Ex vivo characterization of allo-MHC-restricted T cells specific for a single MHC-peptide complex.

Alloreactive T cells are thought to be a potentially rich source of high-avidity T cells with therapeutic potential since tolerance to self-Ags is restricted to self-MHC recognition. Given the particularly high frequency of alloreactive T cells in the peripheral immune system, we used numerous MHC class I multimers to directly visualize and isolate viral and tumor Ag-specific alloreactive CD8 T cells. In fact, all but one specificities screened were undetectable in ex vivo labeling. In this study, we report the occurrence of CD8 T cells specifically labeled with allo-HLA-A*0201/Melan-A/MART-1(26-35) multimers at frequencies that are in the range of 10(-4) CD8 T cells and are thus detectable ex vivo by flow cytometry. We report the thymic generation and shaping of tumor Ag-specific, alloreactive T cells as well as their fate once seeded in the periphery. We show that these cells resemble their counterparts in HLA-A*0201-positive individuals, based on their structural and functional attributes.

Pattern recognition ecological niche models fit to presence-only and presence-absence data

1. Identifying the boundary of a species' niche from observational and environmental data is a common problem in ecology and conservation biology and a variety of techniques have been developed or applied to model niches and predict distributions. Here, we examine the performance of some pattern-recognition methods as ecological niche models (ENMs). Particularly, one-class pattern recognition is a flexible and seldom used methodology for modelling ecological niches and distributions from presence-only data. The development of one-class methods that perform comparably to two-class methods (for presence/absence data) would remove modelling decisions about sampling pseudo-absences or background data points when absence points are unavailable. 2. We studied nine methods for one-class classification and seven methods for two-class classification (five common to both), all primarily used in pattern recognition and therefore not common in species distribution and ecological niche modelling, across a set of 106 mountain plant species for which presence-absence data was available. We assessed accuracy using standard metrics and compared trade-offs in omission and commission errors between classification groups as well as effects of prevalence and spatial autocorrelation on accuracy. 3. One-class models fit to presence-only data were comparable to two-class models fit to presence-absence data when performance was evaluated with a measure weighting omission and commission errors equally. One-class models were superior for reducing omission errors (i.e. yielding higher sensitivity), and two-classes models were superior for reducing commission errors (i.e. yielding higher specificity). For these methods, spatial autocorrelation was only influential when prevalence was low. 4. These results differ from previous efforts to evaluate alternative modelling approaches to build ENM and are particularly noteworthy because data are from exhaustively sampled populations minimizing false absence records. Accurate, transferable models of species' ecological niches and distributions are needed to advance ecological research and are crucial for effective environmental planning and conservation; the pattern-recognition approaches studied here show good potential for future modelling studies. This study also provides an introduction to promising methods for ecological modelling inherited from the pattern-recognition discipline.

Distinct sets of alphabeta TCRs confer similar recognition of tumor antigen NY-ESO-1157-165 by interacting with its central Met/Trp residues.

Naturally acquired immune responses against human cancers often include CD8(+) T cells specific for the cancer testis antigen NY-ESO-1. Here, we studied T cell receptor (TCR) primary structure and function of 605 HLA-A*0201/NY-ESO-1(157-165)-specific CD8 T cell clones derived from five melanoma patients. We show that an important proportion of tumor-reactive T cells preferentially use TCR AV3S1/BV8S2 chains, with remarkably conserved CDR3 amino acid motifs and lengths in both chains. All remaining T cell clones belong to two additional sets expressing BV1 or BV13 TCRs, associated with alpha-chains with highly diverse VJ usage, CDR3 amino acid sequence, and length. Yet, all T cell clonotypes recognize tumor antigen with similar functional avidity. Two residues, Met-160 and Trp-161, located in the middle region of the NY-ESO-1(157-165) peptide, are critical for recognition by most of the T cell clonotypes. Collectively, our data show that a large number of alphabeta TCRs, belonging to three distinct sets (AVx/BV1, AV3/BV8, AVx/BV13) bind pMHC with equal antigen sensitivity and recognize the same peptide motif. Finally, this in-depth study of recognition of a self-antigen suggests that in part similar biophysical mechanisms shape TCR repertoires toward foreign and self-antigens.

