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This study adds to the evidence of the destructive power of social media. Any Tom, Dick or Harry can take to the ether, post whatever they like and be believed by their followers.
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Monday, December 22, 2014
A U.S. judge has cleared the way for consumers to sue Target Corp over the retailer's late 2013 data breach that they say compromised their personal financial information.U.S. District Judge Paul Magnuson in St. Paul, Minnesota, on Thursday dismissed claims by plaintiffs in certain states but largely denied Target's request to toss the proposed class action lawsuit.
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Difference between revisions of "5 Things Everyone Gets Wrong About Spain melanotan store"
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(Created page with "Melanotan Interest then shifted to bremelanotide (PT-141) a metabolite of melanotan-II, and a menalocortin analogue. The first human research related to therapy concerned the...")
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Melanotan Interest then shifted to bremelanotide (PT-141) a metabolite of melanotan-II, and a menalocortin analogue. The first human research related to therapy concerned the usage of melanotan-II, a non-selective melanocyte receptor agonist, administered subcutaneously (Wessells et al 2000 ). If the 2 things you can't live without are a dark, even tan and a fast-acting, lengthy-lasting erection, then add Melanotan II to your holiday purchasing record. This makes Melanotan peptide unique, people do not require as a lot publicity to UV gentle to supply extra melanin, very engaging for individuals wishing to develop greater tanning of the skin in fastest means possible. The incontrovertible fact that tales about attainable side-results amongst some melanotan users are appearing in the media is a fear, and only serves to spotlight the uncertainties around these injections and the necessity for proper research. Melanotan I and Melanotan II have each been tested in small medical trials. First developed within the Nineteen Eighties by researchers at the University of Arizona, Melanotan is principally used for the treatment of skin problems including vitiligo and erythropoietic protoporphyria that have an effect on pores and skin appearance and sensitivity (particularly to sunlight). By promoting melanin within the skin, Melanotan might help ease the signs of these situations and allow these diagnosed to live a extra regular life. Known as "Melâ€, "MT†or "the Barbie drugâ€, Melanotan is an artificial melanocortin, which is a hormone derived from the pituitary gland on the base of the mind that regulates growth and development. What is the difference between Melanotan I and Melanotan II? This protein encourages cells in the skin to supply melanin, so by mimicking it, Melanotan merchandise also lead to more melanin in the skin. Melanotan II (MT2) is a stimulating peptide which induces skin tanning. Melanotan II will increase melanin manufacturing via stimulation of pores and skin pigment cells called melanocytes. Melanotan injections, additionally known as a tan injections, are illegal within the UK and is unlicensed as a result of they haven't been examined for security, quality or effectiveness and no one knows what the attainable unwanted effects are or how serious they might be. It's important to note that the dosages for Melanotan-1and Melanotan-2 are totally different. The increasingly in style self-injecting "Barbie doll drug" Melanotan II is unregulated and, say docs, might be dangerous. Consumers are advised not to use Melanotan-I, Melanotan-II and related injectable tanning merchandise. I've used Melanotan, what should I do now? During clinic trials for its use as a tanning agent, melanotan II was discovered to be a potent stimulator of male erections. A new drug based on melanotan II, bremelanotide, was developed to benefit from this property. Melanotan relies on a drug (afamelanotide) which was developed by the University of Arizona in 1981 for use for photograph protection against sun harm. Melanotan increases the levels of the pigment melanin within the skin. There are two varieties available, Melanotan I and II, which are diluted in water earlier than being injected. There is also concern that melanotan might not be safe when used as a shot under the skin. the place can I buy melanotan 2 uk that is the query we are asked can I buy mt2 ii again all questions and indeed trutan not solely sells melanotan 2 tanning injections it additionally provides full data and steerage on how use tanning injections what's the safest place to purchase melanotan 2 tanning injections uk delight ourselves customer service for purchases this has made us trusted at our online e-store directions dosage merely when you trying any sort of melanotan 2 want a quality product offers resolution sunless from sincere uk primarily based suppliers of melanotan2 supplier free delivery your ensures worth money we offer nasal spray plus every thing need included starter kits including syringes referred being top. Melanotan II binds to receptors aside from those answerable for melanotan.eu tanning and subsequently leads to more aspect-effects. Melanotan I specifically binds to receptors responsible for tanning and so has less side effects. What is Melanotan and the way does it work? "However, some particular person case reports suggest a possible affiliation between Melanotan and melanoma and darkening of moles — there have been different cases documented the place the drug has caused muscle harm. Melanotan.org, a discussion board dedicated to the peptide that shut down in 2011, had thousands of standard posters, many of whom have since migrated to different discussion boards. In 2009, a BBC report tracking simply six needle exchanges discovered that hundreds of individuals had visited these exchanges in order to receive syringes for Melanotan II use. Linn Connie Danielsen, a model and blogger, advised the Norwegian newspaper Verdens Gangthat Melanotan II helps ease the stressful impression of extended winter sun deprivation.


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According to TMZ, Juice WRLD has died at the age of 21 after suffering a seizure in Chicago's Midway airport.1 Comment
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Juice WRLD has enlisted YoungBoy Never Broke Again for his brand new single titled "Bandit". The Chicago artist is currently prepping the release of his new album.
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Frequent collaborators benny blanco and Juice WRLD have teamed up on a brand new song titled "Graduation". No word on what forthcoming project this one will appear.
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UPDATE (March 4th, 2019): Juice WRLD has revealed the tracklist for the album.Leave a Comment


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13 Stranger Things Halloween Decorations Fit For Hawkins
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Chagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease.
To identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener).
Analysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families.
This study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions.
Citation: Brossas J-Y, Gulin JEN, Bisio MMC, Chapelle M, Marinach-Patrice C, Bordessoules M, et al. (2017) Secretome analysis of Trypanosoma cruzi by proteomics studies. PLoS ONE 12(10): e0185504. https://doi.org/10.1371/journal.pone.0185504
Editor: Ana Rodriguez, New York University School of Medicine, UNITED STATES
Received: February 6, 2017; Accepted: September 13, 2017; Published: October 3, 2017
Copyright: © 2017 Brossas et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: The Bruker Society provided support in the form of salary for M. Chapelle, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific role of M. Chapelle is articulated in the 'author contributions' section. The study was also supported by Ecos-Sud (Grant number: A13S03; URL: www.univ-paris13.fr/cofecub-ecos/ecos-sud). Ecos-Sud had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The Bruker Society provided support in the form of salary for M. Chapelle. The cooperation with Bruker society does not alter our adherence to PLOS ONE policies on sharing data and materials.
Trypanosoma cruzi is a protozoan parasite of the order Kinetoplastide and the aetiological agent of Chagas disease a vector-borne infection with a high prevalence in Central and South America. According to recent estimates, nearly 6 million people are infected with this neglected disease in Latin American countries [1]. Moreover, Chagas disease is now an increasing global health problem because of increased migration of infected persons to non-endemic regions [2].
T. cruzi reproduction is mostly clonal, with occasional events of genetic exchange leading to the emergence of hybrid genotypes [3]. These features led to a complex population structure, showing remarkable genetic diversity [4]. Biochemical and genetic typing schemes developed throughout the last decades converged in the delineation of six major T. cruzi evolutionary lineages or discrete typing units (DTUs) termed TcI to TcVI [5]. However the remarkable genetic heterogeneity of T.cruzi could partially account for their wide range of biological features, eco-epidemiological traits, and the large spectrum of clinical manifestations of Chagas disease [6]. No clear correlation between serodiagnostic test reactivity and molecular diversity of T. cruzi has been observed. Instead, discrepancies between serologic test's sensitivity may reflect adaptive immune responses to parasite antigens [7]. In human parasite transmission occurs by vectorial route when metacyclic trypomastigotes present in blood-sucking triatomine bug faeces penetrate the skin or mucous membranes. However, humans can also become infected via blood transfusion or organ transplantation [8], through the ingestion of tainted food and fluids [9], or via vertical transmission from mother-to-child during pregnancy or delivery [10][11].
The clinical course of the T. cruzi infectionhas three phases: acute, chronic without symptoms, and chronic with symptoms [12]. The initial acute phase, lasting for about 2 months, is characterised by high levels of trypomastigotes (the circulating form of T. cruzi) in the blood. This is followed by a chronic phase (with or without symptoms) characterized by low trypomastigotes levels but with many intra-cellular parasites (amastigotes) present in target tissues (cardiac and smooth muscle) that can lead to severe morbidity. During this stage diagnosis is essentially based on the detection for classical (not lytic) anti-T cruzi antibodies in patient serum. At present, there are no diagnostic criteria for treatment response because anti-T. cruzi antibodies can persist many years after parasitological cure. Therefore, there is an urgent need to find a means to improve diagnosis in order to evaluate treatment efficacy.
