. Furthermore, a robust three-step purification pipeline integrated with well-optimized protocols for TEV protease-based H10 tag removal, and recombinant BirA enzyme-based site-specific in vitro biotinylation is described to obtain highly pure biotinylated proteins. Most importantly, the paper demonstrates superior sensitivities in indirect ELISA with directional and efficient immobilization of biotin-tagged proteins on streptavidin-coated surfaces in comparison to passive immobilization. The use of biotin-tagged proteins through specific immobilization also allows more efficient selection of binders from a phage-displayed naïve antibody library. In addition, for both these applications, specific immobilization requires much less amount of protein as compared to passive immobilization and can be easily multiplexed. The simplified strategy described here for the production of highly pure biotin-tagged proteins will find use in numerous applications, including those, which may require immobilization of multiple proteins simultaneously on a solid surface.
Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array
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Settles Matthew L
Full Text Available Abstract Background Natural antisense transcripts (NATs are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded or a different locus (trans-encoded. They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept. Results By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense-antisense
Potentiometric flow injection system for determination of reductants using a polymeric membrane permanganate ion-selective electrode based on current-controlled reagent delivery.
Song, Wenjing; Ding, Jiawang; Liang, Rongning; Qin, Wei
A polymeric membrane permanganate-selective electrode has been developed as a current-controlled reagent release system for potentiometric detection of reductants in flow injection analysis. By applying an external current, diffusion of permanganate ions across the polymeric membrane can be controlled precisely. The permanganate ions released at the sample-membrane interface from the inner filling solution of the electrode are consumed by reaction with a reductant in the sample solution thus changing the measured membrane potential, by which the reductant can be sensed potentiometrically. Ascorbate, dopamine and norepinephrine have been employed as the model reductants. Under the optimized conditions, the potential peak heights are proportional to the reductant concentrations in the ranges of 1.0×10(-5) to 2.5×10(-7)M for ascorbate, of 1.0×10(-5) to 5.0×10(-7)M for dopamine, and of 1.0×10(-5) to 5.0×10(-7)M for norepinephrine, respectively with the corresponding detection limits of 7.8×10(-8), 1.0×10(-7) and 1.0×10(-7)M. The proposed system has been successfully applied to the determination of reductants in pharmaceutical preparations and vegetables, and the results agree well with those of iodimetric analysis. Copyright © 2011 Elsevier B.V. All rights reserved.
Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods
DEFF Research Database (Denmark)
Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E
have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA......Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...
Quantification of selected volatile organic compounds in human urine by gas chromatography selective reagent ionization time of flight mass spectrometry (GC-SRI-TOF-MS) coupled with head-space solid-phase microextraction (HS-SPME).
Mochalski, Paweł; Unterkofler, Karl
Selective reagent ionization time of flight mass spectrometry with NO(+) as the reagent ion (SRI-TOF-MS(NO(+))) in conjunction with gas chromatography (GC) and head-space solid-phase microextraction (HS-SPME) was used to determine selected volatile organic compounds in human urine. A total of 16 volatiles exhibiting high incidence rates were quantified in the urine of 19 healthy volunteers. Amongst them there were ten ketones (acetone, 2-butanone, 3-methyl-2-butanone, 2-pentanone, 3-methyl-2-pentanone, 4-methyl-2-pentanone, 2-hexanone, 3-hexanone, 2-heptanone, and 4-heptanone), three volatile sulphur compounds (dimethyl sulfide, allyl methyl sulfide, and methyl propyl sulfide), and three heterocyclic compounds (furan, 2-methylfuran, 3-methylfuran). The concentrations of the species under study varied between 0.55 nmol L(-1) (0.05 nmol mmol(-1)creatinine) for allyl methyl sulfide and 11.6 μmol L(-1) (1.54 μmol mmol(-1)creatinine) for acetone considering medians. Limits of detection (LODs) ranged from 0.08 nmol L(-1) for allyl methyl sulfide to 1.0 nmol L(-1) for acetone and furan (with RSDs ranging from 5 to 9%). The presented experimental setup assists both real-time and GC analyses of volatile organic compounds, which can be performed consecutively using the same analytical system. Such an approach supports the novel concept of hybrid volatolomics, an approach which combines VOC profiles obtained from two or more body fluids to improve and complement the chemical information on the physiological status of an individual.
First-in-human phase I study of ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2, in subjects with open-angle glaucoma undergoing glaucoma filtration surgery.
Directory of Open Access Journals (Sweden)
Norbert Pfeiffer
Full Text Available To evaluate the safety and tolerability of intravitreal ISTH0036, an antisense oligonucleotide selectively targeting transforming growth factor beta 2 (TGF-β2, in patients with primary open angle glaucoma (POAG undergoing trabeculectomy (TE; glaucoma filtration surgery.In this prospective phase I trial glaucoma patients scheduled for TE with mitomycin C (MMC received a single intravitreal injection of ISTH0036 at the end of surgery in escalating total doses of 6.75 μg, 22.5 μg, 67.5 μg or 225 μg, resulting in calculated intraocular ISTH0036 concentrations in the vitreous humor of approximately 0.3 μM, 1 μM, 3 μM or 10 μM after injection, respectively. Outcomes assessed included: type and frequency of adverse events (AEs, intraocular pressure (IOP, numbers of interventions post trabeculectomy, bleb survival, visual acuity, visual field, electroretinogram (ERG, slit lamp biomicroscopy and optic disc assessment.In total, 12 patients were treated in the 4 dose groups. Main ocular AEs observed were corneal erosion, corneal epithelium defect, or too high or too low IOP, among others. No AE was reported to be related to ISTH0036. All other safety-related analyses did not reveal any toxicities of concern, either. The mean medicated preoperative IOP at decision time-point for surgery was 27.3 mmHg +/- 12.6 mmHg (SD. Mean IOP (±SD for dose levels 1, 2, 3, and 4 were at Day 43 9.8 mmHg ± 1.0 mmHg, 11.3 mmHg ± 6.7 mmHg, 5.5 mmHg ± 3.0 mmHg and 7.5 mmHg ± 2.3 mmHg SD; and at Day 85 9.7 mmHg ± 3.3 mmHg, 14.2 mmHg ± 6.5 mmHg, 5.8 mmHg ± 1.8 mmHg and 7.8 mmHg ± 0.6 mmHg, respectively. In contrast to IOP values for dose levels 1 and 2, IOP values for dose levels 3 and 4 persistently remained below 10 mmHg throughout the observation period.This first-in-human trial demonstrates that intravitreal injection of ISTH0036 at the end of TE is safe. Regarding IOP control, single-dose ISTH0036 administration of 67.5 μg or 225 μg at the time of TE
Transfection of mouse cytotoxic T lymphocyte with an antisense granzyme A vector reduces lytic activity.
Talento, A; Nguyen, M; Law, S; Wu, J K; Poe, M; Blake, J T; Patel, M; Wu, T J; Manyak, C L; Silberklang, M
Murine CTL have seven serine proteases, known as granzymes, in their lytic granules. Despite considerable effort, convincing evidence that these enzymes play an obligatory role in the lytic process has not been presented. To investigate the function of one of these proteases, granzyme A (GA), we utilized an antisense expression vector to lower the level of the enzyme in the cells. An expression vector containing antisense cDNA for GA and the gene for hygromycin B resistance was constructed and electroporated into the murine CTL line, AR1. Transfectants were selected based on resistance to hygromycin B, and a number of stable lines were developed. One of the antisense lines had greatly reduced levels of GA mRNA, when compared to the parental cells or to control lines transfected with the vector lacking the antisense DNA. The message levels for two other CTL granule proteins, granzyme B and perforin, were unaffected by the antisense vector. The amount of GA, as measured by enzymatic activity, was 3- to 10-fold lower in the transfectant. Most significantly, this line also consistently showed 50 to 70% lower ability to lyse nucleated target cells and to degrade their DNA. Furthermore, it exhibited 90 to 95% lower lytic activity to anti-CD3-coated SRBC. Conjugate formation with target cells, however, was normal. These data provide strong evidence that GA plays an important role in the cytolytic cycle, and that the quantity of enzyme is a limiting factor in these cytolytic cells.
International Nuclear Information System (INIS)
A solid phase reagent for use in radioimmunoassay of antigens and antibodies is described. The reagent is prepared by mixing specific antibody or radiolabeled antigen with polyethylene glycol (4000-6000) and gamma globulin in a buffer at pH 4-10 and lyophilizing, the antigens being thyroxine, triiodothyronine, digoxin and digitoxin (1-1000 μCi 125 I/μg). The buffer consists of a 0.08 molar sodium barbital solution containing 0.1% of ox-serum albumin and 0.35% of 8-anilino-1-naftalene sulfonic acid
Application of 2-Trichloromethylbenzimidazole in Analytical Chemistry: A Highly Selective Chromogenic Reagent for Thin-Layer Chromatography and Some Other Analytical Uses
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Leszek Konopski
Full Text Available 2-Trichloromethylbenzimidazole (TCMB was used as a chromogenic reagent in organic or inorganic analysis, mainly in thin-layer chromatography (TLC. In reactions of TCMB with some heteroaromatic nitrogen containing compounds, such as azines, azoles and benzazoles, a formation of high colored products occurred. For azines, the chromogenic reaction was highly regioselective, since the both adjacent α-positions versus the nitrogen atom(s must not be substituted. A TLC method of detection was developed. Thirty azines, azoles, and benzazoles were detected at the detection limit 10 ng to 1 μg. This method was also applied for detection of heteroaromatic pesticides, and the attempts to construct active and passive dosimeters for nicotine were made. In a prechromatographic reaction of aromatic o-diamines with methyl trichloroacetimidate, TCMB or its derivatives were formed in situ. Followed by TLC and visualization in pyridine vapors, this procedure was applied for detection of o-phenylenediamine derivatives. The reaction product of TCMB and pyridine (LI Complex was identified and fully characterized. Two different reaction mechanisms: with electron deficient basic heteroaromatic compounds, like pyridine, and with more acidic compounds, for example, pyrrole, were discussed. In aqueous solutions, the LI Complex may be also used as a new indicator for complexometric, adsorption and acid-base titration of inorganic compounds.
Constructing New Bioorthogonal Reagents and Reactions.
Row, R David; Prescher, Jennifer A
Chemical tools are transforming our understanding of biomolecules and living systems. Included in this group are bioorthogonal reagents-functional groups that are inert to most biological species, but can be selectively ligated with complementary probes, even in live cells and whole organisms. Applications of these tools have revealed fundamental new insights into biomolecule structure and function-information often beyond the reach of genetic approaches. In many cases, the knowledge gained from bioorthogonal probes has enabled new questions to be asked and innovative research to be pursued. Thus, the continued development and application of these tools promises to both refine our view of biological systems and facilitate new discoveries. Despite decades of achievements in bioorthogonal chemistry, limitations remain. Several reagents are too large or insufficiently stable for use in cellular environments. Many bioorthogonal groups also cross-react with one another, restricting them to singular tasks. In this Account, we describe our work to address some of the voids in the bioorthogonal toolbox. Our efforts to date have focused on small reagents with a high degree of tunability: cyclopropenes, triazines, and cyclopropenones. These motifs react selectively with complementary reagents, and their unique features are enabling new pursuits in biology. The Account is organized by common themes that emerged in our development of novel bioorthogonal reagents and reactions. First, natural product structures can serve as valuable starting points for probe design. Cyclopropene, triazine, and cyclopropenone motifs are all found in natural products, suggesting that they would be metabolically stable and compatible with a variety of living systems. Second, fine-tuning bioorthogonal reagents is essential for their successful translation to biological systems. Different applications demand different types of probes; thus, generating a collection of tools that span a continuum of
Identification of sequence motifs significantly associated with antisense activity
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Peek Andrew S
Full Text Available Abstract Background Predicting the suppression activity of antisense oligonucleotide sequences is the main goal of the rational design of nucleic acids. To create an effective predictive model, it is important to know what properties of an oligonucleotide sequence associate significantly with antisense activity. Also, for the model to be efficient we must know what properties do not associate significantly and can be omitted from the model. This paper will discuss the results of a randomization procedure to find motifs that associate significantly with either high or low antisense suppression activity, analysis of their properties, as well as the results of support vector machine modelling using these significant motifs as features. Results We discovered 155 motifs that associate significantly with high antisense suppression activity and 202 motifs that associate significantly with low suppression activity. The motifs range in length from 2 to 5 bases, contain several motifs that have been previously discovered as associating highly with antisense activity, and have thermodynamic properties consistent with previous work associating thermodynamic properties of sequences with their antisense activity. Statistical analysis revealed no correlation between a motif's position within an antisense sequence and that sequences antisense activity. Also, many significant motifs existed as subwords of other significant motifs. Support vector regression experiments indicated that the feature set of significant motifs increased correlation compared to all possible motifs as well as several subsets of the significant motifs. Conclusion The thermodynamic properties of the significantly associated motifs support existing data correlating the thermodynamic properties of the antisense oligonucleotide with antisense efficiency, reinforcing our hypothesis that antisense suppression is strongly associated with probe/target thermodynamics, as there are no enzymatic
Rapid blockade of telomerase activity and tumor cell growth by the DPL lipofection of ribbon antisense to hTR.
Bajpai, Arun K; Park, Jeong-Hoh; Moon, Ik-Jae; Kang, Hyungu; Lee, Yun-Han; Doh, Kyung-Oh; Suh, Seong-Il; Chang, Byeong-Churl; Park, Jong-Gu
Ribbon antisense (RiAS) to the hTR RNA, a component of the telomerase complex, was employed to inhibit telomerase activity and cancer cell growth. The antisense molecule, hTR-RiAS, combined with enhanced cellular uptake was shown to effectively inhibit telomerase activity and cause rapid cell death in various cancer cell lines. When cancer cells were treated with hTR-RiAS, the level of hTR RNA was reduced by more than 90% accompanied with reduction in telomerase activity. When checked for cancer cell viability, cancer cell lines treated with hTR-RiAS using DNA+Peptide+Lipid complex showed 70-80% growth inhibition in 3 days. The reduced cell viability was due to apoptosis as the percentage of cells exhibiting the sub-G0 arrest and DNA fragmentation increased after antisense treatment. Further, when subcutaneous tumors of a colon cancer cell line (SW480) were treated intratumorally with hTR-RiAS, tumor growth was markedly suppressed with almost total ablation of hTR RNA in the tumor tissue. Cells in the tumor tissue were also found to undergo apoptosis after hTR-RiAS treatment. These results suggest that hTR-RiAS is an effective anticancer reagent, with a potential for broad efficacy to diverse malignant tumors.
Reagent-loaded plastic microfluidic chips for detecting homocysteine
International Nuclear Information System (INIS)
Suk, Ji Won; Jang, Jae-Young; Cho, Jun-Hyeong
This report describes the preliminary study on plastic microfluidic chips with pre-loaded reagents for detecting homocysteine (Hcy). All reagents needed in an Hcy immunoassay were included in a microfluidic chip to remove tedious assay steps. A simple and cost-effective bonding method was developed to realize reagent-loaded microfluidic chips. This technique uses an intermediate layer between two plastic substrates by selectively patterning polydimethylsiloxane (PDMS) on the embossed surface of microchannels and fixing the substrates under pressure. Using this bonding method, the competitive immunoassay for SAH, a converted form of Hcy, was performed without any damage to reagents in chips, and the results showed that the fluorescent signal from antibody antigen binding decreased as the SAH concentration increased. Based on the SAH immunoassay, whole immunoassay steps for Hcy detection were carried out in plastic microfluidic chips with all necessary reagents. These experiments demonstrated the feasibility of the Hcy immunoassay in microfluidic devices
Dopamine-induced apoptosis in human neuronal cells: inhibition by nucleic acides antisense to the dopamine transporter
International Nuclear Information System (INIS)
Porat, S.; Gabbay, M.; Tauber, M.; Ratovitski, T.; Blinder, E.; Simantov, R.
Human neuroblastoma NMB cells take up [ 3 H]dopamine in a selective manner indicating that dopamine transporters are responsible for this uptake. These cells were therefore used as a model to study dopamine neurotoxicity, and to elucidate the role of dopamine transporters in controlling cell death. Treatment with 0.05-0.4 mM dopamine changed cells' morphology within 4 h, accompanied by retraction of processes, shrinkage, apoptosis-like atrophy, accumulation of apoptotic particles, DNA fragmentation and cell death. Cycloheximide inhibited dopamine's effect, suggesting that induction of apoptosis by dopamine was dependent upon protein synthesis. Dopamine cytotoxicity, monitored morphologically by flow cytometric analysis, and by lactate dehydrogenase released, was blocked by cocaine but not by the noradrenaline and serotonin uptake blockers desimipramine and imipramine, respectively. Attempting to inhibit dopamine transport and toxicity in a drug-free and highly selective way, three 18-mer dopamine transporter antisense phosphorothioate oligonucleotides (numbers 1, 2 and 3) and a new plasmid vector expressing the entire rat dopamine transporter complementary DNA in the antisense orientation were prepared and tested. Antisense phosphorothioate oligonucleotide 3 inhibited [ 3 H]dopamine uptake in a time- and dose-dependent manner. Likewise, transient transfection of NMB cells with the plasmid expressing dopamine transporter complementary DNA in the antisense orientation partially blocked [ 3 H]dopamine uptake. Antisense phosphorothioate oligonucleotide 3 also decreased, dose-dependently, the toxic effect of dopamine and 6-hydroxydopamine. Western blot analysis with newly prepared anti-human dopamine transporter antibodies showed that antisense phosphorothioate oligonucleotide 3 decreased the transporter protein level. These studies contribute to better understand the mechanism of dopamine-induced apoptosis and neurotoxicity. (Copyright (c) 1996 Elsevier Science B
Fast biosensor with reagent layer
A detection system and a sensor chip for detecting target mols., and thus corresponding analytes in a sample is described. Typically the detection system includes a sensor chip. The sensor chip (1) comprises on its detection surface a dissolvable reagent layer. When the dissolvable reagent layer is
Universal approach for selective trace metal determinations via sequential injection-bead injection-lab-on-valve using renewable hydrophobic bead surfaces as reagent carriers
DEFF Research Database (Denmark)
Long, Xiangbao; Miró, Manuel; Hansen, Elo Harald
involves the use of poly(styrene-divinylbenzene) beads containing pendant octadecyl moieties (C18-PS/DVB), which are preimpregnated with a selective organic metal chelating agent prior to the automatic manipulation of the beads in the microbore conduits of the LOV unit. By adapting this approach...
Synthesis of Alkanethiolate-Capped Metal Nanoparticles Using Alkyl Thiosulfate Ligand Precursors: A Method to Generate Promising Reagents for Selective Catalysis
Directory of Open Access Journals (Sweden)
Khin Aye San
Full Text Available Evaluation of metal nanoparticle catalysts functionalized with well-defined thiolate ligands can be potentially important because such systems can provide a spatial control in the reactivity and selectivity of catalysts. A synthetic method utilizing Bunte salts (sodium S-alkylthiosulfates allows the formation of metal nanoparticles (Au, Ag, Pd, Pt, and Ir capped with alkanethiolate ligands. The catalysis studies on Pd nanoparticles show a strong correlation between the surface ligand structure/composition and the catalytic activity and selectivity for the hydrogenation/isomerization of alkenes, dienes, trienes, and allylic alcohols. The high selectivity of Pd nanoparticles is driven by the controlled electronic properties of the Pd surface limiting the formation of Pd–alkene adducts (or intermediates necessary for (additional hydrogenation. The synthesis of water soluble Pd nanoparticles using ω-carboxylate-S-alkanethiosulfate salts is successfully achieved and these Pd nanoparticles are examined for the hydrogenation of various unsaturated compounds in both homogeneous and heterogeneous environments. Alkanethiolate-capped Pt nanoparticles are also successfully synthesized and further investigated for the hydrogenation of various alkynes to understand their geometric and electronic surface properties. The high catalytic activity of activated terminal alkynes, but the significantly low activity of internal alkynes and unactivated terminal alkynes, are observed for Pt nanoparticles.
Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.
Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira
Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.
Bare eye detection of Hg(II) ions based on enzyme inhibition and using mercaptoethanol as a reagent to improve selectivity.
He, Liuying; Lu, Yuexiang; Wang, Feiyang; Gao, Xinxin; Chen, Ying; Liu, Yueying
The authors describe a colorimetric method for the determination of Hg 2+ ions based on the inhibition of the activity of the enzyme urease. The pH value of solution increases when urease hydrolyzes urea, which can be visualized by adding a pH indicator such as Phenol Red (PhR). Mercaptoethanol as a typical thiol is added to the system to improve selectivity because it binds metal ions and then - unlike the Hg 2+ mercaptoethanol complex - does not inhibit urease. Hence, the color of the pH indicator PhR turns from yellow to pink as the solution becomes alkaline. The Hg 2+ mercaptoethanol complex, in contrast, strongly inhibits urease and the color of the solution remains yellow. The findings were used to design a photometric assay based on the measurement of the ratio of absorptions of PhR at 558 nm and 430 nm. It has a linear response over the 25 to 40 nM Hg 2+ concentration range and a 5 nM detection limit. This is well below the guideline values of Hg 2+ specified by the US Environmental Protection Agency and the World Health Organization for drinking water (10 nM and 30 nM, respectively). The method was employed to the determination of Hg 2+ in water samples spiked with 10 nM levels of Hg 2+ where color changes still can be observed visually. Graphical Abstract Schematic presentation of a colorimetric method for the ultrasensitive detection of Hg 2+ based on the inhibition of urease activity. Mercaptoethanol is used to improve the selectivity. Even at Hg 2+ concentrations as low as 5 nM, the color change still can be easily observed by bare eyes.
Antisense downregulation of mutant huntingtin in a cell model
DEFF Research Database (Denmark)
Hasholt, L.; Abell, K.; Norremolle, A.
or by addition to the culture medium. Results Expression of the fusion protein containing the mutant huntingtin fragment resulted in diffuse green fluorescence in the cytoplasm and formation of aggregates in some of the NT2 cells and NT2-N neurons. We obtained antisense sequence-specific inhibition of expression...... of the fusion protein and/or suppression of the aggregate formation in both cell types. In the NT2 cells the antisense effect was dependent on the way of administration of the oligo. Conclusions The PS-antisense oligo is effective in downregulation of mutant huntingtin, and the reduction of aggregate formation...... is a sensitive biological marker. The findings suggest that antisense knockdown of huntingtin could be a useful strategy for treatment of HD, and could also be suitable for studies of the normal and pathological function of huntingtin in different cellular model systems....
Peptide nucleic acid (PNA) antisense effects in Escherichia coli
DEFF Research Database (Denmark)
Good, L; Nielsen, P E
Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...
Modulation of lipoprotein metabolism by antisense technology: preclinical drug discovery methodology.
Crooke, Rosanne M; Graham, Mark J
Antisense oligonucleotides (ASOs) are a new class of specific therapeutic agents that alter the intermediary metabolism of mRNA, resulting in the suppression of disease-associated gene products. ASOs exert their pharmacological effects after hybridizing, via Watson-Crick base pairing, to a specific target RNA. If appropriately designed, this event results in the recruitment of RNase H, the degradation of targeted mRNA or pre-mRNA, and subsequent inhibition of the synthesis of a specific protein. A key advantage of the technology is the ability to selectively inhibit targets that cannot be modulated by traditional therapeutics such as structural proteins, transcription factors, and, of topical interest, lipoproteins. In this chapter, we will first provide an overview of antisense technology, then more specifically describe the status of lipoprotein-related genes that have been studied using the antisense platform, and finally, outline the general methodology required to design and evaluate the in vitro and in vivo efficacy of those drugs.
Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5.
Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H; Saltzman, W Mark; Glazer, Peter M
The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligomers designed to bind to the selective region of chemokine receptor 5 (CC R5) transcript, induce potent and sequence-specific antisense effects as compared with regular PNA oligomers. In addition, PLGA nanoparticle delivery of MPγPNAs is not toxic to the cells. The findings reported in this study provide a combination of γPNA technology and PLGA-based nanoparticle delivery method for regulating gene expression in live cells via the antisense mechanism.
Application of perfluorinated acids as ion-pairing reagents for reversed-phase chromatography and retention-hydrophobicity relationships studies of selected beta-blockers.
Flieger, J
The addition of the homologous series of perfluorinated acids-trifluoroacetic acid (TFAA), pentafluoropropionic acid (PFPA), heptafluorobutyric acid (HFBA) to mobile phases for reversed-phase high-performance liquid chromatography (RP-HPLC) of beta-blockers was tested. Acidic modifiers were responsible for acidification of mobile phase (pH 3) ensuring the protonation of the beta-blockers and further ion pairs creation. The effect of the type and concentration of mobile phase additives on retention parameters, the efficiency of the peaks, their symmetry and separation selectivity of the beta-blockers mixture were all studied. It appeared that at increasing acid concentration, the retention factor, for all compounds investigated, increased to varying degrees. It should be stressed that the presence of acids more significantly affected the retention of the most hydrophobic beta-blockers. Differences in hydrophobicity of drugs can be maximized through variation of the hydrophobicity of additives. Thus, the relative increase in the retention depends on either concentration and hydrophobicity of the anionic mobile phase additive or hydrophobicity of analytes. According to QSRR (quantitative structure retention relationship) methodology, chromatographic lipophilicity parameters: isocratic log k and log k(w) values (extrapolated retention to pure water) were correlated with the molecular (log P(o/w)) and apparent (log P(app)) octanol-water partition coefficients obtained experimentally by countercurrent chromatography (CCC) or predicted by Pallas software. The obtained, satisfactory retention-hydrophobicity correlations indicate that, in the case of the basic drugs examined in RP-HPLC systems modified with perfluorinated acids, the retention is mainly governed by their hydrophobicity. Copyright 2009 Elsevier B.V. All rights reserved.
Antisense Phosphorodiamidate Morpholino Oligomers as Novel Antiviral Compounds
Directory of Open Access Journals (Sweden)
Yuchen Nan
Full Text Available Phosphorodiamidate morpholino oligomers (PMO are short single-stranded DNA analogs that are built upon a backbone of morpholine rings connected by phosphorodiamidate linkages. As uncharged nucleic acid analogs, PMO bind to complementary sequences of target mRNA by Watson–Crick base pairing to block protein translation through steric blockade. PMO interference of viral protein translation operates independently of RNase H. Meanwhile, PMO are resistant to a variety of enzymes present in biologic fluids, a characteristic that makes them highly suitable for in vivo applications. Notably, PMO-based therapy for Duchenne muscular dystrophy (DMD has been approved by the United States Food and Drug Administration which is now a hallmark for PMO-based antisense therapy. In this review, the development history of PMO, delivery methods for improving cellular uptake of neutrally charged PMO molecules, past studies of PMO antagonism against RNA and DNA viruses, PMO target selection, and remaining questions of PMO antiviral strategies are discussed in detail and new insights are provided.
Profiled support vector machines for antisense oligonucleotide efficacy prediction
Directory of Open Access Journals (Sweden)
Martín-Guerrero José D
Full Text Available Abstract Background This paper presents the use of Support Vector Machines (SVMs for prediction and analysis of antisense oligonucleotide (AO efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1 feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE, and (2 AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions. Results In the first stage, the SVM-RFE technique was most efficient and robust in the presence of low number of samples and high input space dimension. This method yielded an optimal subset of 14 representative features, which were all related to energy and sequence motifs. The second stage evaluated the performance of the predictors (overall correlation coefficient between observed and predicted efficacy, r; mean error, ME; and root-mean-square-error, RMSE using 8-fold and minus-one-RNA cross-validation methods. The profiled SVM produced the best results (r = 0.44, ME = 0.022, and RMSE= 0.278 and predicted high (>75% inhibition of gene expression and low efficacy (http://aosvm.cgb.ki.se/. Conclusions The SVM approach is well suited to the AO prediction problem, and yields a prediction accuracy superior to previous methods. The profiled SVM was found to perform better than the standard SVM, suggesting that it could lead to improvements in other prediction problems as well.
Decontaminating reagents for radioactive systems
International Nuclear Information System (INIS)
Seddon, W.A.
A decontaminating reagent composition has been developed comprising EDTA, citric acid, oxalic acid, and formic acid. Formic acid inhibits the decomposition of both EDTA and citric acid, and yields oxalic acid as a result of its own radiolysis. The invention includes the improvement of initially incorporating formic acid in the mixture and maintaining the presence of formic acid by at least one further addition
Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.
Directory of Open Access Journals (Sweden)
Christopher A Lavender
Full Text Available Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment.
Antisense oligonucleotide inhibition of apolipoprotein C-III reduces plasma triglycerides in rodents, nonhuman primates, and humans.
Graham, Mark J; Lee, Richard G; Bell, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M
Elevated plasma triglyceride levels have been recognized as a risk factor for the development of coronary heart disease. Apolipoprotein C-III (apoC-III) represents both an independent risk factor and a key regulatory factor of plasma triglyceride concentrations. Furthermore, elevated apoC-III levels have been associated with metabolic syndrome and type 2 diabetes mellitus. To date, no selective apoC-III therapeutic agent has been evaluated in the clinic. To test the hypothesis that selective inhibition of apoC-III with antisense drugs in preclinical models and in healthy volunteers would reduce plasma apoC-III and triglyceride levels. Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman primates. We demonstrated the selective reduction of both apoC-III and triglyceride in all preclinical pharmacological evaluations. We also showed that inhibition of apoC-III was well tolerated and not associated with increased liver triglyceride deposition or hepatotoxicity. A double-blind, placebo-controlled, phase I clinical study was performed in healthy subjects. Administration of the human apoC-III antisense drug resulted in dose-dependent reductions in plasma apoC-III, concomitant lowering of triglyceride levels, and produced no clinically meaningful signals in the safety evaluations. Antisense inhibition of apoC-III in preclinical models and in a phase I clinical trial with healthy subjects produced potent, selective reductions in plasma apoC-III and triglyceride, 2 known risk factors for cardiovascular disease. This compelling pharmacological profile supports further clinical investigations in hypertriglyceridemic subjects.
Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis
Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.
Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that
Alteration of rice growth and development via antisense expression ...
African Journals Online (AJOL)
OsGA20ox2 in regulating plant growth and development, we used reverse genomic approach to ... pathways. Similarly, Carmen et al. (2007) suggested that. Carrizo citrange plants have produced antisense ... universal SP6 and T7 primers to conform their reality (Sangon, ..... Optimising the tissue culture conditions for.
Lysine metabolism in antisense C-hordein barley grains
DEFF Research Database (Denmark)
Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A
The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with ...
Advancements of antisense oligonucleotides in treatment of breast cancer
Institute of Scientific and Technical Information of China (English)
YANGShuan-Ping; SONGSan-Tai; 等
Breast cancer is one kind of multi-gene related malignancy.Overexpression of some oncogenes such as HER-2(c-erbB-2,Neu),bcl-2/bcl-xL,protein kinase A(PKA),and transferrin receptor gene(TfR gene),etc significantly affect the prognosis of breast cancer.It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer.Antisense interference.one of useful tools for inhibiting the overexpression of specific oncogenes,was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides(ON)could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases;some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts.Furthermore,the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects,which was probably the best utilization of antisense ON in the treatment of breast cancer.
Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)
DEFF Research Database (Denmark)
Brolin, Camilla; Shiraishi, Takehiko
Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most...
Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics
Directory of Open Access Journals (Sweden)
Moizza Mansoor
Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.
Characterization of Reagent Pencils for Deposition of Reagents onto Paper-Based Microfluidic Devices
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Cheyenne H. Liu
Full Text Available Reagent pencils allow for solvent-free deposition of reagents onto paper-based microfluidic devices. The pencils are portable, easy to use, extend the shelf-life of reagents, and offer a platform for customizing diagnostic devices at the point of care. In this work, reagent pencils were characterized by measuring the wear resistance of pencil cores made from polyethylene glycols (PEGs with different molecular weights and incorporating various concentrations of three different reagents using a standard pin abrasion test, as well as by measuring the efficiency of reagent delivery from the pencils to the test zones of paper-based microfluidic devices using absorption spectroscopy and digital image colorimetry. The molecular weight of the PEG, concentration of the reagent, and the molecular weight of the reagent were all found to have an inverse correlation with the wear of the pencil cores, but the amount of reagent delivered to the test zone of a device correlated most strongly with the concentration of the reagent in the pencil core. Up to 49% of the total reagent deposited on a device with a pencil was released into the test zone, compared to 58% for reagents deposited from a solution. The results suggest that reagent pencils can be prepared for a variety of reagents using PEGs with molecular weights in the range of 2000 to 6000 g/mol.
Targeting antisense mitochondrial ncRNAs inhibits murine melanoma tumor growth and metastasis through reduction in survival and invasion factors.
Lobos-González, Lorena; Silva, Verónica; Araya, Mariela; Restovic, Franko; Echenique, Javiera; Oliveira-Cruz, Luciana; Fitzpatrick, Christopher; Briones, Macarena; Villegas, Jaime; Villota, Claudio; Vidaurre, Soledad; Borgna, Vincenzo; Socias, Miguel; Valenzuela, Sebastián; Lopez, Constanza; Socias, Teresa; Varas, Manuel; Díaz, Jorge; Burzio, Luis O; Burzio, Verónica A
We reported that knockdown of the antisense noncoding mitochondrial RNAs (ASncmtRNAs) induces apoptotic death of several human tumor cell lines, but not normal cells, suggesting this approach for selective therapy against different types of cancer. In order to translate these results to a preclinical scenario, we characterized the murine noncoding mitochondrial RNAs (ncmtRNAs) and performed in vivo knockdown in syngeneic murine melanoma models. Mouse ncmtRNAs display structures similar to the human counterparts, including long double-stranded regions arising from the presence of inverted repeats. Knockdown of ASncmtRNAs with specific antisense oligonucleotides (ASO) reduces murine melanoma B16F10 cell proliferation and induces apoptosis in vitro through downregulation of pro-survival and metastasis markers, particularly survivin. For in vivo studies, subcutaneous B16F10 melanoma tumors in C57BL/6 mice were treated systemically with specific and control antisense oligonucleotides (ASO). For metastasis studies, tumors were resected, followed by systemic administration of ASOs and the presence of metastatic nodules in lungs and liver was assessed. Treatment with specific ASO inhibited tumor growth and metastasis after primary tumor resection. In a metastasis-only assay, mice inoculated intravenously with cells and treated with the same ASO displayed reduced number and size of melanoma nodules in the lungs, compared to controls. Our results suggest that ASncmtRNAs could be potent targets for melanoma therapy. To our knowledge, the ASncmtRNAs are the first potential non-nuclear targets for melanoma therapy.
Bcl-2 antisense therapy in B-cell malignancies.
Chanan-Khan, Asher
Bcl-2 is an apoptosis regulating protein, overexpression of which is associated with chemotherapy resistant disease, aggressive clinical course, and poor survival in patients with B-cell lymphoproliferative disorders. Overexpression of Bcl-2 protein results in an aberrant intrinsic apoptotic pathway that confers a protective effect on malignant cells against a death signal (e.g., chemotherapy or radiotherapy). Downregulation of this oncoprotein, thus, represents a possible new way to target clinically aggressive disease. Preclinical studies have shown that this oncoprotein can be effectively decreased by Bcl-2 antisense in malignant lymphoid cells and can reverse chemotherapy resistance, as well as enhance the anti-apoptotic potential of both chemotherapeutic and biologic agents. Ongoing clinical trials are exploring the role of Bcl-2 downregulation with oblimersen (Bcl-2 antisense) in patients with non-Hodgkin's lymphoma, chronic lymphocytic leukemia and multiple myeloma. Early results from these studies are promising and support the proof of the principle. As these studies are completed and mature data emerges, the role of Bcl-2 antisense therapy in the treatment of B-cell malignancies will become clearer.
A reagent for processing drilling muds
Energy Technology Data Exchange (ETDEWEB)
Polyakov, G.A.; Khon-Pak, A.T.; Khon, A.V.; Normatov, L.N.; Telegin, B.V.
A reagent is proposed for processing drilling muds. It contains an acrylic polymer and potassium permanganate. The reagent is distinguished by the fact that in order to improve the quality of the drilling muds by increasing their salt resistance, the reagent contains hydrolized nitron fiber as the acrylic polymer with the following component relationship (in percent by weight): potassium permanganate, 0.015 to 0.065 and hydrolyzed nitron fiber, the remainder.
A new selective chromogenic reagent for the spectrophotometric determination of thallium(I) and (III) and its separation using flotation and the solid-phase extraction on polyurethane foam.
Abou-El-Sherbini, Khaled S; Mostafa, Gamal A E; Hassanien, Mohamed M
A new sensitive chromogenic reagent, 9,10-phenanthaquinone monoethylthiosemicarbazone (PET), has been synthesized and used in the spectrophotometric determination of Tl(III). In HNO3, H2SO4 or H3PO4 acids, PET can react immediately at room temperature with Tl(III) to form a red 2:1 complex with a maximum absorption at 516 nm. The different analytical parameters affecting the extraction and determination processes have been examined. The calibration curve was found to be linear over the range 0.2-10 microg cm(-3) with a molar absorptivity of 2.2 x 10(4) dm3 mol(-1) cm(-1). Sandell's sensitivity was found to be 0.0093 microg cm(-2). No interference from macroamounts of foreign ions was detected, except for Pd(II). However, Pd(II) does not affect the determination process, because its complex with PET has its lambda(max) at 625 nm. The proposed method has been applied to the determination of Tl(I and III) in synthetic and natural samples after separation by flotation (in oleic acid/kerosene) and solid-phase extraction (on polyurethane foam) techniques. The two methods were found to be accurate and not subject to random error, but solid-phase extraction was preferred because it is cheap, simpler and there is no contamination risk coming from flotation reagents.
Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe
Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription ...
Organic-soluble lanthanide nuclear magnetic resonance shift reagents for sulfonium and isothiouronium salts
International Nuclear Information System (INIS)
Wenzel, T.J.; Zaia, J.
Lanthanide complexes of the formula [Ln(fod) 4 ] - (FOD, 6,6,7,7,8,8,8-heptafluoro-2,2-dimethyl-3,5-octanedione) are effective organic-soluble nuclear magnetic resonance shift reagents for sulfonium and isothiouronium salts. The shift reagent is formed in solution from Ln(fod) 3 and Ag(fod) or K(fod). The selection of Ag(fod) or K(fod) in forming the shift reagent is dependent on the anion of the organic salt. Ag(fod) is more effective with halide salts, whereas K(fod) is preferred with tetrafluoroborate salts. Resolution of diastereotopic hydrogen atoms was observed in the shifted spectra of certain substrates. Enantiomeric resolution was obtained in the spectrum of sec-butylisothiouronium chloride with a chiral shift reagent. The reagents can be employed in solvents such as chloroform and benzene
Effect of some colloid surfactants on spectrophotometric characteristics of metal chelates with chromophore organic reagents
International Nuclear Information System (INIS)
Chernova, R.K.
Theoretical regularities and prospects of using surface active substances (SAS) in spectrophotometric determination of metal ions (including ions of rare-earth elements, transition metals, Be(3)) with chromophore chelating reagents were investigated. The chromophore reagents investigated were pyrocatechol violet, phenolcarboxylic acids of the triarylmethane series, fluorones, phthalexones and azo-compounds. As SAS certain long-chain quaternary ammonium and pyridinium salts (LQAS) were employed. From the results reported it follows that the introduction of LQAS in the system of Mesup(n+)-chromophore reagent is a rather effective method of enhancing the contrast rendition and, in some cases, the sensitivity and selectivity of the reagents. Explanations are suggested as to the factors which cause the changes observed in the contrast of the reactions in the presence of SAS; the underlying phenomena are the ligand-ligand interactions between the organic reagents and SAS and solubilization processes of the reaction products by the micelles of SAS
Biodegradable polymer nanocarriers for therapeutic antisense microRNA delivery in living animals
Paulmurugan, Ramasamy; Sekar, Narayana M.; Sekar, Thillai V.
MicroRNAs are endogenous regulators of gene expression, deregulated in several cellular diseases including cancer. Altering the cellular microenvironment by modulating the microRNAs functions can regulate different genes involved in major cellular processes, and this approach is now being investigated as a promising new generation of molecularly targeted anti-cancer therapies. AntagomiRs (Antisense-miRNAs) are a novel class of chemically modified stable oligonucleotides used for blocking the functions of endogenous microRNAs, which are overexpressed. A key challenge in achieving effective microRNAbased therapeutics lies in the development of an efficient delivery system capable of specifically delivering antisense oligonucleotides and target cancer cells in living animals. We are now developing an effective delivery system designed to selectively deliver antagomiR- 21 and antagomiR-10b to triple negative breast cancer cells, and to revert tumor cell metastasis and invasiveness. The FDA-approved biodegradable PLGA-nanoparticles were selected as a carrier for antagomiRs delivery. Chemically modified antagomiRs (antagomiR-21 and antagomiR-10b) were co-encapsulated in PEGylated-PLGA-nanoparticles by using the double-emulsification (W/O/W) solvent evaporation method, and the resulting average particle size of 150-200nm was used for different in vitro and in vivo experiments. The antagomiR encapsulated PLGA-nanoparticles were evaluated for their in vitro antagomiRs delivery, intracellular release profile, and antagomiRs functional effects, by measuring the endogenous cellular targets, and the cell growth and metastasis. The xenografts of tumor cells in living mice were used for evaluating the anti-metastatic and anti-invasive properties of cells. The results showed that the use of PLGA for antagomiR delivery is not only efficient in crossing cell membrane, but can also maintain functional intracellular antagomiRs level for a extended period of time and achieve
The development of a neutralizing amines based reagent for maintaining the water chemistry for medium and high pressures steam boilers
Butakova, M. V.; Orlov, K. A.; Guseva, O. V.
An overview of the development for neutralizing amine based reagent for water chemistry of steam boilers for medium and high pressures was given. Total values of the neutralization constants and the distribution coefficients of the compositions selected as a main criteria for reagent composition. Experimental results of using this new reagent for water chemistry in HRSG of power plant in oil-production company are discussed.
SINEUPs are modular antisense long-non coding RNAs that increase synthesis of target proteins in cells
Directory of Open Access Journals (Sweden)
Silvia eZucchelli
Full Text Available Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1, is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD while the embedded inverted SINEB2 element is the Effector Domain (ED. By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed towards N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines.In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.
Effective intracellular delivery of oligonucleotides in order to make sense of antisense
Shi, FX; Hoekstra, D
For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the
Inactivation of rabies diagnostic reagents by gamma radiation
International Nuclear Information System (INIS)
Gamble, W.C.; Chappell, W.A.; George, E.H.
Treatment of CVS-11 rabies adsorbing suspensions and street rabies infected mouse brains with gamma radiation resulted in inactivated reagents that are safer to distribute and use. These irradiated reagents were as sensitive and reactive as the nonirradiated control reagents
Developmental transitions in Arabidopsis are regulated by antisense RNAs resulting from bidirectionally transcribed genes.
Krzyczmonik, Katarzyna; Wroblewska-Swiniarska, Agata; Swiezewski, Szymon
Transcription terminators are DNA elements located at the 3' end of genes that ensure efficient cleavage of nascent RNA generating the 3' end of mRNA, as well as facilitating disengagement of elongating DNA-dependent RNA polymerase II. Surprisingly, terminators are also a potent source of antisense transcription. We have recently described an Arabidopsis antisense transcript originating from the 3' end of a master regulator of Arabidopsis thaliana seed dormancy DOG1. In this review, we discuss the broader implications of our discovery in light of recent developments in yeast and Arabidopsis. We show that, surprisingly, the key features of terminators that give rise to antisense transcription are preserved between Arabidopsis and yeast, suggesting a conserved mechanism. We also compare our discovery to known antisense-based regulatory mechanisms, highlighting the link between antisense-based gene expression regulation and major developmental transitions in plants.
Extensive polycistronism and antisense transcription in the mammalian Hox clusters.
Directory of Open Access Journals (Sweden)
Gaëll Mainguy
Full Text Available The Hox clusters play a crucial role in body patterning during animal development. They encode both Hox transcription factor and micro-RNA genes that are activated in a precise temporal and spatial sequence that follows their chromosomal order. These remarkable collinear properties confer functional unit status for Hox clusters. We developed the TranscriptView platform to establish high resolution transcriptional profiling and report here that transcription in the Hox clusters is far more complex than previously described in both human and mouse. Unannotated transcripts can represent up to 60% of the total transcriptional output of a cluster. In particular, we identified 14 non-coding Transcriptional Units antisense to Hox genes, 10 of which (70% have a detectable mouse homolog. Most of these Transcriptional Units in both human and mouse present conserved sizeable sequences (>40 bp overlapping Hox transcripts, suggesting that these Hox antisense transcripts are functional. Hox clusters also display at least seven polycistronic clusters, i.e., different genes being co-transcribed on long isoforms (up to 30 kb. This work provides a reevaluated framework for understanding Hox gene function and dys-function. Such extensive transcriptions may provide a structural explanation for Hox clustering.
Respirable antisense oligonucleotides: a new drug class for respiratory disease
Directory of Open Access Journals (Sweden)
Tanaka Makoto
Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.
21 CFR 866.3780 - Toxoplasma gondii serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3780 Toxoplasma... (immunofluorescent reagents) used to identify Toxoplasma gondii from clinical specimens. The identification aids in...
21 CFR 866.3375 - Mycoplasma spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3375 Mycoplasma... fluorescent dye (immunofluorescent reagents) used to identify Mycoplasma spp. directly from clinical specimens...
21 CFR 866.3220 - Entamoeba histolytica serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3220... fluorescent dye (immunofluorescent reagents) used to identify Entamoeba histolytica directly from clinical...
Identification of mimotopes of Mycobacterium leprae as potential diagnostic reagents
Directory of Open Access Journals (Sweden)
Alban Silvana M
Full Text Available Abstract Background An early diagnostic test for detecting infection in leprosy is fundamental for reducing patients' sequelae. The currently used lepromin is not adequate for disease diagnosis and, so far, no antigen to be used in intradermoreaction has proved to be sensitive and specific for that purpose. Aiming at identifying new reagents to be used in skin tests, candidate antigens were investigated. Methods Random peptide phage display libraries were screened by using antibodies from leprosy patients in order to identify peptides as diagnostic reagents. Results Seven different phage clones were identified using purified antibodies pooled from sera of leprosy patients. When the clones were tested with serum samples by ELISA, three of them, 5A, 6A and 1B, allowed detecting a larger number of leprosy patients when compared to controls. The corresponding peptides expressed by selected phage clones were chemically synthesized. A pilot study was undertaken to assess the use of peptides in skin tests. The intradermal challenge with peptides in animals previously sensitized with Mycobacterium leprae induced a delayed-type hypersensitivity with peptide 5A (2/5 and peptide 1B (1/5. In positive controls, there was a 3/5 reactivity for lepromin and a 4/5 reactivity of the sensitized animals with soluble extract of M. leprae. Conclusions The preliminary data suggest that may be possible to develop reagents with diagnostic potential based on peptide mimotopes selected by phage display using polyclonal human antibodies.
Development of national immunoassay reagent programmes
International Nuclear Information System (INIS)
Sufi, S.B.; Micallef, J.V.; Ahsan, R.; Goncharov, N.P.
Despite the existence of networks of fully equipped laboratories with well-trained staff, the availability of immunodiagnostic services in developing countries is often limited by the high cost of imported kits. There are a number of ways of tackling this problem, ranging from bulk purchase of kits or reagents to local development and production of assay systems. Argentina/Chile, China, Cuba/Mexico, and Thailand are amongst the countries which have established local immunoassay reagent programmes to manufacture low cost, high quality immunoassay reagents. Kits from these projects are now beginning to become available, and it is hoped that they will promote national diagnostic services and research, as well as stimulating the development of reagent programmes for other analytes. (author). 4 refs, 1 tab
Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology
Directory of Open Access Journals (Sweden)
Jutharat Attajarusit
Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants
21 CFR 866.3200 - Echinococcus spp. serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Echinococcus spp. serological reagents. 866.3200... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3200 Echinococcus spp. serological reagents. (a) Identification. Echinococcus spp. serological reagents are devices that...
21 CFR 866.3405 - Poliovirus serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Poliovirus serological reagents. 866.3405 Section... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3405 Poliovirus serological reagents. (a) Identification. Poliovirus serological reagents are devices that consist of antigens...