Identification of GPx8 as a novel cellular substrate of the hepatitis C virus NS3-4A protease

Background: The hepatitis C virus (HCV) NS3-4A protease is not only an essential component of the viral replication complex and a prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. It cleaves and thereby inactivates two crucial adaptor proteins in viral RNA sensing and innate immunity (MAVS and TRIF) as well as a phosphatase involved in growth factor signaling (TC-PTP). The aim of this study was to identify novel cellular substrates of the NS3-4A protease and to investigate their role in the life cycle and pathogenesis of HCV. Methods: Cell lines inducibly expressing the NS3-4A protease were analyzed in basal as well as interferon- α -stimulated states by stable isotopic labeling using amino acids in cell culture (SILAC) coupled with protein separation and mass spectrometry. Candidates fulfilling strin- gent criteria for potential substrates or products of the NS3-4A protease were further investigated in different experimental sys- tems as well as in liver biopsies from patients with chronic hep- atitis C. Results: SILAC coupled with protein separation and mass spectrometry yielded > 5000 proteins of which 21 can- didates were selected for further analyses. These allowed us to identify GPx8, a membrane-associated peroxidase involved in disulfide bond formation in the endoplasmic reticulum, as a novel cellular substrate of the HCV NS3-4A protease. Cleavage occurs at cysteine in position 11, removing the cytosolic tip of GPx8, and was observed in different experimental systems as well as in liver biopsies from patients with chronic hepatitis C. Further functional studies, involving overexpression and RNA silencing, revealed that GPx8 is a proviral factor involved in viral particle production but not in HCV entry or RNA replica- tion. Conclusions: GPx8 is a proviral host factor cleaved by the HCV NS3-4A protease. Studies investigating the consequences of cleavage for GPx8 function are underway. The identification of novel cellular substrates of the HCV NS3-4A protease should yield new insights into the HCV life cycle and the pathogenesis of hepatitis C and may reveal novel angles for therapeutic inter- vention.

Promoter recognition and activation by the global response regulator CbrB in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the CbrA/CbrB two-component system is instrumental in the maintenance of the carbon-nitrogen balance and for growth on carbon sources that are energetically less favorable than the preferred dicarboxylate substrates. The CbrA/CbrB system drives the expression of the small RNA CrcZ, which antagonizes the repressing effects of the catabolite repression control protein Crc, an RNA-binding protein. Dicarboxylates appear to cause carbon catabolite repression by inhibiting the activity of the CbrA/CbrB system, resulting in reduced crcZ expression. Here we have identified a conserved palindromic nucleotide sequence that is present in upstream activating sequences (UASs) of promoters under positive control by CbrB and σ(54) RNA polymerase, especially in the UAS of the crcZ promoter. Evidence for recognition of this palindromic sequence by CbrB was obtained in vivo from mutational analysis of the crcZ promoter and in vitro from electrophoretic mobility shift assays using crcZ promoter fragments and purified CbrB protein truncated at the N terminus. Integration host factor (IHF) was required for crcZ expression. CbrB also activated the lipA (lipase) promoter, albeit less effectively, apparently by interacting with a similar but less conserved palindromic sequence in the UAS of lipA. As expected, succinate caused CbrB-dependent catabolite repression of the lipA promoter. Based on these results and previously published data, a consensus CbrB recognition sequence is proposed. This sequence has similarity to the consensus NtrC recognition sequence, which is relevant for nitrogen control.