A biomarker is defined as a parameter that can be objectively measured as an indicator of normal or pathogenic biological processes, or as an indicator of pharmacological responses to therapeutic interventions[13] [14]. In infectious diseases, there are two main types of markers: markers from the host and markers from the pathogen[15,16]. Only the latter markers provide evidence for the presence of the pathogen. Such markers can be of different types (carbohydrate, peptide and proteins or chemical product, lipids, sugar or nucleic acid) and from different locations (secreted in the serum of infected patients or present inside infected cells). The detection of circulating parasite excreted-secreted antigens (TESA) in chronically infected persons could serve as suitable biomarkers for the infection and treatment follow-up [17,18]. Proteomic approaches present a unique opportunity for identifying new parasite proteins that are potentially detectable in infected patients. We were particularly interested in extracellular proteins (secreted or released by the parasite), as these are often key mediators of host–parasite interactions involved in immune regulation, signalling, or invasion. Many proteomic analysis of T. cruzi secretome are already published [19,20]. These analyses made it possible to obtain a better understanding of the diverse pathways of secretion used by different parasite stages (epimastigote or metacyclic forms) to release many proteins into the extracellular medium and many secreted proteins could be identified. However, these studies have been carried out with parasite stages in the absence of vertebrate host cells, and where secretion was artificially induced. We propose here to complement these studies by analysing the secretome of Vero cells infected with two strains of T. cruzi DTU TcVI [18, 19] CL Brener, the strain used for the first genomic sequence of T. cruzi [21], with preferential tropism for heart and muscle cells [19] and VD, a strain isolated from a case of congenital Chagas disease, with preferential tropism for plancenta [18].
The main objective was to characterize as comprehensively as possible parasitic proteins secreted by T. cruzi (TcVI)-infected cells. The ultimate aim of this first step is to establish a list of antigens with potential usefulness as antigenic markers in the diagnosis and treatment follow-up of Chagas disease.
Materials and methods
Vero cells culture
Vero cells (normal kidney epithelial cells of Cercopithecus aethiops) were obtained from the Virology Laboratory of the Pitié Salpêtrière Hospital (Paris, France). At late exponential growth phase, trypsin-treated Vero cells were subcultured every seven days in RPMI-1640 medium (Life technologies) supplemented with streptomycin/penicillin (Life technologies) and 5% heat-inactivated foetal bovine serum (FBS) (Life technologies). Subcultures were maintained at 37°C in a humidified atmosphere of 5% CO2.
Trypanosoma cruzi culture and stocks
CL Brener strain (collection number: MNHN-CEU- 2016–0159) was a gift from Pr. P. Grellier of the Muséum National d'Histoire Naturelle (Paris, France). VD strain (Tc VI) was isolated from a congenital case of Chagas disease diagnosed at the Parasitology and Chagas Disease Service of the "Dr. Ricardo Gutiérrez Children's' Hospital" (Buenos Aires, Argentina.) Both strains were maintained in CF-1 (Non "Swiss" albino mice from Charles Rivers laboratory) mice prior to their use.
Large-scale trypomastigote production (CL Brener or VD strains) was achieved as follow. At the end of the exponential phase, Vero cells were harvested from subcultures and seeded into 75 cm2 flasks with 104 cells per cm2, followed by 3 days of incubation at 37°C in 5% CO2 in air. The cells were then infected with T. cruzi trypomastigotes at a parasite-cell-ratio 1:5. After 24 hours, the flasks were washed with HBSS buffer in order to remove non-attached cells and free parasites, and were subsequently incubated in RPMI-1640 medium. On day 4 (VD strain) or day 5 (CL Brener strain) post-infection, trypomastigotes were released from the cells. The culture medium was removed and transferred to a centrifuge tube. Attached infected cells were washed with 15 mL of HBSS buffer. The culture medium and wash containing trypomastigotes were mixed and centrifuged at 200 g for 10 minutes at room temperature to remove host cells and their debris. Subsequently, trypomastigotes were collected by supernatant centrifugation at 2000 g for 10 minutes, resuspended in 10 mL and counted in a haemocytometer using a light microscope. The recovered parasites served for the secretome analysis.
Determining the secretome
The parasite suspension was added to each flask containing Vero cells at 7.104 cells per cm2, in a 5:1 parasite-cell ratio. After 24 hours, the cells were washed once with HBSS buffer (without Ca2+ and Mg2+) to remove free parasites from the culture media and they were then incubated for 2 days (VD strain) or 3 days (CL Brener strain) in RPMI-1640 medium-5%SBF. After this, the cells were washed five times with HBSS buffer (with Ca2+ and Mg 2+) to remove all traces of foetal bovine serum (FBS) and then incubated for 24 hours in a medium used for hybridoma culture (PFHM II),.a synthetic culture medium that contains no proteins. After the release of the trypomastigotes into the culture supernatant, the medium was transferred to a 50 mL centrifuge tube and centrifuged at 2000 g for 10 minutes at room temperature to remove any contamination with host cells, T. cruzi and other debris. The supernatant was collected and filtered on a 0.22 μm membrane to remove any residual cells or parasites, and then dialyzed and concentrated 200 -fold on a Vivaspin® 15R centrifugal concentrator (Vivaspin) at 4°C. The resultant concentrate was conserved at -80°C until its use.
One-dimensional electrophoretic analysis
Equal aliquots (40 µg) of proteins obtained from infected Vero cells supernatant were submitted to a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12% SDS-PAGE). Proteins were denaturized by incubating samples with Laemlli buffer (0.5 M Tris–HCl, pH 6.8, 50% glycerol, 10% SDS, 5% β-mercaptoethanol, and 0.05% bromophenol blue) at room temperature for 16 hours before electrophoresis. Electrophoresis was carried out running buffer (50 mM Tris, 192 mM glycine and 0.1% SDS). After protein separation, gels were stained with Instant Blue (Expedeon Ltd., Harston, U.K.) for 20 minutes and transferred to distilled water for direct use.
In-gel digestion
Each track was cut into 9 gel bands using a sterile scalpel and these were individually transferred to 1.5 mL Eppendorf sterile tubes. The gel was covered with 200 μL of destaining solution (50% acetonitrile, 25 mM ammonium bicarbonate in Milli-Q-H2O) and incubated under stirring at room temperature for 10 minutes. This step was repeated until the blue colour was removed. The gel pieces were then dehydrated using 200 μL of acetonitrile for 5 min at room temperature and air dried for 30 min.
Subsequently, 100 μL of buffer (10mM dithiothreitol (DTT), 100 mM ammonium bicarbonate) were added to reduce proteins for 30 minutes at 56°C. Next, 100 μL of 50 mM iodoacetamide buffer was added at room temperature for 15 min. Excess solution was removed and 200 μL of acetonitrile were added to dehydrate gel pieces for 5 min at room temperature until white coloration appeared. Excess acetonitrile was removed and the gel was further air dried until complete dryness (30 min, in general).
Trypsin reagent was prepared by adding 1 mL of ice-cold 50mM ammonium bicarbonate to 20 μg of trypsin (Promega) (final concentration 20 ng/ μL) and the solution was kept on ice. Then, 30 μL of trypsin solution was added to samples to rehydrate gel pieces for 10 min on ice while gently mixing. A total of 30 μL of 50 mM ammonium bicarbonate was added to gel pieces and trypsin digestion was carried out overnight at 37°C. Excess solution was removed and transferred into a new 1.5 mL tube.
30 μL of extraction buffer (50% acetonitrile, 1% tri-fluoro-acetic acid (TFA) in Milli-Q-H2O), was sequentially added to gel pieces for incubation for 10 minutes and transferred into the same tube. After a last incubation with extraction buffer, the samples were completely dried in a vacuum centrifuge and then resuspended in 30 μL of buffer A (98% Milli-Q-H2O, 2% acetonitrile, 0.1% TFA).
Mass spectrometry analysis
Nano LC–MS/MS.
Protein digests were analyzed by a Q-TOF mass spectrometer (Impact II, Bruker Daltonik GmbH; Bremen, Germany) using a Captive Spray source, interfaced with a nano-HPLC RSLC System (Ultimate 3000 Thermo Scientific). Samples were concentrated on a pre-column (Thermo Scientific, C18 PepMap100, 2 cm × 100 μm ID, 5 μm particle size, 100 A) at a flow rate of 10 μL/min using 0.1% tri-fluoro acetic acid. After pre-concentration, peptides were separated on a Thermo Scientific C18 PepMap100 UHPLC column (50 cm x 75 μm ID, 2 μm particles, 100A) at a flow rate of 400 nL/ min using a 90 min linear gradient (buffer A: 2% ACN in 0.1% FA; buffer B 100% ACN in 0.1% FA). Acquisition is data dependent InstantExpertise™ mode (Bruker Daltonik GmbH; Bremen, Germany) for precursor selection based on fixed time (1 MS every 2 sec at 0.25 sec/scan) for MS and as many as possible MSMS in between (1.75 sec) at acquisition time from 0.0625s to 0.25 s/scan based on precursor intensity.
