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Titanium dioxide (TiO2) anatase nanoparticles (NPs) are metal oxide NPs commercialized for several uses of everyday life. However their toxicity has been poorly investigated. Cellular internalization of NPs has been shown to activate macrophages and neutrophils that contribute to superoxide anion production by the NADPH oxidase complex. Transmission electron micrososcopy images showed that the membrane fractions were close to the NPs while fluorescence indicated an interaction between NPs and cytosolic proteins. Using a cell-free system, we have investigated the influence of TiO2 NPs on the behavior of the NADPH oxidase. In the absence of the classical activator molecules of the enzyme (arachidonic acid) but in the presence of TiO2 NPs, no production of superoxide ions could be detected indicating that TiO2 NPs were unable to activate by themselves the complex. However once the NADPH oxidase was activated (i.e., by arachidonic acid), the rate of superoxide anion production went up to 140% of its value without NPs, this effect being dependent on their concentration. In the presence of TiO2 nanoparticles, the NADPH oxidase produces more superoxide ions, hence induces higher oxidative stress. This hyper-activation and the subsequent increase in ROS production by TiO2 NPs could participate to the oxidative stress development.
Citation: Masoud R, Bizouarn T, Trepout S, Wien F, Baciou L, Marco S, et al. (2015) Titanium Dioxide Nanoparticles Increase Superoxide Anion Production by Acting on NADPH Oxidase. PLoS ONE 10(12): e0144829. doi:10.1371/journal.pone.0144829
Editor: Elena A. Rozhkova, Argonne National Laboratory, UNITED STATES
Received: July 22, 2015; Accepted: November 23, 2015; Published: December 29, 2015
Copyright: © 2015 Masoud 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 financial support of ANR 2010-blan-1536-01 is acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Titanium dioxide (TiO2) nanoparticles (NPs) are metal oxides NPs manufactured in large quantities and commercialized for several uses because of their high stability, anticorrosive and photocatalytic properties [1]. For example, they are present in household products, plastics industry, electronics, pharmaceutical additives and food colorants [2,3]. In nanomedicine, TiO2 NPs are under investigation as useful tools in advanced imaging and nanotherapeutics [4]. TiO2 NPs are being explored in cancer diagnosis. They bring many benefits in cancer therapy by absorbing near infrared light [5], and thus being considered as potential photosensitizers for photodynamic therapy [6]. Very promising is the finding that photo-activated nanostructured TiO2 exhibited selective cytotoxicity against breast epithelial cancer cells [7]. Furthermore, the physical properties of TiO2 NPs make them very interesting products for a use in various skin care and cosmetic products such as sunscreens [8]. TiO2 NPs are under investigation as novel treatments for acne vulgaris, atopic dermatitis, hyperpigmented skin lesions, and other non-dermatologic diseases [2,3].
Despite their omnipresence in everyday life, modest research effort has been made in studying their potential adverse effects on living bodies and environment. TiO2 NPs can be absorbed into the human body by inhalation, ingestion, and dermal penetration, then they can be distributed to vital organs, including lymph, brain, lung, liver, and kidney [9–11]. TiO2 NPs can enter not only in cells, but also mitochondria and nuclei [12]. Most work to date has shown that TiO2 NPs toxicity is strongly related to reactive oxygen species (ROS) generation and consequent oxidative stress [12–16].
TiO2 NP-mediated ROS responses have been reported to orchestrate a series of pathological events leading to genotoxicity, immunotoxicity, neurotoxicity and carcinogenicity [17,18]. Neutrophils have been shown to be quickly recruited to titanium dioxide areas [19]. Moreover, cellular internalization of TiO2 and ZnO NPs has been shown to activate immune cells including macrophages and neutrophils that contribute to ROS production [20–24]. TiO2 NPs increased respiratory burst when fish neutrophils were incubated with these NPs [25]. Moreover, they lead to the activation of human ones [23,24]. Recently the same group showed that these nanoparticles enhance the ability of human neutrophils to exert phagocytosis by acting on Syk-dependent signaling pathway [26]. ROS production involves the activation of NADPH oxidase enzymes [22,27], a key player of oxidative stress in immune system cells but also in many other cell types (thyroid, kidney, neurons, and skin) [28–32].
NADPH-oxidase is the only enzyme whose function is to generate superoxide free radicals, which are transformed subsequently into other ROS [33–36]. It is a multi-subunit enzyme complex composed of membrane-bound flavocytochrome b558 (cyt b558), comprising two subunits (Nox2 also known as gp91phox, and p22phox), present in the membranes phagocytes, and four cytosolic components. Nox2 harbors all the redox carriers (bound FAD, two hemes and the NADPH binding site) that transfer electrons from one side of the membrane cell to the other. The cytosolic components include p47phox, p67phox, p40phox, and a small GTPase Rac1 or Rac2 [37].
In resting phagocytes, the components of the complex exist as separated entities but upon cell activation by pro-inflammatory mediators, the cytosolic subunits undergo posttranslational modifications such as phosphorylation and migrate to the membrane bound cyt b558 to constitute the activated NADPH-oxidase complex, the only form able to produced superoxide ions [38].
The aim of our study was to investigate the enzymatic behavior of NADPH oxidase in the presence of TiO2 NPs and to check if NADPH oxidase could be a pathway involved in ROS generation by TiO2 NPs as it has been suggested [27]. We have developed a cell-free system [39–41] that allows controlling the environment, testing and identifying the potential effects of different molecules in various steps of oxidase activation [42,43]. In such cell-free systems, activation is obtained by mixing all proteins with an activator, arachidonic acid (AA). In this study, we have used a construction called trimera, which consisted of the following domains Nter-p47phox (amino acids 1-286) linked to the N-ter p67phox (amino acids 1-212), and the full length Rac1 Q61L [44]. In a previous paper, we have verified that the rates of production of superoxide anions were similar when the classic cytosolic proteins are replaced by trimera protein to activate the cyt b558 and also that the dependences of the activity in function of the enzyme activator AA concentration were also found comparable with the cytosolic fractions and the trimera [45]. Thus, the trimera was chosen in order to avoid complications due to some assembly steps and to activate the cyt b558 in a reproducible manner. We have constantly validated our main conclusions with the separated subunits. We studied not only the effect of TiO2 NPs on the function of NADPH oxidase but we also examined their effects on proteins conformations by different methods (fluorescence, synchrotron radiation circular dichroism (SRCD), transmission electron microscope (TEM), dynamic light scattering (DLS)). The use of these combined methods has provided a broad view of how TiO2 NPs influence NADPH oxidase functioning and hypotheses about the origin of oxidative stress TiO2 NPs dependent.
Material and Methods
Equine heart cytochrome c (cyt c), superoxide dismutase from bovine erythrocyte, arachidonic acid (AA), phenylmethanesulfonyl fluoride (PMSF) and Dulbecco phosphate buffer saline (PBS) and standard PBS were from Sigma (Saint Quentin Fallavier, France). Reduced nicotinamide adenine dinucleotide phosphate (NADPH) was from Acros. Ni-sepharose, superdex 75 and Ficoll-Paque Plus were from GE Healthcare, France. Anatase TiO2 NPs were a generous gift of Dr Hynd Remita. They were suspended in deionized water (1 mg/mL) and sonicated in an ultra-sound bath for 10 min before use. The experiments were performed in phosphate buffer saline (PBS buffer). It is known that TiO2 NPs are affected by the buffer and especially by phosphate [46]. We chose this buffer to be as close as possible to living medium.
Neutrophil membrane preparation
The neutrophils were prepared from human blood from healthy donors (ESF Paris, France) as described in [47]. Briefly, 500 mL of blood was sedimented in 2% dextran solution for 40 min and centrifuged 400 x g 8 min. Dulbecco PBS was added to the pellets, and then the neutrophils were separated from lymphocytes and the red cells by centrifugation for 30 min at 400 x g on Ficoll solution. The red cells were further eliminated after their lysis by centrifugation for 8 min, 400 g, 4°C. The pellet resuspended in PBS pH 7.4 containing 340 mM sucrose, 7 mM magnesium sulphate, 1 mM PMSF, 0.5 mM leupeptin was sonicated in the 30% pulse mode at power pulses (6) in an ice-cooled beaker 6 times during 10 s with resting time of 1 min between the sonications (sonicator XL, Misonix inc.). Neutrophil membranes and cytosol were separated by centrifugation for 1h30 at 200 000 g at 4°C. The membrane fractions were resuspended, aliquoted and stored at -80°C for further experiments.