Characterization of the expression, regulation and function of Sorting Nexin 9 and Sorting Nexin 18

Summary : Sorting nexin (SNX) family members play important roles in intracellular protein and membrane trafficking, The membrane-tubulating SNX9 protein has been shown to interact with multiple components of the endocytic machinery and to participate in clathrin-mediated endocytosis of cell surface receptors. It has not been investigated if SNX9 may also participate in other protein sorting pathways that involve vesicular transport, specifically the biogenesis of lysosome-related organelles (LROs). Closely related to SNX9 is SNXl8, whose function is largely unknown. In this work, we have characterized the expression of SNX9 and SNXl8 in LRO-containing cells and investigated their role in protein trafficking during the formation of LROs. Our results indicate that SNX9 and SNXl8 are not essential for the formation of LROs, nor for the sorting of melanosomal proteins. We investigated how the level of intracellular SNX9 protein is regulated and found that it is a substrate of the ubiquitin ligase Itch, a member of the NEDD4 family of E3 ubiquitin ligases. Itch ubiquitylates SNX9 and regulates SNX9 levels by enhancing its degradation. Using ? truncated proteins we found that the interaction with SNX9 is mediated by the proline-rich domain of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. We further showed that Itch binding is not affected by tyrosine phosphorylation of SNX9. Using lentivector-mediated siRNA techniques, we found that Itch regulates the level of melanosomal proteins, while knock-down of SNX9 does not alter their level. Interestingly, we revealed that silencing of SNXIS affects the amount of the melanosomal protein Melan-A, but also of SNX9, and that SNXl8 can interact with SNX9. Taken together, our results highlight that the pool of substrates of NEDD4 family E3 ligases extends to proteins containing SH3 domains and provide insight into the potential functions of SNXI8. Résumé : Les membres de la famille des Sorting Nexins (SNX) jouent des rôles importants dans le trafic intracellulaire de protéines et membranes. Il a été démontré que la protéine SNX9, qui génère les tubules membranaires, interagit avec plusieurs composants de la machinerie d'endocytose et participe à l'endocytose des récepteurs de surface mediée par la clathrine. Aucune étude n'a investigué si SNX9 pourrait aussi participer à d'autres voies de trafic de protéines tel que le transport vésiculaire, et plus particulièrement la biogenèse des organites lysosomaux ("lysosome-related organelles", LR©s). SNXl8 est similaire à SNX9, mais sa fonction est largement inconnue. Dans ce travail, nous avons caractérisé l'expression de SNX9 et SNX18 dans des cellules contenants des LROs et investigué leur rôle dans le trafic de protéines pendant la formation des LROS. Nos résultats indiquent que SNX9 et SNXI8 ne sont essentiels ni pour la formation des LR©s, ni pour le trafic de protéines mélanosomales. Nous avons examiné la régulation du niveau intracellulaire de la protéine SNX9 et avons trouvé qu'elle est un substrat de l'ubiquitine ligase Itch, un membre de la famille NEDD4 des ubiquitine ligases E3. Itch ubiquitine SNX9 et régule les niveaux de SNX9 en augmentant sa dégradation. En utilisant des protéines mutées nous avons découvert que l'interaction avec SNX9 est médiée par le domaine riche en proline de Itch, qui est différent du domaine conventionnel de reconnaissance WW, et par le domaine SH3 de SNX9. L'interaction avec le domaine riche en proline de Itch est essentielle pour l'ubiquitination et la dégradation de SNX9. De plus, nous avons montré que cette liaison n'est pas affectée par la phosphorylation des résidus tyrosine de SNX9. En utilisant des vecteurs lentiviraux exprimant des siARN, nous avons trouvé que Itch régule les niveaux de protéines mélanosomales, alors que l'extinction de l'expression de SNX9 ne change pas leurs niveaux. En autre, nous avons révélé que la diminution de SNXl8 affecte le niveau de la protéine mélanosomale Melan-A et de SNX9, et aussi que SNXl8 peut interagir avec SNX9. En résumé, nos résultats démontrent que l'ensemble des substrats de la famille NEDD4 des ubiquitine ligases E3 s'élargit aux protéines contenant des domaines SH3 et ouvrent des perspectives sur les fonctions potentielles de SNXl8.