Protein identification with reference to databases.
MS/MS raw data were processed using the Data Analysis software (Bruker Daltonik GmbH; Bremen, Germany) to generate the peak lists. The resulting mgf (Mascot Generic File Format) were submitted to Mascot server version 2.5 (MatrixScience–London, UK) through Proteinscape platform version 4.0 (Bruker Daltonics). Files were then searched against a home-built database (38685 entries) made of compiled Chlorocebus sabeus protein database from UniProtKb (19441 entries) and T. cruzi (strain CL Brener) protein databases from UniProtKb (19244 entries) using trypsin as hydrolysis enzyme. Mass tolerances were set at 10 ppm and 0.01 Da for precursor and product ions, respectively. Two missed cleavages per peptide were allowed considering both tryptic and semi-tryptic cleavages, while the search included fixed carbamidomethylation of Cysteine, and the following variable modifications: N-terminal acetylation, deamidation of asparagines and glutamines and, methionine oxidation. Searches were filtered using both a minimum peptide ion score of 20 and a false discovery rate (FDR) <1% calculated using Percolator.
We opted to analyse all the proteins secreted from Vero cells infected with trypomastigotes of either one of two strains, CL Brener and VD, as a means to derive a list of proteins that could be potential biomarkers for a T. cruzi infection.
In our in vitro infection model, following the multiplication of the intracellular amastigotes and their differentiation into trypomastigotes, the parasites are released from the cells in the culture medium. The culture supernatant is defined in this study as the "secretome", and it includes: 1) soluble proteins from Vero cells, 2) parasite proteins secreted into Vero cells cytoplasm that are then released into the culture media, and 3) all the trypomastigote proteins that have been directly secreted into the culture supernatant.
Comparing the secretome of two T. cruzi strains
To increase the validity of the results we selected either proteins whose LC-MSMS score was over 35 and with at least two peptides, or proteins with a score over 50 but with only one peptide identified. In this way, we identified 441 parasites proteins (Additional File 1) from the secretome derived from Vero cells infected with the CL Brener strain, and, a total of 515 proteins (Additional File 2) from Vero cells infected with the VD strain. Since Vero lineage was isolated from kidney epithelial cells extracted from an African green monkey and this study focuses on parasite proteins, those derived from the Vero cells were excluded from further analyses.
A total of the 591 T. cruzi proteins were found in the secretomes of the 2 strains, 363 were common to both strains (additional file 3), while 78 were exclusive to CL Brener strain and 151 were detected only from the VD strain (see Fig 1).
A total of 591 proteins of T. cruzi were identified. We note that 78 proteins are specific to the CL Brener strain whereas 151 proteins are specific to VD strain. However, 363 proteins are common to both strains.
In order to ascertain the possible role of these secreted proteins, the 363 specific proteins were assigned to 15 functional groups based on information from the literature (Mainly the work of E. Bayer santos et al) and UniprotKB annotation (see Fig 2).
Proteins were classified into 15 functional categories using literature and UniprotKB annotation. Y-axis, categories are indicated. X-axis shows the percentage of each category for each strain.
For both strains, approximately 70% of the proteins belong to the host-parasite interaction category that plays a very important role in virulence. A significant difference is remarkable; the number of identified proteins in the secretome with unknown function for the VD strain (44 proteins) is slightly more twice that for the CL Brener strain (20 proteins). For the VD strain, this subset represents more than 8% of the total proteins identified. A similar difference was noticed for the proteins involved in nucleic acid binding, but with a much lower number of proteins (10 proteins versus 2 proteins, respectively). For the other categories, there was no significant difference between the two strains. The set of T. cruzi proteins identified for each of the strains is provided in Additional Files 1 and 2.
Multigenic superfamilies identified in the secretomes
Among the 591 proteins identified in both T. cruzi secretomes, 379 (64%) correspond to those encoded by different multigenic superfamilies. Trans-sialidase proteins (TcS) and trans-sialidase like proteins are the major families founded in the secretomes of the two strains (315 proteins for TcS, 18 proteins for DGF1, 15 proteins for Masps and 31 proteins for GP63 surface proteins). A total of 315 TcS proteins from the T. cruzi secretomes have been identified for the 2 strains. Among these proteins, 46 are fragments of TcS. We can remark that 184 complete TcS proteins were common to both strains. This implies that 42 proteins are exclusively found in CL Brener strain and 43 for VD strain.
Since, previous studies have shown the presence of 508 complete TcS genes that can be classified into 8 Groups [22], we analysed the expression profile of these TcS in the two secretomes and classified these TcS proteins into groups I to VIII, according to previously described classes. For a better description of the expression of different TcS groups, we derived the ratio between the number of TcS identified in our analysis, and that in each group (see Fig 3).
Classification of TcS proteins for each strain into 8 group previously described [22]. Groups IV, V and VI are less than I, II, III groups VII and VIII for two strains. On the x-axis, the number of group is indicated. The Y-axis shows the percentage of each TcS identified in our analyses.
A reliable biomarker for the T. cruzi infection could be of different nature (protein, lipid, sugar or nucleid acid) but it must be specific to T. cruzi and ubiquitous in the different T. cruzi genotypes. The objective of this work was to obtain and compare the secretomes of two T. cruzi strains infecting Vero celIs, as a way to focus on protein biomarkers for Chagas disease.
For this study we chose to work with two T. cruzi strains of the same DTU (TcVI). The genetic intra-DTU variability in the parasite was poorly studied. The T. cruzi CL Brener genome (TcVI) was published in 2005 [21]. Other genomes, such as that of the Sylvio X10/1 strain and DM28c (TcI), have also been published [23,24]. The VD strain was isolated from a case of congenital Chagas disease and its genome is not sequenced. The comparison of two strains of TcVI genome but with different tropism increases the degree of selection of the most important secreted proteins and eliminated the strain-dependent proteins.
Secretome comparison
We have identified 515 proteins for VD strain compared to 441 proteins for the CL Brener strain. The result of the comparison shows a high proportion (more than 60%) of protein secreted common to both T. cruzi strains. It would seem that gene expression in both T. cruzi strain of the same DTU is relatively homogeneous. This high degree of homology makes possible to identify proteins secreted by all strains of T. cruzi and thus potential markers. This result is very different from that obtained by Geiger et al. who analyzed the secretome of T. brucei and found a small number of proteins common to the two strains of the same group (36 proteins common to the Feo strain and the strain OK out of a total of 270 identified proteins [25].
After functional classification of the parasite proteins, we noted that the proportion of identified proteins involved in proteolysis, signalling, oxydo-reduction, cell structure and motilities was quite different for the two strains. This result suggests that proteins involved in metabolic pathways of stress response, protein folding, proteolysis, and oxidoreductase activity, along with proteins that can interact with different signalling pathways, are important for T. cruzi's capacity to infect its host cell, and that these are probably more conserved in all T. cruzi strains. Furthermore, proteins involved in DNA binding are also more numerous in the secretome of the VD strain as compared to that of the CL Brener strain. This class of proteins plays an essential role in the control of gene expression in T. cruzi, modulating RNA processing stability, turnover, and translation [26].
The VD strain secretome contained more proteins with unknown functions than the CL Brener strain. The VD strain was isolated from a pediatric patient with congenital infection. As previously noted from in vivo investigations [27,28], in our study the VD strain seemed more virulent than the CL Brener strain due the faster emergence from infected Vero cells. The hypothetical proteins of unknown function could be involved in virulence, and also be a source of biomarkers candidates for strain discrimination and for further studies to understand the underlying mechanisms of pathogenicity.
However, more than 70% of the proteins that have been identified in the secretome of the two strains belong to the host-parasite interaction category, and these play a very important role as virulence factors. In this class there are essentially four major multigenic proteins families. A remarkable feature of the T. cruzi genome is the massive expansion of these multigenes family that encodes polymorphic surface proteins such as trans-sialidase (TcS), mucin and mucin-associated surface proteins (MASPs), dispersed gene family 1 (DGF-1), and gp63 peptidases. The gene families coding for these proteins represent about 10% to 30% of the T. cruzi genome [29]. The high sequence variability of these different gene families could confer upon the parasite the ability to invade a significant number of cell types, as well as to have pleiotropic tissue tropisms, as described previously in in vitro, and in in vivo and clinical studies [30] where a wide variety of syndromes associated with T. cruzi infection were described.