21 CFR 866.3415 - Pseudomonas spp. serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Pseudomonas spp. serological reagents. 866.3415... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3415 Pseudomonas spp. serological reagents. (a) Identification. Pseudomonas spp. serological reagents are devices that...
21 CFR 866.3350 - Leptospira spp. serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Leptospira spp. serological reagents. 866.3350... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3350 Leptospira spp. serological reagents. (a) Identification. Leptospira spp. serological reagents are devices that...
21 CFR 866.3500 - Rickettsia serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rickettsia serological reagents. 866.3500 Section... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3500 Rickettsia serological reagents. (a) Identification. Rickettsia serological reagents are devices that consist of antigens...
Functions of chalcogenide electrodes in solutions of complexing reagents and interfering ions
International Nuclear Information System (INIS)
Kiyanskij, V.V.
The possibility to modify chalcogenide electrodes and their behaviour in solutions of complexing reagents for the development of new methods of potentiometric titration has been studied. It is shown that complexing reagents (EDTA, cupferron, 8-hydroxyquinoline, sodium dithiocarbaminate) and Cu(2), Hg(2) produce a strong effect on the functions of Ag, Cu, Cd, Pb - selective electrodes, which is used for titration of potential-determining and non-potential-determining ions ions (Sr 2+ , La 3+ etc.) and also for modification of sulfide-selecting electrode. A method of potentiometric titration of sulfates and chlorides with modified Cd- and Ag-selective electrodes is suggested
Production of reagents for cleaning fluids
Energy Technology Data Exchange (ETDEWEB)
Grunberg, I V; Korostyleva, R N; Pytel, S P; Spasskii, P I; Titarenko, N K; Trachtenberg, S I; Yushkevich, V I
A method for producing reagents for cleaning fluids is proposed using polymerization of acrylonitril, metachrylate or a mixture of the two in water and saponification of the polymers with alakali. To reduce the consumption of monomers and increase the quality of the reagents, 0.4-1.0 parts humic substances, 0.2-1.0 parts hydrolizate from tanning waste products and 1.2-4.0 parts monomers are added to the reaction medium, followed by copolymerization in an acid medium. The proposed method ensures quality reagents which combine lower water yield with a moderate increase in viscosity when acting on clay solutions. Compared with the current method, this method lowers the consumption of an expensive and hard-to-find monomer 1.2-1.4X for one ton of reagent, which lowers the cost of raw material by 1.3-1.7X. This results in a savings of 195-385 rubles per ton of reagent, 600-1200 thousand at 3000 tons/yr.
Industrial detergent wastewater treatment via fenton reagent
International Nuclear Information System (INIS)
Mohd Zairie Mohd Yusuff; Mohd Zulkifli Mohamad Noor; Izirwan Izhab
Production of detergent can generates wastewater containing an organic matter with will consume an oxidation demand, surfactants, suspended solids, fat and oil. Besides, sulfate concentration is high in the most detergent plant effluent because of the sulphonation process that has physiological and toxic effects on marine organisms. Therefore, a research must be conducted to find the solution for this problem. The feasibility of Fentons reagent to treat detergent waste was investigated in this study. The sample of detergent wastewater was taken from FPG Oleo chemicals Sdn. Bhd. This experiment studied the effect of temperature towards the feasibility of Fentons reagent process besides the dosage between hydrogen peroxide (H 2 O 2 ) and ferrous ion (Fe 2+ ) in the reagent. While, evaluated efficiency of Fentons reagent in term of chemical oxygen demand (COD), total suspended solid (TSS) and the turbidity reduction within the experimental design. The result found that overall removal was achieved until 96.2 % in term of COD, 98.1 % in term of TSS and 99.6 % in term of turbidity using Fentons reagent process. Besides, also found that this process is optimum at temperature 35 degree Celsius are able to achieve the Standard A of Parameter Limit of Effluent of Standard A and Standard B were outlined by Department of Environment Malaysia (DOE) based on Environment Quality Act 1974. (author)
Growth inhibition of human pancreatic cancer cells by lipofection mediated IGF-1R antisense oligodeoxynucletides in combination with ionizing radiation
International Nuclear Information System (INIS)
Pan Yaozhen; Sun Chengyi; Wang Yuzhi
Objective: To study the growth inhibition of human pancreatic cancer cells (PC-3) by lipofection-mediated and ionizing radiation improving transfection of IGF-1R antisense oligodeoxynucletides (ASON) in vitro. Methods: Colonigenicity of PC-3 cells in vitro after 60 Co γ-radiation was observed for ascertaining their radiosensitivity and optimal radiation dose was selected according to the radiation sensitivity. PC-3 cells were transfected by two ways: 1) by lipofection-mediated IGF-1R ASON combined with ionizing radiation. 2) by lipo-ASON alone without ionizing radiation. Cell growth was assessed by MTT method. The expression of IGF-1R at mRNA level was examined by RT-PCR. Flow cytometry was used to demonstrate apoptotic changes in lipo-ASON-treated cells. Results: The inhibitory efficiency of lipo-ASON combined with ionizing radiation was higher than that without ionizing radiation (P < 0.05). The apoptotic efficiency and the decreased level of IGF-1R at mRNA were significantly improved (P < 0.05). Conclusion: Lipofection-mediated and ionizing radiation-promoted transfection of IGF-1R antisense oligodeoxynucletides (ASON) significantly decreases IGF-1R at mRNA level and induces apoptosis of human pancreatic cancer cells in vitro
Dual-Specificity Anti-HER-2/neu Antisense DNA Agents for Breast Cancer Therapy
National Research Council Canada - National Science Library
Stein, Stanley
.... To achieve high avidity and specificity, we designed chimeric antisense molecules consisting of a short active DNA fused to a short "anchor" 2'-0-methyl RNA complementary to non-contiguous single...
A Universal Approach for Selective Trace Metal Determinations via Sequential Injection-Bead Injection-Lab-on-Valve (SI-BI-LOV) Using Renewable Reagent-loaded Hydrophobic Beads
DEFF Research Database (Denmark)
Long, Xiangbao; Miró, Manuel; Hansen, Elo Harald
-Lab-on-Valve (SI-LOV) mode. The methodology uses poly(styrene-divinylbenzene) beads containing pendant octadecyl moieties (C18-PS/DVB), which are pre-impregnated with a selective organic metal chelating agent prior to the automatic manipulation of the beads in the microbore conduits of the LOV unit. By adapting...
Reagent for treating clay drilling muds
Energy Technology Data Exchange (ETDEWEB)
Tkachenko, P V; Leshchinskiy, P A; Shnaper, B I; Zinchuk, I F; Zlobin, V P
A reagent is proposed for treating clay drilling muds. It contains lignite, caustic soda and modifying agent. It is distinguished by the fact that in order to reduce the cost of the reagent with simultaneous decrease in the viscosity and static shear stress of the drilling mud, it additionally contains iron sulfate, and the modifying agent contained is wastes of carbonic acid production with the following ratio of components (parts by weight): lignite 10.0-15.0, caustic soda 2.0-3.0, wastes of carbonic acid production 0.5-0.75; iron sulfate 1.0-2.0.
Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.
Chen, Huei-Mei; Rosebrock, Adam P; Khan, Sohail R; Futcher, Bruce; Leatherwood, Janet K
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.
Identification of antisense long noncoding RNAs that function as SINEUPs in human cells.
Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari; Gustincich, Stefano; Carninci, Piero
Mammalian genomes encode numerous natural antisense long noncoding RNAs (lncRNAs) that regulate gene expression. Recently, an antisense lncRNA to mouse Ubiquitin carboxyl-terminal hydrolase L1 (Uchl1) was reported to increase UCHL1 protein synthesis, representing a new functional class of lncRNAs, designated as SINEUPs, for SINE element-containing translation UP-regulators. Here, we show that an antisense lncRNA to the human protein phosphatase 1 regulatory subunit 12A (PPP1R12A), named as R12A-AS1, which overlaps with the 5' UTR and first coding exon of the PPP1R12A mRNA, functions as a SINEUP, increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate for the first time that human natural antisense lncRNAs can up-regulate protein translation, suggesting that endogenous SINEUPs may be widespread and present in many mammalian species.
Repression of Meiotic Genes by Antisense Transcription and by Fkh2 Transcription Factor in Schizosaccharomyces pombe
Chen, Huei-Mei; Rosebrock, Adam P.; Khan, Sohail R.; Futcher, Bruce; Leatherwood, Janet K.
In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression. PMID:22238674
Repression of meiotic genes by antisense transcription and by Fkh2 transcription factor in Schizosaccharomyces pombe.
Directory of Open Access Journals (Sweden)
Huei-Mei Chen
Full Text Available In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. Disruption of the antisense transcription of three of these mid-meiotic genes allowed vegetative sense transcription. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the "unspliced" signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, and we argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that vegetative expression of mid-meiotic genes is repressed at least two independent ways: antisense transcription and Fkh2 repression.
A Snippet of Grignard Reagent's History
Indian Academy of Sciences (India)
Home; Journals; Resonance – Journal of Science Education; Volume 18; Issue 8. A Snippet of Grignard Reagent's History. Sujan Singh Dua. Classroom Volume 18 Issue 8 August 2013 pp 777-780. Fulltext. Click here to view fulltext PDF. Permanent link: https://www.ias.ac.in/article/fulltext/reso/018/08/0777-0780. Keywords.
Combined sewage samples obtained from a wastewater treatment facility were disinfected by the Fenton's Reagent of several different compositions. The pre-settled samples contained both suspended solids (SS) and dissolved organic carbon (DOC) at concentrations of 28 and 290 mg/L,...
Specific RNP capture with antisense LNA/DNA mixmers.
Rogell, Birgit; Fischer, Bernd; Rettel, Mandy; Krijgsveld, Jeroen; Castello, Alfredo; Hentze, Matthias W
RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins. © 2017 Rogell et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
Recent advances in antisense oligonucleotide therapy in genetic neuromuscular diseases
Directory of Open Access Journals (Sweden)
Ashok Verma
Full Text Available Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Mutant genes may reduce expression of wild-type proteins, and strategies to activate expression of the wild-type proteins might provide therapeutic benefits. Also, a toxic mutant protein may cause cell death, and strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO can recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their safety and target delivery, and scientific controlled clinical trials to ascertain their therapeutic safety and efficacy have led to an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. Over the past 1 year, for the first time, the United States Food and Drug Administration has approved two ASO therapies in genetic neuromuscular diseases. This overview summarizes the recent advances in ASO technology, evolution and use of synthetic ASOs as a therapeutic platform, and the mechanism of ASO action by exon-skipping in Duchenne muscular dystrophy and exon-inclusion in spinal muscular atrophy, with comments on their advantages and limitations.
Antisense inhibition of hyaluronan synthase-2 in human osteosarcoma cells inhibits hyaluronan retention and tumorigenicity
International Nuclear Information System (INIS)
Nishida, Yoshihiro; Knudson, Warren; Knudson, Cheryl B.; Ishiguro, Naoki
Osteosarcoma is a common malignant bone tumor associated with childhood and adolescence. The results of numerous studies have suggested that hyaluronan plays an important role in regulating the aggressive behavior of various types of cancer cells. However, no studies have addressed hyaluronan with respect to osteosarcomas. In this investigation, the mRNA expression copy number of three mammalian hyaluronan synthases (HAS) was determined using competitive RT-PCR in the osteoblastic osteosarcoma cell line, MG-63. MG-63 are highly malignant osteosarcoma cells with an abundant hyaluronan-rich matrix. The results demonstrated that HAS-2 is the predominant HAS in MG-63. Accumulation of intracellular hyaluronan increased in association with the proliferative phase of these cells. The selective inhibition of HAS-2 mRNA in MG-63 cells by antisense phosphorothioate oligonucleotides resulted in reduced hyaluronan accumulation by these cells. As expected, the reduction in hyaluronan disrupted the assembly of cell-associated matrices. However, of most interest, coincident with the reduction in hyaluronan, there was a substantial decrease in cell proliferation, a decrease in cell motility and a decrease in cell invasiveness. These data suggest that hyaluronan synthesized by HAS-2 in MG-63 plays a crucial role in osteosarcoma cell proliferation, motility, and invasion
Polymer-supported reagents with enhanced metal ion recognition: Application to separations science
International Nuclear Information System (INIS)
Alexandratos, S.D.
The design and development of polymer-supported reagents with ever-increasing specificities for targeted metal ions remains an important areas of research. The need for efficient separation schemes for both ions and molecules has been outlined in a report by the National Research Council (King) and will gain increased emphasis as environmental restoration is pursued. Polymer-supported reagents are unique in their ability to be applied in an environmentally benign manner to a host of challenges. Such reagents, in the form of beads, can be applied to continuous separation processes ranging from the removal of metal ions in water to the recovery of medicinal drugs produced through biotechnological means. The application of polymer-supported reagents to metal ion separations still requires developing a fundamental understanding of ligand-metal interactions, the role of the polymer in those interactions, and the methods of synthesizing such polymeric reagents in a readily applicable form. Ion exchange resins with sulfonic acid ligands are the prototypical polymer-supported reagents, and their properties have been exhaustively studied (Helfferich). The high acidity of the sulfonic acid group, however, precludes much selectivity, and it displays a very limited range of reaction free energy values with different metal ions (Boyd et al.). The carboxylic acid ligand, present in the acrylate resins, is more selective, though its weak acidity requires relatively high pH solutions for it to be effective. Research has thus been focused on the preparation of polymer-supported reagents with high levels of specificity for targeted metal ions
Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum
Directory of Open Access Journals (Sweden)
López-Barragán María J
Full Text Available Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.
Increased cell survival by inhibition of BRCA1 using an antisense approach in an estrogen responsive ovarian carcinoma cell line
International Nuclear Information System (INIS)
Annab, Lois A; Hawkins, Rebecca E; Solomon, Greg; Barrett, J Carl; Afshari, Cynthia A
protection assay (Fig. 1a). BRCA1 ribonuclease protection probe was made using an in vitro transcription kit (Ambion, Inc, Austin, TX, USA) as previously described [10] and derived clones were tested for protein levels by Western blot analysis using an anti-BRCA1 (Oncogene Research, Ab-1, Cambridge, MA, USA) antibody. Growth curve analysis of Infected populations and were pretreated for 5 days in phenol red-free, Dulbecco's modified eagle medium (DMEM)/F-12 medium (Gibco/Life Technologies) supplemented with 10% charcoal/dextran treated serum (Hyclone, Logan, UT, USA), then plated at 2.5 × 10 6 cells per 100mm dish in triplicate in the absence or presence of estrogen (10 -8 mol/l; 17β-Estradiol; 1,3,5 (10) - Estratriene 3,17β-diol; Sigma, St Louis, MO, USA). For soft agar assay, clones were plated into 10 60-mm dishes at 1 × 10 5 cells/dish containing 0.3% bactopeptone agar with or without added estrogen (10 -8 mol/l) in phenol red-free medium with 10% stripped serum in order to test for anchorage independent growth. BG-1 infected clones were tested for tumorigenicity by injection of cells (10 6 cells in 0.1cm 2 50% matrigel; Collaborative Biomedical Products, Bedford, MA, USA) into subcutaneous sites in 6-week-old athymic Ncr-nude mice (NCI Animal Program, Bethesda, MD, USA) that were ovariectomized at approximately 4 weeks of age. Half of the ovariectomized mice received an implanted 0.18mg estrogen 60-day pellet (Innovative Research of America, Sarasota, FL, USA). Antisense technology was effective in decreasing both RNA and protein levels of BRCA1 in the BG-1 human ovarian adenocarcinoma cells. BRCA1 antisense-infected populations contained significantly less BRCA1 message than control LXSN-infected pools and selected clones contained varying reduced levels of BRCA1 protein compared with control clones (Figs 1a and 1b). Three independent BRCA1 antisense-infected cultures demonstrated a resistance to cell death induced by withdrawal from estrogen over a 6- to 20
PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention
Azeke, John Imuetinyan-Jesu, Jr.
Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that
Chiral reagents in glycosylation and modification of carbohydrates.
Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping
Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.
Antitumor effects of radioiodinated antisense oligonucleotide mediated by VIP receptor
International Nuclear Information System (INIS)
Ou Xiaohong; Tan Tianzhi; Li Yunchun; Kuang Anren
Purpose: we had constructed a targeting delivery system based on intestinal peptide (VIP) for antisense oligonucleotide (ASON) transfer into VIP receptor-positive cells in previous study. The aims of present studies are to observe the antitumor effect of VIP-131I-ASON in HT29 human colon adenocarcinoma xenografts. Methods: A 15-met phosphorothioate ASON, which was complementary to the translation start region of the C-myc oncogene mRNA, was labeled with 131I and the labelled compound was linked to the VIP bound covalently 'to a polylysine chain so as to deliver oligonucleotide into tumor cells. Distribution experiments for evaluating the radiolabeled antisense complexe uptake in tumor tissue were performed in BALB/c nude mice bearing with HT29 tumor xenografts. Nude mice beating HT29 tumor xenografts were adminstered VIP-131I-ASON (3.7,7.4 MBq) or 131I-ASON (3.7 MBq), 131I labeled control sense and nosense DNA (3.7 MBq), or saline. Antitumor effects were assessed using endpoints of tumor growth delay. C-myc-encoded protein expression of tumor was measured by immunocytohistochemical staining. Results: Distribution experiment performed with athymic mice bearing human colon tumor xenografts revealed maximal accumulation of conjugated ASON in the tumor tissue 2 h after administration and significantly higher than that in nude mice injected unconjngated ASON [(5.89±1.03)%ID/g and(1.56±0.31)%ID/g, respectively; t=7.7954 P<0.001]. The radioratio of tumor to muscle was peaked 4h after administration. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing their growth rate 7-fold compare with that in saline-treated mice(tumor growth delay, 25.4±0.89 day). The antitumor effects of unconjugated 131I-ASON were much less profound than VIP-131I-ASON (tumor growth delay, 3.2±1.3 and 25.4±0.89 day, respectively; q=51.4126 P<0.01). Sense, nosense control ON with VIP carder caused no therapeutic effect. There was no progressive weight loss or
21 CFR 660.30 - Reagent Red Blood Cells.
... 21 Food and Drugs 7 2010-04-01 2010-04-01 false Reagent Red Blood Cells. 660.30 Section 660.30...) BIOLOGICS ADDITIONAL STANDARDS FOR DIAGNOSTIC SUBSTANCES FOR LABORATORY TESTS Reagent Red Blood Cells § 660.30 Reagent Red Blood Cells. (a) Proper name and definition. The proper name of the product shall be...
21 CFR 866.3255 - Escherichia coli serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Escherichia coli serological reagents. 866.3255... coli serological reagents. (a) Identification. Escherichia coli serological reagents are devices that consist of antigens and antisera used in serological tests to identify Escherichia coli from cultured...
21 CFR 864.8100 - Bothrops atrox reagent.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Bothrops atrox reagent. 864.8100 Section 864.8100 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8100 Bothrops atrox reagent. (a...
21 CFR 864.8540 - Red cell lysing reagent.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Red cell lysing reagent. 864.8540 Section 864.8540 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8540 Red cell lysing reagent...
A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.
Directory of Open Access Journals (Sweden)
Marcel Veltrop
Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.
Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease.
Monteleone, Giovanni; Neurath, Markus F; Ardizzone, Sandro; Di Sabatino, Antonio; Fantini, Massimo C; Castiglione, Fabiana; Scribano, Maria L; Armuzzi, Alessandro; Caprioli, Flavio; Sturniolo, Giacomo C; Rogai, Francesca; Vecchi, Maurizio; Atreya, Raja; Bossa, Fabrizio; Onali, Sara; Fichera, Maria; Corazza, Gino R; Biancone, Livia; Savarino, Vincenzo; Pica, Roberta; Orlando, Ambrogio; Pallone, Francesco
Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohn's disease. We found that study participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).
Identification of novel non-coding RNAs as potential antisense regulators in the archaeon Sulfolobus solfataricus
DEFF Research Database (Denmark)
tang, T. H.; Polacek, N.; Zywicki, M.
By generating a specialized cDNA library from the archaeon Sulfolobus solfataricus, we have identified 57 novel small non-coding RNA (ncRNA) candidates and confirmed their expression by Northern blot analysis. The majority was found to belong to one of two classes, either antisense or antisense...... elements by inhibiting expression of the transposase mRNA. Surprisingly, the class of antisense RNAs also contained RNAs complementary to tRNAs or sRNAs (small-nucleolar-like RNAs). For the antisense-box ncRNAs, the majority could be assigned to the class of C/D sRNAs, which specify 2'-O-methylation sites...... on rRNAs or tRNAs. Five C/D sRNAs of this group are predicted to target methylation at six sites in 13 different tRNAs, thus pointing to the widespread role of these sRNA species in tRNA modification in Archaea. Another group of antisense-box RNAs, lacking typical C/D sRNA motifs, was predicted...
Antiviral effects of herpes simplex virus specific anti-sense nucleic acids.
Cantin, E M; Podsakoff, G; Willey, D E; Openshaw, H
We have targeted mRNA sequences encompassing the translation initiation codon of the essential herpes simplex virus type 1 (HSV-1) IE3 gene with three kinds of anti-sense molecule. Addition of a 15mer oligodeoxyribonucleoside methylphosphonate to tissue culture cells resulted in suppression of viral replication. HSV-1 replication was also inhibited in cultured cells containing anti-sense vectors expressing transcripts complementary to the IE3 mRNA. We have also constructed a ribozyme which upon base pairing with the target IE3 mRNA induces cleavage at the predicted GUC site. A major obstacle to anti-sense studies in animals is drug delivery of preformed antisense molecules to ganglionic neurons, the site of HSV latency and reactivation. We speculate as to how this may be accomplished through carrier compounds which are taken up by nerve terminals and transported by retrograde axoplasmic flow. By the same route, HSV itself may be used as an anti-sense vector.
Selenium and tellurium reagents in organic synthesis
International Nuclear Information System (INIS)
Comasseto, J.V.
A review of the contribution of the University of Sao Paulo (SP, Brazil) to the organic synthesis of selenium and tellurium reagents is made. Major reactions amoung selenium compounds and insaturated substrates, phosphorus, ester enolates as well as the use of phase transference catalysed reactions to produce arylselenolate are described. For tellurium, interactions of its compounds with organic substrates and reactive intermediates (e.g. benzino diazomethane) are reported. (C.L.B.) [pt
Fenton's Reagent. Innovative Technology Summary Report
International Nuclear Information System (INIS)
The Fenton's Reagent DNAPL treatment process is an in situ oxidation method to destroy DNAPLs in groundwater. Residual industrial solvents, primarily Dense Non-Aqueous Phase Liquids (DNAPLs), are currently the most significant barrier to successful completion of most large groundwater and soil cleanup efforts. DNAPL pools and residues slowly dissolve into surrounding groundwater to create large plumes of organic solvent contamination with concentration levels far above regulatory limits
Organic acids as analytical reagent: Part 1. Estimation of zirconium by gallic acid
International Nuclear Information System (INIS)
Pande, C.S.; Singh, A.K.; Kumar, Ashok
Gallic acid has been found to be a selective reagent for the estimation of zirconium. The acid gives crystalline precipitate at pH of 4.8. The precipitate is ignited and weighed as ZrO 2 . Cations like Ca +2 , Ba +2 , Sr +2 , Mn +2 , Co +2 , Ni +2 , Fe +3 do not interfere in the estimation. (author)
Copper-catalyzed asymmetric ring opening of oxabicyclic alkenes with organolithium reagents
Bos, Pieter H.; Rudolph, Alena; Pérez, Manuel; Fañanás-Mastral, Martín; Harutyunyan, Syuzanna R.; Feringa, Bernard
A highly efficient method is reported for the asymmetric ring opening of oxabicyclic alkenes with organolithium reagents. Using a copper/chiral phosphoramidite complex together with a Lewis acid (BF3·OEt2), full selectivity for the anti isomer and excellent enantioselectivities were obtained for the
Reactivity of lignin and problems of its oxidative destruction with peroxy reagents
International Nuclear Information System (INIS)
Demin, Valerii A; Shereshovets, Valerii V; Monakov, Yurii B
Published data on reactivity and oxidation of lignin and model compounds with hydrogen peroxide, ozone and chlorine dioxide as well as on oxidative destruction of the sulfate pulp lignin with various reagents during bleaching are systematised and generalised. Concepts of lignin activation towards its selective oxidation and kinetic features of sulfate pulp oxidative delignification are considered. The bibliography includes 157 references.
Determination оf Optimum Constructive Parameters for Circulating-Reagent Regeneration Sector Apparatus
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A. M. Sheiko
Full Text Available On the basis of equation analysis for velocity distribution in near filter mudded zone optimal constructional parameters of sector apparatus for circulating-reagent well filter regeneration have been evaluated via angle ratio of forcing and section sectors and number of sectors. The method for determination of sector apparatus length of а selected pump that provides dissolution of mud formation in filter and near filter zone is proposed in the paper. The obtained data would promote upgrading of circulating-reagent water well regeneration technology and it permits to carry out high quality and even rehabilitation of pore space penetration along the full well filter length.
Bcl-2 antisense therapy in B-cell malignant proliferative disorders.
Chanan-Khan, Asher; Czuczman, Myron S
Overexpression of Bcl-2 oncogene has been clinically associated with an aggressive clinical course, chemotherapy and radiotherapy resistance, and poor survival in patients with malignant B-cell disorders. Patients with relapsed or refractory chronic lymphocytic leukemia, multiple myeloma, or non-Hodgkin's lymphoma have limited therapeutic options. Preclinical and early clinical data have shown that Bcl-2 oncoprotein can be decreased by Bcl-2 antisense therapy. Also, downregulation of Bcl-2 protein can result in reversal of chemotherapy resistance and improved antitumor activity of biologic agents. Various clinical trials are evaluating the role of targeting Bcl-2 as a mechanism to enhance the antitumor potential of chemotherapy and immunotherapy. Early results from these clinical studies are encouraging and confirm the proof of principle for antisense therapy. As current data mature, these trials will hopefully validate preliminary results and establish Bcl-2 antisense as an important addition to the current armamentarium used in the treatment of patients with B-cell neoplasms.
Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers
DEFF Research Database (Denmark)
Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E
Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...... is an efficient vector for PNA delivery yielding significantly higher (up to 10-fold) antisense activity than an analogous PNA-octaarginine conjugate, even in the presence of chloroquine, which only slightly enhances the PEI-PNA activity. The PEI-PEG conjugates are preferred due to lower acute cellular toxicity...
Copper-Catalyzed Oxy-Alkynylation of Diazo Compounds with Hypervalent Iodine Reagents.
Hari, Durga Prasad; Waser, Jerome
Alkynes have found widespread applications in synthetic chemistry, biology, and materials sciences. In recent years, methods based on electrophilic alkynylation with hypervalent iodine reagents have made acetylene synthesis more flexible and efficient, but they lead to the formation of one equivalent of an iodoarene as side-product. Herein, a more efficient strategy involving a copper-catalyzed oxy-alkynylation of diazo compounds with ethynylbenziodoxol(on)e (EBX) reagents is described, which proceeds with generation of nitrogen gas as the only waste. This reaction is remarkable for its broad scope in both EBX reagents and diazo compounds. In addition, vinyl diazo compounds gave enynes selectively as single geometric isomers. The functional groups introduced during the transformation served as easy handles to access useful building blocks for synthetic and medicinal chemistry.
Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents.
Katilius, Evaldas; Carmel, Andrew B; Koss, Heidi; O'Connell, Dan; Smith, Breanna C; Sanders, Glenn M; LaBerge, Greggory S
We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (forensic casework. Copyright © 2018. Published by Elsevier B.V.
Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis
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Benoit eBarbeau
Full Text Available The production of antisense transcripts from the 3' long terminal repeat (LTR in human T-lymphotropic retroviruses has now been clearly demonstrated. After the identification of the antisense strand-encoded HTLV-1 bZIP (HBZ factor, we reported that HBZ could interact with CREB transcription factors and consequently turn off the important activating potential of the viral Tax protein on HTLV-1 5' LTR promoter activity. We have recently accumulated new results demonstrating that antisense transcripts also exist in HTLV-2, -3 and -4. Furthermore, our data have confirmed the existence of encoded proteins from these antisense transcripts (termed antisense proteins of HTLVs or APHs. APHs are also involved in the down-regulation of Tax-dependent viral transcription. In this review, we will focus on the different molecular mechanisms used by HBZ and APH-2 to control viral expression. While HBZ interacts with CREB through its basic zipper domain, APH-2 binds to this cellular factor through a five amino acid motif localized in its carboxyl terminus. Moreover, unlike APH-2, HBZ possesses an N-terminal activation domain that also contributes to the inhibition of the viral transcription by interacting with the KIX domain of p300/CBP. On the other hand, HBZ was found to induce T-cell proliferation while APH-2 was unable to promote such proliferation. Interestingly, HTLV-2 has not been causally linked to human T-cell leukemia, while HTLV-1 is responsible for the development of the Adult T-cell Leukemia/Lymphoma (ATLL. We will further discuss the possible role played by antisense proteins in the establishment of pathologies induced by viral infection.
New sorption-reagent materials for decontamination of liquid radioactive waste
International Nuclear Information System (INIS)
Avramenko, V.A.; Golikov, A.P.; Zheleznov, V.V.; Kaplun, E.V.; Marinin, D.V.; Sokolnitskaya, T.A.
Full text: Use of selective sorbents in liquid radioactive waste (LRW) management is widely spread in the field of nuclear power objects liquid waste decontamination, since the main objective there is to remove long-lived radionuclides of the nuclear cycle. The latter include, first of all, cesium-137, strontium-90, cobalt-60 and a number of α-irradiators. In this case LRW composition for most of the nuclear power objects is rather simple, except acidic deactivation solutions. At the same time, liquid radioactive wastes of different research centers have a variable chemical and radiochemical composition depending on objectives and tasks of a given center research activities. As a result, application of sorption technologies in such waste decontamination determines special requirements to these sorbents selectivity: a wide spectrum of radionuclides that can be removed and fairly high selectivity enabling to remove radionuclides from solutions of complex chemical composition (containing surfactants, complexing agents etc.). This paper is concerned with studying properties of new materials selective to different radionuclides. These materials are capable to interact with solution components whether already contained in the waste or deliberately added into resulting solution. Such sorption-reagent materials combine universal character of co-precipitation methods with simplicity of sorption methods. In this work we studied sorption-reagent inorganic ion-exchange materials interacting with sulfate-, carbonate-, oxalate-, sulfide-, and permanganate-ions. Insoluble compounds formed as a result of this interaction increase tens- and hundreds-fold the sorption selectivity of different radionuclides - strontium, cobalt, mercury, iron, and manganese as compared to conventional ion-exchange system. By means of X-ray phase analysis, IR-spectroscopy, chemical and radiochemical analysis, we have studied the mechanism of radionuclide sorption on different sorption-reagent
Role of natural antisense transcripts pertaining to tumor suppressor genes in human carcinomas
International Nuclear Information System (INIS)
Pelicci, G.; Pierotti, M.
Overlapping transcripts in opposite orientations can potentially form perfect sense-antisense duplex RNA. Recently, several studies have revealed the extent of natural antisense transcripts (NATs) and their role in important biological phenomena also in higher organisms. In order to test the hypothesis that the function of NATs in man might represent an essential element in the regulation of gene expression, especially at transcriptional level, in this study we planned to look for, systematically examine, and characterize NATs belonging in the human genome to the tumour suppressor class of genes, so to identify physiological (and potentially pathological) modulators in this gene class
Antimicrobial Effectiveness of Selected Vranac Wines Against Six ...
African Journals Online (AJOL)
glucoside and. Folin - Ciocalteu`s phenol reagent were supplied from Sigma Chemical Co. (St Louis, Mo, USA); used reagents were of analytical quality. Wines samples. Selected Vranac wines from vintage 2009, made from autochthonic Vranac V.
The zebrafish progranulin gene family and antisense transcripts
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Baranowski David
Full Text Available Abstract Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial
A small molecule for a big transformation: Topical application of a 20-nucleotide-long antisense fragment of the DIAP-2 gene inhibits the development of Drosophila melanogaster female imagos
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Nyadar Palmah M.
Full Text Available Several genes have been identified to play important roles associated with sex selection in Drosophila melanogaster. An essential part is attributed to the sex-lethal gene that depends on the expression of the X:A (number of chromosomes to autosomes ratio signal controlling both sex selection and dosage compensation processes in D. melanogaster. Interestingly, for sex selection in D. melanogaster there are no documented data addressing the role of the inhibitor of apoptosis (IAP genes and their signaling influence on this biological process. In this study, we found that topical application of a 20-nucleotide-long antisense DNA fragment (oligoDIAP-2 from the death-associated inhibitor of apoptosis (DIAP-2 gene interferes with D. melanogaster development and significantly decreases the number of female imagos and their biomass. We show that the applied antisense oligoDIAP-2 fragment downregulates the target DIAP-2 gene whose normal concentration is necessary for the development of female D. melanogaster. These data correspond to the results on downregulation of the target host IAP-Z gene of Lymantria dispar L. female imagos after topical treatment with an 18-nucleotide-long antisense DNA fragment from the L. dispar multicapsid nuclear polyhedrosis virus IAP-3 gene at the larval stage. The observed novel phenomenon linking the downregulation of insect IAP genes and the low rate of female imago development could have practical application, especially in insect pest control and molecular pathology.
A Snippet of Grignard Reagent's Histroy There are very few reagents ...
Indian Academy of Sciences (India)
IAS Admin
with bromine, (eq.1). But he failed to notice the formation of phenylmagnesium bromide, because he was using excess bromine which destroyed it. Hence, he could isolate only bromobenzene,. (eq.2). Had he used only one molar equivalent of bromine, perhaps the organomagnesium reagent would bear his name today.
Extraction of uranium(6), transuranium elements and europium by bidentate neutral phosphorus- and phosphorus-nitrogen-containing reagents with substituent in methylene bridge
International Nuclear Information System (INIS)
Kochetkova, N.E.; Kojro, O.Eh.; Nesterova, N.P.; Medved', T.Ya.; Chmutova, M.K.; Myasoedov, B.F.; Kabachnik, M.I.
The influence of substituents in methylene bridge on solubility, extractivity and selectivity of bidentate neutral phosphorus- and phosphorus-nitrogen-containing reagents in the process of U(6), TUE, Eu extraction has been studied. It is ascertained that hydrogen substitution in the bridge of tetraphenylmethylenediphosphine dioxide (1) causes a decrease in the extractivity of reagent as to TPE, uranium (6) and europium. There is no visible regular relation between basicity and extractivity of substituted reagents. Hydrogen substitution in the bridge of diphenyl[diethylcarbamoylmethyl]phosphine oxide (2) causes a decrease in extractivity of the reagent as to TPE, uranium (6) and europium. In contrast to monodentate neutral reagents, when bidentate neutral reagents are used, sometimes no increase in the reagent extractivity with an increase in its basicity is observed. When fragments restricting the conformation mobility of bidentate reagent molecule are introduced in it (here substituents in methylene bridge), it may result in the violation of the regularity, since of all the factors affecting the reagent extractivity the spatial factor may become the prevailing one. On hydrogen substitution in the bridge of 1 separation factors of practically all (with few exceptions) studied pairs of elements increase. Hydrogen substitution in the bridge of 2 causes an increase in separation factor of U (6) /Am pair and it does not affect the separation factor of Am/Eu pair. Hydrogen substitution in the bridge of 1 and 2 does not result in the preparation of more efficient and considerably more selective reagents for extractive isolation and separation of the elements, but some of the substituted reagents (Cl-substituted 1, for instance) may turn out useful for the element separation
Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test
Korte, S.M.; de Kloet, E.R.; Buwalda, B; Bouman, S.D.; Bohus, B
Immobility time of rats in the forced swim test was reduced after bilateral infusion of an 18-mer antisense phosphorothioate oligodeoxynucleotide targeted to the glucocorticoid receptor mRNA into the dentate gyrus of the hippocampus. Vehicle-, sense- and scrambled sequence-treated animals spent
Suppression of cell division by pKi-67 antisense-RNA and recombinant protein.
Duchrow, M; Schmidt, M H; Zingler, M; Anemüller, S; Bruch, H P; Broll, R
The human antigen defined by the monoclonal antibody Ki-67 (pKi-67) is a human nuclear protein strongly associated with cell proliferation and found in all tissues studied. It is widely used as a marker of proliferating cells, yet its function is unknown. To investigate its function we suppressed pKi-67 expression by antisense RNA and overexpressed a partial structure of pKi-67 in HeLa cells. A BrdU-incorporation assay showed a significant decrease in DNA synthesis after antisense inhibition. Cell cycle analysis indicated a higher proportion of cells in G1 phase and a lower proportion of cells in S phase while the number of G(2)/M phase cells remained constant. Overexpression of a recombinant protein encoding three of the repetitive elements from exon 13 of pKi-67 had a similar effect to that obtained by antisense inhibition. The similarity of the effect of expressing 'Ki-67 repeats' and pKi-67 antisense RNA could be explained by a negative effect on the folding of the endogenous protein in the endoplasmatic reticulum. Furthermore excessive self-association of pKi-67 via the repeat structure could inhibit its nuclear transport, preventing it from getting to its presumptive site of action. We conclude that the Ki-67 protein has an important role in the regulation of the cell cycle, which is mediated in part by its repetitive elements. Copyright 2001 S. Karger AG, Basel
Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching
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Kole Ryszard
Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.
Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa
DEFF Research Database (Denmark)
Ghosal, Anubrata; Nielsen, Peter E
Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....
Scintigraphic imaging of oncogenes with antisense probes: does it make sense?
International Nuclear Information System (INIS)
Urbain, J.L.C.; Shore, S.K.; Vekemans, M.C.; Cosenza, S.C.; DeRiel, K.; Patel, G.V.; Charkes, N.D.; Malmud, L.S.; Reddy, E.P.
The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. (orig.)
Tumor delivery of antisense oligomer using trastuzumab within a streptavidin nanoparticle
Energy Technology Data Exchange (ETDEWEB)
Wang, Yi [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Yale University, Yale PET Center, Department of Diagnostic Radiology, New Haven, CT (United States); Liu, Xinrong; Chen, Ling; Cheng, Dengfeng; Rusckowski, Mary [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Hnatowich, Donald J. [University of Massachusetts Medical School, Division of Nuclear Medicine, Department of Radiology, Worcester, MA (United States); Umass Medical School, Department of Radiology, Worcester, MA (United States)
Trastuzumab (Herceptin trademark) is often internalized following binding to Her2+ tumor cells. The objective of this study was to investigate whether trastuzumab can be used as a specific carrier to deliver antisense oligomers into Her2+ tumor cells both in vitro and in vivo. A biotinylated MORF oligomer antisense to RhoC mRNA and its biotinylated sense control were labeled with either lissamine for fluorescence detection or {sup 99m}Tc for radioactivity detection and were linked to biotinylated trastuzumab via streptavidin. The nanoparticles were studied in SUM190 (RhoC+, Her2+) study and SUM149 (RhoC+, Her2-) control cells in culture and as xenografts in mice. As evidence of unimpaired Her2+ binding of trastuzumab within the nanoparticle, accumulations were clearly higher in SUM190 compared to SUM149 cells and, by whole-body imaging, targeting of SUM190 tumor was similar to that expected for a radiolabeled trastuzumab. As evidence of internalization, fluorescence microscopy images of cells grown in culture and obtained from xenografts showed uniform cytoplasm distribution of the lissamine-MORF. An invasion assay showed decreased RhoC expression in SUM190 cells when incubated with the antisense MORF nanoparticles at only 100 nM. Both in cell culture and in animals, the nanoparticle with trastuzumab as specific carrier greatly improved tumor delivery of the antisense oligomer against RhoC mRNA into tumor cells overexpressing Her2 and may be of general utility. (orig.)
Metabolite profiling of Arabidopsis thaliana (L.) plants transformed with an antisense chalcone synthase gene
DEFF Research Database (Denmark)
Le Gall, G.; Metzdorff, Stine Broeng; Pedersen, Jan W.
A metabolite profiling study has been carried out on Arabidopsis thaliana (L.) Heynh. ecotype Wassilewskija and a series of transgenic lines of the ecotype transformed with a CHS (chalcone synthase) antisense construct. Compound identifications by LC/MS and H-1 NMR are discussed. The glucosinolate...
Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)
Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.; Salentijn, E.M.J.
An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.
G3139, a Bcl-2 antisense oligodeoxynucleotide, induces clinical responses in VAD refractory myeloma
van de Donk, N. W. C. J.; de Weerdt, O.; Veth, G.; Eurelings, M.; van Stralen, E.; Frankel, S. R.; Hagenbeek, A.; Bloem, A. C.; Lokhorst, H. M.
Expression of Bcl-2 in multiple myeloma is associated with resistance to chemotherapeutic drugs. Conversely, suppression of Bcl-2 enhanced the chemosensitivity of myeloma cells in vitro. G3139 is an antisense oligodeoxynucleotide targeted to the first six codons of the Bcl-2 mRNA open reading frame.
Antisense silencing of the creA gene in Aspergillus nidulans
DEFF Research Database (Denmark)
Bautista, L. F.; Aleksenko, Alexei Y.; Hentzer, Morten
Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was appr...
Antisense long noncoding RNAs regulate var gene activation in the malaria parasite Plasmodium falciparum.
Amit-Avraham, Inbar; Pozner, Guy; Eshar, Shiri; Fastman, Yair; Kolevzon, Netanel; Yavin, Eylon; Dzikowski, Ron
The virulence of Plasmodium falciparum, the causative agent of the deadliest form of human malaria, is attributed to its ability to evade human immunity through antigenic variation. These parasites alternate between expression of variable antigens, encoded by members of a multicopy gene family named var. Immune evasion through antigenic variation depends on tight regulation of var gene expression, ensuring that only a single var gene is expressed at a time while the rest of the family is maintained transcriptionally silent. Understanding how a single gene is chosen for activation is critical for understanding mutually exclusive expression but remains a mystery. Here, we show that antisense long noncoding RNAs (lncRNAs) initiating from var introns are associated with the single active var gene at the time in the cell cycle when the single var upstream promoter is active. We demonstrate that these antisense transcripts are incorporated into chromatin, and that expression of these antisense lncRNAs in trans triggers activation of a silent var gene in a sequence- and dose-dependent manner. On the other hand, interference with these lncRNAs using complement peptide nucleic acid molecules down-regulated the active var gene, erased the epigenetic memory, and induced expression switching. Altogether, our data provide evidence that these antisense lncRNAs play a key role in regulating var gene activation and mutually exclusive expression.
The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function
Directory of Open Access Journals (Sweden)
Wahlestedt Claes
Full Text Available Abstract Background Mutations in the PTEN induced putative kinase 1 (PINK1 are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila. Results Herein we characterize a novel splice variant of PINK1 (svPINK1 that is homologous to the C-terminus regulatory domain of the protein kinase. Naturally occurring non-coding antisense provides sophisticated mechanisms for diversifying genomes and we describe a human specific non-coding antisense expressed at the PINK1 locus (naPINK1. We further demonstrate that PINK1 varies in vivo when human skeletal muscle mitochondrial content is enhanced, supporting the idea that PINK1 has a physiological role in mitochondrion. The observation of concordant regulation of svPINK1 and naPINK1 during in vivo mitochondrial biogenesis was confirmed using RNAi, where selective targeting of naPINK1 results in loss of the PINK1 splice variant in neuronal cell lines. Conclusion Our data presents the first direct observation that a mammalian non-coding antisense molecule can positively influence the abundance of a cis-transcribed mRNA under physiological abundance conditions. While our analysis implies a possible human specific and dsRNA-mediated mechanism for stabilizing the expression of svPINK1, it also points to a broader genomic strategy for regulating a human disease locus and increases the complexity through which alterations in the regulation of the PINK1 locus could occur.
21 CFR 866.3120 - Chlamydia serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3120 Chlamydia... and antisera used in serological tests to identify antibodies to chlamydia in serum. Additionally...
21 CFR 866.3490 - Rhinovirus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3490 Rhinovirus... and antisera used in serological tests to identify antibodies to rhinovirus in serum. The...
21 CFR 866.3240 - Equine encephalomyelitis virus serological reagents.
... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... these viruses. Equine encephalomyelitis viruses are transmitted to humans by the bite of insects, such...
21 CFR 866.3165 - Cryptococcus neoformans serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3165... clinical specimens or from cultured isolates derived from clinical specimens. The identification aids in...
21 CFR 866.3020 - Adenovirus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3020 Adenovirus... identify adenoviruses directly from clinical specimens. The identification aids in the diagnosis of disease...
21 CFR 866.3480 - Respiratory syncytial virus serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3480... respiratory syncytial viruses from clinical specimens or from tissue culture isolates derived from clinical...
21 CFR 866.3140 - Corynebacterium spp. serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3140.... from clinical specimens. The identification aids in the diagnosis of disease caused by bacteria...
21 CFR 866.3250 - Erysipelothrix rhusiopathiae serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3250... Erysipelothrix rhusiopathiae from cultured isolates derived from clinical specimens. The identification aids in...
21 CFR 866.3270 - Flavobacterium spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3270.... from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of...
21 CFR 866.3205 - Echovirus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3205 Echovirus... echoviruses from clinical specimens or from tissue culture isolates derived from clinical specimens. The...
21 CFR 866.3110 - Campylobacter fetus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3110 Campylobacter... clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the...
21 CFR 866.3320 - Histoplasma capsulatum serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3320... capsulatum from clinical specimens or cultured isolates derived from clinical specimens. The identification...
21 CFR 866.3340 - Klebsiella spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3340 Klebsiella... from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria...
21 CFR 866.3930 - Vibrio cholerae serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3930 Vibrio... from cultured isolates derived from clinical specimens. The identification aids in the diagnosis of...
21 CFR 866.3010 - Acinetobacter calcoaceticus serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3010... this bacterium from cultured isolates derived from clinical specimens. The identification aids in the...
21 CFR 866.3370 - Mycobacterium tuberculosis immunofluorescent reagents.
... HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents... used to identify Mycobacterium tuberculosis directly from clinical specimens. The identification aids...
Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology
Energy Technology Data Exchange (ETDEWEB)
Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer
Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen
Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products
Directory of Open Access Journals (Sweden)
Hsiao Chiu-Bin
Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The
Textile wastewater reuse after additional treatment by Fenton's reagent.
Ribeiro, Marília Cleto Meirelles; Starling, Maria Clara V M; Leão, Mônica Maria Diniz; de Amorim, Camila Costa
This study verifies textile wastewater reuse treated by the conventional activated sludge process and subjected to further treatment by advanced oxidation processes. Three alternative processes are discussed: Fenton, photo-Fenton, and UV/H 2 O 2 . Evaluation of treatments effects was based on factorial experiment design in which the response variables were the maximum removal of COD and the minimum concentration of residual H 2 O 2 in treated wastewater. Results indicated Fenton's reagent, COD/[H 2 O 2 ]/[Fe 2+ ] mass ratio of 1:2:2, as the best alternative. The selected technique was applied to real wastewater collected from a conventional treatment plant of a textile mill. The quality of the wastewater before and after the additional treatment was monitored in terms of 16 physicochemical parameters defined as suitable for the characterization of waters subjected to industrial textile use. The degradation of the wastewater was also evaluated by determining the distribution of its molecular weight along with the organic matter fractionation by ultrafiltration, measured in terms of COD. Finally, a sample of the wastewater after additional treatment was tested for reuse at pilot scale in order to evaluate the impact on the quality of dyed fabrics. Results show partial compliance of treated wastewater with the physicochemical quality guidelines for reuse. Removal and conversion of high and medium molecular weight substances into low molecular weight substances was observed, as well as the degradation of most of the organic matter originally present in the wastewater. Reuse tests indicated positive results, confirming the applicability of wastewater reuse after the suggested additional treatment. Graphical abstract Textile wastewater samples after additional treatment by Fenton's reagent, photo-Fenton and H 2 O 2 /UV tested in different conditions.
New data on masking reagents in complexometry
International Nuclear Information System (INIS)
Yurist, I.M.; Talmud, M.M.; Zajtsev, P.M.
