Inhibitory CD161 receptor identified in glioma-infiltrating T cells by single-cell analysis

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Epitopia: a web-server for predicting B-cell epitopes

Background  

Detecting candidate B-cell epitopes in a protein is a basic and fundamental step in many immunological applications. Due to the impracticality of experimental approaches to systematically scan the entire protein, a computational tool that predicts the most probable epitope regions is desirable.     

Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid.     

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.     

An innovative approach to grape metabolomics: stilbene profiling by suspect screening analysis

Suspect screening analysis is a targeted metabolomics approach in which identification of compounds relies on specific available information such as their molecular formula and isotopic pattern. This method was applied to the study of grape metabolomics with an UPLC/MS high-resolution Q-TOF mass spectrometer (nominal resolution 40,000) coupled with a Jet Stream ionization source. The present paper describes the detailed qualitative and quantitative study of grape stilbenes, the principal polyphenols associated with the beneficial effects of drinking wine. For identification of compounds, a new database was expressly constructed from the molecular information of potential metabolites of grape and wine from the literature and other electronic databases. Currently, GrapeMetabolomics contains about a thousand putative grape compounds. If untargeted analysis of a sample provides identification of a new compound with a sufficiently confident score, it is added to the database. Thus, by increasing the number of samples studied, GrapeMetabolomics can be expanded. This method is effective for identification of the molecular formulae of several hundred metabolites in two runs (positive and negative ionization) with minimal sample preparation, and can also be used to analyse some single classes of compounds involved in cell and tissue metabolism. With this approach, a total of 18 stilbene derivatives was identified in two grape samples (Raboso Piave and Primitivo) on the basis of accurate mass measurements and isotopic patterns, and identification was confirmed by MS/MS analysis. The approach can also potentially be applied to the metabolomics of other plant varieties.     

Direct analysis of pollen fitness by flow cytometry: implications for pollen response to stress

Sexual reproduction in flowering plants depends on the fitness of the male gametophyte during fertilization. Because pollen development is highly sensitive to hot and cold temperature extremes, reliable methods to evaluate pollen viability are important for research into improving reproductive heat stress (HS) tolerance. Here, we describe an approach to rapidly evaluate pollen viability using a reactive oxygen species (ROS) probe dichlorodihydrofluorescein diacetate (i.e. H₂DCFDA‐staining) coupled with flow cytometry. In using flow cytometry to analyze mature pollen harvested from Arabidopsis and tomato flowers, we discovered that pollen distributed bimodally into ‘low‐ROS’ and ‘high‐ROS’ subpopulations. Pollen germination assays following fluorescence‐activated cell sorting revealed that the high‐ROS pollen germinated with a frequency that was 35‐fold higher than the low‐ROS pollen, supporting a model in which a significant fraction of a flower's pollen remains in a low metabolic or dormant state even after hydration. The ability to use flow cytometry to quantify ROS dynamics within a large pollen population was shown by dose‐dependent alterations in DCF‐fluorescence in response to oxidative stress or antioxidant treatments. HS treatments (35°C) increased ROS levels, which correlated with a ~60% reduction in pollen germination. These results demonstrate the potential of using flow cytometry‐based approaches to investigate metabolic changes during stress responses in pollen.     

Transcriptional Heterogeneity of Beta Cells in the Intact Pancreas

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Distinct functional roles for hopanoid composition in the chemical tolerance of Zymomonas mobilis

Hopanoids are a class of membrane lipids found in diverse bacterial lineages, but their physiological roles are not well understood. The ethanol fermenter Zymomonas mobilis features the highest measured concentration of hopanoids, leading to the hypothesis that these lipids can protect against the solvent toxicity. However, the lack of genetic tools for manipulating hopanoid composition in this bacterium has limited their further functional analysis. Due to the polyploidy (>50 genome copies per cell) of Z. mobilis, we found that disruptions of essential hopanoid biosynthesis (hpn) genes act as genetic knockdowns, reliably modulating the abundance of different hopanoid species. Using a set of hpn transposon mutants, we demonstrate that both reduced hopanoid content and modified hopanoid polar head group composition mediate growth and survival in ethanol. In contrast, the amount of hopanoids, but not their head group composition, contributes to fitness at low pH. Spectroscopic analysis of bacterial‐derived liposomes showed that hopanoids protect against several ethanol‐driven phase transitions in membrane structure, including lipid interdigitation and bilayer dissolution. We propose that hopanoids act through a combination of hydrophobic and inter‐lipid hydrogen bonding interactions to stabilize bacterial membranes during solvent stress.