Trimera preparation
The plasmid coding for the trimera was kindly provided by Prof. E. Pick (University of Tel Aviv, Israel). It codes for the Nter-p47phox (amino acids 1-286) linked to the N-ter p67phox (amino acids 1-212), and the full length Rac1 Q61L [44]. The trimera was expressed and isolated from E.coli BL21-(DE)3-plysS. Purification of trimera was performed mainly as previously described [45]. Briefly, after a first step through a nickel affinity chromatography, the protein was further purified by size exclusion chromatography; the protein was then dialyzed overnight against a phosphate buffer (100 mM NaCl and 30 mM Na2HPO4, pH 7.5) and stored at −80°C. The protein concentration was estimated using a NanoDrop2000 spectrophotometer (Thermo scientific, France) and the extinction coefficient of 124,000 mol-1 L cm-1 at 280 nm (Expasy, Protparam). The purity of all proteins were checked by migration on 10% BisTris-NuPAGE SDS gels (Invitrogen), stained with Coomassie Brilliant Blue and quantified by the ImageJ software.
Dynamic light scattering measurements
Dynamic light scattering (DLS) experiments were performed to estimate the NPs size. DLS measurements were performed at room temperature on a Malvern NanoZS equipped with a 633 nm laser. Data were collected with a scattering angle of 173°. A range between 2 and 60 μg/mL of TiO2 NPs suspensions prepared in PBS or water was tested.
Transmission electron microscopy measurement
The morphology and size of NPs were also determined by transmission electron microscopy (TEM). The solutions contained 0.5 mg/mL TiO2 NPs +/-50 μg/mL trimera and +/-1 mg/mL membrane proteins containing 25μg/mL cyt b558. 4 μL of the suspension was deposited onto glow-discharged carbon-coated copper grids and after 1 minute of interaction, the excess of solution was removed with a filter paper (Whatman). As a result, the sample is dried onto the support. Zero-loss (20 eV window) images of TiO2 NPs were acquired on field emission gun transmission electron microscope operating at 200 kV (JEOL 2200FS, JEOL LTD®).
Intrinsic fluorescence Assays
Steady-state fluorescence spectra were performed on Fluorolog3- Horiba spectrofluorimeter at 25°C. Various concentrations of TiO2 NP suspensions (10- 100 μg/mL) were added as indicated to a final volume of 3 mL of buffer (PBS supplemented with 10 mM MgSO4,) containing trimera (5 μg/mL, 60 nM) in a quartz cuvette. The tryptophan fluorescence spectra of trimera were obtained by exciting the samples at 290 nm (2 nm bandwidth) and recorded between 300 to 550 nm (5 nm bandwidth). The excitation wavelength was chosen at 290 nm to optimize the signal to noise ratio and to reduce the contribution of tyrosine residues to the signal [48]. 3 mL of buffer was used as baseline.
Circular dichroism spectroscopy
Synchrotron radiation circular dichroism (SRCD) spectra were measured on the DISCO beam Line at the synchrotron radiation SOLEIL, Gif/Yvette, France. The calibration was made using a solution of camphorsulphonic acid (CSA). Spectra were measured over the wavelength range from 170 to 260 nm. Three scans were measured and averaged for the samples and the baseline. The averaged baseline was subtracted from the samples and the curves obtained smoothed. SRCD spectra were recorded at 25°C. The solutions contained 1.5 mg/mL (18 μM) trimera +/- 60 μg/mL TiO2 NPs, +/- 300 μM AA prepared in 100 mM sodium fluoride; 10 mM sodium phosphate pH 7.0. Spectra are expressed in delta epsilon units, calculated using mean residue weights of 82,681 Da for the trimera. They were fitted using the free software BestSel [49]. Control spectra were recorded with TiO2 NPs.
Measurement of superoxide ion production rates
Superoxide anion production rates were indirectly quantified by the initial rate of cytochrome c (cyt c) reduction, as previously described [50]. The reaction is the following:
Unless indicated, the components of the cell-free system were added as follows: membrane fractions (MF; 2-5 nM cyt b558), trimera (100-200 nM) and arachidonic acid (40 μM) in 500 μl PBS supplemented with 10 mM MgSO4 and incubated for 4 minutes at 25°C in order to allow the NADPH oxidase complex to assemble. The production was initiated by addition of NADPH (250 μM) and the rate of O2-• was quantified by the reduction of cyt c (50 μM). The rate was measured at 550 nm in a Thermo evolution 500 spectrophotometer, using a molar extinction coefficient (Δε of the reduced minus oxidized form of cyt c) of 21 mM-1 cm-1. Control of the production of the O2•- species was performed by addition of 50 μg/mL superoxide dismutase (SOD).
3.1 TiO2 NPs size characterization
The hydrodynamic size of TiO2 NPs in water and PBS was estimated by DLS. The average size of the NPs aggregates in water was about 350 ± 50 nm for the concentration range of 2-80 μg/mL of TiO2. This NP aggregate population was predominant (100%) for TiO2 NPs concentration lower than 20 μg/mL but another population of larger agglomerates whose sizes were estimated to about ~2000 nm (5%) appeared when TiO2 NPs concentration was higher than 20 μg/mL. We noticed also that the size of the NP aggregates in a physiological medium, such as PBS, is similar (460 ± 50 nm) to that in water. By TEM we can also observe particle aggregation (Fig 1A). The aggregates are constituted by particles of about 30± 5 nm (Fig 1B). These results are in accordance with those in the literature where it was shown that TiO2 NPs tend to associate to form relatively strongly bonded aggregates or soft agglomerates [17]. TEM images also showed that TiO2 NPs are in contact with the membrane fractions (Fig 1C). Moreover, aggregation of TiO2 NPs is similar when they were together with the proteins, (Fig 1C). Similarly, DLS measurements showed that the size of TiO2 NPs aggregates did not change when MF (0.5μg/mL cyt b558) and trimera 18 μg/mL were added to 20 μg/mL TiO2 NPs. The concentrations were similar to what we have in the cell free system assays for the measurements of NADPH oxidase activities.
The samples are dried (see materials and methods).
3.2 Tryptophan fluorescence of trimera in the presence of TiO2 NPs
The conformation changes of the trimera were evaluated by measuring the intrinsic fluorescence spectra of tryptophan residues, before and after addition of TiO2 NPs. Trimera contains a total of thirteen tryptophan residues (seven, four and two in the p47phox, p67phox and Rac portions, respectively). The amplitude of the emission spectrum decreased linearly by the addition of TiO2 NPs without any change of the wavelength at the maximum (340 nm) (Fig 2, S1 Fig).The decrease of fluorescence intensity might indicate a quenching due to proximity of TiO2 NPs and some tryptophan residues without change in the surrounding of these residues. The intensity of the shoulder at around 440 nm increased concomitantly with the decrease of the intensity of the 340 nm band and is due to emission from TiO2 NPs (inset of Fig 2). A similar quenching happens with Trp amino acid in solution with a bathochromic effect on the maximum. This indicates some affinity between Trp and TiO2 NPs (S2 Fig)
The solution contains 5 μg/mL (60 nM) trimera and TiO2 NPs at the concentrations of 0, 10, 40, 80 and 100 μg/mL in a final volume of 3 mL of buffer (PBS supplemented with 10 mM MgSO4). The emission spectra were measured using an excitation wavelength of 290 nm as described in the Materials and Methods section. Results are representative of at least three independent experiments. In inset: enlargement of the fluorescence spectrum in the region 360-500 nm for three solutions. Fluorescence spectra of 5 μg/mL trimera alone (green), 100 μg/ mL TiO2 NPs alone (red) 5 μg/mL trimera in the presence of 100 μg/ mL TiO2 NPs (blue).
The eventual changes of the secondary structure due to the NPs were investigated by SRCD spectroscopy. We have recorded the SRCD spectra of 1.5 mg/mL (18μM) trimera in the absence and in the presence of 60 μg/mL TiO2 NPs and 300 μM AA (Fig 3). In S1 Table are gathered the percentages of α-helices and β-sheets obtained by fitting the spectra with the Bestsel software [49]. Analysis of the SRCD spectra of the trimera indicates that this chimeric protein is mostly in random coil (ca. 40%) and that the content of helices is very low (3-4%) (S1 Table). Although it was supposed that the addition of an amphiphile like AA would induce larger changes in the structure of the cytosolic proteins, [48,51] we observe only slight modifications of trimera secondary structure upon addition of AA. Similarly, only slight changes in the structure are observed with NPs. There is a loss of α-helixes and an increase of the disorder (S1 Table).
SRCD spectra of the trimera (18 μM) alone (Blue) and in the presence of TiO2 nanoparticles (60 μg/mL) (sky blue) and AA (300μM) (red). The solvent was NaF 100mM /NaPi 10 mM pH 7, 25°C. The points are experimental, the curves are the fits using BeStSel [49].