Recent literature data on employing inorganic and organic oxygen-, nitrogen- and sulfur-containing substances as masking reagents (MR) in complexonometry of alkali earths, rare earths and transition elements are reviewed for the period of 1971-1983. Effectiveness of any type of MR is shown to be dependent on the electron configuration of a cation being masked. Sr, La, Th, V(6), Zr, Hf, V(5), Nb(5), Ta(5), Mo(6), W(6) a.o. are masked by oxygen-containing ligands. Zn, Cd, Fe(2, 3), Co(2, 3), Ni, etc. are masked by nitrogen- and sulfur-bearing ligands. Thiocompounds mask mainly In, Tl(3), Sn(2), Pb, Bi
Specific Reagent for Cr(III): Imaging Cellular Uptake of Cr(III) in Hct116 Cells and Theoretical Rationalization.
Ali, Firoj; Saha, Sukdeb; Maity, Arunava; Taye, Nandaraj; Si, Mrinal Kanti; Suresh, E; Ganguly, Bishwajit; Chattopadhyay, Samit; Das, Amitava
A new rhodamine-based reagent (L1), trapped inside the micellar structure of biologically benign Triton-X 100, could be used for specific recognition of Cr(III) in aqueous buffer medium having physiological pH. This visible light excitable reagent on selective binding to Cr(III) resulted in a strong fluorescence turn-on response with a maximum at ∼583 nm and tail of that luminescence band extended until 650 nm, an optical response that is desired for avoiding the cellular autofluorescence. Interference studies confirm that other metal ions do not interfere with the detection process of Cr(III) in aqueous buffer medium having pH 7.2. To examine the nature of binding of Cr(III) to L1, various spectroscopic studies are performed with the model reagent L2, which tend to support Cr(III)-η(2)-olefin π-interactions involving two olefin bonds in molecular probe L1. Computational studies are also performed with another model reagent LM to examine the possibility of such Cr(III)-η(2)-olefin π-interactions. Presumably, polar functional groups of the model reagent LM upon coordination to the Cr(III) center effectively reduce the formal charge on the metal ion and this is further substantiated by results of the theoretical studies. This assembly is found to be cell membrane permeable and shows insignificant toxicity toward live colon cancer cells (Hct116). Confocal laser scanning microscopic studies further revealed that the reagent L1 could be used as an imaging reagent for detection of cellular uptake of Cr(III) in pure aqueous buffer medium by Hct116 cells. Examples of a specific reagent for paramagnetic Cr(III) with luminescence ON response are scanty in the contemporary literature. This ligand design helped us in achieving the turn on response by utilizing the conversion from spirolactam to an acyclic xanthene form on coordination to Cr(III).
Alkylation of pyridines at their 4-positions with styrenes plus yttrium reagent or benzyl Grignard reagents.
Mizumori, Tomoya; Hata, Takeshi; Urabe, Hirokazu
A new regioselective alkylation of pyridines at their 4-position was achieved with styrenes in the presence of yttrium trichloride, BuLi, and diisobutylaluminium hydride (DIBAL-H) in THF. Alternatively, similar products were more simply prepared from pyridines and benzyl Grignard reagents. These reactions are not only a useful preparation of 4-substituted pyridines but are also complementary to other relevant reactions usually giving 2-substituted pyridines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Effect of reagent charge on the labeling of erythrocyte membrane proteins by photoactivated reagents
International Nuclear Information System (INIS)
Schaeffer, J.C.; Hakimian, R.; Shimer, M.L.
Leaky erythrocyte ghosts were labeled with 3 H-[2-(4-azido-2-nitroanilino)ethyl]trimethylammonium iodide (cationic label) or 3 H-N-(4-azido-2-nitrophenyl)-β-alanine (anionic label). After the membranes were thoroughly washed, seven times as much cationic label was associated with the membranes as anionic label at 5 μM, whereas at 50 μM the cationic label was favored 15-fold. The distribution of label in the membrane proteins was ascertain by SDS-gel electrophoresis followed by autoradiography. At 50 μM cationic label, erythrocyte membrane protein bands 1,2,3,4.2, and 5 were intensely labeled, while band 6 was labeled weakly. At 5 μM cationic label, bands 1 and 4.2 were heavily labeled, while 2,3 and 5 were labeled less well. At both 50 μM and 5 μM anionic label, bands 1 and 6 were most prominently labeled. Bands 2,3,4.2 and 5 were labeled also at 50 μM, but they were labeled only very weakly at 5 μM. Band 4.1 was labeled very poorly if at all by either reagent. A mixture of the reagents gave an additive pattern. Thus, the charge and concentration of these reagents appear to play a major role in their ability to label membrane proteins indiscriminately. Because these reagents contain the same chromophore, 4-azido-2-nitroaniline, and differ mainly only in their charge, they may prove useful in assessing the location of charged sites on proteins in supramolecular complexes
Preparation and quality test of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe: a preliminary study.
Wen, Ming; Li, Bibo; Ouyang, Yu; Luo, Yi; Li, Shaolin
Molecular imaging of tumor antisense gene techniques have been applied to the study of magnetic resonance (MR) gene imaging associated with malignant tumors. In this study, we designed, synthesized, and tested a novel molecular probe, in which the antisense oligodeoxynucleotide (ASODN) was labeled with superparamagnetic iron oxide (SPIO), and its efficiency was examined by in vitro MR imaging after SK-Br-3 mammary carcinoma cell lines (oncocytes) transfection. The SPIO-labeled ASODN probe was prepared through SPIO conjugated to ASODN using a chemical cross linking method. Its morphology and size were detected by atomic force microscope, size distribution were detected by laser granulometer, the conjugating rate and biological activity were determined by high performance liquid chromatography, and the stability was determined by polyacrylamide gel electrophoresis. After that, the probes were transfected into the SK-Br-3 oncocytes, cellular iron uptake was analyzed qualitatively at light and electron microscopy and was quantified at atomic absorption spectrometry, and the signal change of the transfected cells was observed and measured using MR imaging. The morphology of the SPIO-labeled ASODN probe was mostly spherical with well-distributed scattering, and the diameters were between 25 and 40 nm (95%) by atomic force microscope and laser granulometer, the conjugating rate of the probe was 99%. Moreover, this probe kept its activity under physiological conditions and could conjugate with antisense oligodeoxynucleotide. In addition, light microscopy revealed an intracellular uptake of iron oxides in the cytosol and electron microscopic studies revealed a lysosomal deposition of iron oxides in the transfected SK-Br-3 oncocytes by antisense probes, some of them gathered stacks, and the iron content of the group of transfected SK-Br-3 oncocytes by antisense probe is significantly higher (18.37 +/- 0.42 pg) than other contrast groups, the MR imaging showed that
Review Article: Toxic Effects of Some Reagents Used in Electron ...
African Journals Online (AJOL)
Ultrathin sections prepared for electron microscopy and histochemistry are indispensable in cytological, histological and histochemical studies. The paper discusses the various reagents used in these fields of study. Unfortunately, these reagents and chemicals are hazardous to health. There is wisdom in informing the ...
Physically absorbable reagents-collectors in elementary flotation
Energy Technology Data Exchange (ETDEWEB)
S.A. Kondrat' ev; I.G. Bochkarev [Russian Academy of Sciences, Novosibirsk (Russian Federation). Institute of Mining
Based on the reviewed researches held at the Institute of Mining, Siberian Branch, Russian Academy of Sciences, the effect of physically absorbable reagents-collectors on formation of a flotation complex and its stability in turbulent pulp flows in flotation machines of basic types is considered. The basic requirements for physically absorbable reagents-collectors at different flotation stages are established.
21 CFR 866.3520 - Rubeola (measles) virus serological reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Rubeola (measles) virus serological reagents. 866... Rubeola (measles) virus serological reagents. (a) Identification. Rubeola (measles) virus serological... to rubeola virus in serum. The identification aids in the diagnosis of measles and provides...
21 CFR 866.3355 - Listeria spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3355 Listeria spp... from clinical specimens. Additionally, some of these reagents consist of Listeria spp. antisera... clinical specimens. The identification aids in the diagnosis of listeriosis, a disease caused by bacteria...
21 CFR 866.3550 - Salmonella spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3550 Salmonella... isolates derived from clinical specimens. Additionally, some of these reagents consist of antisera... clinical specimens or cultured isolates derived from clinical specimens. The identification aids in the...
Reagents for the assay of cardenolide glycosides and aglycones
International Nuclear Information System (INIS)
Wilkinson, S.
Some novel reagents are described for use in the radioimmunoassay of the 3-glycone derivatives of cardenolides (cardiac glycosides) and more especially digoxin, digitoxin, gitoxin, periplocin and lanatosides. Using these reagents these cardenolides and their derivatives may be assayed both in aqueous solution and in urine. A method is also described for performing such assays, including a suitable kit. (U.K.)
21 CFR 864.4020 - Analyte specific reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Analyte specific reagents. 864.4020 Section 864.4020 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4020 Analyte specific...
21 CFR 864.8950 - Russell viper venom reagent.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Russell viper venom reagent. 864.8950 Section 864.8950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Reagents § 864.8950 Russell viper venom...
21 CFR 864.4010 - General purpose reagent.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false General purpose reagent. 864.4010 Section 864.4010 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Specimen Preparation Reagents § 864.4010 General purpose...
Use of Competition Kinetics with Fast Reactions of Grignard Reagents
DEFF Research Database (Denmark)
Holm, Torkil
small.This is concluded from experiments in which results obtained by competition kinetics are compared with results obtained directly by flow stream procedures. A clearer picture of the reactivity ratios is obtained when the highly reactive reagent is highly diluted with its competitor. A fast reagent...
This paper evaluates the feasibility of using Fenton's reagents for in-place recovery of spent granular activated carbon (GAC). Fenton's reagents are cycled through spent GAC to degrade sorbed chlorinated hydrocarbons with little loss of carbon capacity. Seven chlorinated compou...
Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers
DEFF Research Database (Denmark)
Shiraishi, Takehiko; Nielsen, Peter E
We have explored the merits of a novel delivery strategy for the antisense oligomers based on cell penetrating peptide (CPP) conjugated to a carrier PNA with sequence complementary to part of the antisense oligomer. The effect of these carrier CPP-PNAs was evaluated by using antisense PNA targeting......-PNA (cPNA1(7)-(D-Arg)8) and hexamer carrier decanoyl-CPP-PNA (Deca-cPNA1(6)-(D-Arg)8), respectively, without showing significant additional cellular toxicity. Most interestingly, the activity reached the same level obtained by enhancement with endosomolytic chloroquine (CQ) treatment, suggesting...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...
Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping
DEFF Research Database (Denmark)
Le, Bao T.; Hornum, Mick; Sharma, Pawan K.
Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...
Spectrophotometric determination of tannins by phosphotungstic-phosphomolybdic reagent
Energy Technology Data Exchange (ETDEWEB)
Reicher, F; Sierakowski, M R; Correa, J B.C. [Parana Univ., Curitiba (Brazil). Dept. de Bioquimica
There are several colorimetric techniques to determine tannins in plant extracts. One frequently used is the Folin method (phosphotungstic acid reagent) that procedures a blue color with phenolic compounds. However, this coloured complex is unstable. With the Folin-Ciocalteau reagent, used in protein determination (Lowry et al. J.B.C. 193: 265, 1951) good results were obtained, even in the absence of cooper solution. Using phosphotungstic-phosphomolybdic reagent (Folin-Denis), it was obtained maximum color with 1,0 ml of the reagent in 20 minutes, after the additon of 10 ml 20% sodium carbonate solution. Tannins samples containing 10 to 200 ..mu..g/ml were analysed. Absorbances are determined at 720 or 600 nm. Tannins of commercial preparations from Acacia negra were analysed by the phosphotungstic-phosphomolybdic reagent before (A) and after (B) treatment with chromate hyde powder. By this procedure hydrolysible tannins were determined (A-B).
Spectrophotometric determination of tannins by phosphotungstic-phosphomolibdic reagent
International Nuclear Information System (INIS)
Reicher, F.; Sierakowski, M.R.; Correa, J.B.C.
There are several colorimetric techniques to determine tannins in plant extracts. One frequently used is the Folin method (phosphotungstic acid reagent) that procedures a blue color with phenolic compounds. However, this coloured complex is unstable. With the Folin-Ciocalteau reagent, used in protein determination (Lowry et al. J.B.C. 193: 265, 1951) good results were obtained, even in the absence of cooper solution. Using phosphotungstic-phosphomolibdic reagent (Folin-Denis), it was obtained maximum color with 1,0 ml of the reagent in 20 minutes, after the adition of 10 ml 20% sodium carbonate solution. Tannins samples containing 10 to 200 μg/ml were analysed. Absorbances are determined at 720 or 600 nm. Tannins of commercial preparations from Acacia negra were analysed by the phosphotungstic-phosphomolibdic reagent before (A) and after (B) treatment with chromate hyde powder. By this procedure hydrolysible tannins were determined (A-B). (Author) [pt
A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin
Štambuk, Nikola; Manojlović, Zoran; Turčić, Petra; Martinić, Roko; Konjevoda, Paško; Weitner, Tin; Wardega, Piotr; Gabričević, Mario
Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense) nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to ...
Organic acids as analytical reagent: Part 1. Estimation of zirconium by gallic acid
Energy Technology Data Exchange (ETDEWEB)
Pande, C S; Singh, A K; Kumar, Ashok [Lucknow Univ. (India). Dept. of Chemistry
Gallic acid has been found to be a selective reagent for the estimation of zirconium. The acid gives a crystalline precipitate at pH of 4.8 which is ignited and weighed as ZrO/sub 2/. Cations like Ca/sup +2/, Ba/sup +2/, Sr/sup +2/, Mn/sup +2/, Co/sup +2/, Ni/sup +2/, Fe/sup +3/ do not interfere in the estimation.
An antisense oligodeoxynucleotide targeted against the type IIβ regulatory subunit mRNA of protein kinase inhibits cAMP-induced differentiation in HL-60 leukemia cells without affecting phorbol ester effects
International Nuclear Information System (INIS)
Tortora, G.; Clair, T.; Cho-Chung, Y.S.
The type II β regulatory subunit of cAMP-dependent protein kinase (RII β ) has been hypothesized to play an important role in the growth inhibition and differentiation induced by site-selective cAMP analogs in human cancer cells, but direct proof of this function has been lacking. To address this tissue, HL-60 human promyelocytic leukemia cells were exposed to RII β antisense synthetic oligodeoxynucleotide, and the effects on cAMP-induced growth regulation were examined. Exposure of these cells to RII β antisense oligodeoxynucleotide resulted in a decrease in cAMP analog-induced growth inhibition and differentiation without apparent effect on differentiation induced by phorbol esters. This loss in cAMP growth regulatory function correlated with a decrease in basal and induced levels of RII β protein. Exposure to RII β sense, RI α and RII α antisense, or irrelevant oligodeoxynucleotides had no such effect. These results show that the RII β regulatory subunit of protein kinase plays a critical role in the cAMP-induced growth regulation of HL-60 leukemia cells
Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.
Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale
Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages.
Conserved alternative and antisense transcripts at the programmed cell death 2 locus
Czech Academy of Sciences Publication Activity Database
Mihola, Ondřej; Forejt, Jiří; Trachtulec, Zdeněk
Roč. 8, - (2007), s. 20 ISSN 1471-2164 R&D Projects: GA ČR(CZ) GA204/01/0997; GA ČR GA301/05/0738; GA AV ČR IAA5052406; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50520514 Keywords : Pdcd2 * antisense * alternative transcript * imprinting Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.180, year: 2007
Antisense expression of a gene encoding a calcium-binding protein ...
Indian Academy of Sciences (India)
using the transgenic approach. The transformation of ... methods using EhCaBP or AtCaM3 gene-specific primers in ... acetone) was added, mixed and incubated for 15–18 h in the dark at .... as expected from the design of the AtCaM3 antisense construct .... Thus, there seems to be a positive qualitative correlation between ...
Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development
International Nuclear Information System (INIS)
Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao; Yu, Xiyan; Wang, Xiufeng
To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.
Review on investigations of antisense oligonucleotides with the use of mass spectrometry.
Studzińska, Sylwia
Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.
Cocaine alters Homer1 natural antisense transcript in the nucleus accumbens.
Sartor, Gregory C; Powell, Samuel K; Velmeshev, Dmitry; Lin, David Y; Magistri, Marco; Wiedner, Hannah J; Malvezzi, Andrea M; Andrade, Nadja S; Faghihi, Mohammad A; Wahlestedt, Claes
Natural antisense transcripts (NATs) are an abundant class of long noncoding RNAs that have recently been shown to be key regulators of chromatin dynamics and gene expression in nervous system development and neurological disorders. However, it is currently unclear if NAT-based mechanisms also play a role in drug-induced neuroadaptations. Aberrant regulation of gene expression is one critical factor underlying the long-lasting behavioral abnormalities that characterize substance use disorder, and it is possible that some drug-induced transcriptional responses are mediated, in part, by perturbations in NAT activity. To test this hypothesis, we used an automated algorithm that mines the NCBI AceView transcriptomics database to identify NAT overlapping genes linked to addiction. We found that 22% of the genes examined contain NATs and that expression of Homer1 natural antisense transcript (Homer1-AS) was altered in the nucleus accumbens (NAc) of mice 2h and 10days following repeated cocaine administration. In in vitro studies, depletion of Homer1-AS lead to an increase in the corresponding sense gene expression, indicating a potential regulatory mechanisms of Homer1 expression by its corresponding antisense transcript. Future in vivo studies are needed to definitely determine a role for Homer1-AS in cocaine-induced behavioral and molecular adaptations. Copyright © 2017 Elsevier Inc. All rights reserved.
Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics
Courtney, Colleen; Chatterjee, Anushree
``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.
Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development
Energy Technology Data Exchange (ETDEWEB)
Tian, Hongmei; Ma, Leyuan; Zhao, Cong; Hao, Hui; Gong, Biao [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Yu, Xiyan, E-mail: email@example.com [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China); Wang, Xiufeng, E-mail: firstname.lastname@example.com [College of Horticulture Science and Engineering, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai' an, Shandong 271018 (China)
To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.
Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.
Directory of Open Access Journals (Sweden)
Xiaolong Wang
Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.
Directory of Open Access Journals (Sweden)
Full Text Available Used the polymeric reagents in oil wastewater treatment is an effective method of eliminate hydrocarbons. The present study aims to finding reagents that lead to lowering of extractible (EXT, suspended solids (SS and chemical oxygen demand (COD of industrial wastewater from washing cars in loading ramps petroleum products. For this purpose five reagents were tested, namely: polyamines, cationic polyacrylamides, polydiallydimethyl ammonium chloride (PolyDADMAC, melamine formaldehyde polymer resin and polydicyandiamide polymer resin. Obtaining removal degrees over 80 % justifies using this method in the industrial practice.
N-tritioacetoxyphthalimide: A new high specific activity tritioacetylating reagent
International Nuclear Information System (INIS)
Saljoughian, M.; Morimoto, Hiromi; Than, Chit
The authors' aim was to develop a nonvolatile, stable, and facile tritioacetylating reagent and to demonstrate its use on simple peptides. Accordingly, the authors made the synthesis of high specific activity N-(tritioacetoxy) derivatives of succinimide, phthalimide, and naphthalimide a major focus. As the preferred approach, N-(tritioacetoxy)phthalimide was prepared by radical dehalogenation of N-(iodoacetoxy)phthalimide using high specific activity tributyltin tritide. This tritiated acetylation reagent was characterized by 3 H and 1 H NMR spectroscopy and by radio-HPLC. Efficacy of the reagent was investigated by tritioacetylation of several peptides at their N-terminal amino group. 26 refs., 1 fig
The reaction of organocerium reagents with easily enolizable ketones
International Nuclear Information System (INIS)
Imamoto, Tsuneo; Kusumoto, Tetsuo; Sugiura, Yasushi; Suzuki, Nobuyo; Takiyama, Nobuyuki
Organocerium (III) reagents were conveniently generated by the reaction of organolithium compounds with anhydrous cerium (III) chloride. The reagents are less basic than organolithiums and Grignard reagents, and they react readily at -78 deg C with easily enolizable ketones such as 2-tetralone to afford addition products in high yields. Cerium (III) enolates were also generated from lithium enolates and cerium (III) chloride. The cerium (III) enolates undergo aldol addition with ketones or sterically crowded aldehyde to give the corresponding β-hydroxy ketones in good to high yields. (author)
Small RNAs and the regulation of cis-natural antisense transcripts in Arabidopsis
Directory of Open Access Journals (Sweden)
Lonardi Stefano
Full Text Available Abstract Background In spite of large intergenic spaces in plant and animal genomes, 7% to 30% of genes in the genomes encode overlapping cis-natural antisense transcripts (cis-NATs. The widespread occurrence of cis-NATs suggests an evolutionary advantage for this type of genomic arrangement. Experimental evidence for the regulation of two cis-NAT gene pairs by natural antisense transcripts-generated small interfering RNAs (nat-siRNAs via the RNA interference (RNAi pathway has been reported in Arabidopsis. However, the extent of siRNA-mediated regulation of cis-NAT genes is still unclear in any genome. Results The hallmarks of RNAi regulation of NATs are 1 inverse regulation of two genes in a cis-NAT pair by environmental and developmental cues and 2 generation of siRNAs by cis-NAT genes. We examined Arabidopsis transcript profiling data from public microarray databases to identify cis-NAT pairs whose sense and antisense transcripts show opposite expression changes. A subset of the cis-NAT genes displayed negatively correlated expression profiles as well as inverse differential expression changes under at least one of the examined developmental stages or treatment conditions. By searching the Arabidopsis Small RNA Project (ASRP and Massively Parallel Signature Sequencing (MPSS small RNA databases as well as our stress-treated small RNA dataset, we found small RNAs that matched at least one gene in 646 pairs out of 1008 (64% protein-coding cis-NAT pairs, which suggests that siRNAs may regulate the expression of many cis-NAT genes. 209 putative siRNAs have the potential to target more than one gene and half of these small RNAs could target multiple members of a gene family. Furthermore, the majority of the putative siRNAs within the overlapping regions tend to target only one transcript of a given NAT pair, which is consistent with our previous finding on salt- and bacteria-induced nat-siRNAs. In addition, we found that genes encoding plastid- or
Specific regulation of point-mutated K-ras-immortalized cell proliferation by a photodynamic antisense strategy.
Higuchi, Maiko; Yamayoshi, Asako; Kato, Kiyoko; Kobori, Akio; Wake, Norio; Murakami, Akira
It has been reported that point mutations in genes are responsible for various cancers, and the selective regulation of gene expression is an important factor in developing new types of anticancer drugs. To develop effective drugs for the regulation of point-mutated genes, we focused on photoreactive antisense oligonucleotides. Previously, we reported that photoreactive oligonucleotides containing 2'-O-psoralenylmethoxyethyl adenosine (2'-Ps-eom) showed drastic photoreactivity in a strictly sequence-specific manner. Here, we demonstrated the specific gene regulatory effects of 2'-Ps-eom on [(12)Val]K-ras mutant (GGT --> GTT). Photo-cross-linking between target mRNAs and 2'-Ps-eom was sequence-specific, and the effect was UVA irradiation-dependent. Furthermore, 2'-Ps-eom was able to inhibit K-ras-immortalized cell proliferation (K12V) but not Vco cells that have the wild-type K-ras gene. These results suggest that the 2'-Ps-eom will be a powerful nucleic acid drug to inhibit the expression of disease-causing point mutation genes, and has great therapeutic potential in the treatment of cancer.
The HLA-DP polymorphism in Denmark investigated by local and international PLT reagents. Definition of two "new" DP antigens
DEFF Research Database (Denmark)
Ødum, Niels; Hartzman, R; Jakobsen, B K
Lymphocytes from highly selected donors were primed for 10 days and subsequently bulk-expanded in IL 2 (TCGF) containing cultures. Two well-discriminatory PLT (CDP = Copenhagen DP) reagents against each of the DPw1-w6 specificities and one against each of the two "new" specificities, CDP4s...
Study of effect of slime processed by ultrasonic on flotation reagent
Energy Technology Data Exchange (ETDEWEB)
Kang, W.; Wang, H.; Hu, J. [Heilongjiang Institute of Science and Technology, Harbin (China)
The changes of wetting heat, oil film thickness, contact angle and absorptive capacity of slime and kerosene before and after ultrasonic treatment were studied by Setaram calorimeter, DCAT21 gauge and a light meter. Batch flotation tests were made. The results show that: the wetting heat from slime and kerosene increases after ultrasonic treatment by 45.85%; the average oil film thickness is reduced by 38.84%; the contact angle of slime and kerosene decreases by 33.56{sup o}; and the absorptive capacity of slime in kerosene decreases by 30.40%. The consumption of flotation reagent after ultrasonic treatment was reduced by 75% while the yield of clean coal was the same. The study shows that ultrasonic treatment decreases the consumption of reagent and increases the efficiency and selectivity of flotation. 8 refs., 3 figs., 4 tabs.
Effect of injection of antisense oligodeoxynucleotides of GAD isozymes into rat ventromedial hypothalamus on food intake and locomotor activity.
Bannai, M; Ichikawa, M; Nishihara, M; Takahashi, M
In the ventromedial hypothalamus (VMH), gamma-aminobutyric acid (GABA) plays a role in regulating feeding and running behaviors. The GABA synthetic enzyme, glutamic acid decarboxylase (GAD), consists of two isozymes, GAD65 and GAD67. In the present study, the phosphorothioated antisense oligodeoxynucleotides (ODNs) of each GAD isozyme were injected bilaterally into the VMH of male rats, and food intake, body weight and locomotor activity were monitored. ODNs were incorporated in the water-absorbent polymer (WAP, 0.2 nmol/microliter) so that ODNs were retained at the injection site. Each antisense ODN of GAD65 or GAD67 tended to reduce food intake on day 1 (day of injection=day 0) though not significantly. An injection combining both antisense ODNs significantly decreased food intake only on day 1, but body weight remained significantly lower than the control for 5 days. This suppression of body weight gain could be attributed to a significant increase in locomotor activity between days 3 and 5. Individual treatment with either ODNs did not change locomotor activity. The increase in daily locomotor activity in the group receiving the combined antisense ODNs occurred mainly during the light phase. Neither vehicle (WAP) nor control ODN affected food intake, body weight and locomotor activity. Histological studies indicated that antisense ODN distributed within 800 micron from the edge of the area where WAP was located 24 h after the injection gradually disappeared within days, but still remained within 300 micron m distance even 7 days after the injection. Antisense ODN was effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias. Further, HPLC analysis revealed that antisense ODNs of GAD isozymes, either alone or combined, decreased the content of GABA by 50% in VMH 24 h after the injection. These results indicate that suppression of GABA synthesis by either of the GAD isozymes is synergistically involved in suppressing food
Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.
Directory of Open Access Journals (Sweden)
Takashi Saito
Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.
Tetrameric DABCO™-Bromine: an Efficient and Versatile Reagent ...
African Journals Online (AJOL)
Reagent for Bromination of Various Organic Compounds. Majid M. Heravi,a* ... aDepartment of Chemistry, School of Sciences, Azzahra University, Vanak, Tehran, Iran. bChemistry ... Synthesis of "-bromo ketones and nitriles has also been ...
21 CFR 866.3470 - Reovirus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3470 Reovirus... and antisera used in serological tests to identify antibodies to reovirus in serum. The identification...
21 CFR 866.3870 - Trypanosoma spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3870 Trypanosoma... consist of antigens and antisera used in serological tests to identify antibodies to Trypanosoma spp. in...
21 CFR 866.3630 - Serratia spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3630 Serratia spp... antigens and antisera used in serological tests to identify Serratia spp. from cultured isolates. The...
21 CFR 866.3850 - Trichinella spiralis serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3850... devices that consist of antigens and antisera used in serological tests to identify antibodies to...
21 CFR 866.3680 - Sporothrix schenckii serological reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3680... devices that consist of antigens and antisera used in serological tests to identify antibodies to...
21 CFR 866.3660 - Shigella spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3660 Shigella spp...), used in serological tests to identify Shigella spp. from cultured isolates. The identification aids in...
21 CFR 866.3400 - Parainfluenza virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3400 Parainfluenza... that consist of antigens and antisera used in serological tests to identify antibodies to parainfluenza...
21 CFR 866.3040 - Aspergillus spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3040 Aspergillus... consist of antigens and antisera used in various serological tests to identify antibodies to Aspergillus...
21 CFR 866.3330 - Influenza virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3330 Influenza... consist of antigens and antisera used in serological tests to identify antibodies to influenza in serum...
21 CFR 866.3125 - Citrobacter spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3125 Citrobacter... isolates derived from clinical specimens. The identification aids in the diagnosis of disease caused by...
21 CFR 866.3940 - West Nile virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3940 West Nile... detection aids in the clinical laboratory diagnosis of viral meningitis/encephalitis caused by West Nile...
21 CFR 866.3740 - Streptococcus spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3740 Streptococcus... derived from clinical specimens. The identification aids in the diagnosis of diseases caused by bacteria...
21 CFR 866.3065 - Bordetella spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3065 Bordetella... serological tests to identify Bordetella spp. from cultured isolates or directly from clinical specimens. The...
21 CFR 866.3035 - Arizona spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3035 Arizona spp... antisera and antigens used to identify Arizona spp. in cultured isolates derived from clinical specimens...
21 CFR 866.3380 - Mumps virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3380 Mumps virus... serological tests to identify mumps viruses from tissue culture isolates derived from clinical specimens. The...
Reagent and process for detaching furfural in petroleum products
Energy Technology Data Exchange (ETDEWEB)
Orelup, R.B.
A two-component liquid reagent and process is provided for detecting the presence of furfural in petroleum products. The reagent comprises two components which are stored separately from each other and are combined prior to admixture with the petroleum product. The process comprises combining the two separate components of the liquid reagent with each other prior to use, admixing the combined components with a petroleum product containing furfural, shaking the resultant mixture, allowing the mixture to separate and observing a red color characteristic of furfural in the lower layer. Alternately, the process may be carried out by combining the second component of the two-component liquid reagent with the petroleum product, followed by admixture with the first component to obtain two separate layers in which the red color characteristic of furfural is observed in the lower layer.
Alkaline phosphatase-fused repebody as a new format of immuno-reagent for an immunoassay
Energy Technology Data Exchange (ETDEWEB)
Seo, Hyo-Deok; Lee, Joong-jae [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of); Kim, Yu Jung [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Hantschel, Oliver [School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne (Switzerland); Lee, Seung-Goo [Industrial Biotechnology and Bioenergy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon (Korea, Republic of); Kim, Hak-Sung, E-mail: email@example.com [Department of Biological Sciences, Korea Advanced Institute of Science and Technology (KAIST), 291 Daehak-ro, Yuseong-gu, Daejeon, 305-701 (Korea, Republic of)
Enzyme-linked immunoassays based on an antibody-antigen interaction are widely used in biological and medical sciences. However, the conjugation of an enzyme to antibodies needs an additional chemical process, usually resulting in randomly cross-linked molecules and a loss of the binding affinity and enzyme activity. Herein, we present the development of an alkaline phosphatase-fused repebody as a new format of immuno-reagent for immunoassays. A repebody specifically binding to human TNF-α (hTNF-α) was selected through a phage display, and its binding affinity was increased up to 49 nM using a modular engineering approach. A monomeric alkaline phosphatase (mAP), which was previously isolated from a metagenome library, was genetically fused to the repebody as a signal generator, and the resulting repebody-mAP fusion protein was used for direct and sandwich immunoassays of hTNF-α. We demonstrate the utility and potential of the repebody-mAP fusion protein as an immuno-reagent by showing the sensitivity of 216 pg mL{sup −1} for hTNF-α in a sandwich immunoassay. Furthermore, this repebody-mAP fusion protein enabled the detection of hTNF-α spiked in a serum-supplemented medium with high accuracy and reproducibility. It is thus expected that a mAP-fused repebody can be broadly used as an immuno-reagent in immunoassays. - Highlights: • A human TNF-α (hTNF-α)-specific repebody was selected using a phage display. • A monomeric alkaline phosphatase (mAP) was genetically fused to the repebody. • mAP-fused repebody enabled detection of hTNF-α with high sensitivity and accuracy. • mAP-fused repebody can be widely used as a new immuno-reagent in immunoassays.
Method for controlled introduction of reagent into a liquid
Energy Technology Data Exchange (ETDEWEB)
Newlove, J.C.; McDougall, L.A.
It is an object of this invention to provide a method for using an article to enhance the production of hydrocarbons from geological reservoirs, more particularly from fractured formations. It is an additional object to devise a method for providing controlled release of a reagent downhole, in a pipeline, or in other oil-containing environments or fluids. Thus, there is provided a method for releasing a treating reagent, such as a wax crystal modifier, scale inhibitor, demulsifier, corrosion inhibitor, antioxidant, and biocide, into a liquid hydrocarbon stream. A plurality of porous, substantially wax-free, plastic particles having a softening point above 60/sup 0/C and being chemically resistant to the hydrocarbon stream are placed in the stream. The said particles contain the treating reagent in their pores, said reagent being insoluble in water and in the particles and being leachable on contact with the stream. The hydrocarbon stream is then flowed past said particles and the reagent is leached from the particle pores. In a specific aspect of this invention, a method is provided for recovering crude oil from an underground formation by means of: depositing the aforementioned particles, containing a suitable reagent, downhole in the oil-producing region of the formation; flowing the oil through the deposited particles, thereby leaching the reagent into the oil; and recovering the oil modified by the presence of an active amount of said reagent. Experiments are described to illustrate ways of producing the polymeric particles of the invention and to illustrate the processes of the invention.
Evaluation of novel derivatisation reagents for the analysis of oxysterols
Energy Technology Data Exchange (ETDEWEB)
Crick, Peter J., E-mail: firstname.lastname@example.com [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Aponte, Jennifer; Bentley, T. William [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Matthews, Ian [College of Engineering, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Wang, Yuqin [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom); Griffiths, William J., E-mail: email@example.com [Institute of Mass Spectrometry, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP (United Kingdom)
Graphical abstract: - Highlights: • New derivatisation reagents for LC–MS analysis of oxysterols. • New reagents based on Girard P give high ion-currents and informative LC–MS{sup n} spectra. • Permanent charge is vital for efficient MS{sup n} fragmentation. • New reagents offer greater scope for incorporation of isotope labels. - Abstract: Oxysterols are oxidised forms of cholesterol that are intermediates in the synthesis of bile acids and steroid hormones. They are also ligands to nuclear and G protein-coupled receptors. Analysis of oxysterols in biological systems is challenging due to their low abundance coupled with their lack of a strong chromophore and poor ionisation characteristics in mass spectrometry (MS). We have previously used enzyme-assisted derivatisation for sterol analysis (EADSA) to identify and quantitate oxysterols in biological samples. This technique relies on tagging sterols with the Girard P reagent to introduce a charged quaternary ammonium group. Here, we have compared several modified Girard-like reagents and show that the permanent charge is vital for efficient MS{sup n} fragmentation. However, we find that the reagent can be extended to include sites for potential stable isotope labels without a loss of performance.
Inhibition of B cell proliferation by antisense DNA to both alpha and beta forms of Fc epsilon R II.
Bhatti, L; Behle, K; Stevens, R H
Epstein-Barr Virus (EBV) infection activates B lymphocyte proliferation through partially understood mechanisms, resulting in phenotypic changes, including the appearance of new antigens. One such antigen is Fc epsilon R II/CD-23 which may be relevant for B cell proliferation. We have used anti-sense oligonucleotides to study the importance of the two forms of this molecule for proliferation in the EBV-transformed, Fc epsilon R II +ve lymphoblastoid B cell line, RPMI 8866. Anti-sense oligodeoxynucleotides were generated to the two forms of Fc epsilon R II; Fc epsilon R IIa (alpha) and IIb (beta) which differ only in their intracytoplasmic domains. Addition of increasing concentrations of anti-sense oligonucleotides, ranging from 1 to 30 microM, significantly decreased cellular proliferation as measured by the incorporation of [3H]thymidine (inhibition range 8-88%). Optimum inhibition of cellular proliferation was apparent at 15 microM concentration of both anti-sense Fc epsilon R IIa and IIb (Fc epsilon R IIa, mean +/- SE = 75 +/- 7% inhibition, p less than 0.001; Fc epsilon R IIb, mean +/- SE = 71 +/- 7% inhibition, p less than 0.001). Anti-sense oligonucleotides complementary to the common part of Fc epsilon R II resulted in a similar inhibition of proliferation. Sense oligonucleotides did not induce significant inhibition. Preincubation of sense and anti-sense oligonucleotides resulted in an abrogation of proliferation inhibition. Moreover, none of these oligonucleotides had any effect on a Fc epsilon R II -ve cell line. Incubation with both anti-sense IIa and IIb resulted in additive, but not synergistic inhibition of proliferation. Addition of soluble Fc epsilon R II did not reverse inhibition of proliferation, suggesting that membrane-bound or intracellular rather than soluble Fc epsilon R II was important for the induced proliferation. Analysis of cell surface expression for Fc epsilon II indicated that while there was a pronounced effect on cell number
Antisense RNA: a genetic approach to cell resistance against Parvovirus; RNA antisentido: una aproximacion de resistencia genetica a Parvovirus
Energy Technology Data Exchange (ETDEWEB)
Ramirez Martinez, J.C.
The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.
Antisense RNA: a genetic approach to cell resistance against Parvovirus. RNA antisentido: una aproximacion de resistencia genetica a Parvovirus
Energy Technology Data Exchange (ETDEWEB)
Ramirez Martinez, J.C.
The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.
Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line.
Leonetti, C; Biroccio, A; Benassi, B; Stringaro, A; Stoppacciaro, A; Semple, S C; Zupi, G
Phosphorothioate c-myc antisense oligodeoxynucleotides [S]ODNs (free INX-6295) were encapsulated in a new liposome formulation and the antitumor activity was compared to the unencapsulated antisense in a human melanoma xenograft. The systemic administration of INX-6295 encapsulated in stabilized antisense lipid particles (SALP INX-6295) improved plasma AUC (area under the plasma concentration-time curve) and initial half-life of free INX-6295, resulting in a significant enhancement in tumor accumulation and improvement in tumor distribution of antisense oligodeoxynucleotides. Animals treated with SALP INX-6295 exhibited a prolonged reduction of c-myc expression, reduced tumor growth and increased mice survival. When administered in combination with cisplatin (DDP), SALP INX-6295 produced a complete tumor regression in approximately 30% of treated mice, which persisted for at least 60 days following the first cycle of treatment. Finally, the median survival of mice treated with DDP/SALP INX-6295 increased by 105% compared to 84% for animals treated with the combination DDP/free INX-6295. These data indicate that the biological activity and the therapeutic efficacy of c-myc antisense therapy may be improved when these agents are administered in lipid-based delivery systems.
Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice
International Nuclear Information System (INIS)
Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.
Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion
The influence of reagent type on the kinetics of ultrafine coal flotation
Read, R.B.; Camp, L.R.; Summers, M.S.; Rapp, D.M.
A kinetic study has been conducted to determine the influence of reagent type on flotation rates of ultrafine coal. Two ultrafine coal samples, the Illinois No. 5 (Springfield) and Pittsburgh No. 8, have been evaluated with various reagent types in order to derive the rate constants for coal (kc), ash (ka), and pyrite (kc). The reagents used in the study include anionic surfactants, anionic surfactant-alcohol mixtures, and frothing alcohols. In general, the surfactant-alcohol mixtures tend to float ultrafine coal at a rate three to four times faster than either pure alcohols or pure anionic surfactants. Pine oil, a mixture of terpene alcohols and hydrocarbons, was an exception to this finding; it exhibited higher rate constants than the pure aliphatic alcohols or other pure anionic surfactants studied; this may be explained by the fact that the sample of pine oil used (70% alpha-terpineol) acted as a frother/collector system similar to alcohol/kerosene. The separation efficiencies of ash and pyrite from coal, as evidenced by the ratios of kc/ka or kc/kp, tend to indicate, however, that commercially available surfactant-alcohol mixtures are not as selective as pure alcohols such as 2-ethyl-1-hexanol or methylisobutylcarbinol. Some distinct differences in various rate constants, or their ratios, were noted between the two coals studied, and are possibly attributable to surface chemistry effects. ?? 1989.
Candidate reagents and procedures for the dissolution of Hanford Site single-shell tank sludges
International Nuclear Information System (INIS)
Schulz, W.W.; Kupfer, M.J.
At least some of the waste in the 149 single-shell tanks (SST) at the US Department of Energy (DOE) Hanford Site will be retrieved, treated, and disposed of. Although the importance of devising efficient and cost-effective sludge dissolution procedures has long been recognized, a concerted bench-scale effort to devise and test such procedures with actual solids representative of those in Hanford Site SSTs has not been performed. Reagents that might be used, either individually or serially, to dissolve sludges include HNO 3 , HNO 3 -oxalic acid, and HNO 3 -HF. This report consolidates and updates perspectives and recommendations concerning reagents and procedures for dissolving Hanford Site SST and selected double-shell tank (DST) sludges. The principal objectives of this report are as follows: (1) Compile and review existing experimental data on dissolution of actual Hanford Site SST and DST sludges. (2) Further inform Hanford Site engineers and scientists concerning the utility of combinations of thermally unstable complexants (TUCS) reagents and various reducing agents for dissolving SST and DST sludges. (This latter technology has recently been explored at the Argonne National Laboratory.) (3) Provide guidance in laying out a comprehensive experimental program to develop technology for dissolving all types of Hanford Site SST and DST sludges. 6 refs., 1 fig., 4 tabs
Assessment and identification of some novel NOx reducing reagents for SNCR process
International Nuclear Information System (INIS)
Mahmood, A.; Javed, M.T.; Irfan, N.; Hamid, A. and K.; Waheed, K.
Nitrogen oxides (NOx) are one of the most hazardous air pollutants arising from the combustion processes. Because of the implementation of strict emission limits many NOx removal technologies have been developed. In the present work post combustion NOx removal technique that is Selective Non-Catalytic Reduction (SNCR) has been investigated in a pilot scale 150 kW combustion rig facility. Investigation has been performed using some novel NOx reducing reagents like urea, ammonium carbonate and mixture of their 50%-50% aqueous solution within the temperature range of 700 to 1200 deg. C., at 1.1% excess oxygen and background NOx level of 500 ppm. The effects of these reagents were determined in term of their temperature characteristics and molar ratio. Among the reducing reagents used urea solution gave the highest NOx removal efficiency (81%) and was attractive due to its superior high temperature (1000 to 1150 deg. C) performance, ammonium carbonate was more effective at lower temperature range (850 to 950 deg. C) though its efficiency (32%) was lower than urea, while 50-50% solution of urea and ammonium carbonate gave higher efficiency than ammonium carbonate but slightly lesser than urea within a wide temperature range (875 to 1125 deg. C). It was also observed that the NOx removal efficiency was increased with increasing the molar ratio. (author)
Hydrogel-Assisted Antisense LNA Gapmer Delivery for In Situ Gene Silencing in Spinal Cord Injury
DEFF Research Database (Denmark)
Moreno, Pedro M.D.; Ferreira, Ana R.; Salvador, Daniela
)-modified AON gapmers in combination with a fibrin hydrogel bridging material to induce gene silencing in situ at a SCI lesion site. LNA gapmers were effectively developed against two promising gene targets aiming at enhancing axonal regeneration—RhoA and GSK3β. The fibrin-matrix-assisted AON delivery system......After spinal cord injury (SCI), nerve regeneration is severely hampered due to the establishment of a highly inhibitory microenvironment at the injury site, through the contribution of multiple factors. The potential of antisense oligonucleotides (AONs) to modify gene expression at different levels...
Nanoparticle for delivery of antisense γPNA oligomers targeting CCR5
Bahal, Raman; McNeer, Nicole Ali; Ly, Danith H.; Saltzman, W. Mark; Glazer, Peter M.
The development of a new class of peptide nucleic acids (PNAs), i.e., gamma PNAs (γPNAs), creates the need for a general and effective method for its delivery into cells for regulating gene expression in mammalian cells. Here we report the antisense activity of a recently developed hydrophilic and biocompatible diethylene glycol (miniPEG)-based gamma peptide nucleic acid called MPγPNAs via its delivery by poly(lactide-co-glycolide) (PLGA)-based nanoparticle system. We show that MPγPNA oligome...
Expression of antisense small RNAs in response to stress in Pseudomonas aeruginosa
DEFF Research Database (Denmark)
Gómez Lozano, María; Marvig, Rasmus Lykke; Tulstrup, Monica Vera-Lise
Background: RNA sequencing technologies reveal that bacteria express RNA molecules other than mRNA, rRNA or tRNA. During the last years genome-wide bacterial transcriptomes have been shown to comprise intergenic RNA, antisense RNA, and untranslated regions, all capable of performing diverse...... that the extent of overlap between the studies is very limited. Conclusions: RNA-seq experiments are revealing hundreds of novel transcripts in all bacterial genomes investigated. The comparison between independent studies that used RNA-seq to detect novel asRNAs in P. aeruginosa shows that the overlap between...
Reagent conditions of the flotation of copper, copper - molybdenum and copper -zinc ores in foreing countries
International Nuclear Information System (INIS)
Nevaeva, L.M.
Reagents-collectors and frothers, used abroad in reagent regimes of flotation of copper, copper-molybdenum and copper zinc ores, have been considered. Xanthogenates, aerofloats, xanthogenformiates, thionocarbamates are mainly used as reagents-collectors. Methylizobutylcarbinol and Daufros are used as reagents-frothers
21 CFR 866.3720 - Streptococcus spp. exo-enzyme reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Streptococcus spp. exo-enzyme reagents. 866.3720... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3720 Streptococcus spp. exo-enzyme reagents. (a) Identification. Streptococcus spp. exoenzyme reagents are devices used...
Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides
DEFF Research Database (Denmark)
Shiraishi, Takehiko; Pankratova, Stanislava; Nielsen, Peter E
Cell-penetrating peptides have been widely used to improve cellular delivery of a variety of proteins and antisense agents. However, recent studies indicate that such cationic peptides are predominantly entering cells via an endosomal pathway. We now show that the nuclear antisense effect in He......La cells of a variety of peptide nucleic acid (PNA) peptide conjugates is significantly enhanced by addition of 6 mM Ca(2+) (as well as by the lysosomotrophic agent chloroquine). In particular, the antisense activities of Tat(48-60) and heptaarginine-conjugated PNAs were increased 44-fold and 8.5-fold......, respectively. Evidence is presented that the mechanism involves endosomal release. The present results show that Ca(2+) can be used as an effective enhancer for in vitro cellular delivery of cationic peptide-conjugated PNA oligomers, and also emphasize the significance of the endosomal escape route...
Reaction of carbohydrates with Vilsmeier reagent
DEFF Research Database (Denmark)
Thota, Niranjan; Mukherjee, Debaraj; Reddy, Mallepally Venkat
A convenient and efficient method for selective replacement of the primary hydroxyl groups of sugars by chlorine with concomitant O-formylation, compatible with the presence of a variety of functional groups, has been developed using the Vilsmeier-Haack reaction. Sugars having free primary hydrox...
Treatment of laundrette wastewater using Starbon and Fenton's reagent.
Tony, Maha A; Parker, Helen L; Clark, James H
The use of grey water for a variety of purposes is gaining increased popularity as a means of preserving scarce freshwater resources. In this work, catalytic oxidation over Fenton's reagent and adsorption techniques using Starbon (mesoporous material derived from polysaccharides) has been applied. These novel techniques are used as an alternative to already studied treatments of grey water such as filtration and/or biological processes. In this study, grey water, collected from a commercial laundrette, has been used. Treatment efficiency was determined by changes in the chemical oxygen demand (COD) of the grey water. Experiments using Fenton's reagent at optimum conditions of Fe(3+) = 40 mg L(-1); H2O2 = 400 mg L(-1) and pH 3 were very successful, resulting in a 95% COD removal after 15 min. Treatment with Starbon adsorption was also effective, reaching up to 81% COD removal at pH 3 within 1 h. The combined treatment with Fenton's reagent and Starbon resulted in a 93% COD removal at a significantly reduced concentration of Fenton's reagent compared to the treatment with solo Fenton's reagent. This lower chemical dose has the advantage of reducing costs and lowering sludge generation.
High-performance reagent modes for flotation recovery of platiniferous copper and nickel sulfides from hard-to-beneficiate ores
Matveeva, T. N.; Chanturiya, V. A.