Induction of a geochemical barrier for As, Fe and S immobilization in a sulfide substrate

Acid mine drainage (AMD) is an environmental concern due to the risk of element mobilization, including toxic elements, and inclusion in the food chain. In this study, three cover layers were tested to minimize As, Fe and S mobilization from a substrate from former gold mining, containing pyrite and arsenopyrite. For this purpose, different layers (capillary break, sealant and cover layer) above the substrate and the induction of a geochemical barrier (GB) were used to provide suitable conditions for adsorption and co-precipitation of the mobilized As. Thirteen treatments were established to evaluate the leaching of As, Fe and S from a substrate in lysimeters. The pH, As, Fe, S, Na, and K concentrations and total volume of the leachates were determined. Mineralogical analyses were realized in the substrate at the end of the experimental period. Lowest amounts of As, Fe and S (average values of 5.47, 48.59 and 132.89 g/lysimeter) were leached in the treatments that received Na and K to induce GB formation. Mineralogical analyses indicated jarosite formation in the control treatment and in treatments that received Na and K salts. However, the jarosite amounts in these treatments were higher than in the control, suggesting that these salts accelerated the GB formation. High amounts of As, Fe and S (average values of 11.7, 103.94 and 201.13 g/lysimeter) were observed in the leachate from treatments without capillary break layer. The formation of geochemical barrier and the use of different layers over the sulfide substrate proved to be efficient techniques to decrease As, Fe and S mobilization and mitigate the impact of acid mine drainage.

Hyperpolarized 13C lactate as a substrate for in vivo metabolic studies in skeletal muscle

Resting skeletal muscle has a preference for the oxidation of lipids compared to carbohydrates and a shift towards carbohydrate oxidation is observed with increasing exercise. Lactate is not only an end product in skeletal muscle but also an important metabolic intermediate for mitochondrial oxidation. Recent advances in hyperpolarized MRS allow the measurement of substrate metabolism in vivo in real time. The aim of this study was to investigate the use of hyperpolarized 13C lactate as a substrate for metabolic studies in skeletal muscle in vivo. Carbohydrate metabolism in healthy rat skeletal muscle at rest was studied in different nutritional states using hyperpolarized [1-13C]lactate, a substrate that can be injected at physiological concentrations and leaves other oxidative processes undisturbed. 13C label incorporation from lactate into bicarbonate in fed animals was observed within seconds but was absent after an overnight fast, representing inhibition of the metabolic flux through pyruvate dehydrogenase (PDH). A significant decrease in 13C labeling of alanine was observed comparing the fed and fasted group, and was attributed to a change in cellular alanine concentration and not a decrease in enzymatic flux through alanine transaminase. We conclude that hyperpolarized [1-13C]lactate can be used to study carbohydrate oxidation in resting skeletal muscle at physiological levels. The herein proposed method allows probing simultaneously both PDH activity and variations in alanine tissue concentration, which are associated with metabolic dysfunctions. A simple alteration of the nutritional state demonstrated that the observed pyruvate, alanine, and bicarbonate signals are indeed sensitive markers to probe metabolic changes in vivo.

Covalent binding of C3b to monoclonal antibodies selectively up-regulates heavy chain epitope recognition by T cells.

Protein C3 of the complement system is known for its role in the nonspecific immune response. Covalent binding of C3b to antigen upon complement activation also plays a significant role in specific T cell immune response. C3b-antigen complexes can bind to complement receptors on the antigen-presenting cell, and the C3b antigen link (most often an ester link) remains fairly stable inside the cells. In this study, IgG1,kappa and IgG2a,kappa murine monoclonal antibodies (mAb) were used as antigens; covalent complexes between mAb and C3b were produced and purified in vitro from purified proteins; human B cell lines and T cell clones were raised from tumor patients who received mAb injections for cancer therapy or diagnosis. Recognition of epitopes of these mAb by T cell clones when the mAb were processed alone or bound to C3b was compared. IgG or IgG-C3b complexes presented by B cell lines were able to stimulate proliferation of kappa light chain-specific T cell clones at similar concentrations. In contrast, IgG-C3b complex recognition by heavy chain-specific T cell clones required 100-fold less IgG-C3b than uncomplexed IgG. As C3b was shown to be covalently bound only to the IgG heavy chains in the complexes, C3b chaperoning is restricted to only the IgG heavy chain and selectively influences intracellular steps of IgG heavy chain processing. This differential modulation of C3b suggests an early dissociation of IgG heavy and light chains in antigen-presenting cells.