The trans-sialidase and tran-sialidase like gene (TcS) super family represents the largest T. cruzi gene family, with more than 1,400 genes. However, only half of these genes are apparently functional [21]. The TcS superfamily is composed of glycosylphosphatidylinositol proteins anchored on T. cruzi surface, but they are also released to the extracellular space. The TcS gene family is highly polymorphic and only a few members have critical residues necessary for catalytic activity [31]. The very large number of different TcS proteins found in both secretomes is not unexpected. It has been shown that TcS expression is highly induced when cells become full of parasites and when amastigotes transform to trypomastigotes [32]. Thus, it would be possible to correlate the presence of large amounts of TcS into culture medium with cell host rupture. Finally, Affranchino JL et al. reported that Vero cells infected with trypomastigotes release vesicles carrying some protein members of the trans-sialidase multigene family [33]. However, our study provides a more detailed description (number and identification) of the TcS members expressed during the life cycle of T. cruzi (DTU TcVI). Recently, a comparative analysis of TcS sequences (508 complete genes) in the T. cruzi CL Brener strain genome led to the differentiation of eight different groups [22]. These authors further showed that the majority of TcS transcripts of all groups are present both in trypomastigotes and amastigotes forms. However, some transcripts are highly expressed in trypomastigote stage whereas other transcripts are expressed in amastigotes. It is interesting to note that Freitas LM and their colleagues found a very low level of expression for two genes from TcS group V in all the developmental stages [22]. The protein structure of the TcS groups V and VI are very similar. Trans-sialidases of these two groups have one signal peptide, a single asp box, a canonical VTV motif and a GPI anchor [22]. Our study appears to confirm these observations since we observed that more than half of the TcS proteins belonging to groups I, II and III, are expressed (19, 117 and 15 proteins, respectively). This percentage increases up to 80% for the VII and VIII groups (17 and 46 sequences, respectively). While for group IV the expression of TcS is less than 40%, and for groups V and VI less than 25% (227 and 39, respectively). This observation is noteworthy, because groups V and VI represent more than 60% of the trans-sialidases (TcS).
Finally, antibodies against two repeated epitopes, SAPA epitope (PVDSSAHG/STPST) and TcD epitope (PKPAE) of trans-sialidases were found in the sera of patients with Chagas' disease in acute and chronic phase [34]. Our sequence analysis shows trans-sialidases that possess the SAPA epitope (Group I) and the TcD epitope (Group IV) in the secretome of both analysed strains. There is a high homology in the TcS group expressed in both strains from same DTU (TcVI), we could suppose that some variations in their expression might be related to the genetic difference between strains. Numerous proteomic studies have revealed that the T. cruzi secretomes contain many proteins. The first secretome analysis has been made from 'reservosome' fraction belonging to Dm28c strain (TcI) epimastigotes [19], suggesting that there are several possible pathways for the secretion of proteins from T. cruzi. In another study [25] the authors identified T. brucei proteins from purified microvesicles extracted from infected rats sera.
In the latest and most comprehensive study [20], the authors identified two types of microvesicles released by epimastigotes and metacyclic forms of Dm28c T. cruzi strain. All these studies pointed to the use of microvesicles to deliver cargo of many proteins into host cells. The role of the proteins released from microvesicles is fundamental in Chagas disease. For example, Cestari et al. observed microvesicles release from infected cells into bloodstream that could allow immune evasion by inhibiting C3 convertase [35]. The protein content of the microvesicles was found to be ubiquitous, contributing to virulence, infectivity and immunity in many pathogens such as Trypanosoma spp. and other related parasites such as Leishmania spp. [36] and Plasmodium spp. [37], but also from bacteria [38] or virus [39].
Nevertheless, all these studies were carried out without the host cell context. In the present study, we analyzed the secretome of T. cruzi in the presence of host cells and particularly at the time when trypomastigotes are released from the cell.
In our model we have identified a set of parasite proteins that have been released from the host cells by secretion, by microvesicles, or directly from the cell cytoplasm to the culture medium. These proteins are linked to biochemical processes that enable the transformation of amastigotes in trypomastigotes, the multiplication of amastigotes, the release of trypomastigotes from host cells and finally invasion of new cells. In the culture medium, we had access to all the proteins secreted or excreted by the parasite.
Our data show that the majority of the proteins identified for both strains were found in the microvesicles of the epimastigote and the metacyclic forms of T.cruzi. However, we have also identified some proteins that were previously found only in microvesicles from epimastigotes, such as the 10 KDa heat shock protein [20]. This result suggests that there are slight differences in the secretome of trypomastigote and amastigote forms of T. cruzi. Although these parasitic proteins are not required for cell invasion (metacyclic form) they may have a role in survival within host cell cytoplasm.
Short list of parasite proteins as a potential marker of infection with T. cruzi
Although the protein superfamilies (TcS, GP63, MASP, and DGF1) are highly represented in the secretome of both strains, they cannot be used as biomarkers for T. cruzi infection. The sequence identity of the TcS is low and is often limited to common motifs (Asp-box, GPI anchor, VTV and FRIP box). These motifs include a maximum of ten amino acids. In addition it should be noted that these proteins have sequences that are fairly variable between the various T. cruzi strains. Pattern searches by the protein BLAST algorithm from National Center for Biotechnology Information (NCBI)show that the TcD epitope is repeated 430 times in the genome of T. cruzi strain CL Brener while it is identified only 5 times in strain DM28C. Similarly we did not find the SAPA epitope In the DM28C strain whereas in the strain CL Brener this pattern is repeated 80 times. So we propose a selected list of proteins (see Table 1) that could potentially serve as markers of an infection with T. cruzi. Indeed, these proteins are secreted, clearly produced abundantly, and are common to at least two strains of TcVI.
The list includes 94 proteins of which 8 are represented by two isoforms and 18 have no known function. These various proteins might also be future therapeutic targets and/or markers of Chagas disease. The protein Q4CVJ1 was not identified in the various secretomes published for T.cruzi, but it has been found in high-throughput screening and is highly expressed and immunogenic; moreover, 68% of sera from patients with Chagas disease recognize this antigen [40]. Finally, the proteins with unknown function, Q4CTF0 and Q4CUB2, have been described as part of the last multigenic family (about 40 members in Cl Brener) subdivided into 3 subfamilies TcTASV-A, B and C. Q4CTF0 is part of the subgroup family TcTASV-A while Q4CUB2 is part of the TcTASV-C family. This subfamily (8 proteins) was identified on the surface of trypomastigotes of T. cruzi and in (strain CL Brener, Sylvio and RA). About 30% of human sera infected by T. cruzi reacted with TcTASV-C [41].
We note that for some proteins in this list, Microtubule-associated protein, Heat shock proteins (HSP70), Complement regulatory protein and Flagellar Calcium-binding protein, specific antibodies can be found in the acute and chronic phase sera of patients with Chagas' disease [38]–[42].
The EMBRARIO laboratory (Brazil) offers a confirmatory test (HBK 740 Imunoblot Linhas) based on a multi-epitope recombinant peptide, including the TC-24 epitope (Flagellar calcium-binding protein) and the MAP repeated epitope (Microtubule-associated protein) [43,44].
Some proteins have already been identified as potential markers in other infections. For example, the fructose biphosphate aldolase enzyme, a key enzyme in the glycolytic pathway, was found in plasma of patients infected with P. falciparum [45]).
The abundance of the parasite proteins and their relative stability in serum are two criteria to help in the identification of suitable markers for T. cruzi infection. In our study the strategy adopted to obtain the T. cruzi secretome may eliminate the most labile proteins since the presence of proteases in the culture supernatant is likely to degrade many of the secreted proteins.
The development and availability of a simple, inexpensive, easy to handle, sensitive and specific method to effectively demonstrate the presence of T. cruzi from a blood sample is a major challenge for the diagnosis of Chagas disease. Identification of one or more proteins (biomarkers) in the patient's serum will be clear evidence for the presence of the parasite.
This study provides the first comparative overview of the secretomes of cells infected by the T. cruzi CL Brenner and the VD strains. Not surprisingly, the majority of excreted proteins belong to the four multigenic gene families (TcS, MASPs, DGF1, and gp63 surface protein), that unfortunately cannot be used as infection marker because of their very high, inter-species, degree of variability.
Some of the proteins identified had not been studied previously, particularly from the VD strain, and some may be new potential therapeutic targets. We have thus established a list of 94 secreted proteins, common to both strains and that do not belong to members of multigene families. These proteins are secreted in large quantities and are relatively stable in presence of many proteases. These conditions are necessary to identify good serum markers. This descriptive study provides the first step toward the identification of potential diagnostic markers in the sera of T. cruzi-infected persons.
Supporting information
S1 Table. Secreted parasites proteins identified in T. cruzi CL Brener strains separated on 1D gel and their classification according to functional categories (informations from literature and UniprotKB annotation).