The paper presents the results of the recent research performed in IPKON Russian Academy of Sciences that deals with development and substantiation of new selective reagents for effective flotation recovery of non-ferrous and noble metals from refractory ores. The choice and development of new selective reagents PTTC, OPDTC, modified butylxanthate (BXm) and modified diethyl-dithiocarbamate (DEDTCm) to float platiniferous copper and nickel sulfide minerals from hard-to-beneficiate ores is substantiated. The mechanism of reagents adsorption and regulation of minerals floatability is discussed. The study of reagent modes indicates that by combining PTTC with the modified xanthate results in 6 - 7 % increase in the recovery of copper, nickel and PGM in the flotation of the low-sulfide platiniferous Cu-Ni ore from the Fedorovo-Panskoye deposit. The substitution of OPDTC for BX makes it possible to increase recovery of Pt by 13 %, Pd by 9 % and 2 - 4 times the noble metal content in the flotation concentrate.
Sense-antisense (complementary) peptide interactions and the proteomic code; potential opportunities in biology and pharmaceutical science.
Miller, Andrew D
A sense peptide can be defined as a peptide whose sequence is coded by the nucleotide sequence (read 5' → 3') of the sense (positive) strand of DNA. Conversely, an antisense (complementary) peptide is coded by the corresponding nucleotide sequence (read 5' → 3') of the antisense (negative) strand of DNA. Research has been accumulating steadily to suggest that sense peptides are capable of specific interactions with their corresponding antisense peptides. Unfortunately, although more and more examples of specific sense-antisense peptide interactions are emerging, the very idea of such interactions does not conform to standard biology dogma and so there remains a sizeable challenge to lift this concept from being perceived as a peripheral phenomenon if not worse, into becoming part of the scientific mainstream. Specific interactions have now been exploited for the inhibition of number of widely different protein-protein and protein-receptor interactions in vitro and in vivo. Further, antisense peptides have also been used to induce the production of antibodies targeted to specific receptors or else the production of anti-idiotypic antibodies targeted against auto-antibodies. Such illustrations of utility would seem to suggest that observed sense-antisense peptide interactions are not just the consequence of a sequence of coincidental 'lucky-hits'. Indeed, at the very least, one might conclude that sense-antisense peptide interactions represent a potentially new and different source of leads for drug discovery. But could there be more to come from studies in this area? Studies on the potential mechanism of sense-antisense peptide interactions suggest that interactions may be driven by amino acid residue interactions specified from the genetic code. If so, such specified amino acid residue interactions could form the basis for an even wider amino acid residue interaction code (proteomic code) that links gene sequences to actual protein structure and function, even
SHAPE selection (SHAPES) enrich for RNA structure signal in SHAPE sequencing-based probing data
DEFF Research Database (Denmark)
Poulsen, Line Dahl; Kielpinski, Lukasz Jan; Salama, Sofie R
transcriptase. Here, we introduce a SHAPE Selection (SHAPES) reagent, N-propanone isatoic anhydride (NPIA), which retains the ability of SHAPE reagents to accurately probe RNA structure, but also allows covalent coupling between the SHAPES reagent and a biotin molecule. We demonstrate that SHAPES...
Defining global gene expression changes of the hypothalamic-pituitary-gonadal axis in female sGnRH-antisense transgenic common carp (Cyprinus carpio.
Directory of Open Access Journals (Sweden)
Jing Xu
Full Text Available BACKGROUND: The hypothalamic-pituitary-gonadal (HPG axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. METHODOLOGY/PRINCIPAL FINDINGS: In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus, 16 and 12 (pituitary, 119 and 93 (ovary, respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. CONCLUSIONS/SIGNIFICANCE: This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the
Construction of a novel chimera consisting of a chelator-containing Tat peptide conjugated to a morpholino antisense oligomer for technetium-99m labeling and accelerating cellular kinetics
Energy Technology Data Exchange (ETDEWEB)
Zhang Yumin [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States)]. E-mail: firstname.lastname@example.com; Tung, C.-H. [Center for Molecular Imaging Research, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA 02129 (United States); He Jiang [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States); Liu Ning [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States); Yanachkov, Ivan [GlSynthesis, Worcester, MA 01605 (United States); Liu Guozheng [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States); Rusckowski, Mary [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States); Vanderheyden, Jean-Luc [Division of Nuclear Medicine, Department of Radiology, University of Massachusetts Medical School, Worcester, MA 01655 (United States)
The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N{sub 2}S{sub 2} chelator for technetium-99m ({sup 99m}Tc) radiolabeling (N{sub 2}S{sub 2}-Tat-MORF). The radiolabeling properties and cellular kinetics of {sup 99m}Tc-N{sub 2}S{sub 2}-Tat-MORF were measured. As hypothesized, the preparation of {sup 99m}Tc-N{sub 2}S{sub 2}-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the {sup 99m}Tc-N{sub 2}S{sub 2}-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG{sub 3}) as chelator for {sup 99m}Tc ({sup 99m}Tc-MAG{sub 3}-MORF). {sup 99m}Tc-N{sub 2}S{sub 2}-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while {sup 99m}Tc-MAG{sub 3}-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and {sup 99m}Tc-N{sub 2}S{sub 2}-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.
Defining Global Gene Expression Changes of the Hypothalamic-Pituitary-Gonadal Axis in Female sGnRH-Antisense Transgenic Common Carp (Cyprinus carpio)
Xu, Jing; Huang, Wei; Zhong, Chengrong; Luo, Daji; Li, Shuangfei; Zhu, Zuoyan; Hu, Wei
Background The hypothalamic-pituitary-gonadal (HPG) axis is critical in the development and regulation of reproduction in fish. The inhibition of neuropeptide gonadotropin-releasing hormone (GnRH) expression may diminish or severely hamper gonadal development due to it being the key regulator of the axis, and then provide a model for the comprehensive study of the expression patterns of genes with respect to the fish reproductive system. Methodology/Principal Findings In a previous study we injected 342 fertilized eggs from the common carp (Cyprinus carpio) with a gene construct that expressed antisense sGnRH. Four years later, we found a total of 38 transgenic fish with abnormal or missing gonads. From this group we selected the 12 sterile females with abnormal ovaries in which we combined suppression subtractive hybridization (SSH) and cDNA microarray analysis to define changes in gene expression of the HPG axis in the present study. As a result, nine, 28, and 212 genes were separately identified as being differentially expressed in hypothalamus, pituitary, and ovary, of which 87 genes were novel. The number of down- and up-regulated genes was five and four (hypothalamus), 16 and 12 (pituitary), 119 and 93 (ovary), respectively. Functional analyses showed that these genes involved in several biological processes, such as biosynthesis, organogenesis, metabolism pathways, immune systems, transport links, and apoptosis. Within these categories, significant genes for neuropeptides, gonadotropins, metabolic, oogenesis and inflammatory factors were identified. Conclusions/Significance This study indicated the progressive scaling-up effect of hypothalamic sGnRH antisense on the pituitary and ovary receptors of female carp and provided comprehensive data with respect to global changes in gene expression throughout the HPG signaling pathway, contributing towards improving our understanding of the molecular mechanisms and regulative pathways in the reproductive system of
Quantitative Antisense Screening and Optimization for Exon 51 Skipping in Duchenne Muscular Dystrophy.
Echigoya, Yusuke; Lim, Kenji Rowel Q; Trieu, Nhu; Bao, Bo; Miskew Nichols, Bailey; Vila, Maria Candida; Novak, James S; Hara, Yuko; Lee, Joshua; Touznik, Aleksander; Mamchaoui, Kamel; Aoki, Yoshitsugu; Takeda, Shin'ichi; Nagaraju, Kanneboyina; Mouly, Vincent; Maruyama, Rika; Duddy, William; Yokota, Toshifumi
Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder, is caused by mutations in the dystrophin (DMD) gene. Exon skipping is a therapeutic approach that uses antisense oligonucleotides (AOs) to modulate splicing and restore the reading frame, leading to truncated, yet functional protein expression. In 2016, the US Food and Drug Administration (FDA) conditionally approved the first phosphorodiamidate morpholino oligomer (morpholino)-based AO drug, eteplirsen, developed for DMD exon 51 skipping. Eteplirsen remains controversial with insufficient evidence of its therapeutic effect in patients. We recently developed an in silico tool to design antisense morpholino sequences for exon skipping. Here, we designed morpholino AOs targeting DMD exon 51 using the in silico tool and quantitatively evaluated the effects in immortalized DMD muscle cells in vitro. To our surprise, most of the newly designed morpholinos induced exon 51 skipping more efficiently compared with the eteplirsen sequence. The efficacy of exon 51 skipping and rescue of dystrophin protein expression were increased by up to more than 12-fold and 7-fold, respectively, compared with the eteplirsen sequence. Significant in vivo efficacy of the most effective morpholino, determined in vitro, was confirmed in mice carrying the human DMD gene. These findings underscore the importance of AO sequence optimization for exon skipping. Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.
Chemical modifications of antisense morpholino oligomers enhance their efficacy against Ebola virus infection.
Swenson, Dana L; Warfield, Kelly L; Warren, Travis K; Lovejoy, Candace; Hassinger, Jed N; Ruthel, Gordon; Blouch, Robert E; Moulton, Hong M; Weller, Dwight D; Iversen, Patrick L; Bavari, Sina
Phosphorodiamidate morpholino oligomers (PMOs) are uncharged nucleic acid-like molecules designed to inactivate the expression of specific genes via the antisense-based steric hindrance of mRNA translation. PMOs have been successful at knocking out viral gene expression and replication in the case of acute viral infections in animal models and have been well tolerated in human clinical trials. We propose that antisense PMOs represent a promising class of therapeutic agents that may be useful for combating filoviral infections. We have previously shown that mice treated with a PMO whose sequence is complementary to a region spanning the start codon of VP24 mRNA were protected against lethal Ebola virus challenge. In the present study, we report on the abilities of two additional VP24-specific PMOs to reduce the cell-free translation of a VP24 reporter, to inhibit the in vitro replication of Ebola virus, and to protect mice against lethal challenge when the PMOs are delivered prior to infection. Additionally, structure-activity relationship evaluations were conducted to assess the enhancement of antiviral efficacy associated with PMO chemical modifications that included conjugation with peptides of various lengths and compositions, positioning of conjugated peptides to either the 5' or the 3' terminus, and the conferring of charge modifications by the addition of piperazine moieties. Conjugation with arginine-rich peptides greatly enhanced the antiviral efficacy of VP24-specific PMOs in infected cells and mice during lethal Ebola virus challenge.
Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.
Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P
Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.
Search for antisense copies of beta-globin mRNA in anemic mouse spleen
Directory of Open Access Journals (Sweden)
Taylor John M
Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.
Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model
Directory of Open Access Journals (Sweden)
Karima Relizani
Full Text Available Antisense oligonucleotides (AONs hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA is considered very promising for the treatment of Duchenne muscular dystrophy (DMD, a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients. Keywords: antisense oligonucleotides, Duchenne muscular dystrophy, preclinical, splice switching, tcDNA-AONs
Determination of tin(II) in reagents for radiopharmaceuticals
International Nuclear Information System (INIS)
Morosanova, E.I.; Loginova, K. A.; Epstein, N.B.
The goal of this work is to elaborate a procedure for rapid and simple determination of tin(II) in reagents for preparation of radiopharmaceuticals based on a system albumin-Tc-99m. Original test tools for the determination of various analytes have been suggested in our lab based on the use of small glass tubes (1-2 mm i.d. - 50-70 mm) filled with indicator powders containing suitable immobilized chromogenic reagents. An analytical signal (a length of colored zone which is proportional to the concentration of an analyte) is detected after a sample passing through the indicator tube. Heteropoly compounds are well-known analytical reagents for a photometric determination of various reductants. For elaboration of indicator tubes abilities of Mo,P-heteropoly compounds to give deeply colored blue compounds after reduction were used. (authors)
Technetium-99m labeled antisense probes uptake in vascular smooth muscle cells
International Nuclear Information System (INIS)
Zhang, Y.X.; Qin, G.M.; An, R.; Cao, G.X.; Cao, W.; Gao, Z.R.
In the arterial wall, smooth muscle cells (SMC) normally exist in a quiescent, differentiated state, representing the contractile phenotype. During the development of atherosclerosis SMC change towards the synthetic phenotype going along with proliferation, chemotactic response and increased monocyte binding. The Fas/Fas ligand/caspase death-signaling pathway, Bcl-2 protein family/mitochondria, the tumor suppressive gene p53, and the proto-oncogene c-myc may be activated in atherosclerotic lesions, and mediates vascular apoptosis during the development of atherosclerosis. The atherosclerotic plaques contained 3-4 fold more c-myc mRNA than those in the normal aortic arteries, while increased Bax and Bak coupled with lack/paucity of Bcl-2 and Bcl-xL are associated with SMC apoptosis in advanced lesions. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. Cell uptake studies: 99m Tc- MAG 3 -DNA radioactivity incorporation into porcine coronary smooth muscle cells in the log and plateau phases, respectively, was determined after different times of incubation at 37. The influence of extracellular 99m Tc- MAG 3 -DNA concentration on SMC uptake was also analyzed. [Results] Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The MAG 3 -DNA was labeled with 99m Tc at room temperature and neutral pH, with a mean labeling efficiency of 80.11%(s.d=2.96%,n=4). The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. After labeling, the stability of the DNA in saline or serum was retained as determined by reverse-phase Sep-Pak C18 chromatography analysis, except a shift at 30 min in serum incubation that suggesting a short time serum protein binding. 99m Tc-MAG 3 -c-myc uptake plateaued at 60 min and was directly proportional to the
Study on biodistribution and imaging of radioiodinated antisense oligonucleotides in nude mice bearing human lymphoma
International Nuclear Information System (INIS)
Wang, R.F.; Shen, J.; Zhang, C.L.; Liu, M.; Guo, F.Q.
The incidence of sporadic lymphoma has risen due to an increase in immunosuppressed patients, particularly those with human immunodeficiency virus (HIV) infection. Sometimes suspect lymphoma has an undetectable location and we can not get the pathological specimen. Management of lymphoma is also difficult because the persistence of a significant number of residual tumor cells after intensive treatment. These relative failures can be attributed to make us choose this study for opening a new diagnostic and therapeutic field of lymphoma from molecular level. Immunoglobulin (Ig) heavy chain framework region (FR) of V1 family have been verified to be a major determinant of malignant phenotype of V1 family B-cell lymphoma. Most of targets for tumor antisense therapy study are protooncogenes, such as c-myc, bc1-2, which are broad -spectrum tumor imaging agents. The aim of this study was to investigate the possibility of using radioiodine labeled FR antisense oligonucleotides (ASONs) as an imaging agent or antisense therapeutic radiopharmaceutical in lymphoma. A 18-mer partial phosphorothioate oligonucleotide sequence was synthesized and grafted in 5 ' with a tyramine group which was further labeled with 125 I or 131 I using the chloramine T method. Normal CD-1 mice were injected via a tail vein with 148 kBq of 125 I-FR-ASON (2∼3 μ g). Animals were sacrificed at 1, 2, 4 and 24 h and tissue samples were studied. Liposome-mediated 3.33 MBq of 131 I-FR-ASON (7 ∼ 9μ g) was injected intratumorally into tumor-bearing BALB/c mice (6 weeks after inoculation of 10 7 Namalwa cells) meanwhile liposome-mediated 131 I labeled sense oligonucleotides served as controls. Biodistribution was monitored by sequential scintigraphy and organ radioactivity measurement 24 h after injection. The percentage of the injected dose per gram (%ID/g) of tumor and tumor/ non-tumor tissue ratios (T/NT) were calculated for each group of mice and the difference between two groups was assessed. The 5
Derivatization reagents in liquid chromatography/electrospray ionization tandem mass spectrometry.
Santa, Tomofumi
Liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) is one of the most prominent analytical techniques owing to its inherent selectivity and sensitivity. In LC/ESI-MS/MS, chemical derivatization is often used to enhance the detection sensitivity. Derivatization improves the chromatographic separation, and enhances the mass spectrometric ionization efficiency and MS/MS detectability. In this review, an overview of the derivatization reagents which have been applied to LC/ESI-MS/MS is presented, focusing on the applications to low molecular weight compounds. 2010 John Wiley & Sons, Ltd.
Durable field resistance to wheat yellow mosaic virus in transgenic wheat containing the antisense virus polymerase gene.
Chen, Ming; Sun, Liying; Wu, Hongya; Chen, Jiong; Ma, Youzhi; Zhang, Xiaoxiang; Du, Lipu; Cheng, Shunhe; Zhang, Boqiao; Ye, Xingguo; Pang, Junlan; Zhang, Xinmei; Li, Liancheng; Andika, Ida B; Chen, Jianping; Xu, Huijun
Wheat yellow mosaic virus (WYMV) has spread rapidly and causes serious yield losses in the major wheat-growing areas in China. Because it is vectored by the fungus-like organism Polymyxa graminis that survives for long periods in soil, it is difficult to eliminate by conventional crop management or fungicides. There is also only limited resistance in commercial cultivars. In this research, fourteen independent transgenic events were obtained by co-transformation with the antisense NIb8 gene (the NIb replicase of WYMV) and a selectable gene bar. Four original transgenic lines (N12, N13, N14 and N15) and an offspring line (N12-1) showed high and durable resistance to WYMV in the field. Four resistant lines were shown to have segregated and only contain NIb8 (without bar) by PCR and herbicide resistance testing in the later generations. Line N12-1 showed broad-spectrum resistance to WYMV isolates from different sites in China. After growing in the infested soil, WYMV could not be detected by tissue printing and Western blot assays of transgenic wheat. The grain yield of transgenic wheat was about 10% greater than the wild-type susceptible control. Northern blot and small RNA deep sequencing analyses showed that there was no accumulation of small interfering RNAs targeting the NIb8 gene in transgenic wheat plants, suggesting that transgene RNA silencing, a common mechanism of virus-derived disease resistance, is not involved in the process of WYMV resistance. This durable and broad-spectrum resistance to WYMV in transgenic wheat will be useful for alleviating the damage caused by WYMV. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.
Directory of Open Access Journals (Sweden)
Keng Boon Wee
Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.
Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.
Directory of Open Access Journals (Sweden)
Blake A Jacobson
Full Text Available BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.
Application of flotational reagents obtained from coke-industry byproducts
Energy Technology Data Exchange (ETDEWEB)
N.I. Nikitin; I.N. Nikitin; N.I. Toporkova [Khar' kov Polytechnic Institute (Ukraine)
Today, the operational efficiency of coal-preparation shops at coke plants largely depends on the flotation process, since flotation is the basic method of regenerating the slurry water in the water-slurry systems and the basic enrichment process for small-grain coal slurries. At The Coal-Chemistry Institute, attempts have been made to address the growing demand for readily available and relatively inexpensive flotational reagents. In particular, a list of promising coke-industry byproducts for use as flotational reagents has been compiled, and the possibility of reducing their toxicity has been established. In addition, various industrial byproducts and wastes have been investigated in terms of flotational activity.
Estimation of 239Pu in urine, influence of Sulkowich reagent
International Nuclear Information System (INIS)
Kalaiselvan, S.; Prasad, M.V.R.; Jeevanram, R.K.
Plutonium is known to be co-precipitated with Sulkowich reagent as calcium ammonium oxalate. In adopting this technique for bio-assay of plutonium, its accuracy depends on the self-absorption of the resulting precipitate in each urine sample. Pu recovery experiments were carried out with varying concentration of Ca and Mg, using different volumes of Sulkowich reagent. When the sample volume is 500 ml, Pu in urine can be estimated with an accuracy and precision of 74.38%+-7.4%, with a detection limit of 0.06 Bq (1.6 pCi) per dm 3 . (author) 3 refs.; 2 figs.; 2 tabs
Microwave-induced electrostatic etching: generation of highly reactive magnesium for application in Grignard reagent formation.
van de Kruijs, Bastiaan H P; Dressen, Mark H C L; Meuldijk, Jan; Vekemans, Jef A J M; Hulshof, Lumbertus A
A detailed study regarding the influence of microwave irradiation on the formation of a series of Grignard reagents in terms of rates and selectivities has revealed that these heterogeneous reactions may display a beneficial microwave effect. The interaction between microwaves and magnesium turnings generates violent electrostatic discharges. These discharges on magnesium lead to melting of the magnesium surface, thus generating highly active magnesium particles. As compared to conventional operation the microwave-induced discharges on the magnesium surface lead to considerably shorter initiation times for the insertion of magnesium in selected substrates (i.e. halothiophenes, halopyridines, octyl halides, and halobenzenes). Thermographic imaging and surface characterization by scanning electron microscopy showed that neither selective heating nor a "specific" microwave effect was causing the reduction in initiation times. This novel and straightforward initiation method eliminates the use of toxic and environmentally adverse initiators. Thus, this initiation method limits the formation of by-products. We clearly demonstrated that microwave irradiation enables fast Grignard reagent formation. Therefore, microwave technology is promising for process intensification of Grignard based coupling reactions.
The establishment of a WHO Reference Reagent for anti-malaria (Plasmodium falciparum) human serum.
Bryan, Donna; Silva, Nilupa; Rigsby, Peter; Dougall, Thomas; Corran, Patrick; Bowyer, Paul W; Ho, Mei Mei
At a World Health Organization (WHO) sponsored meeting it was concluded that there is an urgent need for a reference preparation that contains antibodies against malaria antigens in order to support serology studies and vaccine development. It was proposed that this reference would take the form of a lyophilized serum or plasma pool from a malaria-endemic area. In response, an immunoassay standard, comprising defibrinated human plasma has been prepared and evaluated in a collaborative study. A pool of human plasma from a malaria endemic region was collected from 140 single plasma donations selected for reactivity to Plasmodium falciparum apical membrane antigen-1 (AMA-1) and merozoite surface proteins (MSP-1 19 , MSP-1 42 , MSP-2 and MSP-3). This pool was defibrinated, filled and freeze dried into a single batch of ampoules to yield a stable source of naturally occurring antibodies to P. falciparum. The preparation was evaluated by an enzyme-linked immunosorbent assay (ELISA) in a collaborative study with sixteen participants from twelve different countries. This anti-malaria human serum preparation (NIBSC Code: 10/198) was adopted by the WHO Expert Committee on Biological Standardization (ECBS) in October 2014, as the first WHO reference reagent for anti-malaria (Plasmodium falciparum) human serum with an assigned arbitrary unitage of 100 units (U) per ampoule. Analysis of the reference reagent in a collaborative study has demonstrated the benefit of this preparation for the reduction in inter- and intra-laboratory variability in ELISA. Whilst locally sourced pools are regularly use for harmonization both within and between a few laboratories, the presence of a WHO-endorsed reference reagent should enable optimal harmonization of malaria serological assays either by direct use of the reference reagent or calibration of local standards against this WHO reference. The intended uses of this reference reagent, a multivalent preparation, are (1) to allow cross
Nanomolar Cellular Antisense Activity of Peptide Nucleic Acid (PNA) Cholic Acid ("Umbrella") and Cholesterol Conjugates Delivered by Cationic Lipids
DEFF Research Database (Denmark)
Shiraishi, Takehiko; Nielsen, Peter E
of cholesterol and cholic acid ("umbrella") derivatives of splice correction antisense PNA oligomers. While the conjugates alone were practically inactive up to 1 µM, their activity was dramatically improved when delivered by a cationic lipid transfection agent (LipofectAMINE2000). In particular, PNAs...
Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.
Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram
In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.
Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells
DEFF Research Database (Denmark)
Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E
oligomer. This modification of the PNA does not interfere with the nucleic acid target binding affinity based on thermal stability of the PNA/RNA duplexes. When delivered to cultured HeLa pLuc705 cells by Lipofectamine, the PNAs showed dose-dependent nuclear antisense activity in the nanomolar range...
A new procedure for the treatment of an industrial waste containing flotation reagents
Directory of Open Access Journals (Sweden)
Milosavljević Milutin M.
Full Text Available Flotation reagents can be transformed to industrial waste if they are stored for a long period of time. Also, if synthesis or drying process is not performed under defined conditions in industrial plants, which produce flotation reagents, batch of waste may arise and be stored as a waste. The chemical composition of this waste depends on the phase in which it was created, but typically includes: unreacted alkali hydroxide, solvent - alcohol and trithiocarbonate and oxidation product - dixanthogenate. In this paper a new laboratory procedure for the treatment of such wastes is described. The identification and separation of industrial waste components is also included. From the separated dixantogenate and xanthate a laboratory synthesis of thioncarbamates is given. In addition, a semi-industrial treatment of waste xanthate is presented. Synthesis of N-alkyl and N,N-dialkyl-O-isobutylthioncarbamates were obtained from the filtrate obtained in the first step. As a by-product, sodium thioglycolate was produced. This by-product is transformed to a thioglycolic acid by the addition of an acid. Also, the synthesis of thioncarbamates from dixanthogenates, isolated from industrial waste as a cake, is desribed. Described waste treatment is additionally interesting due to the production of sulphur as another by-product. Laboratory synthesis gave thioncarbamates in yields from 69.7 to 87.7 %, while the semi-industrial process for the selected batches produced thioncarbamates in yields from 74.2 to 80.5 %. Taking into account the importance of the synthesized compounds as selective flotation reagents, a new procedure of their synthesis from industrial waste is characterized by good yields and purity of the obtained compounds, the simplicity of process, low environmental impact and short reaction times of synthesis. [Projekat Ministarstva nauke Republike Srbije, br. III 43007
Toward low-cost affinity reagents: lyophilized yeast-scFv probes specific for pathogen antigens.
Directory of Open Access Journals (Sweden)
Sean A Gray
Full Text Available The generation of affinity reagents, usually monoclonal antibodies, remains a critical bottleneck in biomedical research and diagnostic test development. Recombinant antibody-like proteins such as scFv have yet to replace traditional monoclonal antibodies in antigen detection applications, in large part because of poor performance of scFv in solution. To address this limitation, we have developed assays that use whole yeast cells expressing scFv on their surfaces (yeast-scFv in place of soluble purified scFv or traditional monoclonal antibodies. In this study, a nonimmune library of human scFv displayed on the surfaces of yeast cells was screened for clones that bind to recombinant cyst proteins of Entamoeba histolytica, an enteric pathogen of humans. Selected yeast-scFv clones were stabilized by lyophilization and used in detection assay formats in which the yeast-scFv served as solid support-bound monoclonal antibodies. Specific binding of antigen to the yeast-scFv was detected by staining with rabbit polyclonal antibodies. In flow cytometry-based assays, lyophilized yeast-scFv reagents retained full binding activity and specificity for their cognate antigens after 4 weeks of storage at room temperature in the absence of desiccants or stabilizers. Because flow cytometry is not available to all potential assay users, an immunofluorescence assay was also developed that detects antigen with similar sensitivity and specificity. Antigen-specific whole-cell yeast-scFv reagents can be selected from nonimmune libraries in 2-3 weeks, produced in vast quantities, and packaged in lyophilized form for extended shelf life. Lyophilized yeast-scFv show promise as low cost, renewable alternatives to monoclonal antibodies for diagnosis and research.
Protein-Protein Interaction Reagents | Office of Cancer Genomics
The CTD2 Center at Emory University has a library of genes used to study protein-protein interactions in mammalian cells. These genes are cloned in different mammalian expression vectors. A list of available cancer-associated genes can be accessed below. Emory_CTD^2_PPI_Reagents.xlsx Contact: Haian Fu
[Thermodynamic forecasting of reagents composition for soils decontamination].
Nikolaev, V P; Nikolaevskiĭ, V B; Chirkina, I V; Shcheglov, M Iu
Based on thermodynamic studies, the authors conducted laboratory experiments on searching optimal composition of leaching reagents solution for soils decontamination, when contaminated with Cs-137, of activity coefficient for caesium sulfate microquantities in macrocomponents solutions. The method could be used for modelling the radionuclides phase equillibrium and relocations in soils.
Lanthanide shift reagents, binding, shift mechanisms and exchange
International Nuclear Information System (INIS)
Boer, J.W.M. de
Paramagnetic lanthanide shift reagents, when added to a solution of a substrate, induce shifts in the nuclear magnetic resonance (NMR) spectrum of the substrate molecules. The induced shifts contain information about the structure of the shift reagent substrate complex. The structural information, however, may be difficult to extract because of the following effects: (1) different complexes between shift reagent and substrate may be present in solution, e.g. 1:1 and 1:2 complexes, and the shift observed is a weighed average of the shifts of the substrate nuclei in the different complexes; (2) the Fermi contact interaction, arising from the spin density at the nucleus, contributes to the induced shift; (3) chemical exchange effects may complicate the NMR spectrum. In this thesis, the results of an investigation into the influence of these effects on the NMR spectra of solutions containing a substrate and LSR are presented. The equations describing the pseudo contact and the Fermi contact shift are derived. In addition, it is shown how the modified Bloch equations describing the effect of the chemical exchange processes occurring in the systems studied can be reduced to the familiar equations for a two-site exchange case. The binding of mono- and bifunctional ethers to the shift reagent are reported. An analysis of the induced shifts is given. Finally, the results of the experiments performed to study the exchange behavior of dimethoxyethane and heptafluorodimethyloctanedionato ligands are presented
satl based lesson for teaching grignard reagents in synthetic organic
African Journals Online (AJOL)
Traditionally, Grignard reagent has been very vital component of such synthetic ... knowledge, the systemic methodology of teaching and learning is the key point. Chemistry is ... chosen in particular to enlighten the students about effectiveness of systemic approach to .... Lectures through Systemic Approach to Teaching and.
Investigation of cleaning reagents for calcium chromate spills
International Nuclear Information System (INIS)
Dillard, B.M.
Cleaning of calcium chromate spills can be a problem due to the insolubility of the material and the corrosiveness of several possible cleaning agents on the stainless steel equipment. Because of OSHA Standards for Cr(VI) exposure, it is necessary to remove spills as efficiently as possible in order to prevent the contaminant from becoming airborne. This study involved the comparison of several possible cleaning agents by studying the solubility of calcium chromate in each reagent. Two general types of reagents for dissolution of calcium chromate were investigated; those which act by conversion of the insoluble calcium chromate to a more soluble salt and to H 2 CrO 4 , and those which appear to act as complexing agents and thereby dissolve the calcium chromate. The most efficient of the reagents investigated was hydrochloric acid. However, even dilute solutions of halide acids destroy passivity of stainless steel causing pitting and stress-corrosion. Acetic acid and nitric acid were somewhat less efficient than hydrochloric acid in dissolving calcium chromate. However, both reagents are noncorrosive with stainless steel, nitric acid tending to favor passivity of the materials. Therefore, it is recommended that dilute solutions of either of these two acids be used for removal of calcium chromate spills in conjunction with mechanical methods that might be necessary, depending on the magnitude of the spill
Synthesis and characterization of zwitterionic carbon dioxide fixing reagents
DEFF Research Database (Denmark)
Mikkelsen, Mette; Jørgensen, Mikkel; Krebs, Frederik C
with 13CO2 labeling and medium pressure NMR. The experiments showed that two of the three reagents were able to form carbamates and thus bind CO2. In addition we investigated this particular class of molecules for the possible formation of neutrally charged spiro compounds and we show that these did...
21 CFR 866.3145 - Coxsackievirus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3145... fluorescent dye that are used to identify coxsackievirus from clinical specimens or from tissue culture isolates derived from clinical specimens. The identification aids in the diagnosis of coxsackievirus...
21 CFR 866.3085 - Brucella spp. serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp... from clinical specimens or to identify antibodies to Brucella spp. in serum. Additionally, some of... to identify Brucella spp. directly from clinical specimens or cultured isolates derived from clinical...
21 CFR 866.3510 - Rubella virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3510 Rubella virus... Clinical Laboratory Standards': (i) 1/LA6 "Detection and Quantitation of Rubella IgG Antibody: Evaluation... Products in the Clinical Laboratory, October 1997," (ii) 1/LA18 "Specifications for Immunological Testing...
Manganese-Catalyzed Aerobic Heterocoupling of Aryl Grignard Reagents
DEFF Research Database (Denmark)
Ghaleshahi, Hajar Golshahi; Antonacci, Giuseppe; Madsen, Robert
An improved protocol has been developed for the MnCl2-catalyzed cross-coupling reaction of two arylmagnesium bromides under dioxygen. The reaction was achieved by using the Grignard reagents in a 2:1 ratio and 20 % of MnCl2. Very good yields of the heterocoupling product were obtained when the li...
21 CFR 864.1860 - Immunohistochemistry reagents and kits.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Immunohistochemistry reagents and kits. 864.1860 Section 864.1860 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Biological Stains § 864.1860...
Evaluation of Questionnaire, Reagent Strip and Egg Count as ...
African Journals Online (AJOL)
A longitudinal study covering 55 months evaluated the three diagnostic tools used for confirmation of prevalence of urinary schistosomiasis among 1151 consented primary school pupils in 13 communities of Edo State, Nigeria. Questionnaire, reagent strip method and parasitological examination were employed.
Radioimmunoassay reagent and its use in a radioimmunoassay
International Nuclear Information System (INIS)
Polito, A.J.; Knight, W.S.
A radioimmunoassay to detect or determine a steroid of the cortisone and aldosterone group has been developed. The invention particularly concerns a steroid derivative containing imidazole group which is radioactively labelled. The invented reagents are labelled with iodine 125. (VJ) [de
SATL Based Lesson for Teaching Grignard Reagents in Synthetic ...
African Journals Online (AJOL)
Synthesizing new products from raw materials has been very popular aspects of research in organic chemistry. Traditionally, Grignard reagent has been very vital component of such synthetic procedures. Hence learning of various issues concerning with applications of Grignard reactions in synthetic organic chemistry is ...
Reaction of lupane and oleanane triterpenoids with Lawesson's reagent
Czech Academy of Sciences Publication Activity Database
Kvasnica, Miroslav; Rudovská, I.; Císařová, I.; Šarek, J.
Roč. 64, č. 17 (2008), s. 3736-3743 ISSN 0040-4020 Grant - others:GA ČR(CZ) GP203/05/P025 Institutional research plan: CEZ:AV0Z40550506 Keywords : terpenoids * Lawesson's reagent * ketones * sulfur Subject RIV: CC - Organic Chemistry Impact factor: 2.897, year: 2008
BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity
International Nuclear Information System (INIS)
Lu, Zheng; Ping, Liang; JianBo, Zhou; XiaoBing, Huang; Yu, Wen; Zheng, Wang; Jing, Li
The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG 2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG 2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41% in HepG 2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G 1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+) BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms
Development of versatile universal reagent immunoradiometric assay technique
International Nuclear Information System (INIS)
Hazra, D.K.
Immunoradiometric assays, which make use of labelled antibodies, potentially offer better sensitivity and specificity than do radioimmunoassays, which use labelled antigens. In addition, they can in principle be performed in a particularly convenient scheme wherein the same labelled reagent may be used for many different analytes - thus serving as a ''universal'' labelled reagent. Thus if the specific antibody for every analyte is raised in rabbits, and an anti-rabbit antibody is labelled, the latter may be added after the specific antibody to quantify the amount of specific antibody bound to analyte and thereby the amount of analyte present. The potential greater sensitivity and specificity of the immunoradiometric procedure, coupled with the potential convenience of the ''universal'' labelled reagent, might allow such immunoradiometric techniques to be used effectively in the study of communicable diseases in developing countries. Development of these procedures was the subject of this investigation. Many components of these procedures had to be explored and provisionally optimized, including coating of assay tubes with ''extraction'' antibody, immunological purification of antibodies, labelling of antibodies, and intermediate steps toward these goals. Applications were thereupon tested using those provisionally optimized components. The ''universal'' labelled reagent, a donkey anti-rabbit antiserum, was successfully used in the assay of TSH; however, cross reactions of the reagent with non-rabbit immunoglobulins and other materials present seriously limited the sensitivity of the method. Using conventional immunoradiometric procedures, circulating TB and amoebic antibodies could be detected in patients suffering from these diseases. Similarly, circulating antigens in the same patients could also be detected, but not with sufficient sensitivity and specificity to provide a reliable analytical system. Numerous improvements will be required before these techniques
Central and peripheral administration of antisense oligonucleotide targeting amyloid-β protein precursor improves learning and memory and reduces neuroinflammatory cytokines in Tg2576 (AβPPswe) mice.
Farr, Susan A; Erickson, Michelle A; Niehoff, Michael L; Banks, William A; Morley, John E
Alzheimer's disease (AD) is a progressive neurodegenerative disease. Currently, there are no therapies to stop or reverse the symptoms of AD. We have developed an antisense oligonucleotide (OL-1) against the amyloid-β protein precursor (AβPP) that can decrease AβPP expression and amyloid-β protein (Aβ) production. This antisense rapidly crosses the blood-brain barrier, reverses learning and memory impairments, reduces oxidative stress, and restores brain-to-blood efflux of Aβ in SAMP8 mice. Here, we examined the effects of this AβPP antisense in the Tg2576 mouse model of AD. We administered the OL-1 antisense into the lateral ventricle 3 times at 2week intervals. Seventy-two hours after the third injection, we tested learning and memory in T-maze foot shock avoidance. In the second study, we injected the mice with OL-1 antisense 3 times at 2-week intervals via the tail vein. Seventy-two hours later, we tested learning and memory T-maze, novel object recognition, and elevated plus maze. At the end of behavioral testing, brain tissue was collected. OL-1 antisense administered centrally improved acquisition and retention of T-maze foot shock avoidance. OL-1 antisense administered via tail vein improved learning and memory in both T-maze foot shock avoidance and novel object-place recognition. In the elevated plus maze, the mice which received OL-1 antisense spent less time in the open arms and had fewer entries into the open arms indicating reduced disinhibitation. Biochemical analyses reveal significant reduction of AβPP signal and a reduction of measures of neuroinflammation. The current findings support the therapeutic potential of OL-1 AβPP antisense.
Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.
Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A
The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.
[Subchronic toxicity test of genetically modified rice with double antisense starch-branching enzyme gene].
Li, Min; Piao, Jianhua; Yang, Xiaoguang
To observe the sub-chronic toxic effects of the genetically modified rice with double antisense SBE gene. Based on gender and weight, weanling Wistar rats were randomly sorted into five groups: non-genetically modified rice group (group A), genetically modified rice group (group B), half genetically modified rice group (group C), quarter genetically modified rice group (group D) and AIN-93G normal diet group (group E). Indicators were the followings: body weight, food consumption, blood routine, blood biochemical test, organ weight, bone density and pathological examination of organs. At the middle of the experiment, the percentage of monocyte of female group B was less than that of group E (P 0.05), and no notable abnormity in the pathological examination of main organs (P > 0.05). There were no enough evidence to confirm the sub-chronic toxicity of genetically modified rice on rats.
Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation
DEFF Research Database (Denmark)
Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.
Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...
Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.
van Roon-Mom, Willeke M C; Roos, Raymund A C; de Bot, Susanne T
On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.
Os DNA sintéticos anti-sentido Antisense Synthtetic DNA
Directory of Open Access Journals (Sweden)
Alfredo Cravador
Full Text Available One old dream of the chemist in the field of the drug research is to create molecules capable of reaching their target with the precision of a missile. To accomplish it these molecules must have the propriety of distinguishing qualitative differences between healthy and diseased cells. A therapy based on this principle, able of eradicating specifically defective cells, or cells affected by a pathogen has an enormous advantage with the regard to the classical approach in which the cytotoxic drugs merely exploit quantitative biochemical and kinetic differences between abnormal and normal cells. We present in this article a review on the chemical synthesis of analogues of desoxyribonucleotides and on results obtained on the specific and irreversible inhibition of undesired genetic expression using the antisense principle.
An experimental design approach for optimization of spectrophotometric method for estimation of cefixime trihydrate using ninhydrin as derivatizing reagent in bulk and pharmaceutical formulation
Directory of Open Access Journals (Sweden)
Yogita B. Wani
Full Text Available The aim of the present work is to use experimental design to screen and optimize experimental variables for developing a spectrophotometric method for determining cefixime trihydrate content using ninhydrin as a derivatizing reagent. The method is based on the reaction of the amino group of cefixime with ninhydrin in an alkaline medium to form a yellow-colored derivative (λmax 436 nm. A two-level full factorial design was utilized to screen the effect of ninhydrin reagent concentration (X1, volume of ninhydrin reagent (X2, heating temperature (X3 and heating time (X4 on the formation of the cefixime–ninhydrin complex Y (absorbance. One way ANOVA and Pareto ranking analyses have shown that the ninhydrin reagent concentration (X1, volume of ninhydrin reagent (X2 and heating temperature (X3 were statistically significant factors (P < 0.05 affecting the formation of the cefixime–ninhydrin complex Y (absorbance. A Box-Behnken experimental design with response surface methodology was then utilized to evaluate the main, interaction and quadratic effects of these three factors on the selected response. With the help of a response surface plot and contour plot the optimum values of the selected factors were determined and used for further experiments. These values were a ninhydrin reagent concentration (X1 of 0.2% w/v, volume of ninhydrin reagent (X2 of 1 mL and heating temperature (X3 of 80 °C. The proposed method was validated according to the ICH Q2 (R1 method validation guidelines. The results of the present study have clearly shown that an experimental design concept may be effectively applied to the optimization of a spectrophotometric method for estimating the cefixime trihydrate content in bulk and pharmaceutical formulation with the least number of experimental runs possible.
Highly Stereoselective Gold-Catalyzed Coupling of Diazo Reagents and Fluorinated Enol Silyl Ethers to Tetrasubstituted Alkenes.
Liao, Fu-Min; Cao, Zhong-Yan; Yu, Jin-Sheng; Zhou, Jian
We report a highly stereoselective synthesis of all-carbon or fluorinated tetrasubstituted alkenes from diazo reagents and fluorinated enol silyl ethers, using C-F bond as a synthetic handle. Cationic Au I catalysis plays a key role in this reaction. Remarkable fluorine effects on the reactivity and selectivity was also observed. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Designer HF-Based Fluorination Reagent: Highly Regioselective Synthesis of Fluoroalkenes and gem-Difluoromethylene Compounds from Alkynes
Hydrogen fluoride (HF) and selected nonbasic and weakly coordinating (toward cationic metal) hydrogen-bond acceptors (e.g., DMPU) can form stable complexes through hydrogen bonding. The DMPU/HF complex is a new nucleophilic fluorination reagent that has high acidity and is compatible with cationic metal catalysts. The gold-catalyzed mono- and dihydrofluorination of alkynes using the DMPU/HF complex yields synthetically important fluoroalkenes and gem-difluoromethlylene compounds regioselectively. PMID:25260170
Antisense-induced exon skipping for duplications in Duchenne muscular dystrophy
Directory of Open Access Journals (Sweden)
van Ommen Gert-Jan B
Full Text Available Abstract Background Antisense-mediated exon skipping is currently one of the most promising therapeutic approaches for Duchenne muscular dystrophy (DMD. Using antisense oligonucleotides (AONs targeting specific exons the DMD reading frame is restored and partially functional dystrophins are produced. Following proof of concept in cultured muscle cells from patients with various deletions and point mutations, we now focus on single and multiple exon duplications. These mutations are in principle ideal targets for this approach since the specific skipping of duplicated exons would generate original, full-length transcripts. Methods Cultured muscle cells from DMD patients carrying duplications were transfected with AONs targeting the duplicated exons, and the dystrophin RNA and protein were analyzed. Results For two brothers with an exon 44 duplication, skipping was, even at suboptimal transfection conditions, so efficient that both exons 44 were skipped, thus generating, once more, an out-of-frame transcript. In such cases, one may resort to multi-exon skipping to restore the reading frame, as is shown here by inducing skipping of exon 43 and both exons 44. By contrast, in cells from a patient with an exon 45 duplication we were able to induce single exon 45 skipping, which allowed restoration of wild type dystrophin. The correction of a larger duplication (involving exons 52 to 62, by combinations of AONs targeting the outer exons, appeared problematic due to inefficient skipping and mistargeting of original instead of duplicated exons. Conclusion The correction of DMD duplications by exon skipping depends on the specific exons targeted. Its options vary from the ideal one, restoring for the first time the true, wild type dystrophin, to requiring more 'classical' skipping strategies, while the correction of multi-exon deletions may need the design of tailored approaches.
Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.
Vijayakumar, Sarath; Depreux, Frederic F; Jodelka, Francine M; Lentz, Jennifer J; Rigo, Frank; Jones, Timothy A; Hastings, Michelle L
Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: firstname.lastname@example.com.
Reagent-Free Quantification of Aqueous Free Chlorine via Electrical Readout of Colorimetrically Functionalized Pencil Lines.
Mohtasebi, Amirmasoud; Broomfield, Andrew D; Chowdhury, Tanzina; Selvaganapathy, P Ravi; Kruse, Peter
Colorimetric methods are commonly used to quantify free chlorine in drinking water. However, these methods are not suitable for reagent-free, continuous, and autonomous applications. Here, we demonstrate how functionalization of a pencil-drawn film with phenyl-capped aniline tetramer (PCAT) can be used for quantitative electric readout of free chlorine concentrations. The functionalized film can be implemented in a simple fluidic device for continuous sensing of aqueous free chlorine concentrations. The sensor is selective to free chlorine and can undergo a reagent-free reset for further measurements. Our sensor is superior to electrochemical methods in that it does not require a reference electrode. It is capable of quantification of free chlorine in the range of 0.1-12 ppm with higher precision than colorimetric (absorptivity) methods. The interactions of PCAT with the pencil-drawn film upon exposure to hypochlorite were characterized spectroscopically. A previously reported detection mechanism relied on the measurement of a baseline shift to quantify free chlorine concentrations. The new method demonstrated here measures initial spike size upon exposure to free chlorine. It relies on a fast charge built up on the sensor film due to intermittent PCAT salt formation. It has the advantage of being significantly faster than the measurement of baseline shift, but it cannot be used to detect gradual changes in free chlorine concentration without the use of frequent reset pulses. The stability of PCAT was examined in the presence of free chlorine as a function of pH. While most ions commonly present in drinking water do not interfere with the free chlorine detection, other oxidants may contribute to the signal. Our sensor is easy to fabricate and robust, operates reagent-free, and has very low power requirements and is thus suitable for remote deployment.
Mössbauer spectroscopy research of interaction of alumosilicic reagent and iron dissolved in water
International Nuclear Information System (INIS)
Feklistov, D Y; Filippov, V P; Kurchatov, I M; Laguntsov, N I; Salomasov, V A; Permyakov, Yu V
The aim of this work is to reveal the results of alumosilicic reagent interaction with iron compounds contained in the water. This reagent is simultaneously coagulant-flocculant and adsorbent. The iron atoms state is studied in the reagent and in reacted sediment. The valence state of iron atoms are determined in the reagents and sediments. The existence of iron containing superparamagnetic particles in the sediment is shown. (paper)
A Novel Property of DNA – As a Bioflotation Reagent in Mineral Processing
Vasanthakumar, Balasubramanian; Ravishankar, Honnavar; Subramanian, Sankaran
Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed. PMID:22768298
A novel property of DNA - as a bioflotation reagent in mineral processing.
Vasanthakumar, Balasubramanian; Ravishankar, Honnavar; Subramanian, Sankaran
Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed.
A novel property of DNA - as a bioflotation reagent in mineral processing.
Directory of Open Access Journals (Sweden)
Balasubramanian Vasanthakumar
Full Text Available Environmental concerns regarding the use of certain chemicals in the froth flotation of minerals have led investigators to explore biological entities as potential substitutes for the reagents in vogue. Despite the fact that several microorganisms have been used for the separation of a variety of mineral systems, a detailed characterization of the biochemical molecules involved therein has not been reported so far. In this investigation, the selective flotation of sphalerite from a sphalerite-galena mineral mixture has been achieved using the cellular components of Bacillus species. The key constituent primarily responsible for the flotation of sphalerite has been identified as DNA, which functions as a bio-collector. Furthermore, using reconstitution studies, the obligatory need for the presence of non-DNA components as bio-depressants for galena has been demonstrated. A probable model involving these entities in the selective flotation of sphalerite from the mineral mixture has been discussed.
Kalman filtering applied to a reagent feed system
International Nuclear Information System (INIS)
Griffin, C.D.; Croson, D.V.; Feeley, J.J.
Using a Kalman filter solves a troublesome measurement noise problem and, at the same time, improves nuclear safety by detecting leaks to the process' feed tanks. To demonstrate how this technology of optimal estimation can be exploited, this article presents a systematic plan and example of how a Kalman filter was proven in industrial use on a reagent analyzer. A process to recycle uranium from spent fuel elements uses a reagent stream containing boron to dissolve the fuel. The boron is the neutron poison that prevents a nuclear chain reaction during the uranium dissolution. The purpose of the Kalman filter for this system is to reduce the uncertainty in the boron concentration measurement. The filter also provides incipient fault detection by estimating the unmeasured state of any unpoisoned solution, which would dilute the boron solution, entering the feed vessel
Development of reagents for radioimmunoassay of: triiodothyronine, thyroxine and thyrotrophin
International Nuclear Information System (INIS)
Delgado S, B.; Lavalley E, C.; Ruiz J, A.; Garcia F, C.; Zamorano A, F.