Altogether these results show that the interaction between NPs and trimera, indicated by fluorescence quenching, have no big consequence on the secondary structure of trimera.
3.3 Effects on the functionality
3.3.1 Effects of TiO2 NPs on the NADPH oxidase activity.
First, we have checked that TiO2 NPs alone did not reduce cyt c (data not shown), which means that in these conditions, NPs by themselves do not produce superoxide ions. In order to investigate the effect of NPs on the NADPH oxidase, the rate of superoxide anion production was measured upon addition of TiO2 NPs in the cell free assay conditions previously optimized with trimera [45] (Fig 4). The initial slope of the kinetic curve is equal to the rate of superoxide anion formation. This rate was faster in the presence of TiO2 NP. The identification of O2-• was performed by addition of 50 μg/mL SOD.
Neutrophil membrane fractions (5 nM cyt b558) and trimera 200 nM were incubated together in the presence of 40 μM AA and (0, 20, 30 μg/mL) TiO2 NPs. The production was initiated by addition of NADPH (250 μM) and the rate of O2-• was quantified by the reduction of cyt c (50 μM). Control was performed by the addition of 50 μg/mL SOD. (on the fig: 20μg/mL TiO2 in the presence of SOD). The initial rates of production of superoxide are the following: 92.0±0.3, 134.0±0.5, 119.2±0.4 mol O2•-/s/Mol Cyt b558 for TiO2 NPs 0, 20, 30 μg/m respectively.
The activity of the complex was investigated in parallel with either the trimera or the mix of cytosolic proteins p47phox, p67phox and Rac. All components were incubated together with TiO2 NPs (2-60) μg/mL and 40 μM AA. The rate of superoxide anion production in the absence of NPs was considered as 100% of NADPH oxidase activity. No major difference was noticed between the trimera and the cytosolic proteins (Fig 5). In both cases we clearly observed an increase in the NADPH oxidase activity in the presence of NPs. The curves of Fig 5 exhibit a bell shape profile with a maximum (140% of the reference) at around 20 μg/mL of TiO2 NPs. For higher concentrations of TiO2 NPs (> 20 μg/mL), the rate returned close to the activation level of the control. This result indicates that TiO2 NPs potentiate the NADPH oxidase activity. The activity remains constant for concentrations higher than 40 μg/mL probably due to some aggregations of NPs at higher concentrations.
Neutrophil membrane fractions (5 nM cyt b558) and trimera 200 nM (blue dots) or the cytosolic subunits (p67phox 200 nM, p47phox 260 nM and Rac 580 nM) (red squares) were incubated together in the presence of 40 μM AA and TiO2 NPs. Oxidase activities were expressed as the percent of activity measured in the absence of TiO2 NPs (90 mol O2.-/s/mol cyt b558), and determined as 100%. Points are an average of 3 independent measurements. The dotted curve is a visual fit for both systems.
Thus, we further questioned whether TiO2 NPs alone (20 or 40 μg/mL) could activate the NADPH oxidase complex and thus replace AA as activator (Fig 6). Almost no NADPH oxidase activity (5 ± 2%) was detected with NPs instead of AA (control). Comparable results were obtained using the separated subunits where a maximum activity of 4 ± 2% of AA- dependent activity was reached (data not shown).
Membrane fractions (4 nM cyt b558) with trimera 200 nM were incubated 4 min in the presence of 0, 20 or 40 μg/mL TiO2 NPs. Control experiment representing 100% (83 mol O2•−/s/ mol cyt b558) of the activity was realized in presence of 40 μM AA and in absence of TiO2 NPs. The rates of superoxide production were measured as described in Materials and Methods. Data are the average of 3 independent measurements.
Since TiO2 NPs cannot be considered as activating molecules, the significant increase in the rate of O2•− production with NPs might be due to an indirect effect on the optimized oxidase condition by disturbing the optimal AA concentration. We therefore investigated the effect of TiO2 NPs on the AA activation profile. To probe this effect, we performed titrations of the oxidase activity vs. AA concentration in the absence and in the presence of 20 μg/mL TiO2 NPs added after arachidonic acid (Fig 7). The rate of production with 40 μM AA alone (the concentration used as reference in this paper), was considered as 100%. In agreement with the above -mentioned results, in the presence of TiO2 NPs, the O2•− production rate was higher on the full range of AA concentrations. Both curves exhibited bell-shapes as usual but the optimal concentration of AA was lower (ca. 62 μM) in the presence than in the absence (ca. 90 μM) of NPs. Similar results were obtained when NPs were added before AA (data not shown).
Neutrophil membrane fractions and trimera were incubated together in the presence of different concentration of AA. The TiO2 NPs concentration was as follow, blue dots: no TiO2 NPs; red squares: 20 μg/mL TiO2 NPs. Oxidase activities were expressed as the percent of activity measured in the presence of 40 μM AA (85 mol O2.-/s/mol cyt b558) set as 100%. The curves are visual fits of the experimental points and the maxima have been indicated by crosses. The rate of O2•− production was measured as described in Materials and Methods.
3.3.2 Effect of TiO2 NPs addition at different sequences of cell free system assay.
To examine whether TiO2 NPs have effects on specific steps of the assembly, several concentration of TiO2 NPs (10, 20, 40 μg/mL) were added at different times: (i) to the membrane fractions alone before mixing to the cytosolic subunits; (ii) to mixed membrane fractions and trimera; (iii) to the membrane fractions plus trimera plus AA (Fig 8). Regardless the stages at which TiO2 NPs were added, the rates of production of superoxide were the same within uncertainty. The highest O2•− production was still observed when 20 μg/mL TiO2 NPs were incorporated in the system whatever the sequence of addition of NPs.
Neutrophil membrane fractions (4 nM cyt b558) and 200 nM trimera were incubated together in the presence of 40 μM AA and TiO2 NPs (10, 20, 40 μg/mL). TiO2 NPs was added to the solution either after the membrane fractions or after the membrane fractions and trimera or after the membrane fractions, trimera and AA. Oxidase activity was expressed as the percent of activity measured in the absence of TiO2 NPs (84 mol O2.-/s/mol cyt b558) set as 100%. Results are presented as the mean±SD of 3 independent experiments.
Discussion and Conclusion
Oxide nanoparticles are widely used and their toxicity levels seem to be quite different albeit always related to induction of oxidative stress [27]. Some work has been done on the toxicity of ZnO NPs. A ROS formation enhancement was observed in ZnO-treated liver cells [22,52,53] and on macrophages from wt mice, whereas this formation was impaired in the treated macrophages from the p47phox-/- animals. To our knowledge, this is the only work involving NADPH oxidase. [22]. The use of TiO2 NPs has become widespread including in situations where they can be absorbed by living bodies. The photocatalytic activity of TiO2 is well known [54], however UVA and visible light do not penetrate inside the body. Thus there is no light exposure and no activation of TiO2 NPs by photo-catalysis.
The toxic effects of TiO2 NPs seem to be mainly due to indirect production of ROS and therefore to induction of oxidative stress. One of the first studies about interaction between NPs and neutrophils was done in 1988; Hedenborg demonstrated that TiO2 induced the production of ROS by human neutrophils [55]. It has also been shown that TiO2 NPs can induce oxidative damage to human bronchial epithelial cells in the absence of photoactivation [54,56]. They are known to enhance superoxide production in osteoblasts [16]. TiO2 NPs were shown to interact with proteins and enzymes in hepatic tissues, interfering with antioxidant defense mechanisms and leading to generation of ROS [57]. Since NADPH oxidase is a major actor of oxidative stress by producing superoxide ions, it was evident that investigating the effect of TiO2 NPs on this enzyme constitutes a relevant issue.
The aim of this paper was to obtain comprehensive information on the interaction of TiO2 NPs with the NADPH oxidase. To facilitate such studies, we used a model system that allows performing deeper studies. The different tests performed either with the trimera or with the three separated subunits showed similar results.
It is known that the cytosolic proteins must undergo conformational changes to lead to active enzyme. TiO2 NPs have no significant effect on the secondary structure, as shown by the CD spectra (Fig 3). However the fluorescence of the Tryptophan residues is affected by the presence of NPs. Both results are similar to those obtained with fibrinogen [58]. The quenching of fluorescence of the endogenous tryptophans of the trimera indicates that the NPs are probably close to one or several Trp residues and implies that a complex may be formed between the NPs and the cytosolic protein. In vivo, it was shown that proteins adsorb on TiO2 NPs. In some cases, these NPs induced conformational changes in proteins and affected their functions [12,59,60].