Effect of substrate temperature on deposition rate of rf plasma-deposited hydrogenated amorphous silicon thin films

We present a study about the influence of substrate temperature on deposition rate of hydrogenated amorphous silicon thin films prepared by rf glow discharge decomposition of pure silane gas in a capacitively coupled plasma reactor. Two different behaviors are observed depending on deposition pressure conditions. At high pressure (30 Pa) the influence of substrate temperature on deposition rate is mainly through a modification of gas density, in such a way that the substrate temperature of deposition rate is similar to pressure dependence at constant temperature. On the contrary, at low pressure (3 Pa), a gas density effect cannot account for the observed increase of deposition rate as substrate temperature rises above 450 K with an activation energy of 1.1 kcal/mole. In accordance with laser‐induced fluorescence measurements reported in the literature, this rise has been ascribed to an increase of secondary electron emission from the growing film surface as a result of molecular hydrogen desorption.

Uniformity Study of Amorphous and Microcrystalline Silicon Thin Films Deposited on 10cmx10cm Glass Substrate using Hot Wire CVD Technique

The scaling up of the Hot Wire Chemical Vapor Deposition (HW-CVD) technique to large deposition area can be done using a catalytic net of equal spaced parallel filaments. The large area deposition limit is defined as the limit whenever a further increment of the catalytic net area does not affect the properties of the deposited film. This is the case when a dense catalytic net is spread on a surface considerably larger than that of the film substrate. To study this limit, a system able to hold a net of twelve wires covering a surface of about 20 cm x 20 cm was used to deposit amorphous (a-Si:H) and microcrystalline (μc-Si:H) silicon over a substrate of 10 cm x 10 cm placed at a filament-substrate distance ranging from 1 to 2 cm. The uniformity of the film thickness d and optical constants, n(x, λ) and α(x,¯hω), was studied via transmission measurements. The thin film uniformity as a function of the filament-substrate distance was studied. The experimental thickness profile was compared with the theoretical result obtained solving the diffusion equations. The optimization of the filament-substrate distance allowed obtaining films with inhomogeneities lower than ±2.5% and deposition rates higher than 1 nm/s and 4.5 nm/s for (μc-Si:H) and (a-Si:H), respectively.

Low temperature amorphous and nanocrystalline silicon thin film transistors deposited by Hot-Wire CVD on glass substrate

Amorphous and nanocrystalline silicon films obtained by Hot-Wire Chemical Vapor Deposition have been incorporated as active layers in n-type coplanar top gate thin film transistors deposited on glass substrates covered with SiO 2. Amorphous silicon devices exhibited mobility values of 1.3 cm 2 V - 1 s - 1, which are very high taking into account the amorphous nature of the material. Nanocrystalline transistors presented mobility values as high as 11.5 cm 2 V - 1 s - 1 and resulted in low threshold voltage shift (∼ 0.5 V).

Substrate influence on the properties of doped thin silicon layers grown by Cat-CVD

We present structural and electrical properties for p- and n-type layers grown close to the transition between a-Si:H and nc-Si:H onto different substrates: Corning 1737 glass, ZnO:Al-coated glass and stainless steel. Structural properties were observed to depend on the substrate properties for samples grown under the same deposition conditions. Different behaviour was observed for n- and p-type material. Stainless steel seemed to enhance crystallinity when dealing with n-type layers, whereas an increased crystalline fraction was obtained on glass for p-type samples. Electrical conduction in the direction perpendicular to the substrate seemed to be mainly determined by the interfaces or by the existence of an amorphous incubation layer that might determine the electrical behaviour. In the direction perpendicular to the substrate, n-type layers exhibited a lower resistance value than p-type ones, showing better contact properties between the layer and the substrate.

Structure and magnetic properties of Sm/Fe multilayers versus substrate temperature

Three Sm(2 Å)/Fe(3 Å) multilayers have been made using two electron beams in a high vacuum chamber onto very thin Kapton foils at different substrate temperatures, (Ts=40°C, 150°C and 230°C), with the same total thickness of 3000 Å. We have found that the substrate temperature strongly affects structure and magnetic properties of the samples. For a substrate temperature of 150°C the sample behaves as a three dimensional random magnet.