The radioimmunoassay (RIA) of thyroid hormones it is the but it frequents of all the studies carried out by RIA in the laboratories of Nuclear Medicine, these essays are carried out with imported reagents. In the ININ the reagents and the necessary methodology have been developed for the triiodothyronine (T3), thyroxine (T4) and thyrotrophin (TSH). The good titles of the antibodies (Ac) primary for each hormone were of 1:4,000; 1:750 and 1:1,500. The used separation system was of double Ac with PEG to 10%, with titles of 1:10 for the second Ac of lamb. The specific activity for 125-I-T3 and 125-I-T4 oscillate between 850 at 900 μCi / μ g: being this of 90 μ Ci /μg for TSH. To the first two hormones they were added 1-8 aniline naftalen sulfonic acid (ANS) to concentrations of 3 and 2 mg/ml respectively. As buffer for T3 and T4 it was used Tris-HCl pH 8.6 and PBS with normal serum of rabbit (SNC) for TSH. The standards got ready in buffer or free serum of thyroid hormones. The slope of the standard curves varied between -2.3 to -2.7 and the variation intra and inter assay among 4 to 10%. It is had at the moment in the ININ with standardized reagents for the RIA of T3, T4 and TSH, it is hoped to carry out tests in other laboratories and to establish the conditions of stability more appropriate to begin the preparation of pilot reagents. (Author)
Antisense-mediated suppression of C-hordein biosynthesis in the barley grain results in correlated changes in the transcriptome, protein profile, and amino acid composition
DEFF Research Database (Denmark)
Hansen, Mette; Lange, Marianne; Friis, Carsten
Antisense- or RNAi-mediated suppression of the biosynthesis of nutritionally inferior storage proteins is a promising strategy for improving the amino acid profile of seeds. However, the potential pleiotropic effects of this on interconnected pathways and the agronomic quality traits need...... to be addressed. In the current study, a transcriptomic analysis of an antisense C-hordein line of barley was performed, using a grain-specific cDNA array. The C-hordein antisense line is characterized by marked changes in storage protein and amino acid profiles, while the seed weight is within the normal range...... and no external morphological irregularities were observed. The results of the transcriptome analysis showed excellent correlation with data on changes in the relative proportions of storage proteins and amino acid composition. The antisense line had a lower C-hordein level and down-regulated transcript encoding...
Rapid heteroatom transfer to arylmetals utilizing multifunctional reagent scaffolds
Gao, Hongyin; Zhou, Zhe; Kwon, Doo-Hyun; Coombs, James; Jones, Steven; Behnke, Nicole Erin; Ess, Daniel H.; Kürti, László
Arylmetals are highly valuable carbon nucleophiles that are readily and inexpensively prepared from aryl halides or arenes and widely used on both laboratory and industrial scales to react directly with a wide range of electrophiles. Although C-C bond formation has been a staple of organic synthesis, the direct transfer of primary amino (-NH2) and hydroxyl (-OH) groups to arylmetals in a scalable and environmentally friendly fashion remains a formidable synthetic challenge because of the absence of suitable heteroatom-transfer reagents. Here, we demonstrate the use of bench-stable N-H and N-alkyl oxaziridines derived from readily available terpenoid scaffolds as efficient multifunctional reagents for the direct primary amination and hydroxylation of structurally diverse aryl- and heteroarylmetals. This practical and scalable method provides one-step synthetic access to primary anilines and phenols at low temperature and avoids the use of transition-metal catalysts, ligands and additives, nitrogen-protecting groups, excess reagents and harsh workup conditions.
The adsorption of chelating reagents on oxide minerals
International Nuclear Information System (INIS)
Bryson, M.A.W.
This work constitutes a fundamental study of the interaction between chelating reagents and oxide minerals. The adsorption mechanisms have been elucidated for most of the systems generated by the oxides of copper(II) or iron(III) and chelating reagents octyl hydroxamate, N-phenylbenzohydroxamate, salicylaldoxime, 5-nitro-salicylaldoxime or 8-hydroxyquinoline. In order to better understand the adsorption process associated with copper(II) oxide, the oxide was recrystallized to produce a coarser material with a more uniform surface. This allowed the oxide surface to be viewed under the scanning electron microscope. A detailed investigation of the effect of the system variables; pH, conditioning period, concentration, temperature, surface area and dispersing reagent on the rate of precipitation of the copper chelate species of general form, Cu(chel) 2 , was made. In addition the chemical nature of the adsorbed species and the structural form of the precipitates were determined with the aid of infra-red spectroscopy and the scanning electron microscope. On the basis of these results a model has been formulated for the adsorption processes. The precipitation process was examined in more detail by the study of the adsorption of chelate on copper metal. Contact angle measurements of air bubbles on copper metal conditioned with chelate were related to the adsorption results in an attempt to isolate the optimum conditions for flotation of oxide minerals
Inventory management and reagent supply for automated chemistry.
Kuzniar, E
Developments in automated chemistry have kept pace with developments in HTS such that hundreds of thousands of new compounds can be rapidly synthesized in the belief that the greater the number and diversity of compounds that can be screened, the more successful HTS will be. The increasing use of automation for Multiple Parallel Synthesis (MPS) and the move to automated combinatorial library production is placing an overwhelming burden on the management of reagents. Although automation has improved the efficiency of the processes involved in compound synthesis, the bottleneck has shifted to ordering, collating and preparing reagents for automated chemistry resulting in loss of time, materials and momentum. Major efficiencies have already been made in the area of compound management for high throughput screening. Most of these efficiencies have been achieved with sophisticated library management systems using advanced engineering and data handling for the storage, tracking and retrieval of millions of compounds. The Automation Partnership has already provided many of the top pharmaceutical companies with modular automated storage, preparation and retrieval systems to manage compound libraries for high throughput screening. This article describes how these systems may be implemented to solve the specific problems of inventory management and reagent supply for automated chemistry.
Remediation of polluted soils contaminated with Linear Alkyl Benzenes using Fenton's reagent
Directory of Open Access Journals (Sweden)
Douglas do Nascimento Silva
Full Text Available Linear Alkyl Benzenes (LABs are used as insulating oil for electric cables. When it happens a spill, LABs they are basically sorbed in the soil, because, these compounds have high hidrophobicity and low vapor pressure. The conventional methods of treatment of soils are not efficient. The Fenton's reaction (reaction between a solution of iron II and hydrogen peroxide it generates hydroxyl radicals, not selective, and capable of oxidize a great variety of organic compounds. A study was conducted to evaluate the viability of use of the Fenton's reagents to promote the remediation of polluted soils with Linear Alkyl Benzenes. A column was especially projected for these experiments, packed with a sandy and other soil loamy. The pH of the soil was not altered. The obtained results demonstrated the technical viability of the process of injection of the Fenton's reagents for the treatment of polluted areas with LABs.Os Linear Alquilbenzenos (LABs são usados como fluido refrigerante de cabos elétricos. Quando ocorre um vazamento, os LABs ficam basicamente adsorvidos no solo, pois, são compostos bastante hidrofóbicos e com baixa pressão de vapor. Os métodos convencionais de tratamento de solos não são eficientes. A reação de Fenton (solução de ferro II e peróxido de hidrogênio gera radicais hidroxila, não seletivos, e capazes de oxidar uma grande variedade de compostos orgânicos, chegando a mineralização dos mesmos. Neste trabalho foi estudada a viabilidade de utilização dos reagentes de Fenton para promover a remediação de solos contaminados com LABs. Utilizou-se uma coluna especialmente projetada para estes experimentos, empacotada com um solo arenoso e outro argiloso. O pH do solo não foi alterado. Os resultados obtidos demonstram a viabilidade técnica do processo de injeção dos reagentes de Fenton para o tratamento de áreas contaminadas com LABs.
Methods for the selective detection of alkyne-presenting molecules and related compositions and systems
Valdez, Carlos A.; Vu, Alexander K.
Provided herein are methods for selectively detecting an alkyne-presenting molecule in a sample and related detection reagents, compositions, methods and systems. The methods include contacting a detection reagent with the sample for a time and under a condition to allow binding of the detection reagent to the one or more alkyne-presenting molecules possibly present in the matrix to the detection reagent. The detection reagent includes an organic label moiety presenting an azide group. The binding of the azide group to the alkyne-presenting molecules results in emission of a signal from the organic label moiety.
Analogs of N-cynnamoylphenylhydroxylamine as reagents for amperometric determination of scandium
International Nuclear Information System (INIS)
Shvedene, N.V.; Gallaj, Z.A.; Sheina, N.M.; Zujkova, N.V.
To decrease the detection limit of scandium and increase selectivity of amperometric determination, oxidation of 2-furylacryloyl-N-p-chlorophenylhydroxylamine (FACPhHA) and 3-styrylacryloyl-N-phenylhydroxylamine (SAPhHA) on a graphite electrode has been studied by volt-amperometry. The possibility has been established of using the oxidation current of the reagent for plotting the titration curves. The solubility of scandium complexes with FACPhHA and SAPhHA under conditions of titration against the background with pH 6.0 has been determined and equals (2.1+-0.3)x10 -6 and (5.3+-0.3)x10 -7 , respectively. The methods have been developed of amperometric determination of scandium with the use of the considered reagents against backgrounds with pH 5.5-6.5. The use of SAPhHA has decreased the limit of scandium detection down to 0.1 mgk/ml. Besides, the amperometric method makes it possible to titrate in turbid and coloured media what is an advantage of this method. The developed method is used for determination of scandium in scandium silicide
Inactivation of viable Ascaris eggs by reagents during enumeration.
Nelson, K L; Darby, J L
Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.
International reference reagents: antihuman globulin. An ISBT/ICSH joint working party report. International Society of Blood Transfusion. International Committee for Standardization in Haematology.
Case, J; Ford, D S; Chung, A; Collins, R; Kochman, S; Mazda, T; Overbeeke, M; Perera, R; Sakuldamrongpanich, T; Scott, M; Voak, D; Zupańska, B
An international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti-IgG and anti-C3 (anti-C3c and anti-C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2-ml freeze-dried aliquots from seven assigned distribution centres.
Sense and antisense transcripts of the developmentally regulated murine hsp70.2 gene are expressed in distinct and only partially overlapping areas in the adult brain
Murashov, A. K.; Wolgemuth, D. J.
We have examined the spatial pattern of expression of a member of the hsp70 gene family, hsp70.2, in the mouse central nervous system. Surprisingly, RNA blot analysis and in situ hybridization revealed abundant expression of an 'antisense' hsp70.2 transcript in several areas of adult mouse brain. Two different transcripts recognized by sense and antisense riboprobes for the hsp70.2 gene were expressed in distinct and only partially overlapping neuronal populations. RNA blot analysis revealed low levels of the 2.7 kb transcript of hsp70.2 in several areas of the brain, with highest signal in the hippocampus. Abundant expression of a slightly larger (approximately 2.8 kb) 'antisense' transcript was detected in several brain regions, notably in the brainstem, cerebellum, mesencephalic tectum, thalamus, cortex, and hippocampus. In situ hybridization revealed that the sense and antisense transcripts were both predominantly neuronal and localized to the same cell types in the granular layer of the cerebellum, trapezoid nucleus of the superior olivary complex, locus coeruleus and hippocampus. The hsp70.2 antisense transcripts were particularly abundant in the frontal cortex, dentate gyrus, subthalamic nucleus, zona incerta, superior and inferior colliculi, central gray, brainstem, and cerebellar Purkinje cells. Our findings have revealed a distinct cellular and spatial localization of both sense and antisense transcripts, demonstrating a new level of complexity in the function of the heat shock genes.
Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain
DEFF Research Database (Denmark)
Shiraishi, Takehiko; Nielsen, Peter E
based on a splicing correction of a mutated luciferase gene in HeLa pLuc705 cells by targeting antisense oligonucleotides to a cryptic splice site. Further improvement in the delivery of CatLip-PNA conjugates is achieved by using auxiliary agents/treatments (e.g., chloroquine, calcium ions......Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic....... We have found, however, that this low -bioavailability can be significantly improved by chemical conjugation to a lipid domain ("Lip," such as a fatty acid), thereby creating "CatLip"-conjugates. The cellular uptake of these conjugates is conveniently evaluated using a sensitive cellular assay system...
Novel atomic force microscopy based biopanning for isolation of morphology specific reagents against TDP-43 variants in amyotrophic lateral sclerosis.
Williams, Stephanie M; Venkataraman, Lalitha; Tian, Huilai; Khan, Galam; Harris, Brent T; Sierks, Michael R
Because protein variants play critical roles in many diseases including TDP-43 in Amyotrophic Lateral Sclerosis (ALS), alpha-synuclein in Parkinson's disease and beta-amyloid and tau in Alzheimer's disease, it is critically important to develop morphology specific reagents that can selectively target these disease-specific protein variants to study the role of these variants in disease pathology and for potential diagnostic and therapeutic applications. We have developed novel atomic force microscopy (AFM) based biopanning techniques that enable isolation of reagents that selectively recognize disease-specific protein variants. There are two key phases involved in the process, the negative and positive panning phases. During the negative panning phase, phages that are reactive to off-target antigens are eliminated through multiple rounds of subtractive panning utilizing a series of carefully selected off-target antigens. A key feature in the negative panning phase is utilizing AFM imaging to monitor the process and confirm that all undesired phage particles are removed. For the positive panning phase, the target antigen of interest is fixed on a mica surface and bound phages are eluted and screened to identify phages that selectively bind the target antigen. The target protein variant does not need to be purified providing the appropriate negative panning controls have been used. Even target protein variants that are only present at very low concentrations in complex biological material can be utilized in the positive panning step. Through application of this technology, we acquired antibodies to protein variants of TDP-43 that are selectively found in human ALS brain tissue. We expect that this protocol should be applicable to generating reagents that selectively bind protein variants present in a wide variety of different biological processes and diseases.
The endogenous retroviral insertion in the human complement C4 gene modulates the expression of homologous genes by antisense inhibition.
Schneider, P M; Witzel-Schlömp, K; Rittner, C; Zhang, L
Intron 9 contains the complete endogenous retrovirus HERV-K(C4) as a 6.4-kb insertion in 60% of human C4 genes. The retroviral insertion is in reverse orientation to the C4 coding sequence. Therefore, expression of C4 could lead to the transcription of an antisense RNA, which might protect against exogenous retroviral infections. To test this hypothesis, open reading frames from the HERV sequence were subcloned in sense orientiation into a vector allowing expression of a beta-galactosidase fusion protein. Mouse L cells which had been stably transfected with either the human C4A or C4B gene both carrying the HERV insertion (LC4 cells), and L(Tk-) cells without the C4 gene were transiently transfected either with a retroviral construct or with the wild-type vector. Expression was monitored using an enzymatic assay. We demonstrated that (1) HERV-K(C4) antisense mRNA transcripts are present in cells constitutively expressing C4, (2) expression of retroviral-like constructs is significantly downregulated in cells expressing C4, and (3) this downregulation is further modulated in a dose-dependent fashion following interferon-gamma stimulation of C4 expression. These results support the hypothesis of a genomic antisense strategy mediated by the HERV-K(C4) insertion as a possible defense mechanism against exogenous retroviral infections.
The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice
Directory of Open Access Journals (Sweden)
Ingrid E C Verhaart
Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.
[An evaluation of the China-made HIV antibody test reagents].
Zheng, X W; Zhu, D
This paper reports the results of the evaluation of the China-made HIV antibody screening test reagents, including the IF and IE reagents prepared by the Institute of Virology, CAPM, the ELISA reagent prepared by the Shanghai Institute of Biological Products. Based on the results, the sensitivities of the IF and IE are from 91.2% to 96.9%; the specificities, from 94.6% to 97.3%. Due to the low HIV prevalence in China, the predictive values of negative of these reagents are up to 100%; but the predictive values of positive are very low. It is suggested that these reagents can be used for HIV antibody screen testing in China. The package of some reagents should be improved, the price of some reagents should be decreased.
Analysis of antisense expression by whole genome tiling microarrays and siRNAs suggests mis-annotation of Arabidopsis orphan protein-coding genes.
Directory of Open Access Journals (Sweden)
Casey R Richardson
Full Text Available MicroRNAs (miRNAs and trans-acting small-interfering RNAs (tasi-RNAs are small (20-22 nt long RNAs (smRNAs generated from hairpin secondary structures or antisense transcripts, respectively, that regulate gene expression by Watson-Crick pairing to a target mRNA and altering expression by mechanisms related to RNA interference. The high sequence homology of plant miRNAs to their targets has been the mainstay of miRNA prediction algorithms, which are limited in their predictive power for other kingdoms because miRNA complementarity is less conserved yet transitive processes (production of antisense smRNAs are active in eukaryotes. We hypothesize that antisense transcription and associated smRNAs are biomarkers which can be computationally modeled for gene discovery.We explored rice (Oryza sativa sense and antisense gene expression in publicly available whole genome tiling array transcriptome data and sequenced smRNA libraries (as well as C. elegans and found evidence of transitivity of MIRNA genes similar to that found in Arabidopsis. Statistical analysis of antisense transcript abundances, presence of antisense ESTs, and association with smRNAs suggests several hundred Arabidopsis 'orphan' hypothetical genes are non-coding RNAs. Consistent with this hypothesis, we found novel Arabidopsis homologues of some MIRNA genes on the antisense strand of previously annotated protein-coding genes. A Support Vector Machine (SVM was applied using thermodynamic energy of binding plus novel expression features of sense/antisense transcription topology and siRNA abundances to build a prediction model of miRNA targets. The SVM when trained on targets could predict the "ancient" (deeply conserved class of validated Arabidopsis MIRNA genes with an accuracy of 84%, and 76% for "new" rapidly-evolving MIRNA genes.Antisense and smRNA expression features and computational methods may identify novel MIRNA genes and other non-coding RNAs in plants and potentially other
Anti-sense expression of a metallopeptidase gene enhances nuclear entry of HBV-DNA
International Nuclear Information System (INIS)
Yeh, C.-T.; Lai, H.-Y.; Chu, S.-P.; Tseng, I-Chu
Although several putative hepatitis B virus (HBV) receptors have been identified, none of them is capable of initiating HBV replication in a non-permissive human cell line. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a clone capable of facilitating nuclear transport of HBV-DNA during the early phase of HBV infection. This clone contained a cDNA encoding a metallopeptidase-like protein in anti-sense orientation. Pretreatment of naive HepG2 cells with 1,10-phenanthroline, an inhibitor for liver metallopeptidases, led to nuclear entry of HBV-DNA after HBV infection. However, cccDNA was still undetectable in the nuclei, indicating other cellular factors required to complete the replication cycle were still missing. Our present data suggest that in the initial stage of HBV infection, liver metallopeptidase constitutes a barrier for effective nuclear entry of HBV genomic DNA. Attenuation of metallopeptidase activity may facilitate HBV infection
The successes and future prospects of the linear antisense RNA amplification methodology.
Li, Jifen; Eberwine, James
It has been over a quarter of a century since the introduction of the linear RNA amplification methodology known as antisense RNA (aRNA) amplification. Whereas most molecular biology techniques are rapidly replaced owing to the fast-moving nature of development in the field, the aRNA procedure has become a base that can be built upon through varied uses of the technology. The technique was originally developed to assess RNA populations from small amounts of starting material, including single cells, but over time its use has evolved to include the detection of various cellular entities such as proteins, RNA-binding-protein-associated cargoes, and genomic DNA. In this Perspective we detail the linear aRNA amplification procedure and its use in assessing various components of a cell's chemical phenotype. This procedure is particularly useful in efforts to multiplex the simultaneous detection of various cellular processes. These efforts are necessary to identify the quantitative chemical phenotype of cells that underlies cellular function.
Tuning growth cycles of Brassica crops via natural antisense transcripts of BrFLC.
Li, Xiaorong; Zhang, Shaofeng; Bai, Jinjuan; He, Yuke
Several oilseed and vegetable crops of Brassica are biennials that require a prolonged winter cold for flowering, a process called vernalization. FLOWERING LOCUS C (FLC) is a central repressor of flowering. Here, we report that the overexpression of natural antisense transcripts (NATs) of Brassica rapa FLC (BrFLC) greatly shortens plant growth cycles. In rapid-, medium- and slow-cycling crop types, there are four copies of the BrFLC genes, which show extensive variation in sequences and expression levels. In Bre, a biennial crop type that requires vernalization, five NATs derived from the BrFLC2 locus are rapidly induced under cold conditions, while all four BrFLC genes are gradually down-regulated. The transgenic Bre lines overexpressing a long NAT of BrFLC2 do not require vernalization, resulting in a gradient of shortened growth cycles. Among them, a subset of lines both flower and set seeds as early as Yellow sarson, an annual crop type in which all four BrFLC genes have non-sense mutations and are nonfunctional in flowering repression. Our results demonstrate that the growth cycles of biennial crops of Brassica can be altered by changing the expression levels of BrFLC2 NATs. Thus, BrFLC2 NATs and their transgenic lines are useful for the genetic manipulation of crop growth cycles. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.
Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T
A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.
Changes in Oleic Acid Content of Transgenic Soybeans by Antisense RNA Mediated Posttranscriptional Gene Silencing
Directory of Open Access Journals (Sweden)
Ling Zhang
Full Text Available The Delta-12 oleate desaturase gene (FAD2-1, which converts oleic acid into linoleic acid, is the key enzyme determining the fatty acid composition of seed oil. In this study, we inhibited the expression of endogenous Delta-12 oleate desaturase GmFad2-1b gene by using antisense RNA in soybean Williams 82. By employing the soybean cotyledonary-node method, a part of the cDNA of soybean GmFad2-1b 801 bp was cloned for the construction of a pCAMBIA3300 vector under the soybean seed promoter BCSP. Leaf painting, LibertyLink strip, PCR, Southern blot, qRT-PCR, and fatty acid analysis were used to detect the insertion and expression of GmFad2-1b in the transgenic soybean lines. The results indicate that the metabolically engineered plants exhibited a significant increase in oleic acid (up to 51.71% and a reduction in palmitic acid (to <3% in their seed oil content. No structural differences were observed between the fatty acids of the transgenic and the nontransgenic oil extracts.
Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice
Directory of Open Access Journals (Sweden)
Dominic Jauvin
Full Text Available Myotonic dystrophy type 1 (DM1, a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTGn trinucleotide repeat in the 3′ UTR of the human dystrophia myotonica protein kinase (DMPK gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2′-4′-constrained, ethyl-modified (ISIS 486178 antisense oligonucleotide (ASO targeted to the 3′ UTR of the DMPK gene, which led to a 70% reduction in CUGexp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUGexp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs.
Antisense oligonucleotides suppress cell-volume-induced activation of chloride channels.
Gschwentner, M; Nagl, U O; Wöll, E; Schmarda, A; Ritter, M; Paulmichl, M
Cell volume regulation is an essential feature of most cells. After swelling in hypotonic media, the simultaneous activation of potassium and chloride channels is believed to be the initial, time-determining step in cell volume regulation. The activation of both pathways is functionally linked and enables the cells to lose ions and water, subsequently leading to cell shrinkage and readjustment of the initial volume. NIH 3T3 fibroblasts efficiently regulate their volume after swelling and bear chloride channels that are activated by decreasing extracellular osmolarity. The chloride current elicited in these cells after swelling is reminiscent of the current found in oocytes expressing an outwardly rectifying chloride current termed ICln. Introduction of antisense oligodeoxynucleotides complementary to the first 30 nucleotides of the coding region of the ICln channel into NIH 3T3 fibroblasts suppresses the activation of the swelling-induced chloride current. The experiments directly demonstrate an unambiguous link between a volume-activated chloride current and a cloned protein involved in chloride transport.
Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides
International Nuclear Information System (INIS)
Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren
The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)
RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation.
Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé
Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA-RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners.
Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy
Yokota, Toshifumi; Wood, Matthew J. A.
Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357
PlantNATsDB: a comprehensive database of plant natural antisense transcripts.
Chen, Dijun; Yuan, Chunhui; Zhang, Jian; Zhang, Zhao; Bai, Lin; Meng, Yijun; Chen, Ling-Ling; Chen, Ming
Natural antisense transcripts (NATs), as one type of regulatory RNAs, occur prevalently in plant genomes and play significant roles in physiological and pathological processes. Although their important biological functions have been reported widely, a comprehensive database is lacking up to now. Consequently, we constructed a plant NAT database (PlantNATsDB) involving approximately 2 million NAT pairs in 69 plant species. GO annotation and high-throughput small RNA sequencing data currently available were integrated to investigate the biological function of NATs. PlantNATsDB provides various user-friendly web interfaces to facilitate the presentation of NATs and an integrated, graphical network browser to display the complex networks formed by different NATs. Moreover, a 'Gene Set Analysis' module based on GO annotation was designed to dig out the statistical significantly overrepresented GO categories from the specific NAT network. PlantNATsDB is currently the most comprehensive resource of NATs in the plant kingdom, which can serve as a reference database to investigate the regulatory function of NATs. The PlantNATsDB is freely available at http://bis.zju.edu.cn/pnatdb/.
RNA synthetic biology inspired from bacteria: construction of transcription attenuators under antisense regulation
International Nuclear Information System (INIS)
Dawid, Alexandre; Cayrol, Bastien; Isambert, Hervé
Among all biopolymers, ribonucleic acids or RNA have unique functional versatility, which led to the early suggestion that RNA alone (or a closely related biopolymer) might have once sustained a primitive form of life based on a single type of biopolymer. This has been supported by the demonstration of processive RNA-based replication and the discovery of 'riboswitches' or RNA switches, which directly sense their metabolic environment. In this paper, we further explore the plausibility of this 'RNA world' scenario and show, through synthetic molecular design guided by advanced RNA simulations, that RNA can also perform elementary regulation tasks on its own. We demonstrate that RNA synthetic regulatory modules directly inspired from bacterial transcription attenuators can efficiently activate or repress the expression of other RNA by merely controlling their folding paths 'on the fly' during transcription through simple RNA–RNA antisense interaction. Factors, such as NTP concentration and RNA synthesis rate, affecting the efficiency of this kinetic regulation mechanism are also studied and discussed in the light of evolutionary constraints. Overall, this suggests that direct coupling among synthesis, folding and regulation of RNAs may have enabled the early emergence of autonomous RNA-based regulation networks in absence of both DNA and protein partners
Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology.
Goina, Elisa; Peruzzo, Paolo; Bembi, Bruno; Dardis, Andrea; Buratti, Emanuele
Glycogen storage disease type II (GSDII) is a lysosomal disorder caused by the deficient activity of acid alpha-glucosidase (GAA) enzyme, leading to the accumulation of glycogen within the lysosomes. The disease has been classified in infantile and late-onset forms. Most late-onset patients share a splicing mutation c.-32-13T > G in intron 1 of the GAA gene that prevents efficient recognition of exon 2 by the spliceosome. In this study, we have mapped the splicing silencers of GAA exon 2 and developed antisense morpholino oligonucleotides (AMOs) to inhibit those regions and rescue normal splicing in the presence of the c.-32-13T > G mutation. Using a minigene approach and patient fibroblasts, we successfully increased inclusion of exon 2 in the mRNA and GAA enzyme production by targeting a specific silencer with a combination of AMOs. Most importantly, the use of these AMOs in patient myotubes results in a decreased accumulation of glycogen. To our knowledge, this is the only therapeutic approach resulting in a decrease of glycogen accumulation in patient tissues beside enzyme replacement therapy (ERT) and TFEB overexpression. As a result, it may represent a highly novel and promising therapeutic line for GSDII. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.
Multilevel Regulation of Bacterial Gene Expression with the Combined STAR and Antisense RNA System.
Lee, Young Je; Kim, Soo-Jung; Moon, Tae Seok
Synthetic small RNA regulators have emerged as a versatile tool to predictably control bacterial gene expression. Owing to their simple design principles, small size, and highly orthogonal behavior, these engineered genetic parts have been incorporated into genetic circuits. However, efforts to achieve more sophisticated cellular functions using RNA regulators have been hindered by our limited ability to integrate different RNA regulators into complex circuits. Here, we present a combined RNA regulatory system in Escherichia coli that uses small transcription activating RNA (STAR) and antisense RNA (asRNA) to activate or deactivate target gene expression in a programmable manner. Specifically, we demonstrated that the activated target output by the STAR system can be deactivated by expressing two different types of asRNAs: one binds to and sequesters the STAR regulator, affecting the transcription process, while the other binds to the target mRNA, affecting the translation process. We improved deactivation efficiencies (up to 96%) by optimizing each type of asRNA and then integrating the two optimized asRNAs into a single circuit. Furthermore, we demonstrated that the combined STAR and asRNA system can control gene expression in a reversible way and can regulate expression of a gene in the genome. Lastly, we constructed and simultaneously tested two A AND NOT B logic gates in the same cell to show sophisticated multigene regulation by the combined system. Our approach establishes a methodology for integrating multiple RNA regulators to rationally control multiple genes.
A long natural-antisense RNA is accumulated in the conidia of Aspergillus oryzae.
Tsujii, Masaru; Okuda, Satoshi; Ishi, Kazutomo; Madokoro, Kana; Takeuchi, Michio; Yamagata, Youhei
Analysis of expressed sequence tag libraries from various culture conditions revealed the existence of conidia-specific transcripts assembled to putative conidiation-specific reductase gene (csrA) in Aspergillus oryzae. However, the all transcripts were transcribed with opposite direction to the gene csrA. The sequence analysis of the transcript revealed that the RNA overlapped mRNA of csrA with 3'-end, and did not code protein longer than 60 amino acid residues. We designated the transcript Conidia Specific Long Natural-antisense RNA (CSLNR). The real-time PCR analysis demonstrated that the CSLNR is conidia-specific transcript, which cannot be transcribed in the absence of brlA, and the amount of CSLNR was much more than that of the transcript from csrA in conidia. Furthermore, the csrA deletion, also lacking coding region of CSLNR in A. oryzae reduced the number of conidia. Overexpression of CsrA demonstrated the inhibition of growth and conidiation, while CSLNR did not affect conidiation.
Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.
Jauvin, Dominic; Chrétien, Jessina; Pandey, Sanjay K; Martineau, Laurie; Revillod, Lucille; Bassez, Guillaume; Lachon, Aline; MacLeod, A Robert; Gourdon, Geneviève; Wheeler, Thurman M; Thornton, Charles A; Bennett, C Frank; Puymirat, Jack
Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG) n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.
Antisense pre-treatment increases gene therapy efficacy in dystrophic muscles.
Peccate, Cécile; Mollard, Amédée; Le Hir, Maëva; Julien, Laura; McClorey, Graham; Jarmin, Susan; Le Heron, Anita; Dickson, George; Benkhelifa-Ziyyat, Sofia; Piétri-Rouxel, France; Wood, Matthew J; Voit, Thomas; Lorain, Stéphanie
In preclinical models for Duchenne muscular dystrophy, dystrophin restoration during adeno-associated virus (AAV)-U7-mediated exon-skipping therapy was shown to decrease drastically after six months in treated muscles. This decline in efficacy is strongly correlated with the loss of the therapeutic AAV genomes, probably due to alterations of the dystrophic myofiber membranes. To improve the membrane integrity of the dystrophic myofibers at the time of AAV-U7 injection, mdx muscles were pre-treated with a single dose of the peptide-phosphorodiamidate morpholino (PPMO) antisense oligonucleotides that induced temporary dystrophin expression at the sarcolemma. The PPMO pre-treatment allowed efficient maintenance of AAV genomes in mdx muscles and enhanced the AAV-U7 therapy effect with a ten-fold increase of the protein level after 6 months. PPMO pre-treatment was also beneficial to AAV-mediated gene therapy with transfer of micro-dystrophin cDNA into muscles. Therefore, avoiding vector genome loss after AAV injection by PPMO pre-treatment would allow efficient long-term restoration of dystrophin and the use of lower and thus safer vector doses for Duchenne patients. © The Author 2016. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: email@example.com.
Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa.
Bremer, Jeroen; Bornert, Olivier; Nyström, Alexander; Gostynski, Antoni; Jonkman, Marcel F; Aartsma-Rus, Annemieke; van den Akker, Peter C; Pasmooij, Anna Mg
The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON)-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.
GPR39 splice variants versus antisense gene LYPD1: expression and regulation in gastrointestinal tract, endocrine pancreas, liver, and white adipose tissue
DEFF Research Database (Denmark)
Egerod, Kristoffer L; Holst, Birgitte; Petersen, Pia S
nervous system as characterized with both quantitative RT-PCR and in situ hybridization analysis. A functional analysis of the GPR39 promoter region identified sites for the hepatocyte nuclear factors 1alpha and 4alpha (HNF-1alpha and -4alpha) and specificity protein 1 (SP1) transcription factors as being......G protein-coupled receptor 39 (GPR39) is a constitutively active, orphan member of the ghrelin receptor family that is activated by zinc ions. GPR39 is here described to be expressed in a full-length, biologically active seven-transmembrane form, GPR39-1a, as well as in a truncated splice variant...... five-transmembrane form, GPR39-1b. The 3' exon of the GPR39 gene overlaps with an antisense gene called LYPD1 (Ly-6/PLAUR domain containing 1). Quantitative RT-PCR analysis demonstrated that GPR39-1a is expressed selectively throughout the gastrointestinal tract, including the liver and pancreas...
Assisted delivery of antisense therapeutics in animal models of heritable neurodegenerative and neuromuscular disorders: a systematic review and meta-analysis.
van der Bent, M Leontien; Paulino da Silva Filho, Omar; van Luijk, Judith; Brock, Roland; Wansink, Derick G
Antisense oligonucleotide (AON)-based therapies hold promise for a range of neurodegenerative and neuromuscular diseases and have shown benefit in animal models and patients. Success in the clinic is nevertheless still limited, due to unfavourable biodistribution and poor cellular uptake of AONs. Extensive research is currently being conducted into the formulation of AONs to improve delivery, but thus far there is no consensus on which of those strategies will be the most effective. This systematic review was designed to answer in an unbiased manner which delivery strategies most strongly enhance the efficacy of AONs in animal models of heritable neurodegenerative and neuromuscular diseases. In total, 95 primary studies met the predefined inclusion criteria. Study characteristics and data on biodistribution and toxicity were extracted and reporting quality and risk of bias were assessed. Twenty studies were eligible for meta-analysis. We found that even though the use of delivery systems provides an advantage over naked AONs, it is not yet possible to select the most promising strategies. Importantly, standardisation of experimental procedures is warranted in order to reach conclusions about the most efficient delivery strategies. Our best practice guidelines for future experiments serve as a step in that direction.
Desalting Protein Ions in Native Mass Spectrometry Using Supercharging Reagents
Cassou, Catherine A.; Williams, Evan R.
Effects of the supercharging reagents m-NBA and sulfolane on sodium ion adduction to protein ions formed using native mass spectrometry were investigated. There is extensive sodium adduction on protein ions formed by electrospray ionization from aqueous solutions containing millimolar concentrations of NaCl, which can lower sensitivity by distributing the signal of a given charge state over multiple adducted ions and can reduce mass measuring accuracy for large proteins and non-covalent complexes for which individual adducts cannot be resolved. The average number of sodium ions adducted to the most abundant ion formed from ten small (8.6–29 kDa) proteins for which adducts can be resolved is reduced by 58% or 80% on average, respectively, when 1.5% m-NBA or 2.5% sulfolane are added to aqueous solutions containing sodium compared to without the supercharging reagent. Sulfolane is more effective than m-NBA at reducing sodium ion adduction and at preserving non-covalent protein-ligand and protein-protein interactions. Desalting with 2.5% sulfolane enables detection of several glycosylated forms of 79.7 kDa holo-transferrin and NADH bound to the 146 kDa homotetramer LDH, which are otherwise unresolved due to peak broadening from extensive sodium adduction. Although sulfolane is more effective than m-NBA at protein ion desalting, m-NBA reduces salt clusters at high m/z and can increase the signal-to-noise ratios of protein ions by reducing chemical noise. Desalting is likely a result of these supercharging reagents binding sodium ions in solution, thereby reducing the sodium available to adduct to protein ions. PMID:25133273
A review of reagents for fluorescence microscopy of cellular compartments and structures, Part III: reagents for actin, tubulin, cellular membranes, and whole cell and cytoplasm.
Kilgore, Jason A; Dolman, Nick J; Davidson, Michael W
Non-antibody commercial fluorescent reagents for imaging of cytoskeletal structures have been limited primarily to tubulin and actin, with the main factor in choice based mainly on whether cells are live or fixed and permeabilized. A wider range of options exist for cell membrane dyes, and the choice of reagent primarily depends on the preferred localization in the cell (i.e., all membranes or only the plasma membrane) and usage (i.e., whether the protocol involves fixation and permeabilization). For whole-cell or cytoplasmic imaging, the choice of reagent is determined mostly by the length of time that the cells need to be visualized (hours or days) and by fixation status. Presented here is a discussion on choosing commercially available reagents for these cellular structures, with an emphasis on use for microscopic imaging, with a featured reagent for each structure, a recommended protocol, troubleshooting guide, and example image. Copyright © 2014 John Wiley & Sons, Inc.
Functionalization of epoxy esters with alcohols as stoichiometric reagents.
Pavlović, Dona; Modec, Barbara; Dolenc, Darko
Glycidyl esters, frequently employed as reactive groups on polymeric supports, were functionalized with alcohols as stoichiometric reagents, yielding β-alkoxyalcohols. Among the solvents studied, best results were obtained in ethers in the presence of a strong proton acid as a catalyst. Alcohols include simple alkanols, diols, protected polyols, 3-butyn-1-ol 3-hydroxypropanenitrile and cholesterol. This protocol represents a convenient way for introduction of various functionalities onto epoxy-functionalized polymers. Under the reaction conditions, some side reactions take place, mostly due to the reactive ester group and water present in the reaction mixture.
Improved multiple displacement amplification (iMDA) and ultraclean reagents.
Motley, S Timothy; Picuri, John M; Crowder, Chris D; Minich, Jeremiah J; Hofstadler, Steven A; Eshoo, Mark W
Next-generation sequencing sample preparation requires nanogram to microgram quantities of DNA; however, many relevant samples are comprised of only a few cells. Genomic analysis of these samples requires a whole genome amplification method that is unbiased and free of exogenous DNA contamination. To address these challenges we have developed protocols for the production of DNA-free consumables including reagents and have improved upon multiple displacement amplification (iMDA). A specialized ethylene oxide treatment was developed that renders free DNA and DNA present within Gram positive bacterial cells undetectable by qPCR. To reduce DNA contamination in amplification reagents, a combination of ion exchange chromatography, filtration, and lot testing protocols were developed. Our multiple displacement amplification protocol employs a second strand-displacing DNA polymerase, improved buffers, improved reaction conditions and DNA free reagents. The iMDA protocol, when used in combination with DNA-free laboratory consumables and reagents, significantly improved efficiency and accuracy of amplification and sequencing of specimens with moderate to low levels of DNA. The sensitivity and specificity of sequencing of amplified DNA prepared using iMDA was compared to that of DNA obtained with two commercial whole genome amplification kits using 10 fg (~1-2 bacterial cells worth) of bacterial genomic DNA as a template. Analysis showed >99% of the iMDA reads mapped to the template organism whereas only 0.02% of the reads from the commercial kits mapped to the template. To assess the ability of iMDA to achieve balanced genomic coverage, a non-stochastic amount of bacterial genomic DNA (1 pg) was amplified and sequenced, and data obtained were compared to sequencing data obtained directly from genomic DNA. The iMDA DNA and genomic DNA sequencing had comparable coverage 99.98% of the reference genome at ≥1X coverage and 99.9% at ≥5X coverage while maintaining both balance
Reagents for radioimmunological determination of carcinoembryonic antigen (CEA)
International Nuclear Information System (INIS)
Albert, Z.; Balbierz, H.; Breberowicz, J.
The work was undertaken to prepare the reagents for carcinoembryonic antigen (CEA) radioimmunoassay with double antibody method. The CEA standard of high immunoreactivity was prepared and purified. The purified CEA was used for immunozation of goats. The goat anti - CEA sera were received. IgG fraction from normal goat serum was purified and used for the production of horse anti-goat IgG serum which was then used in the radioimmunoassay of CEA. The labelling of CEA with iodine-125 has been carried out be means of the enzymatic method.(Z.R.)
Determination of the normal range of thyroid hormones in Sudanese by locally produced reagents
International Nuclear Information System (INIS)
Ali, Nagi Ibrahim
In this study serum samples have been collected from 100 volunteers in order to measure serum thyroxine (T 4 ) and serum triiodothyronine (T 3 ). The volunteers were selected carefully in the bases of the thyroid history as they should not complain of any thyroid disorders, no history of thyroid problems. They were males and females covering the range of 10-60 years old. Blood samples were collected, separated and the serum samples were kept frozen in (-20 degree C). Analysis of serum (T 4 and T 3 ) were carried out using sensitive radioimmunoassay (RIA) methods. The reagents were locally produced. The results were analysed by statistical package for social sciences (SPSS) computer program, which specially used for the determination of normal ranges and other medical statistics purposes
Spectrophotometric determination of metformin in pharmaceutical preparations, serum and urine using benzoin as derivatizing reagent
International Nuclear Information System (INIS)
Alamgir, M.; Hayat, A.
A simple and selective spectrophotometric procedure is described for the determination of Metformin based on derivatization with benzoin. The Beers law was obeyed with 2.50-12.50 meu mol L-1 at 290 nm with coefficient of determination (r2) 0.997. The experimental conditions in term of pH, reaction time and temperature, and addition of derivatizing reagent were examined. The pure metformin-benzoin derivative was prepared and characterized by FT-IR and mass spectroscopic techniques. The method was applied for the determination of metformin from pharmaceutical preparations and serum and urine of volunteers after spiking with metformin. The results were checked by standard addition method. A number of pharmaceutical additives and serum or urine matrix did not affect the determination of metformin. (author)
Reverse flow injection spectrophotometric determination of ciprofloxacin in pharmaceuticals using iron from soil as a green reagent
Palamy, Sysay; Ruengsitagoon, Wirat
A novel reverse flow injection spectrophotometric method for the determination of ciprofloxacin was successfully combined with the on-line introduction of an iron solution extracted from soil as green reagent. The assay was optimized by a univariate method to select the optimum conditions for the highest absorbance and highest stability of the complex. Beer-Lambert's law (λmax = 440 nm) is obeyed in the range 0.5-50 μg mL- 1 with a correlation coefficient (r2) of 0.9976 and 0.9996 using soil as green reagent from Khon Kaen, Thailand and Vientiane, Laos, respectively. The average percentage recoveries were in the range of 98.55-102.14% and the precision was in the range of 0.80-1.73%. The limit of detection and the limit of quantitation were 0.20 and 0.69 μg mL- 1, respectively, with a sampling rate of over 46 samples h- 1. The method was successfully applied to the determination of ciprofloxacin in commercial pharmaceutical formulations. The results were in good agreement with those obtained by the reference HPLC method using a t-test at 95% of confidence level for comparison. This method is suitable for laboratories looking for alternative analytical methods using green reagents.
Validity of HydraTrend reagent strips for the assessment of hydration status.
Abbey, Bryce M; Heelan, Kate A; Brown, Gregory A; Bartee, Rodrick T
Hydration is used by athletic governing organizations for weight class eligibility. The measurement of urine specific gravity (USG) as a measure of hydration by reagent strips is a controversial issue. The purpose of this study was to determine the validity of HydraTrend reagent strips that facilitate the correction of USG for alkaline urine samples against refractometry for the assessment of USG. Fifty-one participants (33 males, age = 22.3 ± 1.3 years; 18 females, age = 22.4 ± 1.2 years) provided 84 urine samples. The samples were tested for USG using refractometry and reagent strips and for pH using reagent strips and a digital pH meter. Strong correlation coefficients were found between refractometry and reagent strips for USG (rs(82) = 0.812, p refractometry with USG >1.020, pass reagent strips with USG ≤1.020) occurred 39% (33/84) of the time and false negative results for National Federation of State High School Association (NFHS) requirements (fail refractometry with USG >1.025, pass reagent strips with USG ≤1.025) occurred 14% (12/84) of the time. There were no false positives (pass refractometry and fail reagent strips) for NCAA or NFHS requirements. These data show that refractometry and reagent strips have strong positive correlations. However, the risk of a false negative result leading to incorrect certification of euhydration status outweighs the benefits of the HydraTrend reagent strips for the measurement of USG.
A Green Approach to SNF Reprocessing: Are Common Household Reagents the Answer?
International Nuclear Information System (INIS)
Peper, Shane M.; McNamara, Bruce K.; O'Hara, Matthew J.; Douglas, Matthew
It has been discovered that UO2, the principal component of spent nuclear fuel (SNF), can efficiently be dissolved at room temperature using a combination of common household reagents, namely hydrogen peroxide, baking soda, and ammonia. This rather serendipitous discovery opens up the possibility, for the first time, of considering a non-acidic process for recycling U from SNF. Albeit at the early stages of development, our unconventional dissolution approach possesses many attractive features that could make it a reality in the future. With dissolution byproducts of water and oxygen, our approach poses a minimal threat to the environment. Moreover, the use of common household reagents to afford actinide oxide dissolution suggests a certain degree of economic favorability. With the use of a ''closed'' digestion vessel as a reaction chamber, our approach has substantial versatility with the option of using either aqueous or gaseous reactant feeds or a combination of both. Our approach distinguishes itself from all existing reprocessing technologies in two important ways. First and foremost, it is an alkaline rather than an acidic process, using mild non-corrosive chemicals under ambient conditions to effect actinide separations. Secondly, it does not dissolve the entire SNF matrix, but rather selectively solubilizes U and other light actinides for subsequent separation, resulting in potentially faster head-end dissolution and fewer downstream separation steps. From a safeguards perspective, the use of oxidizing alkaline solutions to effect actinide separations also potentially offers a degree of inherent proliferation resistance, by allowing the U to be selectively removed from the remaining dissolver solution while keeping Pu grouped with the other minor actinides and fission products. This paper will describe the design and general experimental setup of a 'closed' digestion vessel for performing uranium oxide dissolutions under alkaline conditions using gaseous
A Green Approach to SNF Reprocessing: Are Common Household Reagents the Answer?