The proteins secreted have selected with LCMSMS score was over 35 and with at least two peptides identified, or with a score over 50 but with only one peptide identified. For each protein, the number of matched proteins and peptides and the highest score are described.
S2 Table. Secreted parasites proteins identified in T. cruzi VD strains separated on 1D gel and their classification according to functional categories (informations from literature and UniprotKB annotation).
The proteins secreted have selected with LCMSMS score was over 35 and with at least two peptides identified, or with a score over 50 but with only one peptide identified. For each protein, the number of matched proteins and peptides and the highest score are described.
S3 Table. Secreted parasites proteins identified in T. cruzi VD and CL Brener strains (common proteins) separated on 1D gel.
For each protein, the number of matched proteins and peptides and the highest score for CL Brener (white box) and VD (gray box) strain are described.
We would to like to thanks Georges Snounou and Angelita Rebollo for fruitful scientific discussion.
We thank Dr. María Elisa Solana (Facultad de Medicina, Instituto de Investigaciones en Microbiología y Parasitología Médica (IMPAM),UBA-CONICET, Universidad de Buenos Aires) to maintain of the VD strain of T. cruzi".
- 1.
WHO | 6 February 2015, vol. 90, 6 (pp. 33–44) [Internet]. WHO. [cited 2017 Jan 4]. Available from: http://www.who.int/wer/2015/wer9006/en/
- 2. Steverding D. The history of Chagas disease. Parasit Vectors. 2014 Jul 10;7:317. pmid:25011546
- 3. Messenger LA, Miles MA. Evidence and importance of genetic exchange among field populations of Trypanosoma cruzi. Acta Trop. 2015 Nov;151:150–5. pmid:26188331
- 4. Tibayrenc M, Ayala FJ. The population genetics of Trypanosoma cruzi revisited in the light of the predominant clonal evolution model. Acta Trop. 2015 Nov;151:156–65. pmid:26188332
- 5. Zingales B, Andrade SG, Briones MRS, Campbell DA, Chiari E, Fernandes O, et al. A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI. Mem Inst Oswaldo Cruz. 2009 Nov;104(7):1051–4. pmid:20027478
- 6. Zingales B, Miles MA, Campbell DA, Tibayrenc M, Macedo AM, Teixeira MMG, et al. The revised Trypanosoma cruzi subspecific nomenclature: rationale, epidemiological relevance and research applications. Infect Genet Evol J Mol Epidemiol Evol Genet Infect Dis. 2012 Mar;12(2):240–53.
- 7. Martin DL, Marks M, Galdos-Cardenas G, Gilman RH, Goodhew B, Ferrufino L, et al. Regional variation in the correlation of antibody and T-cell responses to Trypanosoma cruzi. Am J Trop Med Hyg. 2014 Jun;90(6):1074–81. pmid:24710614
- 8. Gascon J, Bern C, Pinazo M-J. Chagas disease in Spain, the United States and other non-endemic countries. Acta Trop. 2010 Aug;115(1–2):22–7. pmid:19646412
- 9. Segovia M, Carrasco HJ, Martínez CE, Messenger LA, Nessi A, Londoño JC, et al. Molecular epidemiologic source tracking of orally transmitted Chagas disease, Venezuela. Emerg Infect Dis. 2013 Jul;19(7):1098–101. pmid:23768982
- 10. Carlier Y, Torrico F, Sosa-Estani S, Russomando G, Luquetti A, Freilij H, et al. Congenital Chagas disease: recommendations for diagnosis, treatment and control of newborns, siblings and pregnant women. PLoS Negl Trop Dis. 2011 Oct;5(10):e1250. pmid:22039554
- 11. Freilij H, Altcheh J. Congenital Chagas' disease: diagnostic and clinical aspects. Clin Infect Dis Off Publ Infect Dis Soc Am. 1995 Sep;21(3):551–5.
- 12.
Chagas' Disease—NEJM [Internet]. [cited 2017 Apr 28]. Available from: http://www.nejm.org/doi/full/10.1056/NEJMra1410150
- 13. Biomarkers Definitions Working Group. Biomarkers and surrogate endpoints: preferred definitions and conceptual framework. Clin Pharmacol Ther. 2001 Mar;69(3):89–95. pmid:11240971
- 14. Hulka BS. ASPO Distinguished Achievement Award Lecture. Epidemiological studies using biological markers: issues for epidemiologists. Cancer Epidemiol Biomark Prev Publ Am Assoc Cancer Res Cosponsored Am Soc Prev Oncol. 1991 Dec;1(1):13–9.
- 15. McNerney R, Maeurer M, Abubakar I, Marais B, McHugh TD, Ford N, et al. Tuberculosis diagnostics and biomarkers: needs, challenges, recent advances, and opportunities. J Infect Dis. 2012 May 15;205 Suppl 2:S147–158.
- 16. Jain P, Chakma B, Patra S, Goswami P. Potential Biomarkers and Their Applications for Rapid and Reliable Detection of Malaria. BioMed Res Int [Internet]. 2014 Apr 2 [cited 2017 Apr 26];2014. Available from: https://www.hindawi.com/journals/bmri/2014/852645/abs/
- 17. de Araujo FF, Nagarkatti R, Gupta C, Marino AP, Debrabant A. Aptamer-based detection of disease biomarkers in mouse models for chagas drug discovery. PLoS Negl Trop Dis. 2015 Jan;9(1):e3451. pmid:25569299
- 18. Umezawa ES, Nascimento MS, Stolf AM. Enzyme-linked immunosorbent assay with Trypanosoma cruzi excreted-secreted antigens (TESA-ELISA) for serodiagnosis of acute and chronic Chagas' disease. Diagn Microbiol Infect Dis. 2001 Mar;39(3):169–76. pmid:11337184
- 19. Sant'Anna C, Nakayasu ES, Pereira MG, Lourenço D, de Souza W, Almeida IC, et al. Subcellular proteomics of Trypanosoma cruzi reservosomes. Proteomics. 2009 Apr;9(7):1782–94. pmid:19288526
- 20. Bayer-Santos E, Aguilar-Bonavides C, Rodrigues SP, Cordero EM, Marques AF, Varela-Ramirez A, et al. Proteomic analysis of Trypanosoma cruzi secretome: characterization of two populations of extracellular vesicles and soluble proteins. J Proteome Res. 2013 Feb 1;12(2):883–97. pmid:23214914
- 21. El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran A-N, et al. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science. 2005 Jul 15;309(5733):409–15. pmid:16020725
- 22. Freitas LM, Santos SL dos, Rodrigues-Luiz GF, Mendes TAO, Rodrigues TS, Gazzinelli RT, et al. Genomic Analyses, Gene Expression and Antigenic Profile of the Trans-Sialidase Superfamily of Trypanosoma cruzi Reveal an Undetected Level of Complexity. PLOS ONE. 2011 Oct 19;6(10):e25914. pmid:22039427
- 23. Franzén O, Ochaya S, Sherwood E, Lewis MD, Llewellyn MS, Miles MA, et al. Shotgun sequencing analysis of Trypanosoma cruzi I Sylvio X10/1 and comparison with T. cruzi VI CL Brener. PLoS Negl Trop Dis. 2011 Mar 8;5(3):e984. pmid:21408126
- 24. Grisard EC, Teixeira SMR, de Almeida LGP, Stoco PH, Gerber AL, Talavera-López C, et al. Trypanosoma cruzi Clone Dm28c Draft Genome Sequence. Genome Announc. 2014 Jan 30;2(1).
- 25. Geiger A, Hirtz C, Bécue T, Bellard E, Centeno D, Gargani D, et al. Exocytosis and protein secretion in Trypanosoma. BMC Microbiol. 2010 Jan 26;10:20. pmid:20102621
- 26. De Gaudenzi JG, Noé G, Campo VA, Frasch AC, Cassola A. Gene expression regulation in trypanosomatids. Essays Biochem. 2011;51:31–46. pmid:22023440
- 27. Gulin JEN, Eagleson MA, Postan M, Cutrullis RA, Freilij H, Bournissen FG, et al. Efficacy of voriconazole in a murine model of acute Trypanosoma cruzi infection. J Antimicrob Chemother. 2013 Apr;68(4):888–94. pmid:23212113
- 28. Risso MG, Garbarino GB, Mocetti E, Campetella O, Gonzalez Cappa SM, Buscaglia CA, et al. Differential expression of a virulence factor, the trans-sialidase, by the main Trypanosoma cruzi phylogenetic lineages. J Infect Dis. 2004 Jun 15;189(12):2250–9. pmid:15181573
- 29. De Pablos LM, Osuna A. Multigene families in Trypanosoma cruzi and their role in infectivity. Infect Immun. 2012 Jul;80(7):2258–64. pmid:22431647
- 30. Tonelli RR, Giordano RJ, Barbu EM, Torrecilhas AC, Kobayashi GS, Langley RR, et al. Role of the gp85/trans-sialidases in Trypanosoma cruzi tissue tropism: preferential binding of a conserved peptide motif to the vasculature in vivo. PLoS Negl Trop Dis. 2010 Nov 2;4(11):e864. pmid:21072227
- 31. Schenkman S, Eichinger D, Pereira ME, Nussenzweig V. Structural and functional properties of Trypanosoma trans-sialidase. Annu Rev Microbiol. 1994;48:499–523. pmid:7826016
- 32. Frevert U, Schenkman S, Nussenzweig V. Stage-specific expression and intracellular shedding of the cell surface trans-sialidase of Trypanosoma cruzi. Infect Immun. 1992 Jun;60(6):2349–60. pmid:1375197
- 33. Affranchino JL, Ibañez CF, Luquetti AO, Rassi A, Reyes MB, Macina RA, et al. Identification of a Trypanosoma cruzi antigen that is shed during the acute phase of Chagas' disease. Mol Biochem Parasitol. 1989 May 15;34(3):221–8. pmid:2499788
- 34. Frasch AC, Cazzulo JJ, Aslund L, Pettersson U. Comparison of genes encoding Trypanosoma cruzi antigens. Parasitol Today Pers Ed. 1991 Jun;7(6):148–51.