The CD spectra of the trimera in the presence of TiO2 NPs and in the presence of AA (Fig 1 and S1 Table [61]) do not exhibit much difference. The Nps have no significant effect on the secondary structure of the trimera as suggested by the CD spectra. Yet, we do not observe any ROS production from NADPH complex in the absence of AA suggesting that TiO2 NPs are unable by themselves to activate the enzyme. TEM images showed that membrane fractions are close to TiO2 NPs and suggest also an interaction between TiO2 NPs and the membranous proteins. Obviously this interaction with the MF does not replace that of the trimera and of AA for activation, and does not prevent AA from having access to the MF, however it might be responsible for the hyperactivation.
Surprisingly, the presence of NPs increases the rate of superoxide anion production (up to 140% of its value without NPs), this effect being dependent on the NPs concentration. They do not interact at a specific step of activation, indicating that their targets are indifferently the membrane fraction as well as the cytosolic proteins and that they can work on the system even when the entire complex is assembled and active. Since the presence of TiO2 NPs modifies the AA-dependent activation profile of the enzyme shown in Fig 7, we can postulate that more efficient structure of the NADPH oxidase complex is attained in the presence of NPs. We can exclude a consequence of AA availability due to NPs since the higher NADPH oxidase activity is observed at lower concentration of AA in the presence of NPs than in their absence. This phenomenon cannot be attributed only to an interaction with the sole membrane fraction since the sequence of addition of the NP has no effect on it. An effect on the cytosolic fractions is also likely. Taken together, these facts indicate that the secondary structure of the cytosolic proteins may be conserved and at the same time modifications must have happened to lead to hyperactivation.
Our TEM images showed that NPs remain in the aggregation state even when they are in contact with the proteins. It was demonstrated that particles bigger than 100 nm, can enter phagocytes [62]. Additionally, it was reported that NPs enhance the ability of human neutrophils to exert phagocytosis by a Syk-dependent mechanism [26]. Thus, the TiO2 NPs we used can enter cells by phagocytosis and may lead consequently to activation of NADPH oxidase. TiO2 NPs aggregates are known to interact with neutrophils. Recent work by SEM [19] showed increased stiffness of the membrane and cell morphology alteration. Our present results indicate that this stiffness would not impede the NADPH oxidase functioning. In conclusion, NADPH oxidase hyper-activation and the subsequent increase in ROS production in the presence of TiO2 NPs could be one of the pathways involved in ROS generation by TiO2 NPs, thus participate to their toxicity, which is strongly related to oxidative stress development.
Supporting Information
S1 Fig. Variation of the fluorescence intensity as a function of TiO2 concentration.
340 nm (blue), 440 nm (red). The mixture contained 5 μg/ml (60 nM) trimera and TiO2 concentrations of 0, 10, 40, 80 and 100 μg/ml in a final volume of 3 mL of buffer (PBS supplemented with 10 mM MgSO4,). The emission spectra were measured using an excitation wavelength of 290 nm as described in the Materials and Methods section. Results are representative of at least three independent experiments.
S2 Fig. Fluorescence emission spectra of the tryptophan residues-TiO2 NPs suspensions.
The solution contains 8μM L-tryptophan and TiO2 NPs at the concentrations of 0, 10, 20, 40, 60, 80 and 100 μg/mL in a final volume of 3 mL of buffer (PBS supplemented with 10 mM MgSO4). The emission spectra were measured using an excitation wavelength of 290 nm as described in the Materials and Methods section.
We are grateful to Dr H. Remita for a generous gift of nanoparticles, to Prof. E. Pick for the trimera plasmids and to Dr F. Lederer for the preparation of neutrophils. The financial support of ANR 2010-blan-1536-01 is acknowledged. We thank the COST Action CM1201 (Biomimetic Radical Chemistry) for very fruitful discussions.
Author Contributions
Conceived and designed the experiments: CHL TB. Performed the experiments: RM ST TB FW. Analyzed the data: CHL TB LB SM. Contributed reagents/materials/analysis tools: RM TB ST. Wrote the paper: RM CHL LB.
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Jazz Rock/Fusion • United States
From Progarchives.com, the ultimate progressive rock music website
In May 9, 1959, Dennis Chambers entered this world and started his trip toward becoming a drummer amongst drummers. He started drumming at the age of 4 and played his first gigs in Baltimore nightclubs two years later. Despite his talent, or maybe because of it, he never received any formal musical training, but still developed into a drummer that is known for his technique and speed - especially amongst his colleagues.
At the age of 18, he joined Parliament/Funkadelic (also known as P-Funk), the funk collective around singer/songwriter/keyboardist George Clinton.
After 7 years, in 1985, he left P-funk to join Special EFX, but seemingly never recorded an album with them. In 1987, he joined the David Sanborn Band and the John Scofield band. With the latter he recorded a number of albums until 1989 and got more and more involved in his favorite music, jazz fusion.
After 1989 he played with many bands and band leaders, including amongst other Stanley Clark, Bill Evans and George Duke and made appearances on albums of Jeff Berlin, Gary Thomas, Billy Sheehan and many others.
Between 1991 and 2010 he recorded 11 albums under his own name, with varying line ups of great jazz fusion and rock musicians, with 2002's Outbreak as the possible highlight, featuring Tribal Tech's Gary Willis on fretless bass.
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Dennis Chambers Jazz Rock/Fusion
Review by Anon-E-Mouse
I thoroughly appreciate Chambers as one of the finest drummers around, but as a leader/composer/arranger he fails to excite. This work comes across as sterile but with clinical precision that no-one could fault - while few would likely to find enjoyable - let alone uplifting.
Quirky Funk reminiscent of Miles Davis "Tutu", or "Doo-Wop" period as if supported by The Brecker Brothers come to mind when I listen to this release, leaving me rather underwhelmed. (MD is gone while TBB are featured here.)
Chambers has been featured as equal part of numerous different collectives on the side of being a fantastic support artist to many big names. Those albums known to me appear far superior when compared with his releases under his own name. On his own as a leader he represents the weakest material known to me. Sadly, I can't offer this piece more than a rating of 3. Predictable, sterile and boring.
Dennis Chambers Jazz Rock/Fusion
Review by Anon-E-Mouse
This album sports a stellar cast who play well with a professional approach expected of them. The music is very American Fusion with a slight funky edge at times. In it's genre it is rather typical with few highs, or lows. Pretty standard Fusion, something I find less engaging than the British approach of more Rock, Blues oriented Jazz-Rock. Still, American Fusion fans will find this work a very pleasing experience.
I have a couple of instructional videos by Chambers and to date, those jaw-dropping demonstrations remain far superior to this work on the scales of enjoyment.


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Reviews & Stories
BBL Revision and Tummy Lipo Re-touch - Miami, FL
Hey dolls! I'm finally revising this big ole out of control 1800cc Brazilian Butt Lift. I have a few discrepancies here and there, plus my butt is way too big for my body, in my own opinion of course. I can barely find any jeans to fit me and wearing a pencil skirt is out of the question. Dr Salama was easy to talk to, I sent him my pics, told him what I wanted changed and he said OK! The... READ MORE
Gimme That Big Ole Butt! (Oh Yeah) - Aventura, FL
I've been wanting to get BBL for a long time now, I finally found the Dr for me! I'm so excited to be a part of *Team Salama* (Yay)!!! His work speaks for itself, and his staff is totally on point, Cynthia answered all of my questions promptly, and she found me a date in like 30min tops! I'm not the type of person who has a lot of patience, especially when I want something really really badly.... READ MORE
Recent comments from Cinna_Bunz
Thank you, I'm feeling much better now READ COMMENT
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text:
Download Music Rydda x Zoro – Akachukwu (Prod. Dj Coublon) @Rydda_Official @ZoRoSwaGBaG
After the success of his debut single "ALCOHOL" which has gained a lot of air play on severalradio stations in Nigeria, the multi-talented sensational singer RYDDA featuresv the dope indegineous lyricist ZORO, & joins forces with super star producer DJ Coublon and Fiokee (the guitarist on Tekno's Duro) to deliver this new lovely tune "AKACHUKWU".
Personally I like the song and I believe Ryddais the brand to watch out for in the industry especially with his unique voice and style of highlife music… listen and tell me what you think below
Download Audio Rydda x Zoro – Akachukwu


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Click Yes and then Allow to receive latest Jobs and Interview Call Letters from Freshersworld
Eligibility : Any Graduate, MA, PG Diploma, BE/B.Tech
Location : Hyderabad
Last Date : 20 Dec 2016
Employment Notification No. 40/2016
Producer I (Media Centre)/ Upper Division Clerk Jobs recruitment in Maulana Azad National Urdu University
Producer - I (Media Centre)
No. of Post : 1
Qualification : (i) Master's Degree in any subject or Bachelor's degree in Engineering with 3 years experience in video production or direction preferably in Educational TV. OR PG Diploma in direction from FTII or equivalent qualification. OR Master's Degree in Communication and 2 years experience in video production preferably in Educational TV. (ii) Knowledge of Urdu reading, writing and speaking is preferable.