Energy Technology Data Exchange (ETDEWEB)
Peper, Shane M.; McNamara, Bruce K.; O' Hara, Matthew J.; Douglas, Matthew
It has been discovered that UO2, the principal component of spent nuclear fuel (SNF), can efficiently be dissolved at room temperature using a combination of common household reagents, namely hydrogen peroxide, baking soda, and ammonia. This rather serendipitous discovery opens up the possibility, for the first time, of considering a non-acidic process for recycling U from SNF. Albeit at the early stages of development, our unconventional dissolution approach possesses many attractive features that could make it a reality in the future. With dissolution byproducts of water and oxygen, our approach poses a minimal threat to the environment. Moreover, the use of common household reagents to afford actinide oxide dissolution suggests a certain degree of economic favorability. With the use of a "closed" digestion vessel as a reaction chamber, our approach has substantial versatility with the option of using either aqueous or gaseous reactant feeds or a combination of both. Our approach distinguishes itself from all existing reprocessing technologies in two important ways. First and foremost, it is an alkaline rather than an acidic process, using mild non-corrosive chemicals under ambient conditions to effect actinide separations. Secondly, it does not dissolve the entire SNF matrix, but rather selectively solubilizes U and other light actinides for subsequent separation, resulting in potentially faster head-end dissolution and fewer downstream separation steps. From a safeguards perspective, the use of oxidizing alkaline solutions to effect actinide separations also potentially offers a degree of inherent proliferation resistance, by allowing the U to be selectively removed from the remaining dissolver solution while keeping Pu grouped with the other minor actinides and fission products. This paper will describe the design and general experimental setup of a "closed" digestion vessel for performing uranium oxide dissolutions under alkaline conditions using
Diagnóstico das meningites através de fitas reagentes Diagnosis of meningitis with reagent strips
Directory of Open Access Journals (Sweden)
Roberta M.C. Romanelli
Full Text Available OBJETIVO: determinar a utilidade de fitas reagentes para a avaliação liquórica de pleocitose, glicorraquia e proteinorraquia no diagnóstico precoce e rápido de meningites em crianças. MÉTODOS: Foram incluídas no estudo amostras de líquor provenientes de 164 crianças admitidas no ambulatório de doenças infecto-contagiosas do Centro Geral de Pediatria (CGP-FHEMIG, com suspeita clínica de meningite, no período diurno de Maio/97 à Maio/99. A faixa etária dos pacientes variou de um mês a 12 anos (mediana de 12 meses, sendo obtidos resultados da citobioquímica liquórica (celularidade, glicorraquia e proteinorraquia de 154 desses pacientes. Esses achados foram comparados com reações do líquor em fitas reagentes. RESULTADOS: Através da citobioquímica líquórica foram identificados 43 casos de provável meningite bacteriana, 19 provavelmente viróticas e 83 amostras sem alterações. Pelas fitas reagentes, detectaram-se 41 casos de provável meningite bacteriana, dois casos de infecção meníngea provavelmente virótica, e em 71 exames não se verificaram alterações. Comparando os resultados obtidos por meio das fitas reagentes com a citobioquímica convencional, observou-se sensibilidade, especificidade, valores preditivos positivo e negativo e acurácia (90,7; 98,1; 95,1; 96,4; 96,1%, respectivamente. Ademais, a análise estatística pelo teste de Mc Nemar não evidenciou discordância significativa no diagnóstico de meningite bacteriana obtido através de ambos os métodos (p=0,68 e, pela estatística Kappa, verificou-se elevado grau de concordância entre os testes (pOBJECTIVE: to determine the usefulness of reagent strips in the evaluation of pleocytosis, cerebrospinal fluid glucose and protein levels for early and rapid diagnosis of meningitis in children. METHODS: We included cerebrospinal fluid samples of 164 children admitted to the outpatient clinic of Communicable Diseases of the General Pediatric Center (Funda
Optically transparent, superhydrophobic methyltrimethoxysilane based silica coatings without silylating reagent
International Nuclear Information System (INIS)
Kavale, Mahendra S.; Mahadik, D.B.; Parale, V.G.; Wagh, P.B.; Gupta, Satish C.; Rao, A.Venkateswara; Barshilia, Harish C.
The superhydrophobic surfaces have drawn lot of interest, in both academic and industries because of optically transparent, adherent and self-cleaning behavior. Surface chemical composition and morphology plays an important role in determining the superhydrophobic nature of coating surface. Such concert of non-wettability can be achieved, using surface modifying reagents or co-precursor method in sol-gel process. Attempts have been made to increase the hydrophobicity and optical transparency of methyltrimethoxysilane (MTMS) based silica coatings using polymethylmethacrylate (PMMA) instead of formal routes like surface modification using silylating reagents. The optically transparent, superhydrophobic uniform coatings were obtained by simple dip coating method. The molar ratio of MTMS:MeOH:H 2 O was kept constant at 1:5.63:1.58, respectively with 0.5 M NH 4 F as a catalyst and the weight percent of PMMA varied from 1 to 8. The hydrophobicity of silica coatings was analyzed by FTIR and contact angle measurements. These substrates exhibited 91% optical transmittance as compared to glass and water drop contact angle as high as 171 ± 1 deg. The effect of humidity on hydrophobic nature of coating has been studied by exposing these films at relative humidity of 90% at constant temperature of 30 deg. C for a period of 45 days. The micro-structural studies carried out by transmission electron microscopy (TEM).
The blocking reagent optimization for the magnetoelastic biosensor
Hu, Jiajia; Chai, Yating; Horikawa, Shin; Wikle, Howard C.; Wang, Feng'en; Du, Songtao; Chin, Bryan A.; Hu, Jing
The wireless phage-based magnetoelastic (ME) biosensor has proven to be promising for real-time detection of pathogenic bacteria on fresh produces. The ME biosensor consists of a freestanding ME resonator as the signal transducer and filamentous phage as the biomolecular-recognition element, which can specifically bind to a pathogen of interest. Due to the Joule magnetostriction effect, the biosensors can be placed into mechanical resonance when subjected to a time-varying magnetic field alternating at the sensor's resonant frequency. Upon the attachment of the target pathogen, the mass of the biosensor increases, thereby decreasing its resonant frequency. This paper presents an investigation of blocking reagents immobilization for detecting Salmonella Typhimurium on fresh food surfaces. Three different blocking reagents (BSA, SuperBlock blocking buffer, and blocker BLOTTO) were used and compared. The optical microscope was used for bacterial cells binding observation. Student t-test was used to statistically analysis the experiment results. The results shows that SuperBlock blocking buffer and blocker BLOTTO have much better blocking performance than usually used BSA.
Radiation chemical technology of industrial polymer reagents development
International Nuclear Information System (INIS)
Kudaibergenov, S.; Nurkeeva, Z.; Mun, G.; Sigitov, V.; Maltzeva, R.; Petukhov, V.; Tchekushin, A.
The goal of this project is to develop the technology of producing of polymeric reagents from the raw materials of Kazakstan for application in medicine, agriculture, enhanced oil recovery and ecology. To achieve the objectives the next technological lines or operations (Blocks) should be realized: 1. Rectification column and distilling apparatus for purification of monomers and solvents including analytical equipment to control the quality of the final product; 2. Irradiation of reaction mixture by either gamma-irradiation source Co-60; 3. Purification of polymer reagents; 4. Producing of commercial products. It is supposed that the power irradiation devices for producing of hydrogels will be mounted on the research atomic reactor of the Almaty Branch of the Institute of Atomic Energy of the National Nuclear Center. There are high qualification personal which has much experience in radioactive materials operating. Irradiation technologies will provide the low cost of hydrogels, approximately 250-300 US$ per 1 ton. Expected results. One can expect that the realization of this project allows to produce hydrogels in industrial scale to cover partly the requirements of medicine, agriculture, oil industry and ecology
Total Synthesis of Natural Products Using Hypervalent Iodine Reagents
Directory of Open Access Journals (Sweden)
Gaetan eMaertens
Full Text Available We present a review of natural product syntheses accomplished in our laboratory during the last five years. Each synthetic route features a phenol dearomatization promoted by an environmentally benign hypervalent iodine reagent. The dearomatizations demonstrate the aromatic ring umpolung concept, and involve stereoselective remodeling of the inert unsaturations of a phenol into a highly functionalized key intermediate that may contain a quaternary carbon center and a prochiral dienone system. Several new oxidative strategies were employed, including transpositions (1,3-alkyl shift and Prins-pinacol, a polycyclization, an ipso rearrangement, and direct nucleophilic additions at the phenol para position. Several alkaloids, heterocyclic compounds, and a polycyclic core have been achieved, including sceletenone (a serotonin reuptake inhibitor, acetylaspidoalbidine (an antitumor agent, fortucine (antiviral and antitumor, erysotramidine (curare-like effect, platensimycin (an antibiotic, and the main core of a kaurane diterpene (immunosuppressive agent and stimulator of apoptosis. These concise and in some cases enantioselective syntheses effectively demonstrate the importance of hypervalent iodine reagents in the total synthesis of bioactive natural products.
Classical kinematic model for direct reactions of oriented reagents
International Nuclear Information System (INIS)
Schechter, I.; Prisant, M.G.; Levine, R.D.
A simple kinematic model based on the concept of an orientation-dependent critical configuration for reaction is introduced and applied. The model serves two complementary purposes. In the predictive mode the model provides an easily implemented procedure for computing the reactivity of oriented reagents (including those actually amenable to measure) from a given potential energy surface. The predictions of the model are compared against classical trajectory results for the H + D 2 reaction. By use of realistic potential energy surfaces the model is applied to the Li + HF and O + HCl reactions where the HX molecules are pumped by a polarized laser. A given classical trajectory is deemed reactive or not according to whether it can surmount the barrier at that particular orientation. The essential difference with the model of Levine and Bernstein is that the averaging over initial conditions is performed by using a Monte Carlo integration. One can therefore use the correct orientation-dependent shape (and not only height) of the barrier to reaction and, furthermore, use oriented or aligned reagents. Since the only numerical step is a Monte Carlo sampling of initial conditions, very many trajectories can be run. This suffices to determine the reaction cross section for different initial conditions. To probe the products, they have employed the kinematic approach of Elsum and Gordon. The result is a model where, under varying initial conditions, examining final-state distributions or screening different potential energy surfaces can be efficiently carried out
Tunable, Quantitative Fenton-RAFT Polymerization via Metered Reagent Addition.
Nothling, Mitchell D; McKenzie, Thomas G; Reyhani, Amin; Qiao, Greg G
A continuous supply of radical species is a key requirement for activating chain growth and accessing quantitative monomer conversions in reversible addition-fragmentation chain transfer (RAFT) polymerization. In Fenton-RAFT, activation is provided by hydroxyl radicals, whose indiscriminate reactivity and short-lived nature poses a challenge to accessing extended polymerization times and quantitative monomer conversions. Here, an alternative Fenton-RAFT procedure is presented, whereby radical generation can be finely controlled via metered dosing of a component of the Fenton redox reaction (H 2 O 2 ) using an external pumping system. By limiting the instantaneous flux of radicals and ensuring sustained radical generation over tunable time periods, metered reagent addition reduces unwanted radical "wasting" reactions and provides access to consistent quantitative monomer conversions with high chain-end fidelity. Fine tuning of radical concentration during polymerization is achieved simply via adjustment of reagent dose rate, offering significant potential for automation. This modular strategy holds promise for extending traditional RAFT initiation toward more tightly regulated radical concentration profiles and affords excellent prospects for the automation of Fenton-RAFT polymerization. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys
Directory of Open Access Journals (Sweden)
Colby S Shemesh
Full Text Available Triantennary N-acetyl galactosamine (GalNAc3 is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA-C6 cluster significantly enhances antisense oligonucleotide (ASO potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of 3H-radiolabeled (3H placed in THA or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5′-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.
Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals
Directory of Open Access Journals (Sweden)
Punit P Seth
Full Text Available We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs modified with α-L-locked nucleic acid (LNA and related modifications targeting phosphatase and tensin homologue (PTEN messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.
Regulation of human heme oxygenase in endothelial cells by using sense and antisense retroviral constructs.
Quan, S; Yang, L; Abraham, N G; Kappas, A
Our objective was to determine whether overexpression and underexpression of human heme oxygenase (HHO)-1 could be controlled on a long-term basis by introduction of the HO-1 gene in sense (S) and antisense (AS) orientation with an appropriate vector into endothelial cells. Retroviral vector (LXSN) containing viral long terminal repeat promoter-driven human HO-1 S (LSN-HHO-1) and LXSN vectors containing HHO-1 promoter (HOP)-controlled HHO-1 S and AS (LSN-HOP-HHO-1 and LSN-HOP-HHO-1-AS) sequences were constructed and used to transfect rat lung microvessel endothelial cells (RLMV cells) and human dermal microvessel endothelial cells (HMEC-1 cells). RLMV cells transduced with HHO-1 S expressed human HO-1 mRNA and HO-1 protein associated with elevation in total HO activity compared with nontransduced cells. Vector-mediated expression of HHO-1 S or AS under control of HOP resulted in effective production of HO-1 or blocked induction of endogenous human HO-1 in HMEC-1 cells, respectively. Overexpression of HO-1 AS was associated with a long-term decrease (45%) of endogenous HO-1 protein and an increase (167%) in unmetabolized exogenous heme in HMEC-1 cells. Carbon monoxide (CO) production in HO-1 S- or AS-transduced HMEC-1 cells after heme treatment was increased (159%) or decreased (50%), respectively, compared with nontransduced cells. HO-2 protein levels did not change. These findings demonstrate that HHO-1 S and AS retroviral constructs are functional in enhancing and reducing HO activity, respectively, and thus can be used to regulate cellular heme levels, the activity of heme-dependent enzymes, and the rate of heme catabolism to CO and bilirubin.
Uptake of DNA by cancer cells without a transfection reagent
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Yanping Kong
Full Text Available Abstract Background Cancer cells exhibit elevated levels of glucose uptake and may obtain pre-formed, diet-derived fatty acids from the bloodstream to boost their rapid growth; they may also use nucleic acid from their microenvironment. The study of processing nucleic acid by cancer cells will help improve the understanding of the metabolism of cancer. DNA is commonly packaged into a viral or lipid particle to be transferred into cells; this process is called transfection in laboratory. Cancer cells are known for having gene mutations and the evolving ability of endocytosis. Their uptake of DNAs might be different from normal cells; they may take in DNAs directly from the environment. In this report, we studied the uptake of DNAs in cancer cells without a transfection reagent. Methods A group of DNA fragments were prepared with PCR and labeled with isotope phosphorous-32 to test their uptake by Huh 7 (liver cancer and THLE3 (normal liver cells after incubation overnight by counting radioactivity of the cells' genomic DNA. Multiple cell lines including breast cancer and lung cancer were tested with the same method. DNA molecules were also labeled with fluorescence to test the location in the cells using a kit of "label it fluorescence in situ hybridization (FISH" from Mirus (USA. Results The data demonstrated that hepatocellular carcinoma cells possess the ability to take in large DNA fragments directly without a transfection reagent whereas normal liver cells cannot. Huh7 and MDA-MB231 cells displayed a significantly higher Rhodamine density in the cytoplasmic phagosomes and this suggests that the mechanism of uptake of large DNA by cancer cells is likely endocytosis. The efficacy of uptake is related to the DNA's size. Some cell lines of lung cancer and breast cancer also showed similar uptake of DNA. Conclusions In the present study, we have revealed the evidence that some cancer cells, but not nontumorigenic cells, can take DNA
Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.
Yu, Xing Xian; Watts, Lynnetta M; Manchem, Vara Prasad; Chakravarty, Kaushik; Monia, Brett P; McCaleb, Michael L; Bhanot, Sanjay
Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4) in peripheral tissues. Treatment of diet-induce obese (DIO) mice with FGFR4 antisense oligonucleotides (ASO) specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW) and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.
Peripheral reduction of FGFR4 with antisense oligonucleotides increases metabolic rate and lowers adiposity in diet-induced obese mice.
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Xing Xian Yu
Full Text Available Obesity is a primary risk factor for multiple metabolic disorders. Many drugs for the treatment of obesity, which mainly act through CNS as appetite suppressants, have failed during development or been removed from the market due to unacceptable adverse effects. Thus, there are very few efficacious drugs available and remains a great unmet medical need for anti-obesity drugs that increase energy expenditure by acting on peripheral tissues without severe side effects. Here, we report a novel approach involving antisense inhibition of fibroblast growth factor receptor 4 (FGFR4 in peripheral tissues. Treatment of diet-induce obese (DIO mice with FGFR4 antisense oligonucleotides (ASO specifically reduced liver FGFR4 expression that not only resulted in decrease in body weight (BW and adiposity in free-feeding conditions, but also lowered BW and adiposity under caloric restriction. In addition, combination treatment with FGFR4 ASO and rimonabant showed additive reduction in BW and adiposity. FGFR4 ASO treatment increased basal metabolic rate during free-feeding conditions and, more importantly, prevented adaptive decreases of metabolic rate induced by caloric restriction. The treatment increased fatty acid oxidation while decreased lipogenesis in both liver and fat. Mechanistic studies indicated that anti-obesity effect of FGFR4 ASO was mediated at least in part through an induction of plasma FGF15 level resulted from reduction of hepatic FGFR4 expression. The anti-obesity effect was accompanied by improvement in plasma glycemia, whole body insulin sensitivity, plasma lipid levels and liver steatosis. Therefore, FGFR4 could be a potential novel target and antisense reduction of hepatic FGFR4 expression could be an efficacious therapy as an adjunct to diet restriction or to an appetite suppressant for the treatment of obesity and related metabolic disorders.
Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft
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Baldi Alfonso
Full Text Available Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139 as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy. Results The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50% accompanied by a marked tumor re-growth delay (TRD, about 20 days. The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days. Conclusions These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.
Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.
Semple, S C; Klimuk, S K; Harasym, T O; Dos Santos, N; Ansell, S M; Wong, K F; Maurer, N; Stark, H; Cullis, P R; Hope, M J; Scherrer, P
Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.
Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe
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Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)
Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.
Evaluation of selected neutralizing agents for the treatment of uranium tailings leachates. Laboratory progress report
International Nuclear Information System (INIS)
Sherwood, D.R.; Serne, R.J.
Laboratory experiments were conducted to evaluate the performance of selected neutralizing agents for the treatment of uranium tailings solutions. Highly acidic tailings solutions (pH 3 ) reagent grade; Calcium hydroxide [Ca(OH) 2 ] reagent grade; Magnesium oxide (MgO) reagent grade; Sodium carbonate (Na 2 CO 3 ) reagent grade; and Sodium hydroxide (NaOH) reagent grade. Evaluation of the effectiveness for the treatment of uranium tailings solutions for the selected neutralizing agents under controlled laboratory conditions was based on three criteria. The criteria are: (1) treated effluent water quality, (2) neutralized sludge handling and hydraulic properties, and (3) reagent costs and acid neutralizing efficiency. On the basis of these limited laboratory results calcium hydroxide or its dehydrated form CaO (lime) appears to be the most effective option for treatment of uranium tailings solutions
A new method for quasi-reagent-free biomonitoring of mercury in human urine.
Schlathauer, Maria; Reitsam, Verena; Schierl, Rudolf; Leopold, Kerstin
A novel analytical method for sampling and extraction of mercury (Hg) from human urine is presented in this work. The method is based on selective accumulation and separation of Hg from fresh urine sample onto active nanogold-coated silica material by highly efficient solid-phase extraction. After thermal desorption of Hg from the extractant, detection is performed by atomic fluorescence spectrometry (AFS). The feasibility and validity of the optimized, quasi-reagent-free approach was confirmed by recovery experiments in spiked real urine (recovery rate 96.13 ± 5.34%) and by comparison of found Hg concentrations in real urine samples - originating from occupationally exposed persons - with values obtained from reference methods cold vapor - atomic absorption spectrometry (CVAAS) and cold vapor - atomic fluorescence spectrometry (CV-AFS). A very good agreement of the found values reveals the validity of the proposed approach. The limit of detection (LOD) was found to be as low as 0.004 μg Hg L -1 and a high reproducibility with a relative standard deviations ≤4.2% (n = 6) is given. Moreover, storage of the samples for up to one week at an ambient temperature of 30 °C reveals no analyte losses or contamination. In conclusion, the proposed method enables easy-to-handle on-site extraction of total Hg from human urine ensuring at the same time reagent-free sample stabilization, providing quick and safe sampling, which can be performed by untrained persons. Copyright © 2017 Elsevier B.V. All rights reserved.
Simultaneous Expression from Both the Sense and Antisense Strand of the Erythropoietin Receptor Gene Mitigates Acute Lung Injury
concept efficacy that increasing EpoR or RopE expression by cDNA delivery to lung cells in vitro enhances cytoprotection against hyperoxia-induced injury...oxidative damage, cell culture, rodent model, inhalation cDNA delivery, sense and antisense erythropoietin receptor transcripts 16. SECURITY...prevention of acute lung injury. 1-6 50% Subtask 1: Prepare plasmid cDNA of EpoR and RopE in nanoparticle formulation. 1 Completed 06.2017 Subtask 2
Endogenous Msx1 antisense transcript: In vivo and in vitro evidences, structure, and potential involvement in skeleton development in mammals
Blin-Wakkach, C.; Lezot, F.; Ghoul-Mazgar, S.; Hotton, D.; Monteiro, S.; Teillaud, C.; Pibouin, L.; Orestes-Cardoso, S.; Papagerakis, P.; Macdougall, M.; Robert, B.; Berdal, A.
Msx1 is a key factor for the development of tooth and craniofacial skeleton and has been proposed to play a pivotal role in terminal cell differentiation. In this paper, we demonstrated the presence of an endogenous Msx1 antisense RNA (Msx1-AS RNA) in mice, rats, and humans. In situ analysis revealed that this RNA is expressed only in differentiated dental and bone cells with an inverse correlation with Msx1 protein. These in vivo data and overexpression of Msx1 sense and AS RNA in an odontob...
Modification of the mitochondrial sulfonylurea receptor by thiol reagents.
Szewczyk, A; Wójcik, G; Lobanov, N A; Nalecz, M J
The purpose of this study was to investigate the effects exerted by thiol-modifying reagents on themitochondrial sulfonylurea receptor. The thiol-oxidizing agents (timerosal and 5, 5'-dithio-bis(2-nitrobenzoic acid)) were found to produce a large inhibition (70% to 80%) of specific binding of [(3)H]glibenclamide to the beef heart mitochondrial membrane. Similar effects were observed with membrane permeable (N-ethylmaleimide) and non-permeable (mersalyl) thiol modifying agents. Glibenclamide binding was also decreased by oxidizing agents (hydrogen peroxide) but not by reducing agents (reduced gluthatione, dithiothreitol and the 2,3-dihydroxy-1,4-dithiolbutane). The results suggest that intact thiol groups, facing the mitochondrial matrix, are essential for glibenclamide binding to the mitochondrial sulfonylurea receptor. Copyright 1999 Academic Press.
Oxidation of cashew tree gum exudate polysaccharide with TEMPO reagent
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Cunha, Pablyana L.R.; Maciel, Jeanny S.; Paula, Regina C.M. de; Feitosa, Judith P.A. [Universidade Federal do Ceara, Fortaleza, CE (Brazil). Dept. de Quimica Organica e Inorganica; Sierakowski, Maria Rita [Universidade Federal do Parana, Curitiba, PR (Brazil). Dept. de Quimica]. E-mail: firstname.lastname@example.com
Cashew gum (CG), an exudate polysaccharide from Anacardium occidentale trees, was oxidized with TEMPO reagent and the product (CGOX) characterized by spectroscopic techniques (FTIR and NMR), chromatographic analyses (HPLC and GPC), viscosity measurements and thermal analysis (TGA). The yield of the reaction product was 96%. The uronic acid content in starting gum (7.2 m%) was increased to 36 m%. The degree of oxidation based on free galactose and glucose units was 68%. NMR data show that oxidation occurred preferentially at primary carbons of galactose units. High degradation degree after oxidation was estimated by the difference on the expected and observed {eta}{sub CGOX}/{eta}{sub CG} ratio. The presence of organic and inorganic impurities in the new polyelectrolyte was detected by TGA. A less thermally stable cashew gum is formed after the oxidation with TEMPO based on initial decomposition temperature and IPDT. (author)
Oxidation of cashew tree gum exudate polysaccharide with TEMPO reagent
International Nuclear Information System (INIS)
Cunha, Pablyana L.R.; Maciel, Jeanny S.; Paula, Regina C.M. de; Feitosa, Judith P.A.; Sierakowski, Maria Rita
Cashew gum (CG), an exudate polysaccharide from Anacardium occidentale trees, was oxidized with TEMPO reagent and the product (CGOX) characterized by spectroscopic techniques (FTIR and NMR), chromatographic analyses (HPLC and GPC), viscosity measurements and thermal analysis (TGA). The yield of the reaction product was 96%. The uronic acid content in starting gum (7.2 m%) was increased to 36 m%. The degree of oxidation based on free galactose and glucose units was 68%. NMR data show that oxidation occurred preferentially at primary carbons of galactose units. High degradation degree after oxidation was estimated by the difference on the expected and observed η CGOX /η CG ratio. The presence of organic and inorganic impurities in the new polyelectrolyte was detected by TGA. A less thermally stable cashew gum is formed after the oxidation with TEMPO based on initial decomposition temperature and IPDT. (author)
Mandelazo I as a reagent for Zr(IV) determination
International Nuclear Information System (INIS)
Rakha, T.H.; Filip, P.; Stefan, N.
A spectrometric study of the reaction of the Zr(IV) ions with Mandelazo I was carried out. Absorption spectra revealed that the maximum absorption of the zirconium compound appears at a wavelength (316 nm) different from the maxima of the reagent (253 and 390 nm). Beer-Lambert law is followed for zirconium concentrations of the order of 8.8 x 10 -5 M (i.e. 8 μg Zr(IV)/mL). Possible interferences of ions such as Be(II), Cu(II), Zn(II), Al(III), Th(IV), U(VI), Mn(II), Fe(III), Co(II) and Ni(II) were investigated in connection with some masking agents such as SO 4 2- and C 2 O 4 2- . Also, the solid state Zr(IV)- Mandelazo I compound was prepared and characterized by nitrogen and thermogravimetric analyses
Recovery of uranium low grade ores by froth flotation: study of the texture and synergetic effects of flotation reagents
International Nuclear Information System (INIS)
Duverger, Agathe
Due to the energy growing demand, uranium low grade ores may be those exploited in the future. Uranium ores conventional treatment does not often use mineral processing such as concentration methods for reducing leaching reagent consumption. The aim of this work is to develop an upgrading process to improve the operating process (alkaline heap leaching) taking into account the mineralogical and textural variability of the ore. The Trekkopje deposit is composed of calcrete and a gypscrete. The uranium bearing mineral is carnotite (K 2 (UO 2 ) 2 [VO 4 ] 2 .3H 2 O). The gangue minerals are composed by silicates, such as quartz, feldspars, micas and Ca-minerals, calcite and gypsum (XRD and ICP-MS analysis). A SEM image processing was used to study the textural properties and the exposed free surface of mineral inclusions in clay clusters. In calcrete milled to -200 μm, 50 % of all carnotite is associated with clay clusters, which are composed by 98 % of palygorskite, 2 % of illite, montmorillonite, and interbedded clays (XRD and microprobe analysis). The carnotite grain size is 95 % less than 70 μm. Calcite is the main inclusion in clay clusters. Indeed, the calcite inclusions average rate in the clay clusters is 12 % and 5 % for carnotite inclusion. And the free exposed surface percentage of these minerals in clay clusters is 3 % and 6 %, thus indicating that the inclusions should not affect the behavior of mixed clay particles. However, ore flotation essays did not verify this hypothesis. Three minerals separation have been proposed based on the mineral ability to consume leaching reagents: separating Ca-minerals from silicates, palygorskite from gangue minerals and carnotite from gangue minerals. A study of silicates and Ca-minerals electrokinetic properties (electrophoresis) was carried out to select the collectors and the optimum pH range for selective flotation. Basic pH near neutral was proved to be optimal for the separation of gangue minerals with cationic
SiRNAs conjugated with aromatic compounds induce RISC-mediated antisense strand selection and strong gene-silencing activity
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Kubo, Takanori, E-mail: firstname.lastname@example.com [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Yanagihara, Kazuyoshi [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan); Division of Genetics, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045 (Japan); Takei, Yoshifumi [Department of Biochemistry, Nagoya University Graduate School of Medicine, 65 Tsurumi-cho, Showa-ku, Nagoya 466-8550 (Japan); Mihara, Keichiro [Department of Hematology and Oncology, Research Institute for Radiation Biology and Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553 (Japan); Sato, Yuichiro; Seyama, Toshio [Faculty of Pharmacy, Yasuda Women' s University, 6-13-1 Yasuhigashi, Asaminami-ku, Hiroshima 731-0153 (Japan)
Highlights: Black-Right-Pointing-Pointer SiRNAs conjugated with aromatic compounds (Ar-siRNAs) at 5 Prime -sense strand were synthesized. Black-Right-Pointing-Pointer Ar-siRNAs increased resistance against nuclease degradation. Black-Right-Pointing-Pointer Ar-siRNAs were thermodynamically stable compared with the unmodified siRNA. Black-Right-Pointing-Pointer High levels of cellular uptake and cytoplasmic localization were found. Black-Right-Pointing-Pointer Strong gene-silencing efficacy was exhibited in the Ar-siRNAs. -- Abstract: Short interference RNA (siRNA) is a powerful tool for suppressing gene expression in mammalian cells. In this study, we focused on the development of siRNAs conjugated with aromatic compounds in order to improve the potency of RNAi and thus to overcome several problems with siRNAs, such as cellular delivery and nuclease stability. The siRNAs conjugated with phenyl, hydroxyphenyl, naphthyl, and pyrenyl derivatives showed strong resistance to nuclease degradation, and were thermodynamically stable compared with unmodified siRNA. A high level of membrane permeability in HeLa cells was also observed. Moreover, these siRNAs exhibited enhanced RNAi efficacy, which exceeded that of locked nucleic acid (LNA)-modified siRNAs, against exogenous Renilla luciferase in HeLa cells. In particular, abundant cytoplasmic localization and strong gene-silencing efficacy were found in the siRNAs conjugated with phenyl and hydroxyphenyl derivatives. The novel siRNAs conjugated with aromatic compounds are promising candidates for a new generation of modified siRNAs that can solve many of the problems associated with RNAi technology.
A review of reagents for fluorescence microscopy of cellular compartments and structures, part I: BacMam labeling and reagents for vesicular structures.
Dolman, Nick J; Kilgore, Jason A; Davidson, Michael W
Fluorescent labeling of vesicular structures in cultured cells, particularly for live cells, can be challenging for a number of reasons. The first challenge is to identify a reagent that will be specific enough where some structures have a number of potential reagents and others very few options. The emergence of BacMam constructs has allowed more easy-to-use choices. Presented here is a discussion of BacMam constructs as well as a review of commercially-available reagents for labeling vesicular structures in cells, including endosomes, peroxisomes, lysosomes, and autophagosomes, complete with a featured reagent for each structure, recommended protocol, troubleshooting guide, and example image. © 2013 by John Wiley & Sons, Inc.
Combination of vascular endothelial growth factor antisense oligonucleotide therapy and radiotherapy increases the curative effects against maxillofacial VX2 tumors in rabbits
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Zheng Linfeng, E-mail: firstname.lastname@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Li Yujie, E-mail: email@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Han, E-mail: firstname.lastname@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhao Jinglong, E-mail: email@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Wang Xifu, E-mail: firstname.lastname@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Hu Yunsheng, E-mail: email@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China); Zhang Guixiang, E-mail: firstname.lastname@example.com [Department of Radiology, Shanghai First People' s Hospital, Medical College, Shanghai Jiaotong University, Hanning Road, 100, 200080 Shanghai (China)
Purpose: To study the effects of combination of vascular endothelial growth factor (VEGF) antisense oligonucleotide therapy and radiotherapy on maxillofacial VX2 tumors in rabbits. Methods: We used 24 New Zealand white rabbits as a model to induce maxillofacial VX2 tumor. The rabbits were randomly divided into the following 4 groups: radiotherapy group (group A), treated with 16 Gy of radiotherapy; VEGF antisense oligonucleotide treatment group (group B), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor; VEGF antisense oligonucleotide combined with radiotherapy group (group C), treated with an injection of 150 {mu}g of VEGF antisense oligonucleotide into the local tumor immediately after 16 Gy of radiotherapy; and control group (group D), treated with an injection of 300 {mu}l 5% aqueous glucose solution into the local tumor. On days 3 and 14 after treatment, dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was performed to calculate maximal enhancement ratio (MER), slope of enhancement (SLE), and tumor volume change. Rabbits were killed on day 14 to obtain samples for pathological examination and immunohistochemical staining for VEGF. Results: In group C, tumor volume was significantly reduced on day 14 after treatment, and the difference was statistically different as compared to that before treatment, on day 3 after treatment and other groups (P < 0.01). Values of both MER and SLE after treatment were significantly lower than the values before treatment (P < 0.05). Pathological specimen revealed tumor cell edema, bleeding, necrosis, vascular wall thickening and occlusion, and decreased VEGF expression. The immunohistochemical score (IHS) of group C was significantly different from groups A and D respectively (P < 0.05). Conclusion: Injecting the tumor with VEGF antisense oligonucleotide immediately after radiotherapy can enhance the curative effect on rabbit maxillofacial VX2 tumor, and DCE-MRI can serve
A specific Tween-80-Rhodamine S-MWNTs phosphorescent reagent for the detection of trace calcitonin
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Liu Jiaming, E-mail: email@example.com [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Huang Xiaomei; Zhang Lihong; Zheng Zhiyong [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Department of Food and Biological Engineering, Zhangzhou Institute of Technology, Zhangzhou, 363000 (China); Lin Xuan; Zhang Xiaoyang; Jiao Li; Cui Malin [Department of Chemistry and Environmental Science, Zhangzhou Normal College, Zhangzhou, 363000 (China); Jiang Shulian [Fujian Provincial Bureau of Quality and Technical Supervision, Zhangzhou, 363000 (China); Lin Shaoqin [Department of Biochemistry, Fujian Education College, Fuzhou 350001 (China)
dramatically enhance the RTP signal of the system. The developed TRM phosphorescent reagent was used to label anti-calcitonin antibody (Ab{sub CT}) to form the TRM-Ab{sub CT} labelling product, which could take high specific immunoreaction with CT, and the {Delta}I{sub p} (= I{sub p2} - I{sub p1}, I{sub p2} and I{sub p1} were the phosphorescence intensity of the test solution and the blank sample, respectively) of the system was linear to the content of CT. Hence, a new solid substrate room temperature phosphorescence immunoassay (SSRTPIA) was established for the determination of CT in human serum. This sensitive (limit of quantification (LOQ) was 8.0 Multiplication-Sign 10{sup -14} g mL{sup -1}), accurate, selective and precise method has been applied to determine CT in human serum and predict primary osteoporosis and fractures, with the results in good agreement with those obtained by chemiluminescence immunoassay (CLIA). Simultaneously, the structure of MWNTs was characterized with scanning electron microscopy (SEM) and infrared spectroscopy (IR), and the reaction mechanisms of both labelling Ab{sub CT} with TRM and SSRTPIA for the determination of trace CT were discussed.
Chemosensitization of Human Renal Cell Cancer Using Antisense Oligonucleotides Targeting the Antiapoptotic Gene Clusterin
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Tobias Zellweger
Full Text Available BACKGROUND: Renal cell cancer (RCC is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO, has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98% and an overexpression, compared to normal tissue, in a majority of RCC (69%. Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dosedependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo
Unravelling the Secrets of Mycobacterial Cidality through the Lens of Antisense.
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Parvinder Kaur
Full Text Available One of the major impediments in anti-tubercular drug discovery is the lack of a robust grammar that governs the in-vitro to the in-vivo translation of efficacy. Mycobacterium tuberculosis (Mtb is capable of growing both extracellular as well as intracellular; encountering various hostile conditions like acidic milieu, free radicals, starvation, oxygen deprivation, and immune effector mechanisms. Unique survival strategies of Mtb have prompted researchers to develop in-vitro equivalents to simulate in-vivo physiologies and exploited to find efficacious inhibitors against various phenotypes. Conventionally, the inhibitors are screened on Mtb under the conditions that are unrelated to the in-vivo disease environments. The present study was aimed to (1. Investigate cidality of Mtb targets using a non-chemical inhibitor antisense-RNA (AS-RNA under in-vivo simulated in-vitro conditions.(2. Confirm the cidality of the targets under in-vivo in experimental tuberculosis. (3. Correlate in-vitro vs. in-vivo cidality data to identify the in-vitro condition that best predicts in-vivo cidality potential of the targets. Using cidality as a metric for efficacy, and AS-RNA as a target-specific inhibitor, we delineated the cidality potential of five target genes under six different physiological conditions (replicating, hypoxia, low pH, nutrient starvation, nitrogen depletion, and nitric oxide.In-vitro cidality confirmed in experimental tuberculosis in BALB/c mice using the AS-RNA allowed us to identify cidal targets in the rank order of rpoB>aroK>ppk>rpoC>ilvB. RpoB was used as the cidality control. In-vitro and in-vivo studies feature aroK (encoding shikimate kinase as an in-vivo mycobactericidal target suitable for anti-TB drug discovery. In-vitro to in-vivo cidality correlations suggested the low pH (R = 0.9856 in-vitro model as best predictor of in-vivo cidality; however, similar correlation studies in pathologically relevant (Kramnik mice are warranted. In
Isolation and antisense suppression of flavonoid 3', 5'-hydroxylase modifies flower pigments and colour in cyclamen
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Patel Deepa
Full Text Available Abstract Background Cyclamen is a popular and economically significant pot plant crop in several countries. Molecular breeding technologies provide opportunities to metabolically engineer the well-characterized flavonoid biosynthetic pathway for altered anthocyanin profile and hence the colour of the flower. Previously we reported on a genetic transformation system for cyclamen. Our aim in this study was to change pigment profiles and flower colours in cyclamen through the suppression of flavonoid 3', 5'-hydroxylase, an enzyme in the flavonoid pathway that plays a determining role in the colour of anthocyanin pigments. Results A full-length cDNA putatively identified as a F3'5'H (CpF3'5'H was isolated from cyclamen flower tissue. Amino acid and phylogeny analyses indicated the CpF3'5'H encodes a F3'5'H enzyme. Two cultivars of minicyclamen were transformed via Agrobacterium tumefaciens with an antisense CpF3'5'H construct. Flowers of the transgenic lines showed modified colour and this correlated positively with the loss of endogenous F3'5'H transcript. Changes in observed colour were confirmed by colorimeter measurements, with an overall loss in intensity of colour (C in the transgenic lines and a shift in hue from purple to red/pink in one cultivar. HPLC analysis showed that delphinidin-derived pigment levels were reduced in transgenic lines relative to control lines while the percentage of cyanidin-derived pigments increased. Total anthocyanin concentration was reduced up to 80% in some transgenic lines and a smaller increase in flavonol concentration was recorded. Differences were also seen in the ratio of flavonol types that accumulated. Conclusion To our knowledge this is the first report of genetic modification of the anthocyanin pathway in the commercially important species cyclamen. The effects of suppressing a key enzyme, F3'5'H, were wide ranging, extending from anthocyanins to other branches of the flavonoid pathway. The results
Inhibition of enterovirus 71 infection by antisense octaguanidinium dendrimer-conjugated morpholino oligomers.
Tan, Chee Wah; Chan, Yoke Fun; Quah, Yi Wan; Poh, Chit Laa
Enterovirus 71 (EV-71) infections are generally manifested as mild hand, foot and mouth disease, but have been reported to cause severe neurological complications with high mortality rates. Treatment options remain limited due to the lack of antivirals. Octaguanidinium-conjugated morpholino oligomers (vivo-MOs) are single-stranded DNA-like antisense agents that can readily penetrate cells and reduce gene expression by steric blocking of complementary RNA sequences. In this study, inhibitory effects of three vivo-MOs that are complementary to the EV-71 internal ribosome entry site (IRES) and the RNA-dependent RNA polymerase (RdRP) were tested in RD cells. Vivo-MO-1 and vivo-MO-2 targeting the EV-71 IRES showed significant viral plaque reductions of 2.5 and 3.5 log10PFU/ml, respectively. Both vivo-MOs reduced viral RNA copies and viral capsid expression in RD cells in a dose-dependent manner. In contrast, vivo-MO-3 targeting the EV-71 RdRP exhibited less antiviral activity. Both vivo-MO-1 and 2 remained active when administered either 4h before or within 6h after EV-71 infection. Vivo-MO-2 exhibited antiviral activities against poliovirus (PV) and coxsackievirus A16 but vivo-MO-1 showed no antiviral activities against PV. Both the IRES-targeting vivo-MO-1 and vivo-MO-2 inhibit EV-71 RNA translation. Resistant mutants arose after serial passages in the presence of vivo-MO-1, but none were isolated against vivo-MO-2. A single T to C substitution at nucleotide position 533 was sufficient to confer resistance to vivo-MO-1. Our findings suggest that IRES-targeting vivo-MOs are good antiviral candidates for treating early EV-71 infection, and vivo-MO-2 is a more favorable candidate with broader antiviral spectrum against enteroviruses and are refractory to antiviral resistance. Copyright © 2014 Elsevier B.V. All rights reserved.
21 CFR 864.9650 - Quality control kit for blood banking reagents.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Quality control kit for blood banking reagents... SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Products Used In Establishments That Manufacture Blood and Blood Products § 864.9650 Quality control kit for blood banking reagents. (a...
A new achiral reagent for the incorporation of multiple amino groups into oligonucleotides
DEFF Research Database (Denmark)
Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael
The synthesis of a new functionalized achiral linker reagent (10) for the incorporation of multiple primary amino groups into oligonucleotides is described. The linker reagent is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and the linker can be incorporated...
21 CFR 864.9225 - Cell-freezing apparatus and reagents for in vitro diagnostic use.
... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Cell-freezing apparatus and reagents for in vitro diagnostic use. 864.9225 Section 864.9225 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH... Establishments That Manufacture Blood and Blood Products § 864.9225 Cell-freezing apparatus and reagents for in...
Facile synthesis of aliphatic isothiocyanates and thioureas on solid phase using peptide coupling reagents
DEFF Research Database (Denmark)
Boas, Ulrik; Andersen, Heidi Gertz; Christensen, Jørn B.
Peptide coupling reagents can be used as versatile reagents for the formation of aliphatic isothiocyanates and thioureas on solid phase from the corresponding solid-phase anchored aliphatic primary amines. The formation of the thioureas is fast and highly chemoselective, and proceeds via formatio...
Exploring the Potential for Using Inexpensive Natural Reagents Extracted from Plants to Teach Chemical Analysis
Hartwell, Supaporn Kradtap
A number of scientific articles report on the use of natural extracts from plants as chemical reagents, where the main objective is to present the scientific applications of those natural plant extracts. The author suggests that natural reagents extracted from plants can be used as alternative low cost tools in teaching chemical analysis,…
In situ generation of the Ohira-Bestmann Reagent from stable sulfonyl azide
DEFF Research Database (Denmark)
Jepsen, Tue Heesgaard; Kristensen, Jesper Langgaard
We report an improved method for in situ generation of the Ohira-Bestmann reagent. Using the recently reported bench stable imidazole-1-sulfonyl azide as diazotransfer reagent, this new method represents a safe and scalable approach for the transformation of aldehydes into terminal alkynes...
Storage Conditions of Conjugated Reagents Can Impact Results of Immunogenicity Assays
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Robert J. Kubiak
Full Text Available Consistent performance of anti-drug antibody (ADA assays through all stages of clinical development is critical for the assessment of immunogenicity and interpretation of PK, PD, safety, and efficacy. The electrochemiluminescent assays commonly employed for ADA measurement use drug conjugated with ruthenium and biotin to bind ADA in samples. Here we report an association between high nonspecific ADA responses in certain drug-naïve individuals and the storage buffer of the conjugated reagents used in a monoclonal antibody ADA assay. Ruthenylated reagents stored in phosphate-buffered saline (PBS buffer had increased levels of aggregate and produced variable and high baseline responses in some subjects. Reagents stored in a histidine-sucrose buffer (HSB had lower aggregate levels and produced low sample responses. In contrast to PBS, conjugated reagents formulated in HSB remained low in aggregate content and in sample response variability after 5 freeze/thaw cycles. A reagent monitoring control (RMC serum was prepared for the real-time evaluation of conjugated reagent quality. Using appropriate buffers for storage of conjugated reagents together with RMCs capable of monitoring of reagent aggregation status can help ensure consistent, long-term performance of ADA methods.
Systematic trends in photonic reagent induced reactions in a homologous chemical family.
Tibbetts, Katharine Moore; Xing, Xi; Rabitz, Herschel
The growing use of ultrafast laser pulses to induce chemical reactions prompts consideration of these pulses as "photonic reagents" in analogy to chemical reagents. This work explores the prospect that photonic reagents may affect systematic trends in dissociative ionization reactions of a homologous family of halomethanes, much as systematic outcomes are often observed for reactions between homologous families of chemical reagents and chemical substrates. The experiments in this work with photonic reagents of varying pulse energy and linear spectral chirp reveal systematic correlations between observable ion yields and the following set of natural variables describing the substrate molecules: the ionization energy of the parent molecule, the appearance energy of each fragment ion, and the relative strength of carbon-halogen bonds in molecules containing two different halogens. The results suggest that reactions induced by photonic reagents exhibit systematic behavior analogous to that observed in reactions driven by chemical reagents, which provides a basis to consider empirical "rules" for predicting the outcomes of photonic reagent induced reactions.
Application of cyclic phosphonamide reagents in the total synthesis of natural products and biologically active molecules
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Thilo Focken
Full Text Available A review of the synthesis of natural products and bioactive compounds adopting phosphonamide anion technology is presented highlighting the utility of phosphonamide reagents in stereocontrolled bond-forming reactions. Methodologies utilizing phosphonamide anions in asymmetric alkylations, Michael additions, olefinations, and cyclopropanations will be summarized, as well as an overview of the synthesis of the employed phosphonamide reagents.
Fenton's Reagent was used to treat soil from a wood-treating site in southeastern Ohio which had been contaminated with creosote. Slurries, consisting of 10 µg of contaminated soil and 30 mL water were treated with 40 mL of Fenton's Reagent (1:1 of 30% H2O2 ...
Antisense-MDM2 Sensitizes LNCaP Prostate Cancer Cells to Androgen Deprivation, Radiation, and the Combination In Vivo
International Nuclear Information System (INIS)
Stoyanova, Radka; Hachem, Paul; Hensley, Harvey; Khor, L.-Y.; Mu Zhaomei; Hammond, M. Elizabeth H.; Agrawal, Sudhir; Pollack, Alan
Purpose: To test the effects of antisense (AS)-MDM2 alone and with androgen deprivation (AD), radiotherapy (RT), and AD + RT on wild-type LNCaP cells in an orthotopic in vivo model. Methods: Androgen-sensitive LNCaP cells were grown in the prostates of nude mice. Magnetic resonance imaging-based tumor volume and serum prostate-specific antigen (PSA) measurements were used to assess effects on tumor response. Tumor response was measured by biochemical and tumor volume failure definitions and doubling time estimates from fitted PSA and tumor volume growth curves. Expression of MDM2, p53, p21, and Ki-67 was quantified using immunohistochemical staining and image analysis of formalin-fixed tissue, analogous to methods used clinically. Results: Antisense-MDM2 significantly inhibited the growth of LNCaP tumors over the mismatch controls. The most significant increase in tumor growth delay and tumor doubling time was from AS-MDM2 + AD + RT, although the effect of AS-MDM2 + AD was substantial. Expression of MDM2 was significantly reduced by AS-MDM2 in the setting of RT. Conclusions: This is the first in vivo investigation of the effects of AS-MDM2 in an orthotopic model and the first to demonstrate incremental sensitization when added to AD and AD + RT. The results with AD underscore the potential to affect micrometastatic disease, which is probably responsible for treatment failure in 30-40% of men with high-risk disease
Quantitative Analysis of Survivin Protein Expression and Its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors
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Anna L Olsen
Full Text Available RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB of non-small cell lung cancer (NSCLC patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO (LY2181308, we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.
PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice
Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.
The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659
Long-term Exon Skipping Studies With 2′-O-Methyl Phosphorothioate Antisense Oligonucleotides in Dystrophic Mouse Models
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Christa L Tanganyika-de Winter
Full Text Available Antisense-mediated exon skipping for Duchenne muscular dystrophy (DMD is currently tested in phase 3 clinical trials. The aim of this approach is to modulate splicing by skipping a specific exon to reframe disrupted dystrophin transcripts, allowing the synthesis of a partly functional dystrophin protein. Studies in animal models allow detailed analysis of the pharmacokinetic and pharmacodynamic profile of antisense oligonucleotides (AONs. Here, we tested the safety and efficacy of subcutaneously administered 2′-O-methyl phosphorothioate AON at 200 mg/kg/week for up to 6 months in mouse models with varying levels of disease severity: mdx mice (mild phenotype and mdx mice with one utrophin allele (mdx/utrn+/−; more severe phenotype. Long-term treatment was well tolerated and exon skipping and dystrophin restoration confirmed for all animals. Notably, in the more severely affected mdx/utrn+/− mice the therapeutic effect was larger: creatine kinase (CK levels were more decreased and rotarod running time was more increased. This suggests that the mdx/utrn+/− model may be a more suitable model to test potential therapies than the regular mdx mouse. Our results also indicate that long-term subcutaneous treatment in dystrophic mouse models with these AONs is safe and beneficial.
REM sleep enhancement and behavioral cataplexy following orexin (hypocretin)-II receptor antisense perfusion in the pontine reticular formation.