- 35. Cestari I, Ansa-Addo E, Deolindo P, Inal JM, Ramirez MI. Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles. J Immunol Baltim Md 1950. 2012 Feb 15;188(4):1942–52.
- 36. Silverman JM, Chan SK, Robinson DP, Dwyer DM, Nandan D, Foster LJ, et al. Proteomic analysis of the secretome of Leishmania donovani. Genome Biol. 2008;9(2):R35. pmid:18282296
- 37. Mantel P-Y, Hoang AN, Goldowitz I, Potashnikova D, Hamza B, Vorobjev I, et al. Malaria-infected erythrocyte-derived microvesicles mediate cellular communication within the parasite population and with the host immune system. Cell Host Microbe. 2013 May 15;13(5):521–34. pmid:23684304
- 38. Ståhl A, Arvidsson I, Johansson KE, Chromek M, Rebetz J, Loos S, et al. A novel mechanism of bacterial toxin transfer within host blood cell-derived microvesicles. PLoS Pathog. 2015 Feb;11(2):e1004619. pmid:25719452
- 39. Mercier SK, Donaghy H, Botting RA, Turville SG, Harman AN, Nasr N, et al. The microvesicle component of HIV-1 inocula modulates dendritic cell infection and maturation and enhances adhesion to and activation of T lymphocytes. PLoS Pathog. 2013 Oct;9(10):e1003700. pmid:24204260
- 40. Cooley G, Etheridge RD, Boehlke C, Bundy B, Weatherly DB, Minning T, et al. High throughput selection of effective serodiagnostics for Trypanosoma cruzi infection. PLoS Negl Trop Dis. 2008 Oct 8;2(10):e316. pmid:18841200
- 41. Bernabó G, Levy G, Ziliani M, Caeiro LD, Sánchez DO, Tekiel V. TcTASV-C, a Protein Family in Trypanosoma cruzi that Is Predominantly Trypomastigote-Stage Specific and Secreted to the Medium. PLOS ONE. 2013 juil;8(7):e71192. pmid:23923058
- 42. Meira WSF, Galvão LMC, Gontijo ED, Machado-Coelho GLL, Norris KA, Chiari E. Use of the Trypanosoma cruzi recombinant complement regulatory protein to evaluate therapeutic efficacy following treatment of chronic chagasic patients. J Clin Microbiol. 2004 Feb;42(2):707–12. pmid:14766840
- 43. Krautz GM, Galvão LM, Cançado JR, Guevara-Espinoza A, Ouaissi A, Krettli AU. Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease. J Clin Microbiol. 1995 Aug;33(8):2086–90. pmid:7559953
- 44. Fernández-Villegas A, Pinazo MJ, Marañón C, Thomas MC, Posada E, Carrilero B, et al. Short-term follow-up of chagasic patients after benzonidazole treatment using multiple serological markers. BMC Infect Dis. 2011 Jul 31;11:206. pmid:21801456
- 45. Thézénas ML, Huang H, Njie M, Ramaprasad A, Nwakanma DC, Fischer R, et al. PfHPRT: a new biomarker candidate of acute Plasmodium falciparum infection. J Proteome Res. 2013 Mar 1;12(3):1211–22. pmid:23339668


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Best quality and factory Washer aluminum anodized cnc machinig for Morocco Manufacturers
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Best quality and factory Washer aluminum anodized cnc machinig for Morocco Manufacturers Detail:
1. Name: CNC machining
2. Process: cnc machining, cnc turning, cnc milling,
3. Material grade: Steel, Ss303, brass, aluminum A356, Al6061, A357, metal and variety
4. Machining type: turning, drilling, tapping, milling
5. Finish: Polishing, coating, anodizing etc
6. Dimension: According to customer's drawing or sample
7. Certification: ISO9001: 2000 /TS16949
8. Application: Luxury yacht, machinery part, truck part, Auto part, Industry.
9. Quality control: 100% inspected
10. Color: Per customer's requirement
11.Tolerance: +/-0.01mm
|CNC machining
|Stainless steel, metal, brass, aluminium and variety
|R&D capacity:
|1) Mould/Tooling analysis, design & manufacturing
2) Mould/Tooling testing & confirm the sample
3) CNC machining
4) Surface treatment: Trimming, polishing, cleaning & power coating
5) Full Inspection
6) Packing
|Production Equipments:
|Photo show as below
|Machining Equipments:
|1) CNC milling and turning, grinding, honing, lapping, broaching and other secondary machining.
2) integral CNC machineries & four machining centers, such as milling, broaching, drilling, turning, planning, grinding machinery and three-axle machining centers.
3) CNC Milling Machine
4) CNC Lathe Machine
5) 4-axis machine
|Test Equipments:
|1) Hardness tester
2) Chemical analysis
3) Digital Measuring Projector
4) Dynamic Balancing Tester
5) Universal testing machine & Coordinate-Detector for CNC machining
All test machining can ensure high precision CNC machining parts
|Quality Certification:
|Surface Treatment:
|1) Sand blasting
2) Zinc/chrome plating
3) Powder coating
4) Anodizing,
5) Heat treatment etc for CNC machining
6) Other customer requirement
|Production usage:
|1) Auto parts
2) motorcycle parts
3) pump parts
4) locomotive parts
5) machinery parts
6) metal parts
7) CNC machining
8) machined parts
9) precision machining, etc.
Our Advantage:
1. Professional experience
2. All kinds of material is available
3. SGS Audited
4. High quality with competitive price
5. Fast delivery time
6. Sample available
7. Have export experience to all over the world
8. Precision cnc machining part with competitive price
Quality Control
1.Material: All of our material will be tested when coming.
2.Manufacture Process: Full control of process
1) The worker will do self-inspection first during production, and scrap the part with poor material quality, out of tolerance.
2) The QC will inspect the part dimension random about 1-2 hour, in order to control the dimension and found mistake to make corrective action immediately.
3) When the parts finished machining process, they will be arranged to have surface treatment (such as anodize or powder coated), then, the QC will inspect the parts again,because after surface treatment, the tolerance of products will changed little,if there is any defective parts ,we will pick them out directly.
4) Before shipping,our package worker will check the part surface and see if there is any scratch or other not good items,if yes,pick them out.
We are professional in cnc machining part.
1. For sea shipment, all of our products are packed in standard export wooden case to avoid any damage during shipment.
2. For air shipment, all of our products are packed in PVC single to avoid the damage.
After sale service
1. The quality of products is the most import thing of company, our company always pay most attention in this point, even we have very strictly quality control system, but we still can`t promise every part you received will 100% perfect, so if there is any defective part you received, you just need to offer us the evidence (such as picture), we will check and confirm it. After that, we will repair or redo them.
2.Because of our strictly quality control system,so we have the confidence to promise our customer with this.Please kindly noted that it`s our advantage compare with others,we realize that only the high qualityand good service can keep the friendly and longterm business relationship with our customer and it`s also the only way for an enterprise to be existed.
Welcome to offer a trial order with your drawings!
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Reliable good quality and very good credit standing are our principles, which will help us at a top-ranking position. Adhering to your tenet of "quality 1st, purchaser supreme" for Best quality and factory Washer aluminum anodized cnc machinig for Morocco Manufacturers, The product will supply to all over the world, such as: Italy , Muscat , kazakhstan , With many years good service and development, we have a qualified international trade sales team. Our goods have exported to North America, Europe, Japan, Korea, Australia, New Zealand, Russia and other countries. Looking forward to build up a good and long term cooperation with you in coming future!