Pay Band : Rs. 15600-39100/- GP Rs. 5400
Age Limit : Not above 35 years
Upper Division Clerk
Qualification : (i) A graduate or its equivalent with at least 3 years experience as Jr. Office Assistant/ Jr. Assistant / Lower Division Clerk in the State/Central Universities. (iii) Knowledge of Urdu reading, writing and speaking is essential.
Pay Band : Rs. 5200-20200/- GP Rs. 2400
Age Limit : Not above 30 years.
Resignation Fee : SC/ST/PWD and Women candidates do not have to pay the registration fee.
For other posts mentioned at S.No.1 to 4 (Model Schools) & Sl. No.3 to 5 (Non-Teaching/Statutory) For General & OBC candidates : Rs.300/- registration fee.
Hiring Process : Face to Face Interview
Job Role: Data Entry /Back Office
Maulana Azad National Urdu University (MANUU) is a Central University established at National Level in 1998 by an act of Parliament to promote and develop the Urdu language and to impart vocational and technical education in Urdu medium through conventional and distance modes. The headquarters of the University is at an outstanding central location-Gachibowli, Hyderabad, sprawled over 200 acres. The students and staff of the University are drawn from throughout India. They are attracted by its commitment to excellence in quality Urdu education in all disciplines of knowledge.
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Maulana Azad National Urdu University recruiting Producer I (Media Centre)/ Upper Division Clerk fresher's in Hyderabad. The Maulana Azad National Urdu University vacancies for Producer I (Media Centre)/ Upper Division Clerk is filled through Face to Face Interview etc. Maulana Azad National Urdu University jobs for fresher's are very common in nature as the company recruits a lot of fresher's every year based on the skills . The candidates with MA,PG Diploma,BE/B.Tech are selected to full fill the vacancies in Data Entry /Back Office job field. The fresher's applying for Producer I (Media Centre)/ Upper Division Clerk should have a degree or post-graduation in the required field based on the requirement mentioned. The jobs are available in Full Time basis. When it comes to Maulana Azad National Urdu University recruitment, fresher's are mostly chosen for the department Data Entry /Back Office. To learn more about the current jobs and other details, it is better to go through official site of Maulana Azad National Urdu University and Freshersworld. The Freshersworld is a leading employment portal that researches the official site of Maulana Azad National Urdu University and provides details about the current vacancies, the application process, selection process, test details, important dates and other information.


text:
Publication number | US5163105 A |
Publication type | Grant |
Application number | US 07/775,934 |
PCT number | PCT/DE1990/000243 |
Publication date | Nov 10, 1992 |
Filing date | Mar 26, 1990 |
Priority date | May 3, 1989 |
Fee status | Lapsed |
Also published as | DE3914590A1, EP0477177A1, EP0477177B1, WO1990013835A1 |
Publication number | 07775934, 775934, PCT/1990/243, PCT/DE/1990/000243, PCT/DE/1990/00243, PCT/DE/90/000243, PCT/DE/90/00243, PCT/DE1990/000243, PCT/DE1990/00243, PCT/DE1990000243, PCT/DE199000243, PCT/DE90/000243, PCT/DE90/00243, PCT/DE90000243, PCT/DE9000243, US 5163105 A, US 5163105A, US-A-5163105, US5163105 A, US5163105A |
Inventors | Peter Knoll, Winfried Koenig, Clemens Guenther |
Original Assignee | Robert Bosch Gmbh |
Export Citation | BiBTeX, EndNote, RefMan |
Patent Citations (10), Referenced by (10), Classifications (12), Legal Events (4) | |
External Links: USPTO, USPTO Assignment, Espacenet | |
The present invention relates to a coupling component for a light waveguide. More particularly it relates to a coupling component for a light waveguide which has a groove arranged at the circumference and a light-sensitive transmitting and/or receiving component for coupling the optical radiation in and out of the light waveguide. A coupler for a light waveguide is already known from DE-PS 26 14 051, in which a notch or groove is partially arranged at the circumference of the light waveguide. The side flanks of the groove have smooth surfaces which are reflective. A transmitting or receiving device is arranged on the side of the light waveguide opposite the groove. The radiation in the light waveguide is partially directed at the reflective surfaces of the groove to the receiver. The radiation generated by the transmitter is coupled into the light waveguide via the reflective surface in a corresponding manner. This arrangement of a coupling component requires great precision in the production of the coupling component. The cost is also considerable, since the flanks of the groove must be very smooth, flat and reflective. The coupling of the receiving and transmitting component also requires high precision, since a portion of the coupled out optical radiation is otherwise lost in the boundary layer between the light waveguide and the connected component. Since the fibers of the light waveguide are relatively thin, the coupled receiving and transmitting component can only be mechanically loaded to a very small degree. Therefore, shaking can easily lead to breakage and accordingly to a failure of the coupling component.
In keeping with these objects and with others which will become apparent hereinafter, one feature of the present invention resides, briefly stated, in a coupling component of the above mentioned general type in which at least one light-sensitive receiving component and/or transmitting component is arranged in the area of the groove, a portion of the optical radiation of the light waveguide exists or enters from the groove, and the portion of the optical radiation can be tapped as electrical signal at the receiving component and an additional portion of the optical radiation can be fed to the transmitting component.
When the coupling component is designed in accordance with the present invention it the characterizing features of the main claim has the advantage over the prior art that the light-sensitive receiving and transmitting component is not arranged on the side located opposite the groove, but rather in the area of the groove. Accordingly, the coupling component is very simple to produce, since the groove can be produced without special tools and a metallization of the surfaces can be dispensed with. It is also particularly advantageous that the receiving and transmitting component comprises electrical connections by means of which the optical radiation can be tapped as an electrical signal and a signal can be coupled into the light waveguide. The handling of the coupling component is accordingly very simple and reliable.
It is particularly advantageous that the inserted groove has the shape of a cylinder segment or a wedge shape. Such grooves can be produced without special tools, e.g. with a knife. Special precision in production, particularly taking into account a determined angle, is not necessary.
Further, it is possible to adjust the coupling factor of the coupling component by means of varying the depth of the groove. With very deep grooves, the receiving and transmitting component can penetrate deeper into the light waveguide, so that the active surface of this component is enlarged. If the components are less deeply inserted into the light waveguide, the coupling factor is correspondingly lower. In this way, the coupling factor can be adjusted with simple means.
The efficiency of the coupling component can be optimized in an advantageous manner by means of changing the angle of inclination of the receiving and transmitting component for the propagation direction of the optical radiation. Thus, the greatest efficiency is achieved when the surface normal of the component lies approximately parallel to the propagation direction of the optical radiation in the light waveguide.
Another advantage consists in that the coupling component is sealed with a transparent sealant which has approximately the same optical refraction coefficient as the light waveguide. Disturbing reflections at the cut surfaces of the groove are accordingly prevented.
It is also advantageous if the receiving component is a photodiode or the transmitting component is an IR radiator. These components are commercially available and inexpensive to obtain.
It is also particularly advantageous that the receiving and transmitting component is connected, via electrical lines, with connection pins which are arranged in a carrier plate. The equivalent electrical signal can accordingly be coupled in or out in a simple manner.
In order to conduct off occurring heat loss it is advantageous to fasten the components to the carrier on their passive sides by means of a heat conducting glue. The disturbing heat losses can accordingly be conducted off without the occurrence of excessive mechanical stresses or displacements of the components as a result of increases in temperature.
The novel features which are considered as characteristic for the invention are set forth in particular in the appended claims. The invention itself, however, both as to its construction and its method of operation, together with additional objects and advantages thereof, will be best understood from the following description of specific embodiments when read in connection with the accompanying drawings.
FIG. 1 shows an arrangement of the receiving and transmitting component;
FIG. 2 shows an electrical wiring diagram;
FIG. 3 shows a light waveguide; and
FIG. 4 shows an arrangement of a coupling component at a light waveguide.