Thakkar, M M; Ramesh, V; Cape, E G; Winston, S; Strecker, R E; McCarley, R W
Orexin (hypocretin)-containing neurons of the hypothalamus project to brainstem sites that are involved in the neural control of REM sleep, including the locus coeruleus, the dorsal raphe nucleus, the cholinergic zone of the mesopontine tegmentum, and the pontine reticular formation (PRF). Orexin knockout mice exhibit narcolepsy/cataplexy, and a mutant and defective gene for the orexin type II receptor is present in dogs with an inherited form of narcolepsy/cataplexy. However, the physiological systems mediating these effects have not been described. We reasoned that, since the effector neurons for the majority of REM sleep signs, including muscle atonia, were located in the PRF, this region was likely implicated in the production of these orexin-related abnormalities. To test this possibility, we used microdialysis perfusion of orexin type II receptor antisense in the PRF of rats. Ten to 24 hours after antisense perfusion, REM sleep increased two- to three-fold during both the light period (quiescent phase) and the dark period (active phase), and infrared video showed episodes of behavioral cataplexy. Moreover, preliminary data indicated no REM-related effects following perfusion with nonsense DNA, or when perfusion sites were outside the PRF. More work is needed to provide precise localization of the most effective site of orexin-induced inhibition of REM sleep phenomena.
In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts
Ling, King-Hwa; Brautigan, Peter J.; Moore, Sarah; Fraser, Rachel; Leong, Melody Pui-Yee; Leong, Jia-Wen; Zainal Abidin, Shahidee; Lee, Han-Chung; Cheah, Pike-See; Raison, Joy M.; Babic, Milena; Lee, Young Kyung; Daish, Tasman; Mattiske, Deidre M.; Mann, Jeffrey R.; Adelson, David L.; Thomas, Paul Q.; Hahn, Christopher N.; Scott, Hamish S.
SRY (Sex Determining Region Y)-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1], [2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH), Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR), gain-of-function and in situ hybridization (ISH) experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1. PMID:26958646
Data in support of transcriptional regulation and function of Fas-antisense long noncoding RNA during human erythropoiesis
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Olga Villamizar
Full Text Available This paper describes data related to a research article titled, "Fas-antisense long noncoding RNA is differentially expressed during maturation of human erythrocytes and confers resistance to Fas-mediated cell death" [1]. Long noncoding RNAs (lncRNAs are increasingly appreciated for their capacity to regulate many steps of gene expression. While recent studies suggest that many lncRNAs are functional, the scope of their actions throughout human biology is largely undefined including human red blood cell development (erythropoiesis. Here we include expression data for 82 lncRNAs during early, intermediate and late stages of human erythropoiesis using a commercial qPCR Array. From these data, we identified lncRNA Fas-antisense 1 (Fas-AS1 or Saf described in the research article. Also included are 5′ untranslated sequences (UTR for lncRNA Saf with transcription factor target sequences identified. Quantitative RT-PCR data demonstrate relative levels of critical erythroid transcription factors, GATA-1 and KLF1, in K562 human erythroleukemia cells and maturing erythroblasts derived from human CD34+ cells. End point and quantitative RT-PCR data for cDNA prepared using random hexamers versus oligo(dT18 revealed that lncRNA Saf is not effectively polyadenylated. Finally, we include flow cytometry histograms demonstrating Fas levels on maturing erythroblasts derived from human CD34+ cells transduced using mock conditions or with lentivirus particles encoding for Saf.
Intravenous administration of stabilized antisense lipid particles (SALP) leads to activation and expansion of liver natural killer cells.
Bramson, J L; Bodner, C A; Johnson, J; Semple, S; Hope, M J
Stabilized antisense lipid particles (SALP) have been developed for the systemic delivery of oligonucleotides. The impact of intravenous SALP administration was measured with respect to activation of natural killer (NK) and NK1.1+ T (NKT) cells in the livers of immunocompetent mice. Treatment with a SALP containing a highly mitogenic oligonucleotide (INX-6295) generated an increase in NK cytolytic activity and cell number within the liver but did not appear to affect the number of hepatic NKT cells or their cytolytic activity. The same results were observed after intravenous administration of the mitogenic oligonucleotide alone. Interestingly, treatment with a SALP containing a weakly mitogenic oligonucleotide (INX-6300) also activated the liver NK cells, whereas the oligonucleotide alone was unable to elicit these effects. The NK stimulatory activity of a SALP containing INX-6300 required both lipid and oligonucleotide components. These results demonstrate that in addition to modifying the pharmacokinetics and biodistribution of intravenously administered oligonucleotides, SALP possess immunostimulatory activity independent of oligonucleotide mitogenicity, which can serve as an adjuvant to antisense therapies for cancer.
In depth analysis of the Sox4 gene locus that consists of sense and natural antisense transcripts
Directory of Open Access Journals (Sweden)
King-Hwa Ling
Full Text Available SRY (Sex Determining Region Y-Box 4 or Sox4 is an important regulator of the pan-neuronal gene expression during post-mitotic cell differentiation within the mammalian brain. Sox4 gene locus has been previously characterized with multiple sense and overlapping natural antisense transcripts [1,2]. Here we provide accompanying data on various analyses performed and described in Ling et al. [2]. The data include a detail description of various features found at Sox4 gene locus, additional experimental data derived from RNA-Fluorescence in situ Hybridization (RNA-FISH, Western blotting, strand-specific reverse-transcription quantitative polymerase chain reaction (RT-qPCR, gain-of-function and in situ hybridization (ISH experiments. All the additional data provided here support the existence of an endogenous small interfering- or PIWI interacting-like small RNA known as Sox4_sir3, which origin was found within the overlapping region consisting of a sense and a natural antisense transcript known as Sox4ot1.
Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA
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Margot Martinez-Moreno
Full Text Available Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA. During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS biology.
Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity
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Beena eKadakkuzha
Full Text Available Despite the advances in our understanding of transcriptome, regulation and function of its noncoding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN, a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN has a key role in learning and long-term memory storage in Aplysia. We have identified NAT-SRN in the central nervous system (CNS and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduced levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.
Asymmetric localization of natural antisense RNA of neuropeptide sensorin in Aplysia sensory neurons during aging and activity.
Kadakkuzha, Beena M; Liu, Xin-An; Narvaez, Maria; Kaye, Alexandra; Akhmedov, Komolitdin; Puthanveettil, Sathyanarayanan V
Despite the advances in our understanding of transcriptome, regulation and function of its non-coding components continue to be poorly understood. Here we searched for natural antisense transcript for sensorin (NAT-SRN), a neuropeptide expressed in the presynaptic sensory neurons of gill-withdrawal reflex of the marine snail Aplysia californica. Sensorin (SRN) has a key role in learning and long-term memory storage in Aplysia. We have now identified NAT-SRN in the central nervous system (CNS) and have confirmed its expression by northern blotting and fluorescent RNA in situ hybridization. Quantitative analysis of NAT-SRN in micro-dissected cell bodies and processes of sensory neurons suggest that NAT-SRN is present in the distal neuronal processes along with sense transcripts. Importantly, aging is associated with reduction in levels of NAT-SRN in sensory neuron processes. Furthermore, we find that forskolin, an activator of CREB signaling, differentially alters the distribution of SRN and NAT-SRN. These studies reveal novel insights into physiological regulation of natural antisense RNAs.
Effect of antisense c-raf-1 on tumorigenicity and radiation sensitivity of a human squamous carcinoma
International Nuclear Information System (INIS)
Kasid, U.; Pfeifer, A.; Brennan, T.; Beckett, M.; Weichselbaum, R.R.; Dritschilo, A.; Mark, G.E.
Antisense RNA-mediated inhibition of gene expression was used to investigate the biological function of the c-raf-1 gene in a radiation-resistant human squamous carcinoma cell line, SQ-20B. S1 nuclease protection assays revealed that transfection of full-length raf complementary DNA in the antisense orientation (AS) leads to a specific reduction (greater than tenfold) of steady-state levels of the endogenous c-raf-1 sense (S) transcript in SQ-20B cells. In nude mice, the malignant potential of SQ-20B cells transfected with raf (S) was significantly increased relative to that of SQ-20B cells transfected with raf (AS). SQ-20B cells containing transfected raf (S) maintained a radiation-resistant phenotype as compared to those cells harboring the AS version, which appeared to have enhanced radiation sensitivity. These data indicate that the reduced expression of endogenous c-raf-1 is sufficient to modulate the tumorigenicity and the radiation-resistant phenotype of SQ-20B cells, thus implicating c-raf-1 in a pathway important to the genesis of this type of cancer
Antisense RNA Controls LRP1 Sense Transcript Expression through Interaction with a Chromatin-Associated Protein, HMGB2
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Yasunari Yamanaka
Full Text Available Long non-coding RNAs (lncRNAs, including natural antisense transcripts (NATs, are expressed more extensively than previously anticipated and have widespread roles in regulating gene expression. Nevertheless, the molecular mechanisms of action of the majority of NATs remain largely unknown. Here, we identify a NAT of low-density lipoprotein receptor-related protein 1 (Lrp1, referred to as Lrp1-AS, that negatively regulates Lrp1 expression. We show that Lrp1-AS directly binds to high-mobility group box 2 (Hmgb2 and inhibits the activity of Hmgb2 to enhance Srebp1a-dependent transcription of Lrp1. Short oligonucleotides targeting Lrp1-AS inhibit the interaction of antisense transcript and Hmgb2 protein and increase Lrp1 expression by enhancing Hmgb2 activity. Quantitative RT-PCR analysis of brain tissue samples from Alzheimer's disease patients and aged-matched controls revealed upregulation of LRP1-AS and downregulation of LRP1. Our data suggest a regulatory mechanism whereby a NAT interacts with a ubiquitous chromatin-associated protein to modulate its activity in a locus-specific fashion.
Hydrazine reagents as derivatizing agents in environmental analysis--a critical review.
Vogel, M; Büldt, A; Karst, U
Hydrazine reagents are a well-known group of derivatizing agents for the determination of aldehydes and ketones in liquid and gaseous samples. Within this article, the most important hydrazine reagents are critically summarized, and their major applications in different fields, including environmental analysis, food chemistry and industrial analysis are introduced. As 2,4-dinitrophenylhydrazine (DNPH) is the basic reagent for several international standard procedures, its properties are discussed in detail. Particular focus is directed on the chemistry of the hydrazine reagents, and chemical interferences are considered. Recent methods for the determination of various oxidants using hydrazine reagents are presented as well. Due to limited space, this review does not cover the related field of carbohydrate analysis, although many chemical aspects are similar.
A Catalyst-Free Amination of Functional Organolithium Reagents by Flow Chemistry.
Kim, Heejin; Yonekura, Yuya; Yoshida, Jun-Ichi
Reported is the electrophilic amination of functional organolithium intermediates with well-designed aminating reagents under mild reaction conditions using flow microreactors. The aminating reagents were optimized to achieve efficient C-N bond formation without using any catalyst. The electrophilic amination reactions of functionalized aryllithiums were successfully conducted under mild reaction conditions, within 1 minute, by using flow microreactors. The aminating reagent was also prepared by the flow method. Based on stopped-flow NMR analysis, the reaction time for the preparation of the aminating reagent was quickly optimized without the necessity of work-up. Integrated one-flow synthesis consisting of the generation of an aryllithium, the preparation of an aminating reagent, and their combined reaction was successfully achieved to give the desired amine within 5 minutes of total reaction time. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Adsorption of a novel reagent scheme on scheelite and calcite causing an effective flotation separation.
Gao, Yuesheng; Gao, Zhiyong; Sun, Wei; Yin, Zhigang; Wang, Jianjun; Hu, Yuehua
The efficient separation of scheelite from calcium-bearing minerals, especially calcite, remains a challenge in practice. In this work, a novel reagent scheme incorporating a depressant of sodium hexametaphosphate (SHMP) and a collector mixture of octyl hydroxamic acid (HXMA-8) and sodium oleate (NaOl) was employed in both single and mixed binary mineral flotation, and it proved to be highly effective for the separation. Furthermore, the role of the pH value in the separation was evaluated. Additionally, the mechanism of the selective separation was investigated systemically via zeta potential measurements, fourier transform infrared (FTIR) spectroscopy analysis, X-ray photoelectron (XPS) spectroscopy analysis and crystal chemistry calculations. It turns out that the selective chemisorption of SHMP on calcite (in the form of complexation between H 2 PO 4 - /HPO 4 2- and Ca 2+ ) over scheelite is ascribed to the stronger reactivity and higher density of Ca ions on the commonly exposed surfaces of calcite minerals. The intense adsorption of HXMA-8 on scheelite over calcite due to the match of the OO distances in WO 4 2- of scheelite and CONHOH of HXMA-8 holds the key to the successful separation. We were also interested in warranting the previous claim that NaOl is readily adsorbed on both minerals via chemisorption. Our results provided valuable insights into the application of mixed collectors and an effective depressant for flotation separation. Copyright © 2017 Elsevier Inc. All rights reserved.
Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells.
Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas
Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Development of IRMA reagent and methodology for PSA
International Nuclear Information System (INIS)
Najafi, R.
The PSA test is a solid phase two-site immunoassay. Rabbit anti PSA is coated or bound on surface of solid phase and monoclonal anti PSA labeled with 1-125. The PSA molecules present in the standard solution or serum are 'Sandwiched' between the two antibodies. After formation of coated antibody-antigen-labeled antibody complex, the unbound labeled antibody will removed by washing. The complex is measured by gamma counter. The concentration of analyte is proportional to the counts of test sample. In order to develop kits for IRMA PSA, it should be prepared three essential reagents Antibody coated solid phase, labeled antibody, standards and finally optimizing them to obtain an standard curve fit to measure specimen PSA in desired range of concentration. The type of solid phase and procedure(s) to coat or bind to antibody, is still main debatable subject in development and setting up RIA/IRMA kits. In our experiments, polystyrene beads, because of their easy to coat with antibody as well as easy to use, can be considered as a desired solid phase. Most antibodies are passively adsorbed to a plastic surface (e.g. Polystyrene, Propylene, and Polyvinyl chloride) from a diluted buffer. The antibody coated plastic surface, then acts as solid phase reagent. Poor efficiency and time required to reach equilibrium and also lack of reproducibility especially batch-to-batch variation between materials, are disadvantages in this simple coating procedure. Improvements can be made by coating second antibody on surface of beads, and reaction between second and primary antibodies. There is also possible to enhance more coating efficiency of beads by using Staphylococcus ureus-Protein A. Protein A is a major component of staphylococcus aureus cell wall which has an affinity for FC segment of immunoglobulin G (IgG) of some species, including human; rabbit; and mice. This property of Staphylococcal Protein A has made it a very useful tool in the purification of classes and subclasses
Enrichment of coal pulps by selective flocculation
Energy Technology Data Exchange (ETDEWEB)
Blaschke, Z
The results are presented of selective flocculation of coal pulps using different reagents. In some tests the coal particles were flocculated, and in others the coal remained in suspension and the dirt was flocculated. Selective flocculation makes it possible to obtain coal concentrates with a very low ash content from slurries with a high ash content. (In Polish)
Enrichment of coal pulps by selective flocculation
Energy Technology Data Exchange (ETDEWEB)
Blaschke, Z
The results are presented of selective flocculation of coal pulps using different reagents. In some tests the coal particles were flocculated, and in others the coal remained in suspension and the dirt was flocculated. Selective flocculation makes it possible to obtain coal concentrates with a very low ash content from slurries with a high ash content.
A Perspective on Reagent Diversity and Non-covalent Binding of Reactive Carbonyl Species (RCS and Effector Reagents in Non-enzymatic Glycation (NEG: Mechanistic Considerations and Implications for Future Research
Directory of Open Access Journals (Sweden)
Kenneth J. Rodnick
Full Text Available This perspective focuses on illustrating the underappreciated connections between reactive carbonyl species (RCS, initial binding in the nonenzymatic glycation (NEG process, and nonenzymatic covalent protein modification (here termed NECPM. While glucose is the central species involved in NEG, recent studies indicate that the initially-bound glucose species in the NEG of human hemoglobin (HbA and human serum albumin (HSA are non-RCS ring-closed isomers. The ring-opened glucose, an RCS structure that reacts in the NEG process, is most likely generated from previously-bound ring-closed isomers undergoing concerted acid/base reactions while bound to protein. The generation of the glucose RCS can involve concomitantly-bound physiological species (e.g., inorganic phosphate, water, etc.; here termed effector reagents. Extant NEG schemes do not account for these recent findings. In addition, effector reagent reactions with glucose in the serum and erythrocyte cytosol can generate RCS (e.g., glyoxal, glyceraldehyde, etc.. Recent research has shown that these RCS covalently modify proteins in vivo via NECPM mechanisms. A general scheme that reflects both the reagent and mechanistic diversity that can lead to NEG and NECPM is presented here. A perspective that accounts for the relationships between RCS, NEG, and NECPM can facilitate the understanding of site selectivity, may help explain overall glycation rates, and may have implications for the clinical assessment/control of diabetes mellitus. In view of this perspective, concentrations of ribose, fructose, Pi, bicarbonate, counter ions, and the resulting RCS generated within intracellular and extracellular compartments may be of importance and of clinical relevance. Future research is also proposed.
High throughput screening method for identification of new lipofection reagents.
Regelin, A E; Fernholz, E; Krug, H F; Massing, U
Lipofection, the transfer of genetic material into cells by means of cationic lipids, is of growing interest for in vitro and in vivo approaches. In order to identify ideal lipofection reagents in a HTS, we have developed an automated lipofection method for the transfer of reporter genes into cells and for determination of the lipofection results. The method has specifically been designed and optimized for 96-well microtiter plates and can successfully be carried out by a pipetting robot with accessory equipment. It consists of two separate parts: (1) pretransfection (preparation of liposomes, formation of lipoplexes, and lipoplex transfer to the cells) and (2) posttransfection (determination of the reporter enzyme activity and the protein content of the transfected cells). Individual steps of the lipofection method were specifically optimized - for example, lipoplex formation and incubation time as well as cell lysis, cell cultivating, and the reporter gene assay. The HTS method facilitates characterization of the transfection properties (efficiency and cytotoxicity) of large numbers of (cationic) lipids in various adherent cell types.
Quercetin as colorimetric reagent for determination of zirconium
Grimaldi, F.S.; White, C.E.
Methods described in the literature for the determination of zirconium are generally designed for relatively large amounts of this element. A good procedure using colorimetric reagent for the determination of trace amounts is desirable. Quercetin has been found to yield a sensitive color reaction with zirconium suitable for the determination of from 0.1 to 50?? of zirconium dioxide. The procedure developed involves the separation of zirconium from interfering elements by precipitation with p-dimethylaminoazophenylarsonic acid prior to its estimation with quercetin. The quercetin reaction is carried out in 0.5N hydrochloric acid solution. Under the operating conditions it is indicated that quercetin forms a 2 to 1 complex with zirconium; however, a 2 to 1 and a 1 to 1 complex can coexist under special conditions. Approximate values for the equilibrium constants of the complexes are K1 = 0.33 ?? 10-5 and K2 = 1.3 ?? 10-9. Seven Bureau of Standards samples of glass sands and refractories were analyzed with excellent results. The method described should find considerable application in the analysis of minerals and other materials for macro as well as micro amounts of zirconium.
Degradation of ion spent resin using the Fenton's reagent
International Nuclear Information System (INIS)
Araujo, Leandro Goulart de
The most common method for spent radioactive ion exchange resin treatment is its immobilization in cement, which reduces the radionuclides release into the environment. Although this method is efficient, it increases considerably the final volume of the waste due to the low incorporation capacity. The objective of this work was to develop a degradation method of spent resins arising from the nuclear research reactor located at the Nuclear and Energy Research Institute (IPEN-CNEN/SP), using an Advanced Oxidation Process (AOP) with Fenton's reagents. This method would allow a higher incorporation in cement. Three different resins were evaluated: cationic, anionic and a mixture of both resins. The reactions were conducted varying the catalyst concentration (25, 50, 100 and 150 mM), the volume of hydrogen peroxide (320 to 460 mL), and three different temperatures, 50, 60 and 70 deg C. Degradation of about 98% was achieved using a 50 mM catalyst solution and 330 mL of hydrogen peroxide solution. The most efficient temperature was 60 deg C. (author)
The NIH-NIAID Filariasis Research Reagent Resource Center.
Directory of Open Access Journals (Sweden)
Michelle L Michalski
Full Text Available Filarial worms cause a variety of tropical diseases in humans; however, they are difficult to study because they have complex life cycles that require arthropod intermediate hosts and mammalian definitive hosts. Research efforts in industrialized countries are further complicated by the fact that some filarial nematodes that cause disease in humans are restricted in host specificity to humans alone. This potentially makes the commitment to research difficult, expensive, and restrictive. Over 40 years ago, the United States National Institutes of Health-National Institute of Allergy and Infectious Diseases (NIH-NIAID established a resource from which investigators could obtain various filarial parasite species and life cycle stages without having to expend the effort and funds necessary to maintain the entire life cycles in their own laboratories. This centralized resource (The Filariasis Research Reagent Resource Center, or FR3 translated into cost savings to both NIH-NIAID and to principal investigators by freeing up personnel costs on grants and allowing investigators to divert more funds to targeted research goals. Many investigators, especially those new to the field of tropical medicine, are unaware of the scope of materials and support provided by the FR3. This review is intended to provide a short history of the contract, brief descriptions of the fiilarial species and molecular resources provided, and an estimate of the impact the resource has had on the research community, and describes some new additions and potential benefits the resource center might have for the ever-changing research interests of investigators.
Evaluation of the resin oxidation process using Fenton's reagent
International Nuclear Information System (INIS)
Araujo, Leandro G.; Goes, Marcos M.; Marumo, Julio T.
The ion exchange resin is considered radioactive waste after its final useful life in nuclear reactors. Usually, this type of waste is treated with the immobilization in cement Portland, in order to form a solid monolithic matrix, reducing the possibility of radionuclides release in to environment. Because of the characteristic of expansion and contraction of the resins in presence of water, its incorporation in the common Portland cement is limited in 10% in direct immobilization, causing high costs in the final product. A pre-treatment would be able to reduce the volume, degrading the resins and increasing the load capacity of this material. This paper is about a method of degradation of ion spent resins from the nuclear research reactor of Nuclear and Energy Research Institute (IPEN/CNEN-SP), Brazil, using the Fenton's reagent. The resin evaluated was a mixture of cationic and anionic resins. The reactions were conducted by varying the concentration of the catalyst (25 to 80 mM), with and without external heat. The time of reaction was two hours. The concentration of 50 mM of catalyst was the most effective in degrading approximately 99%. The resin degradation was confirmed by the presence of CaCO 3 as a white precipitate resulting from the reaction between the Ca(OH) 2 and the CO 2 from the resin degradation. It was possible to degrade the resins without external heating. The calcium carbonates showed no correlation with the residual resin mass. (author)
Deep soil mixing for reagent delivery and contaminant treatment
International Nuclear Information System (INIS)
Korte, N.; Gardner, F.G.; Cline, S.R.; West, O.R.
Deep soil mixing was evaluated for treating clay soils contaminated with TCE and its byproducts at the Department of Energy's Kansas City Plant. The objective of the project was to evaluate the extent of limitations posed by the stiff, silty-clay soil. Three treatment approaches were tested. The first was vapor stripping. In contrast to previous work, however, laboratory treatability studies indicated that mixing saturated, clay soil was not efficient unless powdered lime was added. Thus, powder injection of lime was attempted in conjunction with the mixing/stripping operation. In separate treatment cells, potassium permanganate solution was mixed with the soil as a means of destroying contaminants in situ. Finally, microbial treatment was studied in a third treatment zone. The clay soil caused operational problems such as breakage of the shroud seal and frequent reagent blowouts. Nevertheless, treatment efficiencies of more than 70% were achieved in the saturated zone with chemical oxidation. Although expensive ($1128/yd 3 ), there are few alternatives for soils of this type
Anti-sense suppression of epidermal growth factor receptor expression alters cellular proliferation, cell-adhesion and tumorigenicity in ovarian cancer cells.
Alper, O; De Santis, M L; Stromberg, K; Hacker, N F; Cho-Chung, Y S; Salomon, D S
Over-expression of epidermal growth factor receptor (EGFR) in ovarian cancer has been well documented. Human NIH:OVCAR-8 ovarian carcinoma cells were transfected with an expression vector containing the anti-sense orientation of truncated human EGFR cDNA. EGFR anti-sense over-expression resulted in decreased EGFR protein and mRNA expression, cell proliferation and tumor formation in nude mice. In accordance with the reduced levels of EGFR in EGFR anti-sense-expressing cells, tyrosine phosphorylation of EGFR was decreased compared to untransfected parental cells treated with EGF. In EGFR anti-sense-transfected cells, expression of erbB-3, but not erbB-2, was increased. In addition, basal and heregulin-beta 1-stimulated tyrosine phosphorylation of erbB-3 was higher in EGFR anti-sense vector-transfected cells. A morphological alteration in EGFR anti-sense gene-expressing cells was correlated with a decrease in the expression of E-cadherin, alpha-catenin and, to a lesser extent, beta-catenin. Changes in the expression of these proteins were associated with a reduction in complex formation among E-cadherin, beta-catenin and alpha-catenin and between beta-catenin and EGFR in EGFR anti-sense-expressing cells compared to sense-transfected control cells. These results demonstrate that EGFR expression in ovarian carcinoma cells regulates expression of cell adhesion proteins that may enhance cell growth and invasiveness. Copyright 2000 Wiley-Liss, Inc.
Plant 7SL RNA and tRNA(Tyr) genes with inserted antisense sequences are efficiently expressed in an in vitro transcription system from Nicotiana tabacum cells
Czech Academy of Sciences Publication Activity Database
Yukawa, Y.; Matoušek, Jaroslav; Grimm, M.; Vrba, Lukáš; Steger, G.; Sugiura, M.; Beier, H.
Roč. 50, - (2002), s. 713-723 ISSN 0167-4412 R&D Projects: GA ČR GA521/99/1591; GA MŠk ME 463 Keywords : antisense RNA Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.529, year: 2002
Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study
International Nuclear Information System (INIS)
Kiani, Melika; Mirzazadeh Tekie, Farnaz Sadat; Dinarvand, Meshkat; Soleimani, Masoud; Dinarvand, Rassoul; Atyabi, Fatemeh
Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~ 115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. - Highlights: • Formation of stable nanoparticle with dextran and chitosan derivatives for oral and intravenous gene delivery. • Satifactory cellular uptake of nanoparticles and approximately complete suppression of hSET1 expression in SW480 cell lines • Prolonged stability of nanoparticles against biorelevent media with desirable release rate.
Preliminary studies on gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats
International Nuclear Information System (INIS)
Liu Chunjie; Wang Dewen; Zhang Zhaoshan; Gao Yabing; Xiong Chengqi; Long Jianyin; Wang Huixin; Peng Ruiyun; Cui Xuemei
Objective: To observed the efficiency of gene therapy with TGF β1 antisense gene/liposome complexes and adenovirus transfer vector in RPF rats. Methods: TGFβ1 sense and antisense gene expression vectors and adenovirus transfer vector were introduced into rat bronchus by way of intratracheal instillation. Results: At day 1.5 after TGFβ1 sense and antisense gene transfer, PCR amplification using neo gene-specific primer from lung tissue DNA was all positive. After day 5.5, 67% (2/3) of lung tissue DNA was positive. RNA dot blot hybridization indicated that TGFβ1 mRNA content of lung tissue transfected with pMAMneo-antiTGFβ1 gene decreased. Detection of lung hydroxyproline (Hyp) content after day 35 of gene transfer showed that even in lung of rats received pMAMneo-AntiTGFβ1 lipid complexes it raised remarkably (P 9 pfu/ml were instilled into bronchus at 0.5 ml per rat. After day 2 day 6, the lung tissues of all six rats (three per each group )expressed the transfected luciferase gene by luminometer. Conclusion: Cationic lipid-mediated TGFβ1 antisense gene therapy was a simple and easy method. It can slow down the course of pathogenesis of lung fibrosis. Replication-deficient recombinant adenovirus-mediated gene therapy of lung diseases is a good and efficient method
Thiolated carboxymethyl dextran as a nanocarrier for colon delivery of hSET1 antisense: In vitro stability and efficiency study
Energy Technology Data Exchange (ETDEWEB)
Kiani, Melika, E-mail: email@example.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Mirzazadeh Tekie, Farnaz Sadat, E-mail: firstname.lastname@example.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Dinarvand, Meshkat, E-mail: email@example.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Soleimani, Masoud, E-mail: email@example.com [Stem Cell Technology Research Centre, P.O. Box 14155-3174, Tehran (Iran, Islamic Republic of); Department of Hematology, School of Medical Sciences, Tarbiat Modares University, P.O. Box: 14115-111, Tehran (Iran, Islamic Republic of); Dinarvand, Rassoul, E-mail: email@example.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Atyabi, Fatemeh, E-mail: firstname.lastname@example.com [Department of Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, P.O. Box 14155-6451, Tehran (Iran, Islamic Republic of); Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)
Gene therapy is an optimistic approach in cancer treatment. However, for efficient delivery of gene materials, designing an appropriate vector is necessary. Polyelectrolyte complexes (PECs) of chitosan and dextran could be considered a proper nanoparticulate carrier for sensitive biomaterials. In this study, PECs of chitosan and thiolated dextran were used as either an injectable or oral gene delivery system. hSET1 antisense was loaded into the PECs to suppress proliferation of colon cancer cell line. The prepared nanoparticles have ~ 115 nm diameter size and positive zeta potential with high mucoadhesion properties. They are able to protect antisense from degradation in serum and biorelevant fluids (FaSSIF and FaSSGF). Furthermore, prepared nanoparticles demonstrated superior cellular penetration and inhibitory effect on SW480 colon cancer cell proliferation. All nanoparticles significantly down regulated hSET1 in comparison with naked antisense. It can be concluded that thiolated PECs have potential use for injectable or oral delivery of nucleic acids such as antisense. - Highlights: • Formation of stable nanoparticle with dextran and chitosan derivatives for oral and intravenous gene delivery. • Satifactory cellular uptake of nanoparticles and approximately complete suppression of hSET1 expression in SW480 cell lines • Prolonged stability of nanoparticles against biorelevent media with desirable release rate.
Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene
Directory of Open Access Journals (Sweden)
Shumei Liang
Conclusion: Our results demonstrate that the potent effects of PNAs on bacterial growth and cell viability were mediated by the down-regulation or even knock-out of ftsZ gene expression. This highlights the utility of ftsZ as a promising target for the development of new antisense antibacterial agents to treat MRSA infections.
Oxacillin sensitization of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius by antisense peptide nucleic acids in vitro.
Goh, Shan; Loeffler, Anette; Lloyd, David H; Nair, Sean P; Good, Liam
Antibiotic resistance genes can be targeted by antisense agents, which can reduce their expression and thus restore cellular susceptibility to existing antibiotics. Antisense inhibitors can be gene and pathogen specific, or designed to inhibit a group of bacteria having conserved sequences within resistance genes. Here, we aimed to develop antisense peptide nucleic acids (PNAs) that could be used to effectively restore susceptibility to β-lactams in methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP). Antisense PNAs specific for conserved regions of the mobilisable gene mecA, and the growth essential gene, ftsZ, were designed. Clinical MRSA and MRSP strains of high oxacillin resistance were treated with PNAs and assayed for reduction in colony forming units on oxacillin plates, reduction in target gene mRNA levels, and cell size. Anti-mecA PNA at 7.5 and 2.5 μM reduced mecA mRNA in MRSA and MRSP (p resistance in staphylococci. Further studies are warranted as clinical treatment alternatives are needed.
Feasibility of SPECT-CT imaging to study the pharmacokinetics of antisense oligonucleotides in a mouse model of Duchenne muscular dystrophy
Steeg, E. van de; Läppchen, T.; Aguilera, B.; Jansen, H.T.; Muilwijk, D.; Vermue, R.; Hoorn, J.W. van der; Donato, K.; Rossin, R.; Visser, P.C. de; Vlaming, M.L.H.
Antisense oligonucleotides (AONs) are promising candidates for treatment of Duchenne muscular dystrophy (DMD), a severe and progressive disease resulting in premature death. However, more knowledge on the pharmacokinetics of new AON drug candidates is desired for effective application in the clinic.
Determination of dopamine in presence of ascorbic acid and uric acid using poly (Spands Reagent) modified carbon paste electrode
Energy Technology Data Exchange (ETDEWEB)
Veera Manohara Reddy, Y.; Prabhakara Rao, V.; Vijaya Bhaskar Reddy, A.; Lavanya, M.; Venu, M.; Lavanya, M.; Madhavi, G., E-mail: email@example.com
In this paper, we have fabricated a modified carbon paste electrode (CPE) by electropolymerisation of spands reagent (SR) onto surface of CPE using cyclic voltammetry (CV). The developed electrode was abbreviated as poly(SR)/CPE and the surface morphology of the modified electrode was studied by using scanning electron microscopy (SEM). The developed electrode showed higher electrocatalytic properties towards the detection of dopamine (DA) in 0.1 M phosphate buffer solution (PBS) at pH 7.0. The effect of pH, scan rate, accumulation time and concentration of dopamine was studied at poly(SR)/CPE. The poly(SR)/CPE was successfully used as a sensor for the selective determination of DA in presence of ascorbic acid (AA) and uric acid (UA) without any interference. The poly(SR)/CPE showed a good detection limit of 0.7 μM over the linear dynamic range of 1.6 μM to 16 μM, which is extremely lower than the reported methods. The prepared poly(SR)/CPE exhibited good stability, high sensitivity, better reproducibility, low detection limit towards the determination of DA. The developed method was also applied for the determination of DA in real samples. - Highlights: • Electropolymerization of spands reagent was fabricated by cyclic voltammetry • The Poly (spands reagent) electrode shows excellent electrocatalytic activity for the detection of dopamine. • The detection limit for dopamine was found to be 0.7 μM. • The proposed method can be applied for DA in injection and human blood serum samples.
Ir/Sn dual-reagent catalysis towards highly selective alkylation of ...
Indian Academy of Sciences (India)
Organometallic; bimetallic; catalysis; alkylation; benzyl alcohol; iridium, tin. 1. Introduction ... cording to our proposal, the oxidative addition of tin(IV) halides across a ..... 33. 4. Conclusion. In summary, we have demonstrated here an Ir/Sn.
Irreversible Cysteine-Selective Protein Labeling Employing Modular Electrophilic Tetrafluoroethylation Reagents
Czech Academy of Sciences Publication Activity Database
Václavík, Jiří; Zschoche, R.; Klimánková, Iveta; Matoušek, V.; Beier, Petr; Hilvert, D.; Togni, A.
Roč. 23, č. 27 (2017), s. 6490-6494 ISSN 0947-6539 R&D Projects: GA ČR(CZ) GA17-00598S Institutional support: RVO:61388963 Keywords : bioconjugation * cysteine * enzymes * fluorine * fluoroalkylation * hypervalent iodine Subject RIV: CC - Organic Chemistry OBOR OECD: Organic chemistry Impact factor: 5.317, year: 2016
A new ecologically friendly process for the synthesis of selective flotation reagents
Directory of Open Access Journals (Sweden)
Aleksandar D. Marinković
Full Text Available A new ecologically friendly synthesis of N-alkyl-O-ethylthioncarbamates from ethyl dixanthogenates, waste material from the xanthate production plant, and monoalkylamines has been performed in this work. Structure determination of the synthesized compounds and also corresponding intermediates has been performed by IR, 1H-NMR and MS spectrometric methods. Residual dixanthogenates and thioncarbamates in waste water have been done by the gas chromatographic method and corresponding standard curve. It was confirmed that the reaction product was not present in water, while the concentration of dixanthogenates has been determined to be under the maximum contamination limit.
Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
Directory of Open Access Journals (Sweden)
Helena Sork
Full Text Available The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
Stability study for magnetic reagent assaying Hb and HbA1c
Energy Technology Data Exchange (ETDEWEB)
Hsieh, Wen-Pin [Actherm Inc., Hsinchu 200, Taiwan (China); Chieh, J.J.; Yang, C.C. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Yang, S.Y. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); MagQu Co., Ltd., Sindian Dist., New Taipei City 231, Taiwan (China); Chen, Po-Yu; Huang, Yu-Hao [Actherm Inc., Hsinchu 200, Taiwan (China); Hong, Y.W. [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China); Horng, H.E., E-mail: email@example.com [Institute of Electro-optical Science and Technology, National Taiwan Normal University, Taipei 116, Taiwan (China)
Reagents for magnetically labeled immunoassay on human Hb and human HbA1c have been synthesized. The reagents consist of Fe{sub 3}O{sub 4} magnetic particles biofunctionalized with antibodies against Hb and HbA1c. It has been demonstrated that the reagents can be applied to quantitatively detect Hb and HbA1c by using immunomagnetic reduction assay. In addition to characterizing the assay properties, such as the standard curve and the low-detection limit, the stability of reagents is investigated. To do this, the temporal dependence of particle sizes and the bio-activity of reagents are monitored. The results show that the reagents are highly stable when stored at 2-8 Degree-Sign C. This means that the reagents synthesized in this work are promising for practical applications. - Highlights: Black-Right-Pointing-Pointer The properties of assaying Hb and HbA1c using immunomagnetic reduction are studied. Black-Right-Pointing-Pointer The magnetic nanoparticles with antibodies are highly stable in solutions. Black-Right-Pointing-Pointer No significant mutual interference between Hb and HbA1c in assays is observed. Black-Right-Pointing-Pointer High-sensitivity assays on Hb and HbA1c using immunomagnetic reduction are achieved.
Stability study for magnetic reagent assaying Hb and HbA1c
International Nuclear Information System (INIS)
Hsieh, Wen-Pin; Chieh, J.J.; Yang, C.C.; Yang, S.Y.; Chen, Po-Yu; Huang, Yu-Hao; Hong, Y.W.; Horng, H.E.
Reagents for magnetically labeled immunoassay on human Hb and human HbA1c have been synthesized. The reagents consist of Fe 3 O 4 magnetic particles biofunctionalized with antibodies against Hb and HbA1c. It has been demonstrated that the reagents can be applied to quantitatively detect Hb and HbA1c by using immunomagnetic reduction assay. In addition to characterizing the assay properties, such as the standard curve and the low-detection limit, the stability of reagents is investigated. To do this, the temporal dependence of particle sizes and the bio-activity of reagents are monitored. The results show that the reagents are highly stable when stored at 2–8 °C. This means that the reagents synthesized in this work are promising for practical applications. - Highlights: ► The properties of assaying Hb and HbA1c using immunomagnetic reduction are studied. ► The magnetic nanoparticles with antibodies are highly stable in solutions. ► No significant mutual interference between Hb and HbA1c in assays is observed. ► High-sensitivity assays on Hb and HbA1c using immunomagnetic reduction are achieved.
[Research of regional medical consumables reagent logistics system in the modern hospital].
Wu, Jingjiong; Zhang, Yanwen; Luo, Xiaochen; Zhang, Qing; Zhu, Jianxin
To explore the modern hospital and regional medical consumable reagents logistics system management. The characteristics of regional logistics, through cooperation between medical institutions within the region, and organize a wide range of special logistics activities, to make reasonable of the regional medical consumable reagents logistics. To set the regional management system, dynamic management systems, supply chain information management system, after-sales service system and assessment system. By the research of existing medical market and medical resources, to establish the regional medical supplies reagents directory and the initial data. The emphasis is centralized dispatch of medical supplies reagents, to introduce qualified logistics company for dispatching, to improve the modern hospital management efficiency, to costs down. Regional medical center and regional community health service centers constitute a regional logistics network, the introduction of medical consumable reagents logistics services, fully embodies integrity level, relevance, purpose, environmental adaptability of characteristics by the medical consumable reagents regional logistics distribution. Modern logistics distribution systems can increase the area of medical consumables reagent management efficiency and reduce costs.
Strand-specific RNA-seq reveals widespread occurrence of novel cis-natural antisense transcripts in rice
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Lu Tingting
Full Text Available Abstract Background Cis-natural antisense transcripts (cis-NATs are RNAs transcribed from the antisense strand of a gene locus, and are complementary to the RNA transcribed from the sense strand. Common techniques including microarray approach and analysis of transcriptome databases are the major ways to globally identify cis-NATs in various eukaryotic organisms. Genome-wide in silico analysis has identified a large number of cis-NATs that may generate endogenous short interfering RNAs (nat-siRNAs, which participate in important biogenesis mechanisms for transcriptional and post-transcriptional regulation in rice. However, the transcriptomes are yet to be deeply sequenced to comprehensively investigate cis-NATs. Results We applied high-throughput strand-specific complementary DNA sequencing technology (ssRNA-seq to deeply sequence mRNA for assessing sense and antisense transcripts that were derived under salt, drought and cold stresses, and normal conditions, in the model plant rice (Oryza sativa. Combined with RAP-DB genome annotation (the Rice Annotation Project Database build-5 data set, 76,013 transcripts corresponding to 45,844 unique gene loci were assembled, in which 4873 gene loci were newly identified. Of 3819 putative rice cis-NATs, 2292 were detected as expressed and giving rise to small RNAs from their overlapping regions through integrated analysis of ssRNA-seq data and small RNA data. Among them, 503 cis-NATs seemed to be associated with specific conditions. The deep sequence data from isolated epidermal cells of rice seedlings further showed that 54.0% of cis-NATs were expressed simultaneously in a population of homogenous cells. Nearly 9.7% of rice transcripts were involved in one-to-one or many-to-many cis-NATs formation. Furthermore, only 17.4-34.7% of 223 many-to-many cis-NAT groups were all expressed and generated nat-siRNAs, indicating that only some cis-NAT groups may be involved in complex regulatory networks. Conclusions
Efficacy comparative of different laboratory test reagents for hepatitis C virus antibody
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GUO Feibo
Full Text Available Objective To investigate the effects of different laboratory test reagents for hepatitis C virus (HCV antibody through a comparative analysis. Methods A total of 207 samples which tested positive by four anti-HCV screening reagents commonly used in the laboratories in China (Kehua, Xinchuang, Wantai, and Abbott were included. HCV RNA nucleic acid amplification (NAT was performed, and if NAT results were negative, recombinant immunoblot assay (RIBA was performed for further confirmation. The test results of these four screening reagents were compared, and their S/CO values and true positive rates were analyzed. Results Of all the 205 samples testing positive by any one reagent, 191 (93.2% tested positive by the four reagents, and 14 (6.8% were tested inconsistently by the four reagents. The positive predictive values of Xinchuang, Kehua, Wantai, and Abbott reagents were 88.2% (180/204, 93.8% (180/192, 91.4% (180/197, and 90.0% (180/200, respectively. The S/CO thresholds with a positive predictive value of ≥95% for Xinchuang, Kehua, Wantai, and Abbott reagents were 9.0, 4.0, 5.0, and 7.0, respectively. Conclusion Xinchuang, Kehua, Wantai, and Abbott reagents have significantly different S/CO thresholds with a positive predictive value of ≥95%, which are significantly different from those in other domestic laboratories. Each laboratory should establish an applicable S/CO threshold with a positive predictive value of ≥95%, in order to reduce the sample size for confirmatory test.
Degradation of ion spent resin using the Fenton's reagent; Degradacao da resina de troca ionica utilizando o reagente de Fenton
Energy Technology Data Exchange (ETDEWEB)
Araujo, Leandro Goulart de
The most common method for spent radioactive ion exchange resin treatment is its immobilization in cement, which reduces the radionuclides release into the environment. Although this method is efficient, it increases considerably the final volume of the waste due to the low incorporation capacity. The objective of this work was to develop a degradation method of spent resins arising from the nuclear research reactor located at the Nuclear and Energy Research Institute (IPEN-CNEN/SP), using an Advanced Oxidation Process (AOP) with Fenton's reagents. This method would allow a higher incorporation in cement. Three different resins were evaluated: cationic, anionic and a mixture of both resins. The reactions were conducted varying the catalyst concentration (25, 50, 100 and 150 mM), the volume of hydrogen peroxide (320 to 460 mL), and three different temperatures, 50, 60 and 70 deg C. Degradation of about 98% was achieved using a 50 mM catalyst solution and 330 mL of hydrogen peroxide solution. The most efficient temperature was 60 deg C. (author)
Influence of different chelators (HYNIC, MAG3 and DTPA) on tumor cell accumulation and mouse biodistribution of technetium-99m labeled to antisense DNA
International Nuclear Information System (INIS)
Zhang, Y.M.; Liu, N.; Zhu, Z.-H.; Rusckowski, M.; Hnatowich, D.J.
We have shown recently that cell accumulation in culture of antisense DNA is strongly influenced by the presence of a 99m Tc-MAG 3 group for radiolabeling. We have now compared the in vitro and mouse in vivo behavior of 99m Tc when radiolabeled to one antisense phosphorothioate DNA by three different methods. The 18-mer antisense DNA against the RIα subunit of PKA was conjugated via a primary amine on the 5'-end with the NHS esters of HYNIC and MAG 3 and by the cyclic anhydride of DTPA. Surface plasmon resonance measurements revealed that the association rate constant for hybridization was unchanged for all three chelators as compared with that of the native DNA. Size exclusion HPLC showed rapid and quantitative protein binding for all three chelators upon incubation of labeled DNAs in 37 C serum and cell culture medium. However, in each case, radiolabeled and intact oligonucleotide was still detectable after 24 h. Cellular uptake was tested in an RIα mRNA-positive cancer cell line. The order of cellular accumulation of 99m Tc was DTPA>HYNIC(tricine)>MAG 3 , with the differences increasing with time between 4 and 24 h. The rate of 99m Tc egress from cells was found to be MAG 3 >HYNIC>DTPA, which may explain the order of cellular accumulation. The biodistribution in normal mice was heavily influenced by the labeling method and followed a pattern similar to that seen previously by us for peptides labeled with the same chelators. In conclusion, although these studies concerned only one antisense DNA in one cell line, the results suggest that the success of antisense imaging may depend, in part, on the method of radiolabeling. (orig.)
Physicochemical and biological properties of self-assembled antisense/poly(amidoamine dendrimer nanoparticles: the effect of dendrimer generation and charge ratio
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Alireza Nomani
Full Text Available Alireza Nomani1,6, Ismaeil Haririan1,5, Ramin Rahimnia2,4, Shamileh Fouladdel2, Tarane Gazori1, Rassoul Dinarvand1, Yadollah Omidi3, Ebrahim Azizi2,41Department of Pharmaceutics, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 2Molecular Research Lab, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 3Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, Iran; 4Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 5Biomaterials Research Center (BRC Tehran, Iran; 6Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, IranAbstract: To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine dendrimer (PAMAM dendrimer and a short-stranded DNA (antisense oligonucleotide, multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS; zeta potential measurement; and atomic force microscopy (AFM. PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller molecules produce more heterodisperse and large nanoparticles when they are condensed with a cationic dendrimer. AFM images also showed that such nanoparticles were spherical. The stability of the antisense content of the nanoparticles was investigated over different charge ratios using polyacrylamide gel electrophoresis. It was clear from such analyses that much more than charge neutrality point was required to obtain stable nanoparticles. For cell uptake, self-assembled nanoparticles were prepared with PAMAM G5 and 5'-FITC labeled antisense and the uptake experiment was carried out in T47D cell culture. This investigation also shows that the cytotoxicity of the nanoparticles was
Reagent precipitation of copper ions from wastewater of machine-building factories
Porozhnyuk, L. A.; Lupandina, N. S.; Porozhnyuk, E. V.
The article presents the results of reagent removal of copper ions from wastewater of machine-building factories. The urgency of the study is conditioned by the widening of the range of effective reagents through the implementation of industrial waste. The investigation covers mineralogical and fractional composition of chalk enrichment waste. In the work, the conditions of thermal activation of chalk enrichment waste used for reagent removal of copper ions from wastewater were elaborated. It was shown that the thermal activation of waste facilitates the increased treatment efficacy up to the set sanitation, hygiene and technological standards.
Quality control of radioimmunoassay reagents for T4
International Nuclear Information System (INIS)
Wayan Radiatning, S.
Quality control of radioimmunoassay reagents for T4. A program of quality control testing has been carried out for 125 I-T4, T4 standards and T4 antisera. 125 I-labelled T4 has been tested for its specific activity, radiochemical purity using a Sephadex G-25 column, immunological activity, based on the immunological reaction between labelled antigen and excess T4 antibody, and non-specific binding. The useful shelf-life of the labelled compound was determined by monitoring the decrease in radiochemical purity and immunological activity, and the increase in non-specific binding. T4 standards were calibrated by means of T4 RIA kit manufactured by DPC (Diagnostic Products Corporation). A test on parallelism was also performed using hyperthyroid sera. T4 antisera were evaluated with respect to titre, avidity and specifity. The test results on 125 I-T4 show a specific activity varying between 1830-2020 uCi/ug, a radiochemical purity above 90%, an immunological more than 80% and a non-specific binding of less than 5%. The standard curve for T4 was found to coincide well with the standard curve of the DPC kit and parallel with the curve for hyperthyroid sera. The titre of T4 antisera obtained was 1:300, the avidity was about 4.8 x 10 7 and the cross-reaction for T3 was 1.6%. It can be concluded from the experimental results, that the 125 I-T4, T4 standards and T4 antisera prepared meet the requirements for the manufacture of T4 kits. (author). 5 refs.; 14 figs
The antisense expression of AhPEPC1 increases seed oil production in peanuts (Arachis hypogaea L.)