Our company has the whole series about pvc profile extrusion machine . Welcome to contact our company directly . Please send your inquiry to the e-mail; firstname.lastname@example.com
By Pamela from Pretoria - 2015.06.19 10:42
We have worked with many companies, but this time is the best,detailed explanation, timely delivery and quality qualified, nice!
By Sabina from Belgium - 2015.11.02 11:11


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Peter Boyce II
Type: Associate
Since joining General Catalyst in 2013, Peter has focused on making early stage investments & working closely with portfolio companies including: Cadre, Outdoor Voices, Mark43, Roman, Rebag, and Major League Hacking. Other investments he has supported include Giphy, Circle, Freebird, Jet.com (acquired by Walmart), and Workflow (acquired by Apple).
In addition, Peter co-founded & oversees Rough Draft Ventures, through which General Catalyst invests in promising university entrepreneurs. With a portfolio of over 150 startups backed on college campuses, RDV has expanded access to capital and mentorship for student entrepreneurs across the country.
Peter graduated from Harvard College with an A.B. in Applied Mathematics and Secondary in Computer Science. While on campus, he co-founded Harvard Ventures & HackHarvard: two student-led organizations that build community around engineers and future founders. Peter grew up in NYC and is a proud alum of Stuyvesant High School.


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I want to hire Trumpet Players in Merced, CA
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Bay Area All Strings & Brass(7)
String Quartet / Trumpet Player
Mountain View, CA
88 miles from Merced
1 Verified Booking
Recent review: I would like to thank Bay Area Strings & Brass for providing a musician to play the trumpet…
Honor Brass Band(4)
Brass Band / Trumpet Player
San Francisco, CA
112 miles from Merced
5 Verified Bookings
Recent review: Honor Brass Band was quick to respond and excellent at communicating throughout the planning…
Dan Zemelman: Award Winning Pianist(11)
Pianist / Trumpet Player
San Francisco, CA
112 miles from Merced
Recent review: Dan did an amazing job for my partners birthday party. I found him initially through Craigslist…
The Gumbo Band(0)
Brass Band / Trumpet Player
San Francisco, CA
112 miles from Merced
: The Gumbo Band; The Gumbo Band is a Mardi Gras Marching and Funeral Band . We also do Zydeco stage shows,…
The Bayou Bump(4)
New Orleans Style Entertainment / Trumpet Player
Los Angeles, CA
257 miles from Merced
6 Verified Bookings
Recent review: We wholeheartedly recommend this amazing band. We hired them to do a 2nd line for our dear friend…
Cloud 9 String Quartet(19)
String Quartet / Trumpet Player
Denver, CO
Serves Merced
39 Verified Bookings
Recent review: Cloud nine did an absolutely fonominal job playing at our wedding Mass. Each one of our guests…
New Orleans Potholes Brass Band(6)
Brass Band / Trumpet Player
New Orleans, LA
Serves Merced
5 Verified Bookings
Recent review: Potholes were so easy to work with! They took care of everything and led an amazing second line…
New Orleans Avengers(24)
Brass Band / Trumpet Player
New Orleans, LA
Serves Merced
48 Verified Bookings
Recent review: The whole band was amazing! They were on time and played great music. They communicated well…
Hacienda Brass Band(19)
Brass Band / Trumpet Player
New Orleans, LA
Serves Merced
35 Verified Bookings
Recent review: When we came out of the church, the Hacienda Brass Band was there and they started the party…
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Quick facts
Trumpet Players in Merced, CA
779 quotes sent
In the past year, Trumpet Players have sent 779 quotes to event planners
194th most popular
Trumpet Players rank #194 in popularity out of 543 categories on GigSalad
4.9 out of 5 stars
273 Trumpet Player reviews on GigSalad with an average rating of 4.9 stars
1.4 days on average to book
From request to booking, it takes 1.4 days (on average) to hire a Trumpet Player
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Trumpet Players in Merced, CA
It's ok for these guys to toot their own horn because when they do it sounds good! Think of the music of Miles Davis and Louis Armstrong. Now bring that music and energy to your event with one of these fantastic trumpet players! Want the trumpet use it's music to bring you down the aisle, or maybe you need a trumpet for a jazz night at your club? Well GigSalad is here to fix all your trumpeting needs! With trumpeters from jazz to herald trumpeteers you are sure to find the right trumpet player for your event. Search for Trumpet Players in Merced, California above.
Please note that these Trumpet Players may also travel to Planada, Atwater, Winton, El Nido, Le Grand, Cressey, Snelling, Livingston, Ballico, Chowchilla, Hornitos, Santa Rita Park, Delhi, and Denair.


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Nick Jonas & Priyanka Chopra's Casual Look Should Be The New Style Code For Engagement Bash
Priyanka Chopra and Nick Jonas kept it simple, real, and fun at their engagement bash. All the guests were dressed to party and Priyanka and Nick were themselves dressed so casually. Well, they clearly redefined engagement bash and encouraged us to dress down for the big occasion.
Priyanka wore a plain-hued structured dress for the function. It was a classy dress, which accentuated her slender frame. Her outfit of the night was deep-necked and was enhanced by a tight bodice. It was a stunning number and Priyanka looked elegant as ever in her dress.
The 'Baywatch' actress teamed her dress with humble pencil heels, which went perfectly well with her engagement bash ensemble. She mostly kept her look jewellery-free but her stunning diamond choker, which was coiled around her neck, was certainly a statement piece.
Her makeup was on the dewy side and highlighted by a wine red lip shade and subtle kohl. Her hairdo was side-swept, which gave her look a romantic touch.
Nick, on the other hand, wore a collared shirt and teamed it with a geometric-patterned sweater and formal pants. He rounded off his look with white-hued loafers.
Well, we loved their unusual party style and certainly looking forward to their wedding.


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Install twrp Recovery and Root Samsung Galaxy S10e
November 30, 2019 | Rooting | No Comments|
We will teach you how to Install twrp Recovery and Root Samsung Galaxy S10e with Odin Windows Application on your Computer
- Take a backup of everything on your Samsung phone, sometimes rooting may cause bootloop i.e Device stuck at the welcome logo screen.
- If your device gets stuck on the boot screen, you need to flash in the stock ROM to fix it.
- Follow this page to get the Samsung stock ROMS
- Download the original mobile driver for Samsung phones
- A working detectable USB Cord.
- The firmware you want to flash.
- A Windows PC.
- A Good Battery Backup
- Odin
- Driver
- Twrp for Samsung Galaxy S10e
How to Install twrp Recovery on Samsung Galaxy S10e
Step 1- Run odin and Check AP/PDA under AP Section Load Downloaded Recovery File [Must compress it in tar Format]
Step 2. Check auto Reboot. Reboot Phone to Download Mode and Connect to PC..
Step 3. You can See COM/added Option. Click Start
Step 4. Wait For Success. Done
How to Root Samsung Galaxy S10e–
- Step 1. Download Magisk 18.1.zip & Move it to Device storage.
- Step 2. Switch of Phone and together Press Power and Volume up button to boot Device into twrp or use code
adb reboot recovery
- Step 3. In the twrp Go to backup tab and Create a nandroid backup. Don't forgot to Include NVRAM or IMEI aka ESF.
- Step 4. Go to Install tab and Locate Download Magisk 18.1.zip and confirm Install, Wait For Success Done.
- Step 5. Flash Noverity opt Encrypt.zip to Disable boot Verification.to fix RMM State Flash this
- Now, disconnect your Samsung phone from your computer and restart it
- Your Samsung device is hopefully rooted. To confirm the root, check if the SuperSU App is installed on your Samsung device. if the app is present on your phone is root. You can also download Root Checker APP from Playstore and check with it.
For stock roms and official firmware for Android smartphone follow the link below.
Theses stock roms can be rooted in using many ways, rooting phones gives you access to many powerful features that are hidden on the phone


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Supports PDF, DOC, DOCX, TXT, XLS, WPD, HTM, HTML files up to 5 MB
Job Description:
Contribute to a safer, secure and more sustainable World!
At UL, we know why we come to work. Thousands of us around the world wake up every day ..
... and implementation of enterprise wide communication strategies. To support communications excellence ... frameworks to ensure high-quality, aligned, brand compliant communication is delivered to the market. ... across function Defines..
... Visual Basic SharePoint Media Relations Communication Skills Organizational Skills Description Developing ... on a variety of strategic communication programs that improve organizational alignment ... such as internal and external..
Job Description
Reporting to the Vice President of Internal Communications, you will lead the team responsible for editorial strategy, campaign support, project management, and the best practices playbook for the internal ..
... driving employee engagement and corporate communication with an emphasis on patient ... effectiveness through the creation of communication processes and guidelines. Identifies and ... look and feel of Astellas..