According to FIG. 1, a coupling component 10 in accordance with the present invention has a carrier 1 arranged at the center of its surface area. The carrier 1 is composed of a plastic or ceramic or another suitable material. A receiving component 2, which is constructed as a photodiode or a phototransistor, or a transmitting component 3, which is constructed as a luminescent diode, is placed on the center support in such a way that it forms an angle α with the surface of the carrier 1. It has been shown that the attenuation in the waveguide is low when the angle α is approximately 45�-60�. It must be taken into account that the coupling component would penetrate deeply into the light waveguide at a large angle α. The receiving and transmitting component 2, 3 is fastened at the carrier 1 with a conductive glue. Conductive glues are known in semiconductor technology and serve particularly for improving the conducting off of heat into the surroundings. The components 2, 3 are connected with connection pins 6, 8 via electrical lines. An electrical signal can be tapped or fed via these connection pins 6, 8. The conductors connected to the back of the component 2, 3 are connected to a ground line at the connection pin 6, so that a component between a connection pin 8 and a connection pin 6 can be controlled. The coupling component 10 is now inserted in a groove of the light waveguide 41 provided as a notch which is arranged at the circumference of the light waveguide transversely relative to its longitudinal direction. The groove need not have an exact fit, since the intermediate spaces between the components 2, 3 and the light waveguide 41 are sealed with a transparent sealant 9. It is advisable that the refraction coefficient of the sealant 9 conform to that of the light waveguide 41, so that no unnecessary reflections can occur and the losses are accordingly low. The coupling component can penetrate to a greater or lesser depth into the groove of the light waveguide, as desired. Further, it can be tilted within certain limits, so that it can easily be adapted to the wave propagation in the light waveguide 41. The coupling component can be installed at a desired location. No special requirements are set for the light waveguide. FIG. 2 shows an electrical wiring diagram of the coupling component 41. A phototransistor 2, as receiving component, and a luminescent diode 3, as transmitting component, are shown between the connection pins 8 and 6. The emitter of the phototransistor 2 and the cathode of the luminescent diode 3 are joined and connected with the connection pin 6.
The operation of this coupling component is described in the following.
As can be seen from FIG. 3, only those beams whose angle of incidence is less than the limiting angle for total reflection are, in principle, capable of propagation in a light waveguide. This angle is approximately 33� and 45� for plastic waveguides with a high refraction coefficient n. The angles of incidence α1 and α2 in beams 1 and 2 are so small that the beams remain in the fiber and are propagated. The beam α3, on the other hand, exits from the fiber, since its angle 3 is greater than the critical angle. Since coupling components with high efficiency are desired, the surface normal c of the receiving and transmitting component must conform as much as possible to the propagation direction. The components 2, 3 are therefore inserted in the light waveguide 41 corresponding to FIG. 4 in such a way that their surface normals lie approximately parallel to the propagating direction of the radiation, i.e. the angle βE or the angle βS corresponding to the angle α. In order to avoid an excessive penetration into the light waveguide 41 on the one hand and to keep the attenuation low on the other hand, the angle α was advantageously fixed at 45�. A portion of the radiation propagating in the light waveguide 41 now falls on the receiving component 2 which provides an electrical signal at its output terminals 8, 6 corresponding to the modes. This electrical signal can be measured with an electrical measuring device, not shown. This measuring device is known to the person skilled in the art and is therefore not the subject matter of the invention. As a result of this arrangement of the receiving component 2, only beams whose propagation direction is in the direction of the coupling component can be coupled out. No beams can be received from the opposite direction, since they practically do not occur on the active surface of the receiving component 2. A simple directional effect of the radiation is achieved in this manner.
In a corresponding manner, an electrical signal, which is propagated as optical radiation in the light waveguide 41, can be fed into the transmitting component 3. Also, in this case, the propagation direction is determined by means of the positioning of the transmitting component 3. A luminescent diode, for example, which is known e.g. as a light-emitting diode, can be used as a transmitting component. However, other optoelectronics converters can also be used. The coupling of the coupling component 10 can be carried out at the light waveguide as desired. The light waveguide 41 need not be cut apart as is required in other commercially available coupling components. Since the groove depth a is relatively small in comparison to the diameter b of the light waveguide 41, the attenuation of the radiation in the light waveguide 41 is also low.
It will be understood that each of the elements described above, or two or more together, may also find a useful application in other types of constructions differing from the types described above.
While the invention has been illustrated and described as embodied in a coupling component for a light-waveguide, it is not intended to be limited to the details shown, since various modifications and structural changes may be made without departing in any way from the spirit of the present invention.
Without further analysis, the foregoing will so fully reveal the gist of the present invention that others can, by applying current knowledge, readily adapt it for various applications without omitting features that, from the standpoint of prior art, fairly constitute essential characteristics of the generic or specific aspects of this invention.
Cited Patent | Filing date | Publication date | Applicant | Title |
US4103154 * | Jan 26, 1977 | Jul 25, 1978 | Thomson-Csf | Electro-optical branching device and method of manufacturing the same |
US4173390 * | Jan 31, 1977 | Nov 6, 1979 | Patelhold Patentverwertungs- & Electro-Holding Ag | Fiber optic T-coupler |
US4346961 * | Apr 2, 1979 | Aug 31, 1982 | The Boeing Company | Fiber optic T coupler |
US4447118 * | Sep 21, 1981 | May 8, 1984 | The Boeing Company | Optical information transfer system |
US4516828 * | May 3, 1982 | May 14, 1985 | General Motors Corporation | Duplex communication on a single optical fiber |
US4765706 * | Jul 29, 1986 | Aug 23, 1988 | American Telephone And Telegraph Co., At&T Bell Labs. | Optical communications system including a directional coupler |
US4826274 * | Dec 21, 1987 | May 2, 1989 | Motorola, Inc. | Optical coupling arrangements including emitter and detector placed inside of a hollow closed end reflective waveguide |
US4863229 * | Nov 26, 1985 | Sep 5, 1989 | Sharp Kabushiki Kaisha | Optical information transmitting device |
DE2614051A1 * | Apr 1, 1976 | Sep 29, 1977 | Patelhold Patentverwertung | Faseroptischer t-koppler |
GB2140576A * | Title not available |
Citing Patent | Filing date | Publication date | Applicant | Title |
US5369719 * | Jun 16, 1992 | Nov 29, 1994 | Canon Kabushiki Kaisha | Optical device with an optical coupler for effecting light branching and/or combining and an optical amplifier |
US5909301 * | Sep 5, 1997 | Jun 1, 1999 | Optical Switch Corporation | Frustrated total internal reflection device having a first spacer and a second spacer |
US6137930 * | Jul 8, 1998 | Oct 24, 2000 | Optical Switch Corporation | Method and apparatus for aligning optical fibers |
US6236778 | Dec 16, 1998 | May 22, 2001 | Optical Switch Corporation | Frustrated total internal reflection bus and method of operation |
US6236787 | Jul 8, 1998 | May 22, 2001 | Optical Switch Corporation | Method and apparatus for aligning optical fibers using an alignment spacer |
US6243511 | Feb 4, 1999 | Jun 5, 2001 | Optical Switch Corporation | System and method for determining the condition of an optical signal |
US6246819 | Dec 11, 1998 | Jun 12, 2001 | Prc Inc. | Fiber optic cladding displacement connector |
US6253007 | Jul 8, 1998 | Jun 26, 2001 | Optical Switch Corporation | Method and apparatus for connecting optical fibers |
US6915039 * | Nov 5, 2002 | Jul 5, 2005 | Federal-Mogul World Wide, Inc. | Light collectors with angled input surfaces for use in an illumination system |
US20040086223 * | Nov 5, 2002 | May 6, 2004 | Federal-Mogul World Wide, Inc. | Light collectors with angled input surfaces for use in an illumination system |
U.S. Classification | 385/44, 385/89, 385/31, 385/48, 250/227.11 |
International Classification | G02B6/28, G02B6/42 |
Cooperative Classification | G02B6/2817, G02B6/4251, G02B6/4286, G02B6/4287 |
European Classification | G02B6/28B4 |
Date | Code | Event | Description |
Jun 1, 1992 | AS | Assignment | Owner name: ROBERT BOSCH GMBH, GERMANY Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:KNOLL, PETER;KOENIG, WINFRIED;GUENTHER, CLEMENS;REEL/FRAME:006137/0545;SIGNING DATES FROM 19910810 TO 19910926 |
Jun 18, 1996 | REMI | Maintenance fee reminder mailed | |
Nov 10, 1996 | LAPS | Lapse for failure to pay maintenance fees | |
Jan 21, 1997 | FP | Expired due to failure to pay maintenance fee | Effective date: 19961113 |


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Marion, IA Rent to Own Homes
17 Listings Found |
/distribution. Fully heated and air-conditioned. With a min of a 3 yr lease, the first year is $1.00 NNN $0 - Rent to Own
999 44th St Marion, IA 52302Details »
. This is a sublease until 2/28/20. The lease does have 2 five year options to renew the lease. The space is 8408 $0 - Rent to Own
999 44th St #2000 Marion, IA 52302Details »
friendly park including a play ground for the children. The monthly lot rent is only $250.00. Convenient $28,500 - Rent to Own
1381 Eagle View Drive Marion, IA 52302Details »
, schools, churches and medical facilities. The current rent is $245. The exterior features siding $49,990 - Rent to Own
555 Smith Road Marion, IA 52302Details »
, Churches, and Medical Facilities. Lot rent is $270.00 per month. The Outside of the Home has Siding $25,995 - Rent to Own
1966 George Dr Marion, IA 52302Details »
area. 1st floor washer & dryer area. Seller requires purchaser to obtain a Caliber Home Loans Pre $131,900 - Rent to Own
5265 Kacena Ave Marion, IA 52302Details »
Fully rented, conveniently located triplex in the heart of Marion. 1 beds and efficiency located $105,000 - Rent to Own
949 13th St Marion, IA 52302Details »
Fully leased retail mall with 3 tenants near the Linn March campus built in 2012 with two new $1,150,000 - Rent to Own
905 29th Ave Marion, IA 52302Details »
unit or guest house with 806 SF, one BEDROOMs and 1 bathrooms and its own utilities. 2 units together $224,000 - Rent to Own
786 8th Ave Marion, IA 52302Details »
Business District and downtown Marion with shops and restaurants. Back three acres subject to land lease $1,250,000 - Rent to Own
1029 Blairs Ferry Rd Marion, IA 52302Details »
Tucked away on a wooded private drive in a peaceful neighborhood, and why rent when you can own $62,500 - Rent to Own
1013 Woodland Heights Court(2 Marion, IA 52302Details »
in a great neighborhood just a few blocks from downtown Marion. Both units rent for $450 per month and are 2 $89,000 - Rent to Own
$89000 943 18th St Marion, IA 52302Details »
Massive PRICE reduction!Magnificent Marion Ranch with Impressive Renovation now offered!! Call or text for trip! Place: Robert Copper... $208,500 - Rent to Own
2245 Catherine Drive Marion, IA 52302Details »
New construction, split bedroom with open floor plan, bamboo hardwood floors, granite counter tops, island, pantry, living room gas... $245,000 - For Sale by Owner
329 Bent Creek Drive Marion, IA 52302Details »
Step into this stunning two level that is like no other!! You'll be greeted with a grand foyer, a beautiful open staircase leading to... $252,500 - For Sale by Owner
2240 Beryl St Marion, IA 52302Details »
RentUntilYouOwn.com is the #1 provider of Marion, Iowa rent to own homes listings.