Energy Technology Data Exchange (ETDEWEB)
Pan, L.; Zhang, J.; Chi, X.; Chen, N.; Chen, M.; Wang, M.; Wang, T.; Yang, Z.; Zhang, Z.; Wan, Y.; Yu, S.; Liu, F.
Although phosphoenolpyruvate carboxylases (PEPCs) are reported to be involved in fatty acid accumulation, nitrogen assimilation, and salt and drought stresses, knowledge regarding PEPC gene functions is still limited, particularly in peanuts (Arachis hypogaea L.). In this study, the antisense expression of the peanut PEPC isoform 1 (AhPEPC1) gene increased the lipid content by 5.7%–10.3%. This indicated that AhPEPC1 might be related to plant lipid accumulation. The transgenic plants underwent more root elongation than the wild-type under salinity stress. Additionally, the specific down regulation of the AhPEPC1 gene improved the salt tolerance in peanuts. This is the first report on the role of PEPC in lipid accumulation and salt tolerance in peanuts.
Effects of Antisense Oligonucleotides against C-Reactive Protein on the Development of Atherosclerosis in WHHL Rabbits
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Qi Yu
Full Text Available Increased plasma levels of C-reactive protein (CRP are closely associated with cardiovascular diseases, but whether CRP is directly involved in the pathogenesis of atherosclerosis is still under debate. Many controversial and contradictory results using transgenic mice and rabbits have been published but it is also unclear whether CRP lowering can be used for the treatment of atherosclerosis. In the current study, we examined the effects of the rabbit CRP antisense oligonucleotides (ASO on the development of atherosclerosis in WHHL rabbits. CRP ASO treatment led to a significant reduction of plasma CRP levels; however, both aortic and coronary atherosclerotic lesions were not significantly changed compared to those of control WHHL rabbits. These results suggest that inhibition of plasma CRP does not affect the development of atherosclerosis in WHHL rabbits.
Down-regulation of Survivin by Antisense Oligonucleotides Increases Apoptosis, Inhibits Cytokinesis and Anchorage-Independent Growth
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Jun Chen
Full Text Available Survivin, a member of the inhibitor of apoptosis protein (IAP family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.
Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain
DEFF Research Database (Denmark)
Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir
Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway...... for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...... with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates...
Formulation and drug-content assay of microencapsulated antisense oligonucleotide to NF-κB using ATR-FTIR
International Nuclear Information System (INIS)
Siwale, Rodney; Meadows, Fred; Mody, Vicky V; Shah, Samit
Antisense oligonucleotide to NF-κB sequence: 5′-GGA AAC ACA TCC TCC ATG-3′, was microencapsulated in an albumin matrix by the method of spray drying TM . Spectral analysis was performed on varying drug loading formulations of both drugs by mid-IR attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR). An out of plane O–H bending vibration at 948 cm −1 , unique to both the native and microencapsulated drugs was identified. The calculated peak areas corresponded to the drug loadings in the microsphere formulations. A standard curve could then be used to determine the drug content of an unknown microsphere formulation. Accuracy and precision were determined to be comparable to other analytical techniques such as HPLC. (paper)
Some aspects of surfactant action mechanism in the organic reagents - metal ions systems
Energy Technology Data Exchange (ETDEWEB)
Chernova, R K; Shtykov, S N; Beloliptseva, G M; Sukhova, L K; Amelin, V G; Kulapina, E G [Saratovskij Gosudarstvennyj Univ. (USSR)
Results are reviewed of investigations into the interaction of ions of Mo(6), W, Zr, Be, Sc, Nb, Ta, J, rare earths, a. o. with organic reagents of triphenylmethane class in the 8M H/sub 2/SO/sub 4/-pH14 acidity range and the 1x10/sup -3/-5x10/sup -6/ M concentration range both in the presence and absence of different surfactant type (cetylpyridine, methyltrimethylammonium, synthanols, etc). Three types of effects, determining enhancement of the sensitivity and selectivity of reactions jn the Me-R-surfactant systems, were determined: an increase in the number of coordinated ligands, the activating effect of cation surfactants resulting in a potential complexing in acid media, multicenter interaction of polydentate ligands both via chelating groups and auxochrome groups in the presence of cation surfactants. Protolytic and flotation properties of ionic associates are considered. The observed effects are explained from the viewpoint of electrostatic and hydrophobic interactions in the R-surfactant systems, observed by the methods of NMR, polarography amperometry, conductometry. A possible use of the investigated M-R-surfactant systems as complexonometric indicators was evaluated. A possibility was shown of using them for direct titrimetric determination of hundredth milligram portions of Cu, Ga, In and Sc at a titrant concentratjon of less than 0.01 M. It follows from the estimation of basic optical parameters of the Me-R-surfactant systems that detection.
Some aspects of surfactant action mechanism in the organic reagents - metal ions systems
International Nuclear Information System (INIS)
Chernova, R.K.; Shtykov, S.N.; Beloliptseva, G.M.; Sukhova, L.K.; Amelin, V.G.; Kulapina, E.G.
Results are reviewed of investigations into the interaction of ions of Mo(6), W, Zr, Be, Sc, Nb, Ta, J, rare earths, a. o. with organic reagents of triphenylmethane class in the 8M H 2 SO 4 -pH14 acidity range and the 1x10 -3 -5x10 -6 M concentration range both in the presence and absence of different surfactant type (cetylpyridine, methyltrimethylammonium, synthanols, etc). Three types of effects, determining enhancement of the sensitivity and selectivity of reactions jn the Me-R-surfactant systems, were determined: an increase in the number of coordinated ligands, the activating effect of cation surfactants resulting in a potential complexing in acid media, multicenter interaction of polydentate ligands both via chelating groups and auxochrome groups in the presence of cation surfactants. Protolytic and flotation properties of ionic associates are considered. The observed effects are explained from the viewpoint of electrostatic and hydrophobic interactions in the R-surfactant systems, observed by the methods of NMR, polarography amperometry, conductometry. A possible use of the investigated M-R-surfactant systems as complexonometric indicators was evaluated. A possibility was shown of using them for direct titrimetric determination of hundredth milligram portions of Cu, Ga, In and Sc at a titrant concentratjon of less than 0.01 M. It follows from the estimation of basic optical parameters of the Me-R-surfactant systems that detection
Spectroscopic studies on surface reactions between minerals and reagents in flotation systems
International Nuclear Information System (INIS)
Giesekke, E.W.
A study of the adsorbed species at the interface between the minerals and the aqueous solution is reported in the hope that it will contribute to a better understanding of selective mineral flotation by various reagents. The results of infrared spectroscopic studies are cited from the author's investigation on the fluorite-sodium oleate and fluorite-linoleate systems. Electron-spectroscopic techniques, e.g., electron spectroscopy for chemical analysis (ESCA) have also been useful in the identification of adsorbed species on mineral surfaces. Some experimental data from the literature are discussed. These studies have the disadvantage that they are not in situ investigations of the interface between the mineral and the aqueous solution. The potential use of other spectroscopic techniques are discussed, photo-acoustic, Raman, and electron-spin-resonance spectroscopy being considered as possible alternatives. It is suggested that the relatively small surface areas of minerals used in flotation (i.e. smaller than 2m 2 .g- 1 ) impose severe restrictions on the use of such techniques
Application of Neesler reagent in the ammonium quantification used in the fermentations of biotechnology products
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Dinorah Torres-Idavoy
Full Text Available The ammonium salts are used in fermentations to supplement the deficient amounts of nitrogen and stabilize the pH of the culture medium. The excess ammonium ion exerts a detrimental effect on the fermentation process inhibiting microbial growth. An analytical method based on Neesler reagent was developed for monitoring and controlling the concentration of ammonium during the fermentation process. The test was standardized, by means of the selection of measuring equipment, and the reaction time as well as comparing standards of ammonium salts. The method was characterized with the evaluation of the next parameters: Specificity, Linearity and Range, Quantification Limit, Accuracy and Precision. The method proved to be specific. Two linear curves were defined in the ranges of concentrations of ammonium chloride salt (2-20 μg/ml and ammonium sulfate salt (5-30 μg/ml. The limits of quantification were the lowest points of each one. The method proved to be accurate and precise. This assay was applied to samples of the yeast culture and bacteria of the genus Saccharomyces and E. coli respectively. A novel method in micro plate for quantification and analytical control of ammonia was developed. This method is used to control this fundamental chemical component in the fermentations, to optimize the culture medium. Thus, an appropriate expression of recombinant proteins and proper vaccine candidates for clinical use are achieved
Diazo Reagents with Small Steric Footprints for Simultaneous Arming/SAR Studies of Alcohol-Containing Natural Products via O–H Insertion
Chamni, Supakarn; He, Qing-Li; Dang, Yongjun; Bhat, Shridhar; Liu, Jun O.; Romo, Daniel
Natural products are essential tools for basic cellular studies leading to the identification of medically relevant protein targets and the discovery of potential therapeutic leads. The development of methods that enable mild and selective derivatization of natural products continues to be of significant interest for mining their information-rich content. Herein, we describe novel diazo reagents for simultaneous arming and structure-activity relationship (SAR) studies of alcohol-containing natural products with a small steric footprint, namely an α-trifluoroethyl (HTFB) substituted reagent. The Rh(II)-catalyzed O–H insertion reaction of several natural products, including the potent translation inhibitor lactimidomycin, was investigated and useful reactivity and both chemo- and site (chemosite) selectivities were observed. Differential binding to the known protein targets of both FK506 and fumagillol was demonstrated, validating the advantage of the smaller steric footprint of trifluoroethyl derivatives. A p-azidophenyl diazo reagent is also described that will prove useful for photoaffinity labeling of low affinity small molecule protein receptors. PMID:21894934
Thiolated chitosan nanoparticles as a delivery system for antisense therapy: evaluation against EGFR in T47D breast cancer cells
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Talaei F
Full Text Available Fatemeh Talaei1, Ebrahim Azizi2, Rassoul Dinarvand3, Fatemeh Atyabi31Novel Drug Delivery Systems Lab, 2Molecular Research Lab, Department of Pharmacology and Toxicology, 3Nanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, IranAbstract: Thiolated chitosan has high transfection and mucoadhesive properties. We investigated the potential of two recently synthesized polymers: NAC-C (N-acetyl cysteine-chitosan and NAP-C (N-acetyl penicillamine-chitosan in anticancer drug delivery targeting epidermal growth factor receptor (EGFR. Doxorubicin (DOX and antisense oligonucleotide (ASOND-loaded polymer nanoparticles were prepared in water by a gelation process. Particle characterization, drug loading, and drug release were evaluated. To verify drug delivery efficiency in vitro experiments on a breast cancer cell line (T47D were performed. EGFR gene and protein expression was analyzed by real time quantitative polymerase chain reaction and Western blotting, respectively. A loading percentage of 63% ± 5% for ASOND and 70% ± 5% for DOX was achieved. Drug release data after 15 hours showed that ASOND and DOX were completely released from chitosan-based particles while a lower and more sustained release of only 22% ± 8% was measured for thiolated particles. In a cytosol simulated release medium/reducing environment, such as found intracellularly, polymer-based nanoparticles dissociated, liberating approximately 50% of both active substances within 7 hours. ASOND-loaded polymer nanoparticles had higher stability and high mucoadhesive properties. The ASOND-loaded thiolated particles significantly suppressed EGFR gene expression in T47D cells compared with ASOND-loaded chitosan particles and downregulated EGFR protein expression in cells. This study could facilitate future investigations into the functionality of NAP-C and NAC-C polymers as an efficient ASOND delivery system in vitro and in vivo
HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role.
Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G
Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4 + /CD25 + T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins.
HTLV Deregulation of the NF-κB Pathway: An Update on Tax and Antisense Proteins Role
Fochi, Stefania; Mutascio, Simona; Bertazzoni, Umberto; Zipeto, Donato; Romanelli, Maria G.
Human T-cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL), an aggressive CD4+/CD25+ T-cell malignancy and of a severe neurodegenerative disease, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). The chronic activation or deregulation of the canonical and non-canonical nuclear factor kappa B (NF-κB) pathways play a crucial role in tumorigenesis. The HTLV-1 Tax-1 oncoprotein is a potent activator of the NF-κB transcription factors and the NF-κB response is required for promoting the development of HTLV-1 transformed cell lines. The homologous retrovirus HTLV-2, which also expresses a Tax-2 transforming protein, is not associated with ATL. In this review, we provide an updated synopsis of the role of Tax-1 in the deregulation of the NF-κB pathway, highlighting the differences with the homologous Tax-2. Special emphasis is directed toward the understanding of the molecular mechanisms involved in NF-κB activation resulting from Tax interaction with host factors affecting several cellular processes, such as cell cycle, apoptosis, senescence, cell proliferation, autophagy, and post-translational modifications. We also discuss the current knowledge on the role of the antisense viral protein HBZ in down-regulating the NF-κB activation induced by Tax, and its implication in cellular senescence. In addition, we review the recent studies on the mechanism of HBZ-mediated inhibition of NF-κB activity as compared to that exerted by the HTLV-2 antisense protein, APH-2. Finally, we discuss recent advances aimed at understanding the role exerted in the development of ATL by the perturbation of NF-κB pathway by viral regulatory proteins. PMID:29515558
A chemiluminescence reagent free method for the determination of captopril in medicine and urine samples by using trivalent silver
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Zhaofu Fu
Full Text Available A novel flow-injection chemiluminescence (FI-CL method free of CL reagent was developed for the determination of captopril based on its enhancing effect on the CL derived from diperiodatoargentate(III-sulfuric acid system. Compared with the conventional CL system, the CL system based on trivalent silver was characterized of good selectivity for the absence of CL reagent. The CL mechanism was discussed through CL spectra and UV–vis absorption spectra. The conditions of the FI-CL system were investigated and optimized. Under the optimal conditions, the relative CL intensity was linear with the captopril concentration in the range of 0.3–15.0 μg/mL. The detection limit for captopril was 0.05 μg/mL, and the relative standard deviation (n=11 was 2.0% for 5.0 μg/mL captopril. The proposed method was applied to the analysis of captopril in tablet and human urine with the recoveries of 83.1%–112.5%, and the relative standard deviations of 0.5%–4.4%. The results obtained by the proposed method agreed well with those obtained from HPLC method. The proposed method is fast, convenient, and cost-effective for the determination of captopril in medicine and biological samples.
Hemorrhagic Shock-Induced Vascular Hyporeactivity in the Rat: Relationship to Gene Expression of Nitric Oxide Synthase, Endothelin-1, and Select Cytokines in Corresponding Organs
the Selected Genes Sense Antisense Product length (bp) G3PDH 5=-TCCTGCACCACCAACTGCTTAG-3= 5=-TGCTTCACCACCTTCTTGATGTC-3= 341 iNOS 5...GAPDH, as a housekeeping gene, was not affected significantly by the hemorrhage protocol. The results showed that mRNA levels of all enzymes and
21 CFR 866.3235 - Epstein-Barr virus serological reagents.
... (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3235 Epstein-Barr... consist of antigens and antisera used in serological tests to identify antibodies to Epstein-Barr virus in...
Small-scale one -pot reductive alkylation of unprotected aminocyclitols with supported reagents
Czech Academy of Sciences Publication Activity Database
Šíša, Miroslav; Trapero, A.; Llebaria, A.; Delgado, A.
-, č. 19 (2008), s. 3167-3170 ISSN 0039-7881 Institutional research plan: CEZ:AV0Z40550506 Keywords : alkylation s * aldehydes * supported reagents * aminocyclitols Subject RIV: CC - Organic Chemistry Impact factor: 2.470, year: 2008
21 CFR 866.3390 - Neisseria spp. direct serological test reagents.
... SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3390... Neisseria spp. directly from clinical specimens. The identification aids in the diagnosis of disease caused...
Improvements to parallel plate flow chambers to reduce reagent and cellular requirements
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Larson Richard S
Full Text Available Abstract Background The parallel plate flow chamber has become a mainstay for examination of leukocytes under physiologic flow conditions. Several design modifications have occurred over the years, yet a comparison of these different designs has not been performed. In addition, the reagent requirements of many designs prohibit the study of rare leukocyte populations and require large amounts of reagents. Results In this study, we evaluate modifications to a newer parallel plate flow chamber design in comparison to the original parallel plate flow chamber described by Lawrence et al. We show that modifications in the chamber size, internal tubing diameters, injection valves, and a recirculation design may dramatically reduce the cellular and reagent requirements without altering measurements. Conclusions These modifications are simple and easily implemented so that study of rare leukocyte subsets using scarce or expensive reagents can occur.
TAPIR: a device for automatic titration with incremental weighing of the titration reagent
International Nuclear Information System (INIS)
Ganivet, Michel
TAPIR is a titration device enabling automatic analyses with weighting of the titration reagent. The titration method used can be based on potentiometry, amperometry, color indicator change... The reproducibility is about 3.10 -4 [fr
Evaluation of Tropaeolin 000-1 as a Colorimetric Reagent for Assay ...
African Journals Online (AJOL)
for Assay of Duloxetine and Escitalopram in Solid Dosage ... Purpose: To explore the application of tropaeolin 000-1 reagent for the rapid, ..... The effects of placebo interference testing are ... oppositely charged TL and TO ions form a stable.
Reagents and fractions impact on sulphide ore heap bioleaching at Smolnik mine
Oros, L. M.; Zavada, J.
Mine Smolnik is one of the oldest sulphide ore mines in Europe and it is also an important part of bioleaching development. This paper follows previous attempts to extract residual metals from nearby heaps via variations in bioleaching reagents with regard to recent findings and needs in the related industry. Furthermore, economic and process relations between reagents and chosen heap fractions were also investigated in this case study.
Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips.
Stevens, J F; Tsang, W; Newall, R G
Two new methods for the assay of total activities of lactate dehydrogenase and creatine kinase are described, in which the enzyme activities are measured from a solid-state reagent strip during a kinetic reaction, the reaction being monitored in the ultra-violet region of the spectrum by reflectance spectroscopy. The performances of these methods are evaluated, and compared to conventional "wet" chemistry methods. The solid-phase reagent methods demonstrated precision and accuracy acceptable ...
Method of Generating Hydrocarbon Reagents from Diesel, Natural Gas and Other Logistical Fuels
Herling, Darrell R [Richland, WA; Aardahl, Chris L [Richland, WA; Rozmiarek, Robert T [Middleton, WI; Rappe, Kenneth G [Richland, WA; Wang, Yong [Richland, WA; Holladay, Jamelyn D [Kennewick, WA
The present invention provides a process for producing reagents for a chemical reaction by introducing a fuel containing hydrocarbons into a flash distillation process wherein the fuel is separated into a first component having a lower average molecular weight and a second component having a higher average molecular weight. The first component is then reformed to produce synthesis gas wherein the synthesis gas is reacted catalytically to produce the desire reagent.
Synthesis and flotation activity of reagent-collectors based on dithiocarbonyl and phosphoryl derivatives of amino alcohols
Directory of Open Access Journals (Sweden)
Nazgul Orazbaevna Akimbaeva
Full Text Available Research has been carried out to study the flotation activity of new surface active substances among dithiocarbonyl and phosphoryl derivatives of monoethanolamine and diethanolamine on the gold-bearing sulphide ore of the Bestobinskoye deposit of Kazakhstan. Among the first synthesized compounds, effective collectors for selective enrichment of sulfide polymetallic gold ores were found. Sodium N-octyl-N-2-hydroxyethyldithiocarbamate (AA-41 and sodium O-2-(dimethoxyphosphoryl-2-hydroxyethylaminoethylxanthate (GF-2 were proposed as new flotation reagents. They were tested in the flotation of gold-bearing polymetallic ore as additional collectors and showed good results, contributing to an increase in the percentage of gold extraction in the collective concentrate in comparison with the factory basic regime in which a mixture of butyl xanthate (BX and ditiophosphate butyl ether or butyl airoflot (BAF was used. The results of flotation tests indicate that the collective ability of AA-41 flotation agent as an additional collector is at the level of BX. While the GF-2 flotation agent as a collector in combination with BAF provides a higher gold recovery to the concentrate of 90.8%, which is higher than gold recovery when combined with BX and BAF collectors (87.7%. It should be specially emphasized that the consumption of collectors AA-41 and GF-2 in comparison with the BX consumption in the base mode is much lower, so the AA-41 consumption is 11% lower and the consumption of GF-2 is lower by 33%. Methods for synthesizing the AA-41 flotation reagents and GF-2 flotation agents were developed, and the evidence of their structure was given with the help of physicochemical methods (IRS and NMR 1Н, 13С. The principal technological scheme of the process of obtaining the flotation agent-collector GF-2, which showed the best results in the flotation of gold-bearing polymetallic ore, was developed. In the synthesis of the flotation reagent AA-41, one of the
Optimal lipofection reagent varies with the molecular modifications of the DNA.
Conrad, A H; Behlke, M A; Jaffredo, T; Conrad, G W
Cationic lipid reagents differ in their cytofection efficacy with different cell types. No evidence has addressed whether the same lipid reagent is best for different DNAs in a single cell line. Immortalized avian embryonic cardiomyocytes cultured in vitro were tested with 15 cationic lipid reagents using (A) a beta-gal expression plasmid, (B) a fluorescein-tagged, phosphorothioate-modified ODN B, (C) a fluorescein-tagged, ethoxy-modified ODN C with the same nucleotide sequence as ODN B, and (D) a fluorescein-tagged, phosphorothioate-modified ODN D with a different nucleotide sequence from ODNs B and C. Cytofection was scored as percent of cells expressing beta-gal activity or showing diffuse cellular fluorescence. The best lipid reagents for the phosphorothioate-modified ODNs were ODN-specific and markedly different from the best lipid reagents for the expression plasmid or for the ethoxy-modified ODN. These results suggest that the best cationic lipid reagent for a particular cell type varies with the physical and chemical form of the DNA being transfected into the cells.
Hydrogen sulfide deactivates common nitrobenzofurazan-based fluorescent thiol labeling reagents.
Montoya, Leticia A; Pluth, Michael D
Sulfhydryl-containing compounds, including thiols and hydrogen sulfide (H2S), play important but differential roles in biological structure and function. One major challenge in separating the biological roles of thiols and H2S is developing tools to effectively separate the reactivity of these sulfhydryl-containing compounds. To address this challenge, we report the differential responses of common electrophilic fluorescent thiol labeling reagents, including nitrobenzofurazan-based scaffolds, maleimides, alkylating agents, and electrophilic aldehydes, toward cysteine and H2S. Although H2S reacted with all of the investigated scaffolds, the photophysical response to each scaffold was significantly different. Maleimide-based, alkylating, and aldehydic thiol labeling reagents provided a diminished fluorescence response when treated with H2S. By contrast, nitrobenzofurazan-based labeling reagents were deactivated by H2S addition. Furthermore, the addition of H2S to thiol-activated nitrobenzofurazan-based reagents reduced the fluorescence signal, thus establishing the incompatibility of nitrobenzofurazan-based thiol labeling reagents in the presence of H2S. Taken together, these studies highlight the differential reactivity of thiols and H2S toward common thiol-labeling reagents and suggest that sufficient care must be taken when labeling or measuring thiols in cellular environments that produce H2S due to the potential for both false-positive and eroded responses.
Maternal urogenital schistosomiasis; monitoring disease morbidity by simple reagent strips.
Directory of Open Access Journals (Sweden)
Oyetunde T Oyeyemi
Full Text Available Urine analysis is one of the recommended antenatal guidelines for early diagnosis of pregnancy-associated complications. While in practice, urine analysis by dipstick had been used to provide useful information on other urinary tract infections, its applications for early detection of urogenital schistosomiasis in pregnant women is often times not given due attention in most endemic areas. Our study therefore assessed the performance of some common urinalysis parameters in the diagnosis of maternal urogenital schistosomiasis in endemic rural communities of Nigeria.The cross-sectional epidemiologic survey of urogenital schistosomiasis was conducted among pregnant women in Yewa North Local Government, Ogun State, Nigeria. The women were microscopically examined for infection with Schistosoma haematobium, visually observed for macrohematuria, and screened for microhematuria and proteinuria using standard urine chemical reagent strips. Of 261 volunteered participants, 19.9% tested positive for S. haematobium infection. The proportion of microhematuria (23.8% was significantly higher than that of macrohematuria (3.8% and proteinuria (16.8% (P<0.05. Microhematuria with sensitivity (82.7% and specificity (89.0% was the best diagnostic indicator of urogenital schistosomiasis. Macrohematuria with the least sensitivity (11.8% was however the most specific (98.1% for diagnosing urogenital schistosomiasis in pregnant women. Maximum microhematuria sensitivity (100.0% was observed in women between 15-19 years but sensitivity was consistently low in older age groups. Maximum sensitivity, specificity and predictive values (100.0% were recorded for microhematuria in first trimester women. Diagnostic efficiency of proteinuria and macrohematuria was also better in the first trimester women except the 25.0% specificity recorded for proteinuria. The overall diagnostic performance of microhematuria and proteinuria was better in secundigravidae.Microhematuria can be
Frozen Chirality of Tertiary Aromatic Amides: Access to Enantioenriched Tertiary α-Amino Acid or Amino Alcohol without Chiral Reagent.
Mai, Thi Thoa; Viswambharan, Baby; Gori, Didier; Guillot, Régis; Naubron, Jean-Valère; Kouklovsky, Cyrille; Alezra, Valérie
One of the fundamental and intriguing aspects of life is the homochirality of the essential molecules. In this field, the absolute asymmetric synthesis of α-amino acids is a major challenge. Herein, we report access, by chemical means, to tertiary α-amino acid derivatives in up to 96 % ee without using any chiral reagent. In our strategy, the dynamic axial chirality of tertiary aromatic amides is frozen in a crystal and is responsible for the stereoselectivity of the subsequent steps. Furthermore, we could control the configuration of the final product by manually sorting and selecting the initial crystals. Based on vibrational circular dichroism studies, we could rationalize the observed stereoselectivity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Pentobarbital quantitation using EMIT serum barbiturate assay reagents: application to monitoring of high-dose pentobarbital therapy.
Pape, B E; Cary, P L; Clay, L C; Godolphin, W
Pentobarbital serum concentrations associated with a high-dose therapeutic regimen were determined using EMIT immunoassay reagents. Replicate analyses of serum controls resulted in a within-assay coefficient of variation of 5.0% and a between-assay coefficient of variation of 10%. Regression analysis of 44 serum samples analyzed by this technique (y) and a reference procedure (x) were y = 0.98x + 3.6 (r = 0.98; x = ultraviolet spectroscopy) and y = 1.04x + 2.4 (r = 0.96; x = high-performance liquid chromatography). Clinical evaluation of the results indicates the immunoassay is sufficiently sensitive and selective for pentobarbital to allow accurate quantitation within the therapeutic range associated with high-dose therapy.
Simultaneous determination of glucose, triglycerides, urea, cholesterol, albumin and total protein in human plasma by Fourier transform infrared spectroscopy: direct clinical biochemistry without reagents.
Jessen, Torben E; Höskuldsson, Agnar T; Bjerrum, Poul J; Verder, Henrik; Sørensen, Lars; Bratholm, Palle S; Christensen, Bo; Jensen, Lene S; Jensen, Maria A B
Direct measurement of chemical constituents in complex biologic matrices without the use of analyte specific reagents could be a step forward toward the simplification of clinical biochemistry. Problems related to reagents such as production errors, improper handling, and lot-to-lot variations would be eliminated as well as errors occurring during assay execution. We describe and validate a reagent free method for direct measurement of six analytes in human plasma based on Fourier-transform infrared spectroscopy (FTIR). Blood plasma is analyzed without any sample preparation. FTIR spectrum of the raw plasma is recorded in a sampling cuvette specially designed for measurement of aqueous solutions. For each analyte, a mathematical calibration process is performed by a stepwise selection of wavelengths giving the optimal least-squares correlation between the measured FTIR signal and the analyte concentration measured by conventional clinical reference methods. The developed calibration algorithms are subsequently evaluated for their capability to predict the concentration of the six analytes in blinded patient samples. The correlation between the six FTIR methods and corresponding reference methods were 0.87albumin and total protein in human plasma. Copyright © 2014 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
The potential of P1 site alterations in peptidomimetic protease inhibitors as suggested by virtual screening and explored by the use of C-C-coupling reagents.
Weik, Steffen; Luksch, Torsten; Evers, Andreas; Böttcher, Jark; Sotriffer, Christoph A; Hasilik, Andrej; Löffler, Hans-Gerhard; Klebe, Gerhard; Rademann, Jörg
A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.
Beneficial metabolic effects of CB1R anti-sense oligonucleotide treatment in diet-induced obese AKR/J mice.
Directory of Open Access Journals (Sweden)
Yuting Tang
Full Text Available An increasing amount of evidence supports pleiotropic metabolic roles of the cannibinoid-1 receptor (CB1R in peripheral tissues such as adipose, liver, skeletal muscle and pancreas. To further understand the metabolic consequences of specific blockade of CB1R function in peripheral tissues, we performed a 10-week-study with an anti-sense oligonucleotide directed against the CB1R in diet-induced obese (DIO AKR/J mice. DIO AKR/J mice were treated with CB1R ASO Isis-414930 (6.25, 12.5 and 25 mg/kg/week or control ASO Isis-141923 (25 mg/kg/week via intraperitoneal injection for 10 weeks. At the end of the treatment, CB1R mRNA from the 25 mg/kg/week CB1R ASO group in the epididymal fat and kidney was decreased by 81% and 63%, respectively. Body weight gain was decreased in a dose-dependent fashion, significantly different in the 25 mg/kg/week CB1R ASO group (46.1±1.0 g vs veh, 51.2±0.9 g, p<0.05. Body fat mass was reduced in parallel with attenuated body weight gain. CB1R ASO treatment led to decreased fed glucose level (at week 8, 25 mg/kg/week group, 145±4 mg/dL vs veh, 195±10 mg/dL, p<0.05. Moreover, CB1R ASO treatment dose-dependently improved glucose excursion during an oral glucose tolerance test, whereas control ASO exerted no effect. Liver steatosis was also decreased upon CB1R ASO treatment. At the end of the study, plasma insulin and leptin levels were significantly reduced by 25 mg/kg/week CB1R ASO treatment. SREBP1 mRNA expression was decreased in both epididymal fat and liver. G6PC and fatty acid translocase/CD36 mRNA levels were also reduced in the liver. In summary, CB1R ASO treatment in DIO AKR/J mice led to improved insulin sensitivity and glucose homeostasis. The beneficial effects of CB1R ASO treatment strongly support the notion that selective inhibition of the peripheral CB1R, without blockade of central CB1R, may serve as an effective approach for treating type II diabetes, obesity and the metabolic syndrome.
Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles
Energy Technology Data Exchange (ETDEWEB)
Murphy, Jane; Hall, William W. [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland); Ratner, Lee [Department of Medicine, Division of Molecular Oncology, Washington University School of Medicine, Saint Louis, Missouri, United States of America (United States); Sheehy, Noreen [Centre for Research in Infectious Diseases, School of Medicine and Medical Science, University College Dublin, Belfield, Dublin 4 (Ireland)
The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.
Retroviral gene transfer of an antisense construct against membrane type 1 matrix metalloproteinase reduces the invasiveness of rheumatoid arthritis synovial fibroblasts.
Rutkauskaite, Edita; Volkmer, Dagmar; Shigeyama, Yukio; Schedel, Jörg; Pap, Geza; Müller-Ladner, Ulf; Meinecke, Ingmar; Alexander, Dorothea; Gay, Renate E; Drynda, Susanne; Neumann, Wolfram; Michel, Beat A; Aicher, Wilhelm K; Gay, Steffen; Pap, Thomas
Membrane type 1 matrix metalloproteinase (MT1-MMP) is expressed prominently in rheumatoid arthritis synovial fibroblasts (RASFs), but the specific contribution of MT1-MMP to fibroblast-mediated destruction of articular cartilage is incompletely understood. This study used gene transfer of an antisense expression construct to assess the effects of MT1-MMP inhibition on the invasiveness of RASFs. Retroviral gene transfer of a pLXIN vector-based antisense RNA expression construct (MT1-MMPalphaS) to MT1-MMP was used to stably transduce RASFs. Levels of MT1-MMP RNA and protein were determined by quantitative polymerase chain reaction, Western blotting, and immunocytochemistry in MT1-MMPalphaS-transduced RASFs as well as in control cells, with monitoring for 60 days. The effects of MT1-MMPalphaS on the invasiveness of RASFs were analyzed in the SCID mouse co-implantation model of RA. MT1-MMPalphaS-transduced RASFs produced high levels of antisense RNA that exceeded endogenous levels of MT1-MMP messenger RNA by 15-fold and resulted in a down-regulation of MT1-MMP at the protein level. Inhibition of MT1-MMP production was maintained for 60 days and significantly reduced the invasiveness of RASFs in the SCID mouse model. Whereas prominent invasion into cartilage by non-transduced and mock-transduced RASFs was observed (mean invasion scores 3.0 and 3.1, respectively), MT1-MMPalphaS-transduced cells showed only moderate invasiveness (mean invasion score 1.8; P < 0.05). The data demonstrate that an antisense RNA expression construct against MT1-MMP can be generated and expressed in RASFs for at least 60 days. Inhibition of MT1-MMP significantly reduces the cartilage degradation by RASFs.
Effective photo-enhancement of cellular activity of fluorophore-octaarginine antisense PNA conjugates correlates with singlet oxygen formation, endosomal escape and chromophore lipophilicity
DEFF Research Database (Denmark)
Yarani, Reza; Shiraishi, Takehiko; Nielsen, Peter E.
Photochemical internalization (PCI) is a cellular drug delivery method based on the generation of light-induced reactive oxygen species (ROS) causing damage to the endosomal membrane and thereby resulting in drug release to the cytoplasm. In our study a series of antisense fluorophore octaarginin...... indicate that efficient photodynamic endosomal escape is strongly dependent on the quantum yield for photochemical singlet oxygen formation, photostability as well as the lipophilicity of the chromophore....
The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles
Negin Saffarzadeh; Seyed Mehdi Kalantar; Ali Jebali; Seyed Hossein Hekmatimoghaddam; Mohammad Hassan Sheikhha; Ehsan Farashahi
Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium...
Novel interactions between the HTLV antisense proteins HBZ and APH-2 and the NFAR protein family: Implications for the HTLV lifecycles
International Nuclear Information System (INIS)
Murphy, Jane; Hall, William W.; Ratner, Lee; Sheehy, Noreen
The human T-cell leukaemia virus type 1 and type 2 (HTLV-1/HTLV-2) antisense proteins HBZ and APH-2 play key roles in the HTLV lifecycles and persistence in the host. Nuclear Factors Associated with double-stranded RNA (NFAR) proteins NF90/110 function in the lifecycles of several viruses and participate in host innate immunity against infection and oncogenesis. Using GST pulldown and co-immunoprecipitation assays we demonstrate specific novel interactions between HBZ/APH-2 and NF90/110 and characterised the protein domains involved. Moreover we show that NF90/110 significantly enhance Tax mediated LTR activation, an effect that was abolished by HBZ but enhanced by APH-2. Additionally we found that HBZ and APH-2 modulate the promoter activity of survivin and are capable of antagonising NF110-mediated survivin activation. Thus interactions between HTLV antisense proteins and the NFAR protein family have an overall positive impact on HTLV infection. Hence NFARs may represent potential therapeutic targets in HTLV infected cells. - Highlights: • This study demonstrates for the first time interactions between NF90/110 and the HTLV antisense proteins HBZ and APH-2. • We show that NF90/110 significantly enhance LTR activation by the HTLV Tax protein, an effect that is abolished by HBZ but enhanced by APH-2. • The study shows that even though the HTLV antisense proteins activate survivin expression they antagonize the ability of NF90/110 to do so. • Overall we found that NF90/110 positively regulate HTLV infection and as such might represent a therapeutic target in infected cells.
Physicochemical and biological properties of self-assembled antisense/poly(amidoamine) dendrimer nanoparticles: the effect of dendrimer generation and charge ratio
Nomani, Alireza; Haririan, Ismaeil; Rahimnia, Ramin; Fouladdel, Shamileh; Gazori, Tarane; Dinarvand, Rassoul; Omidi, Yadollah; Azizi, Ebrahim
To gain a deeper understanding of the physicochemical phenomenon of self-assembled nanoparticles of different generations and ratios of poly (amidoamine) dendrimer (PAMAM) dendrimer and a short-stranded DNA (antisense oligonucleotide), multiple methods were used to characterize these nanoparticles including photon correlation spectroscopy (PCS); zeta potential measurement; and atomic force microscopy (AFM). PCS and AFM results revealed that, in contrast to larger molecules of DNA, smaller mol...
Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts
Directory of Open Access Journals (Sweden)
Herington Adrian C
Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin
Diversity of antisense and other non-coding RNAs in Archaea revealed by comparative small RNA sequencing in four Pyrobaculum species
Directory of Open Access Journals (Sweden)
David L Bernick
Full Text Available A great diversity of small, non-coding RNA molecules with roles in gene regulation and RNA processing have been intensely studied in eukaryotic and bacterial model organisms, yet our knowledge of possible parallel roles for small RNAs in archaea is limited. We employed RNA-seq to identify novel small RNA across multiple species of the hyperthermophilic genus Pyrobaculum, known for unusual RNA gene characteristics. By comparing transcriptional data collected in parallel among four species, we were able to identify conserved RNA genes fitting into known and novel families. Among our findings, we highlight three novel cis-antisense small RNAs encoded opposite to key regulatory (ferric uptake regulator, metabolic (triose-phosphate isomerase, and core transcriptional apparatus genes (transcription factor B. We also found a large increase in the number of conserved C/D box small RNA genes over what had been previously recognized; many of these genes are encoded antisense to protein coding genes. The conserved opposition to orthologous genes across the Pyrobaculum genus suggests similarities to other cis-antisense regulatory systems. Furthermore, the genus-specific nature of these small RNAs indicates they are relatively recent, stable adaptations.
Widespread anti-sense transcription in apple is correlated with siRNA production and indicates a large potential for transcriptional and/or post-transcriptional control.
Celton, Jean-Marc; Gaillard, Sylvain; Bruneau, Maryline; Pelletier, Sandra; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Navarro, Lionel; Laurens, François; Renou, Jean-Pierre
Characterizing the transcriptome of eukaryotic organisms is essential for studying gene regulation and its impact on phenotype. The realization that anti-sense (AS) and noncoding RNA transcription is pervasive in many genomes has emphasized our limited understanding of gene transcription and post-transcriptional regulation. Numerous mechanisms including convergent transcription, anti-correlated expression of sense and AS transcripts, and RNAi remain ill-defined. Here, we have combined microarray analysis and high-throughput sequencing of small RNAs (sRNAs) to unravel the complexity of transcriptional and potential post-transcriptional regulation in eight organs of apple (Malus × domestica). The percentage of AS transcript expression is higher than that identified in annual plants such as rice and Arabidopsis thaliana. Furthermore, we show that a majority of AS transcripts are transcribed beyond 3'UTR regions, and may cover a significant portion of the predicted sense transcripts. Finally we demonstrate at a genome-wide scale that anti-sense transcript expression is correlated with the presence of both short (21-23 nt) and long (> 30 nt) siRNAs, and that the sRNA coverage depth varies with the level of AS transcript expression. Our study provides a new insight on the functional role of anti-sense transcripts at the genome-wide level, and a new basis for the understanding of sRNA biogenesis in plants. © 2014 INRA. New Phytologist © 2014 New Phytologist Trust.
Tribromoisocyanuric Acid/NaNO : a New Reagent for Mononitration ...
African Journals Online (AJOL)
reaction,1 as the nitrated products are important intermediates for fine ... the presence of solid acids9 has received attention with regard to regioselectivity. In some ... tive procedure for the selective nitration of phenols with TBCA/. NaNO2 in the ...
Nanomechanical identification of liquid reagents in a microfluidic channel
DEFF Research Database (Denmark)
Khan, Faheem; Kim, Seonghwan; Lee, Dongkyu
Integration of promising technologies that can enhance sensitivity, selectivity, and throughput into micro total analysis systems (μTAS) are important in making them useful in precise screening of reaction byproducts in analytical chemistry, cellular biology and pharmaceutical industries. But unf......, and petrochemical analysis....
Sorption-reagent treatment of brines produced by reverse osmosis unit for liquid radioactive waste management
International Nuclear Information System (INIS)
Avramenko, V. A.; Zheleznov, V. V.; Sergienko, V. I.; Chizhevsky, I. Yu
The results of the pilot plant tests (2002-2003) of the sorption-reagent decontamination of high salinity radioactive waste (brines) remaining after the low-salinity liquid radioactive waste (LRW) treatment in the reverse-osmosis unit from long-lived radionuclides are presented. The sorption-reagent materials used in this work were developed in the Institute of Chemistry FEDRAS. They enable one to decontaminate brines with total salt content up to 50 g/l from long-lived radionuclides of Cs, Sr and Co. At joint application of the reverse-osmosis and sorption-reagent technologies total volume of solid radioactive waste (SRW) decreases up to 100-fold as compared to the technology of cementation of reverse osmosis brines. In this case total cost of LRW treatment and SRW disposal decreases more than 10-fold. Brines decontaminated from radionuclides are then diluted down to the ecologically safe total salts content in water to be disposed of. Tests were performed to compare the efficiency of technologies including evaporation of brines remaining after reverse osmosis process and their decontamination by means of the sorption-reagent method. It was shown that, as compared to evaporation, the sorption-reagent technology provides substantial advantages as in regard to radioactive waste total volume reduction as in view of total cost of the waste management
Remotely controlled reagent feed system for mixed waste treatment Tank Farm
International Nuclear Information System (INIS)
Dennison, D.K.; Bowers, J.S.; Reed, R.K.
LLNL has developed and installed a large-scale. remotely controlled, reagent feed system for use at its existing aqueous low-level radioactive and mixed waste treatment facility (Tank Farm). LLNL's Tank Farm is used to treat aqueous low-level and mixed wastes prior to vacuum filtration and to remove the hazardous and radioactive components before it is discharged to the City of Livermore Water Reclamation Plant (LWRP) via the sanitary sewer in accordance with established limits. This reagent feed system was installed to improve operational safety and process efficiency by eliminating the need for manual handling of various reagents used in the aqueous waste treatment processes. This was done by installing a delivery system that is controlled either remotely or locally via a programmable logic controller (PLC). The system consists of a pumping station, four sets of piping to each of six 6,800-L (1,800-gal) treatment tanks, air-actuated discharge valves at each tank, a pH/temperature probe at each tank, and the PLC-based control and monitoring system. During operation, the reagents are slowly added to the tanks in a preprogrammed and controlled manner while the pH, temperature, and liquid level are continuously monitored by the PLC. This paper presents the purpose of this reagent feed system, provides background related to LLNL's low-level/mixed waste treatment processes, describes the major system components, outlines system operation, and discusses current status and plans
Decolorizing textile wastewater with Fenton's reagent electrogenerated with a solar photovoltaic cell.
Figueroa, Sandra; Vázquez, Leticia; Alvarez-Gallegos, A
In this work it is demonstrated that Fenton's reagent can be electroproduced with abundant and cheap feedstock: oxygen saturated wastewater and solar energy. Experiments were carried out in a divided electrochemical flow cell using two electrodes: a three dimensional reticulated vitreous carbon cathode and stainless steel anode. Fenton's reagent is produced by oxygen reduction on the cathode in the presence of 1mM Fe(2+). The influence of electrolyte nature and its concentration and potential difference on the current efficiency, as well as the rate of Fenton's reagent electroproduction is discussed and it is concluded that over this extended range of conditions the current efficiency, for Fenton's reagent production, fell within the range 50-70%. It is possible to electroproduce a stoichiometric amount of Fenton reagent for the oxidation of 0.061mM Reactive Black 5 (in tap water+0.05M Na(2)SO(4), approximately pH 2.8). Similar results were obtained for solutions containing 0.1mM Acid Green 25. Some practical applications in the field of water treatment are included. The energy required for drive electrochemical reaction is supplied to the flow cell by means of a commercial solar panel.
Microdetermination of Sucrose in Plasma with the Anthrone Reagent.
polysaccharides . The initial attempts to use it for selective determinations of monosaccharides in a mixture, however, were frustrated by a mutual...disaccharides and polysaccharides are hydrolyzed to form monosaccharides . In addition, water is split off from the latter to form hydroxaldehyde...supernate were then concentrated to dryness at 80 C with a manifold evaporator after which endogenous monosaccharides were destroyed by the addition of
Aerobic and Electrochemical Oxidations with N-Oxyl Reagents
Miles, Kelsey C.
Selective oxidation of organic compounds represents a significant challenge for chemical transformations. Oxidation methods that utilize nitroxyl catalysts have become increasingly attractive and include Cu/nitroxyl and nitroxyl/NO x co-catalyst systems. Electrochemical activation of nitroxyls is also well known and offers an appealing alternative to the use of chemical co-oxidants. However, academic and industrial organic synthetic communities have not widely adopted electrochemical methods. Nitroxyl catalysts facilitate effective and selective oxidation of alcohols and aldehydes to ketones and carboxylic acids. Selective benzylic, allylic, and alpha-heteroatom C-H abstraction can also be achieved with nitroxyls and provides access to oxygenated products when used in combination with molecular oxygen as a radical trap. This thesis reports various chemical and electrochemical oxidation methods that were developed using nitroxyl mediators. Chapter 1 provides a short review on practical aerobic alcohol oxidation with Cu/nitroxyl and nitroxyl/NO x systems and emphasizes the utility of bicyclic nitroxyls as co-catalysts. In Chapter 2, the combination of these bicyclic nitroxyls with NOx is explored for development of a mild oxidation of alpha-chiral aryl aldehydes and showcases a sequential asymmetric hydroformylation/oxidation method. Chapter 3 reports the synthesis and characterization of two novel Cu/bicyclic nitroxyl complexes and the electronic structure analysis of these complexes. Chapter 4 highlights the electrochemical activation of various nitroxyls and reports an in-depth study on electrochemical alcohol oxidation and compares the reactivity of nitroxyls under electrochemical or chemical activation. N-oxyls can also participate in selective C-H abstraction, and Chapter 5 reports the chemical and electrochemical activation of N-oxyls for radical-mediated C-H oxygenation of (hetero)arylmethanes. For these electrochemical transformations, the development of
Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy
Directory of Open Access Journals (Sweden)
Silvana M G Jirka
Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS and 2'-O-Me phosphorothioate (2OMePS on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.
Targeting TGF-β Signaling by Antisense Oligonucleotide-mediated Knockdown of TGF-β Type I Receptor
Directory of Open Access Journals (Sweden)
Dwi U Kemaladewi
Full Text Available Duchenne muscular dystrophy (DMD is caused by lack of functional dystrophin and results in progressive myofiber damage and degeneration. In addition, impaired muscle regeneration and fibrosis contribute to the progressive pathology of DMD. Importantly, transforming growth factor-β (TGF-β is implicated in DMD pathology and is known to stimulate fibrosis and inhibit muscle regeneration. In this study, we present a new strategy to target TGF-β signaling cascades by specifically inhibiting the expression of TGF-β type I receptor TGFBR1 (ALK5. Antisense oligonucleotides (AONs were designed to specifically induce exon skipping of mouse ALK5 transcripts. AON-induced exon skipping of ALK5 resulted in specific downregulation of full-length receptor transcripts in vitro in different cell types, repression of TGF-β activity, and enhanced C2C12 myoblast differentiation. To determine the effect of these AONs in dystrophic muscles, we performed intramuscular injections of ALK5 AONs in mdx mice, which resulted in a decrease in expression of fibrosis-related genes and upregulation of Myog expression compared to control AON-injected muscles. In summary, our study presents a novel method to target TGF-β signaling cascades with potential beneficial effects for DMD.