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... Summary Were looking for a communication professional who is strategic and ... for developing and implementing integrated communication plans. This role is on ... at all levels. The Communications..
... Summary We're looking for a communication professional who is strategic and ... for developing and implementing integrated communication plans. This role is on ... at all levels. The Communications..


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Star Stories
A beautiful book with stories from different cultures. A stellar gift for the myths, monsters, and folktales fan.
A beautiful book with stories from different cultures. A stellar gift for the myths, monsters, and folktales fan.
A tour-de-force tour of the human body - organs, systems, glands, everything you could imagine. Written for the layperson but chock-full of amazing anatomy explanations as well as factoids that you will want to remember for future conversation but of course will never be able to keep track of (the liver is part of 500 different metabolic processes and contains a quarter of all your blood). Fascinating digressions about particular diseases, scientists, discoveries and yet more facts. (And if you're an audio-book lover, Bryson's own reading of the book on Libro.fm is fabulous.)
With regards to the coming catastrophic effects of climate change the first sentence of this powerful polemic says it all: "It is worse, much worse, than you think."
This left me both frightened of all the problems to come, but at the same time optimistic of all the problems that'll finally be be solved.
Before reading this book, I thought I had a decent idea of how the world works. Shortly into the first chapter, I realized how little I understood. From networks to blockchains, Greenfield masterfully critiques nine foundational technologies that often invisibly shape our lives. An important book for anyone wanting a thoughtful relationship with the world around them.
Nobel Prize winning researcher and teacher Eric R. Kandel, with profound empathy, essentially delivers a survey of current knowledge of the workings of the brain using the frame of brain disorders. Our understanding of the biology of the mind is changing rapidly and Kandel charts these advances in clear and very comprehensible language explaining how they are leading to new insights about human nature and consciousness itself.
Introduces you to all the people who watched the moon landing and said "That's what I want to do with my life," with or without NASA.
This is a fantastic nonfiction picture book about one of the champions of coral reef restoration. Beautiful illustrations and an inspiring true-life story.
In July of 2015, the New Horizons spacecraft completed the first ever flyby of Pluto. For me and many others, this was the first I'd heard of New Horizons, but Alan Stern and the Pluto Underground had been working towards this since 1989, and the process was far from easy. Follow the epic journey from idea to proposal to actual mission to the furthest and final unexplored planet in our solar system. You'll learn about planetary science, space exploration, and Pluto itself.
Never mind what you've heard about the coming effects of climate change. As Elizabeth Rush disturbingly documents, we are already seeing widespread effects of sea level rise. With great empathy and a lyrical voice she closely examines the response of various American coastal communities to the dramatic changes they struggle to face.


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Constance from Fairfield Age: 28. Pretty slut.I meet when a man is nice, then I also get pleasure from it.
Actually there really was a Manuela, who remained lame all of her life after she threw herself down the stairs. There is a meeting asking the teachers in the school and the headmistress. US VHS video release cover. An International Bio-Critical Dictionary.
Patricia from Fairfield Age: 22. Looking for an experienced man, hope for a passionate night)
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From Wikipedia, the free encyclopedia. Ilsa is to play another major role in the play, but is barred from performing after her letter to her parents denouncing the school is returned because of a wrong address. Thiele also said, "However, I really don't want to make a great deal of [ Films directed by Carl Froelich. It was also distributed in Japan, the United States where it was first banned, then released in a heavily cut version , England and France.
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An International Bio-Critical Dictionary. Having mostly played the same roles on stage, the cast was able to produce the film at speed and on a low budget of RM 55, Although sound had only been used for two years in cinema, it was used artfully. That's far from my mind, because the whole thing of course is also a revolt against the cruel Prussian education system. After Leipzig the play was produced on the stage in Berlin as Gestern und heute [2] with a different cast and a more prominent lesbian theme, which was again toned down somewhat for the film.
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20 Apr 2018, Aleen Joy Jarasa, EMIS Editor
Tencent invests $100 million in India's Dream11
The development comes shortly after the High Courts ruled in favor of Dream11, judging that playing online games on the platform does not equal to gambling. The ruling has cleared the decks for a substantial investment from global investors and potentially giving a major boost to the sports gaming industry, The Economic Times has added.
Once the deal is sealed, it will be the second largest deal closed by Tencent in India over the last two months, with the $115-million investment in Gaana being the first.
Sign in and read the full article in emis.com Original source: The Economic Times


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The third autumn wave of the project is aimed at the consumer preferences in the purchase and operation of winter tires.
Ford Mondeo sedan has received a technological upgrade aimed at the safety improving of the car and improving of the intelligent driver assistance systems. People can order updated Ford Mondeo at all official Ford dealers in Russia.05 julymore info
On 1 October AvtoVAZ is launching the new action, which will decrease prices for the Lada Samara family by almost 20,000 roubles. This information was given by Alexander Bredihin, director of AvtoVAZ marketing department, during the interview for Eho Moskvy radiostation. He also said, that special prices for the Lada Samara will be active from 1 October till 31 December.
At that he mentioned, that the cheapest car of the Lada Samara family, three-door hatchback with Standard package will cost 239,900 roubles. "It is a very good price and a very good offer. We hope that the demand for the Lada Samara cars will grow significantly during these three months", - A. Bredihin said. The director of AvtoVAZ marketing department also mentioned, that on 1 January 2012 AvtoVAZ will stop production of the Lada Samara sedans.
From January till August 2011 the Lada Samara sales in the Russian market came up to 74,755 cars – that is 10% more than a year ago.
|Source: www.autostat.ru
AvtoVAZ signed an agreement with General Motors on the repurchase of a 50% stake in Togliatti joint venture GM-AvtoVAZ from an American company.more
The Italian car market grew by 2.2% and reached 150,587 cars in November. According to the results of 11 months of 2019, car sales fell by 0.6% and reached 1 million 775 thousand 884 units in Italy.more
The car market grew by 0.7% to 172,735 cars in France in November. Thus, positive dynamics is recorded for the third month in a row.more
Experts of the analytical agency AUTOSTAT conducted a study of the financial capacity of the market of new cars in RF regions in 2011-2013.more
In 2012 the number of cars in Russia grew more than 10% in comparison to last year.more
According to data of analytic agency AUTOSTAT, the number of sales of cars was 2,7 million units in the after sales market in January-June of 2012 and it was more by 24% than in January-June of the previous year, when it was sold 2,1 million of new carsmore


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Both equally incoming and outgoing phone calls can be recorded with the Simply call Recorder Plus. Click here: Get Simply call recorder In addition for iOS. Call Recorder Totally free. An iOS consumer can simply just history each, the incoming and outgoing calls with the enable of Get in touch with Recorder Totally free. The most wonderful function of the Application? It's totally free.
All you are essential to do is tap the record button on the application and the application alone will dial the selection and therefore merge the phone calls in get to begin recording. While for outgoing calls, you need to have to do the exact same by the dialing the recording variety to start with http://callrecorderios.com/ followed by dialing the human being you want to chat to and merge these phone proceedings. Click listed here: Get Phone Recorder Absolutely free for iOS. Call Recorder – IntCall. This is a further no cost Voice Recording app out there for iOS system. This app will get integrated with one's mobile phone and even to his VoIP software program and can record calls.
The recordings will be placed beneath Get in touch with Recorder – IntCall's servers of VoIP. The recordings can be retrieved at any stage of time and can be heard specifically from the machine. The recordings can even be despatched to a desired staff by the medium of E-mail.
How will i record on my small apple iphone
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Numerous people today assume that there is no Voice Recording attribute in Google Voice, but if observed closely then it can be noticed that Google Voice, which is just one of the best VoIP apps also comprise of Connect with Recording facility. All one has to do is enable this aspect in his Apple iphone and press 4 although on call.
It will start recording, and urgent 4 yet again will stop the recording. iPadio. iPadio is a little totally free app available on the Apple Application Retailer and can be applied to document not only calls, but also films and even audio. The calls recorded by this app get saved to the iPadio On the net Account and can be accessed on a later on stage each time necessary. This application, however isn't going to deliver a incredibly easy way of recording calls, but overall is a useful and great-to-use app. In buy to record calls, one has to initial phone the iPadio's quantity, punch in the Unique PIN sent to the person, and then merge the call which is to be recorded.
While this could seem to be to be a cumbersome endeavor, but neither does it consider time, nor is it a headache. Call Recording by NoNotes. com. This is just one of individuals apps that offer dictation providers far too to the recorded call. NoNotes. com is a really renowned contact recording and ECommerce transcription provider. The app furnished by them is a multi-intent app and saves all the recorded calls on the cloud server. The application allows the recording of incoming as perfectly as outgoing calls and help you save them on the Cloud server, from where they can be retrieved whenever needed.