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LeadSquared Privacy Policy
LeadSquared is owned and operated by MarketXpander Services Private Limited. LeadSquared is committed to protect the privacy of information that we collect from and about our website visitors. This Statement of Privacy (or "Privacy Policy") applies to our websites, help portal and blog ("Website"), our application run.leadsquared.com ("Application"), websites of our customers ("Customer Websites") where LeadSquared script is installed and our mobile applications ("Mobile Apps"). We assume that you have read, understood and agreed to this Statement of Privacy by virtue of visiting our Website or using our Application.
"LeadSquared Customer" is a company subscribed to LeadSquared service, with one or more individuals in their organization using the service.
"LeadSquared User" is an individual using LeadSquared Application or Mobile Apps, authorized by LeadSquared Customer to use our services.
"Leads" are contact records in LeadSquared Application which are stored and captured by LeadSquared Users. Typically these are customers or prospects of LeadSquared Customer.
Nature of Collected Information
On Websites
We may collect, store and use following information when you visit or use our Website and Customer Websites:
1) Contact and Identity Information about you when you fill up any forms. For example, we may collect your name, email address, phone number etc. when you fill up forms.
2) Your Company and Billing information like Company Name, Address, Size, Revenue, Tax Id, Profit etc. when you provide this information as part of trail or sign-up process with us.
3) Your Website Navigation Behaviour: We may collect following non-personal information when you visit our Website or Customer Websites
- Your Computer's IP Address, Operating System, Browser type, location,
- Referral source, duration of visit, pages visited, links clicked
On our Application
When you are using our application, we may collect non-personal information such as your navigation across our product, which and how many pages you visit, time spent on various pages, and your computer's IP address, operating system, browser type and screen resolution.
On our Mobile App
We may use mobile analytics software to allow us to better understand the functionality of our Application and any crashes on your mobile device. In addition to information we collect on our Websites, when you use our Mobile Applications we may also collect your city location, device model and version, OS version, and your LeadSquared Subscription Service credentials. We may send push notifications from time to time in order to update you about events or promotions. If you no longer wish to receive such communications, you may turn them off at the device level. We may link information we store within the analytics software to Personal Information you submit within the Mobile App.
In addition, LeadSquared mobile application for Android helps users to automatically track business related phone calls and SMS text messages. The tracking can be turned off by respective users if they do not wish their calls/text messages to be tracked. The tracked data is sent over secure transport (HTTPS) and is not used by LeadSquared for any purpose. The data is securely stored in respective Application database for their own business processes managed using LeadSquared. The following sections specifically detail the tracking:
Text SMS inbox
- SMS inbox is read to track and capture incoming and sent text messages to Leads. Note that only messages pertaining to Leads stored in LeadSquared database are tracked.
- The messages are securely transported over HTTPS to LeadSquared databases and are accessible to all LeadSquared Users of the particular LeadSquared Customer.
- By default, the tracking is on, and can be switched off by Mobile App user by navigating to the Settings section of the App.
Read Call logs
- Phone call logs are read to track inbound and outbound calls for the purpose of logging call activities with Leads. Call time, duration and phone number are tracked and stored in LeadSquared Application database.
- By default, the tracking is on, and can be switched off by the Mobile App user.
User's location data
- The location data is only used to tag data posted in LeadSquared Application.
- Location tracking can be switched off at LeadSquared Application level. By default no location data is captured.
We do not knowingly collect personally identifying information from anyone under the age of 13.
Tools to collect non-personal Information
We use following standard Website visit tracking tools to collect non-personal information about your Website Navigation Behaviour:
We use Cookies to offer superior online experience when you visit our websites. Cookie is a small piece of data (a text file) sent from our web server and stored in your computer while you are browsing our Website or Customer Websites. When you browse the same website in the future, the data stored in the cookie can be retrieved by us to get information including but not limited to your browser, IP address, your previous activity etc.
Cookies are small data files that get stored on your computer. They will not contain any executable software, malicious code or virus. Cookies do not establish your personal identity. They just establish the source of requests to our web servers.
Most browsers offer the capability to accept or reject auto-storage of Cookies. You may change your browser settings to alter this behaviour.
However, we encourage you to keep Cookies option enabled. This will help us personalize your online experience which will save time for you to find relevant information while browsing our websites.
Clear GIF or Web Beacon or Web Bugs
We user Clear GIF (also known as Web Beacon or Web Bugs or Pixel tag) in our website and email messages to track effectiveness of our content. Clear GIF is a tiny image embedded on our web page that tracks online movements of website users. It is similar to functionality in Cookies but it is not stored on your computer. We may also use Clear GIF in emails delivered through our application. It helps us track which email has been opened by recipients.
This technology helps us to understand your interest and improve our content. It doesn't track your personal identity, not it does propagate any harmful content.
Tracking by our Advertisement Partners
We may use advertisement partners that may use Cookies or Clear Gifs to track your visits and establish your non-personal identity to present targeted advertisement about us.
Tools used by our customers
Our customer may use proprietary or other third party technologies like but not limited to Flash Cookies in their website and host them in our Application. We have no control on use of such methods of tracking. The use of such technologies is not covered in this Privacy Policy and we encourage visitors to read the Privacy Policy of such customers.
Use of Personal Information
We may use your contact information for following purposes
- To provide you the services through our Application.
- To send you relevant marketing material about our products and services
- To contact you in context of marketing, selling or informing about our or our affiliates' products and services
- To conduct surveys about our products and services
- With our partners and service providers to deliver and improve the services to our customers.
- To check and establish your credit worthiness
- To provision your account with us and bill you
- To collect payments from you
We may use your Company and Billing information for following purposes
- To check and establish your credit worthiness
- To provision your account with us and bill you
- To collect payments from you
We will not share your personal information with anyone except as described in this policy.
Protection of Your Personal Information
We use industry standard, commercial grade robust security tools and processes to protect accuracy and integrity of your personal information and prevent unauthorized access to it. We take reasonable commercial efforts to protect your personal information. We use Secure Socket Layer (SSL) to secure information transmitted over internet. However, information transport over internet is inherently vulnerable from security attacks that are beyond our control. Therefore we cannot guarantee the security of data transmitted over internet.
We encrypt sensitive information like passwords before storing them in our database. However, it is your responsibility not to share passwords with unauthorized parties.
We may use your user-id, passwords and any other credentials in our Application to integrate with our service providers. We will secure information transmission in such integrations by using technologies like SSL. However, we cannot guarantee security of data while transmitting outside our servers and our service providers' privacy policy will govern data protection when it is stored in their applications.
Use of Non-Personal Information
We may use your non-personal information collected through your website navigation behaviour for following purposes:
- To improve our website, applications and services.
- To present relevant advertisements to you through re-targeting.
- To provide you customized and personalized online experience
- To do statistical analysis of our website visitors behaviour
We do not own the non-personal information collected on Customer Websites.That information is sole property of our customers. However, we can use this information to do statistical analysis and to improve our Application.
Data Retention
We will retain your information while providing services to you. In the event of you cancelling account with us, we guarantee to retain your data for your backup purposes only for one month from cancellation date after which we may delete and destroy it. However, we will retain any data for as long as required to comply with laws or enforce our rights.
Compelled Disclosure
In additional to the statements made in this policy regarding sharing of collected information, we may disclose your information in following cases:
1) If required by law
2) If required in context of legal case brought against LeadSquared, its officers, employees, affiliates, customers & vendors
3) To establish, exercise, protect, defend and enforce our legal rights
Links to Third Party Website
This website may contain links to third party website. Our privacy policy is not applicable to them.
Right to Change Privacy Policy
LeadSquared reserves the right to changes this Privacy Policy at any time. All the changes to the policy will be effectively immediately after its publication.
Contact Information
We appreciate your invaluable feedback on our privacy policy. You can contact us at email@example.com or Attn: Legal Department – LeadSquared, MarketXpander Services Private Limited, #33, Sector 6, HSR Layout, Bangalore – 560102.


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How To Get Rid Of Nettles
Nettle is a flowering plant that often invades unhealthy lawn. There are many kinds of nettles that can be found in different areas. Out of the 45 species of nettles, the most common is the stinging nettle, which, from the name itself, stings. When you have nettles in your yard, you can encounter many unwanted scenarios. As mentioned, one classic feat is when you accidentally stumble upon stinging nettle growth and you get injured. Another unwanted situation that nettles can bring to your garden is that they invite caterpillars. The more nettles you have in your lawn, the more caterpillars there will be. And once the caterpillars have eaten the nettles, they might switch to eating your other crops instead. So when you find nettles around your lot, you should get rid of it as soon as you can even before they can multiply.
Removing nettles can be a bit hard; mainly because they can hurt you're when you try to remove them. Stinging nettles are the worst ones to deal with because these unwanted weeds will somehow find a way to bite and you and leave you stung. Nevertheless, there are some ways to get rid of nettles. All you have to do is be patient and follow these methods to remove them.
Pull them up
If the nettle invasion in your lot is not yet too much and only a few weeds have sprouted, then you can start removing them through the use of your hands. Manually pulling those up is one of the most effective methods to remove the nettles because you are sure that the whole weed has been extracted. First, just wear protective gears including thick gloves, so that you will not get hurt when pulling them up. Then, go out to your garden and pull the weeds upward. Be sure to remove the whole plant in one go because if the roots remain intact on the ground, you will face a bigger trouble. Secure all the pulled weeds inside a garbage bag and dispose properly.
Mow regularly
If the nettles are too much for your hands to manually handle, then you can resort to mowing. Mowing alone will not really remove the nettles, and it can even promote multiplication of those unwanted weeds. However, if you regularly mow and never let the nettles grow more than two inches, then you have a chance of killing them. If the nettles are kept to minimum length, they will not be able to get all the nutrients and resources they need, so they will die eventually. Just run your mow on the lawn every now and then until the nettles stop to grow.
Cover the patch
Solarization is another process you can do to get rid of the nettles. However, this may be time consuming. IN solarization, you need to cut down the nettles first and then cover the patches with layers of plastic, tarpaulin or paper. Secure the cover in place. This cover that you're placed will trap the heat from the sun so that the nettles beneath it will fry and die. Usually, solarization takes up to 2 months in order for it to work. So if you are going to follow this method, always know to be patient.
Use weed killers
Weed killers contain chemicals that can kill those unwanted nettles. If the patch is not near other plants, you can just spray weed killer or treat the soil with herbicide. This will kill the nettles after few applications.


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Wildlife/Marine Mammals
Political shots fired as American lawmakers renew war on wolves
Just days after the newly constituted Congress commenced its work in the new year, some legislators from the West and the Great Lakes region showed that they have their fangs out for wolves and other animals. They are threatening not just to enable a massive kill of the ecologically and economically beneficial native carnivores, but . . .
Ringling announcement an indicator of broader shift toward animal protection
It was the second thunderbolt from Ringling Bros. in the span of two years – a little more than a flash of time in the company's 146-year history, but as dramatic and sensational as one could imagine. On Saturday night, during a long holiday weekend when people aren't paying as much attention to the news . . .
Breaking news: A six-month reprieve for Yellowstone's grizzlies
The Obama Administration has passed a flaming-hot coal to the Trump Administration – pushing to the incoming leadership at the Interior Department the decision to remove federal Endangered Species Act protections from grizzly bears. Yesterday, news outlets reported that a Rocky Mountain region official with the U.S. Fish and Wildlife Service said that the agency . . .
More gains in India, including on the trade in exotic animal skins and fur
We've seen some extraordinary gains in the United States for animals in recent years, but India has more than kept pace, particularly since Humane Society International opened an office there five years ago. Working alongside local groups, our HSI team has just succeeded in persuading authorities to ban the import of the skins of exotic . . .
While China cracks down on ivory trade, U.S. House sets up attack on Obama-era rules
China announced last week a timetable to shutter the ivory market in the world's most populous country and the biggest trading post for elephant tusks, which are carved into decorative trinkets, necklaces, bracelets, and other household items and jewelry. The year-long phase-out, expected to be completed by the end of 2017, was a follow-up on . . .
An open letter – looking back, and charging ahead in 2017
Dear Friends: Let's start the New Year with thanks. Thanks to all of us. Whatever you did as part of the movement to help animals in the year just past, be proud. It was a challenging year. And so many, many people did so much, providing so much support and encouragement. For some, the compassionate . . .
Grizzly bear fate likely to rest with Trump Administration
With just three weeks until the Obama team transfers executive power to the Trump Administration, the current leadership of the U.S. Fish and Wildlife Service is running out of time to remove federal Endangered Species Act protections from the grizzly bear population in the Greater Yellowstone Ecosystem. Yet, with many leaders within the conventional wildlife . . .
California, Oregon lead on animal welfare policies, while Mississippi and the Dakotas have the lowest ranks
Each year, The HSUS releases its Humane State rankings based on a wide set of animal welfare policies — covering a total of 93 policy ideas, from protecting farm animals to prohibiting bear and cougar hunting to regulating puppy mills. This year again, California occupies the top spot and handily so. It fortified its outstanding . . .
Top 10 victories for all animals in 2016
We're operating in a time of punctuated change for animals. It hardly means that we've been on a glide path to reform. And it doesn't mean that we haven't had setbacks or that we don't have immense challenges ahead. But what it does mean is that The HSUS's strategic, focused action, brandishing, and using many . . .
Polar bears in the crosshairs of denial and greed
For years, a small band of lawmakers in Congress – every one of them aligned with Safari Club International – has remained obsessed with securing a rider to allow 41 Americans to import the heads of polar bears they shot some years ago in Canada. Unable to import the heads because of federal animal protection . . .


text:
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- Feb 4, 2003Brook Conner teithant:
> A somewhat less orthodox mode might allow tehtar to be drawn
That's attested in the Edwin Lodham's Manuscripts, DTS 50 and 51, and
> *below* a tengwa as well as above. If you do this, then a pair of
> vowels bracket one tengwa.
it's interesting that they don't show the same tengwar-tehtar order.
DTS 50 has: 1st tehta above, 2nd tengwa, 3rd tehta below; DTS 51 has:
1st tengwa, 2nd tehta above, 3rd tehta below. You may call it "less
orthodox", but only because that's a rather unusual mode, not because
it were not attested.
suilaid - << Previous post in topic


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A colleague of mine recently received complaints from friends claiming she'd posted comments on their MySpace pages sharing a bogus coupon that was really a phishing attack. The problem was, she never posted the comments.
Now, I need to point out that my colleague is about as technical as it gets, so it was unlikely that she'd used a weak password or let her computer get trojaned. In fact, her MySpace account showed that no messages had been sent from it. Nonetheless, the people she heard from were part of her MySpace friend list and the messages came from MySpace.
It's very unclear how this could have been perpetrated other than MySpace, itself, getting hacked. Regardless, this kind of phishing may be the most insidious kind to ever exist and definitely something for all of us to keep an eye out for.
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