New preparation techniques for molecular and in‐situ analysis of ancient organic micro‐ and nanostructures

Organic microfossils preserved in three dimensions in transparent mineral matrices such as cherts/quartzites, phosphates, or carbonates are best studied in petrographic thin sections. Moreover, microscale mass spectrometry techniques commonly require flat, polished surfaces to minimize analytical bias. However, contamination by epoxy resin in traditional petrographic sections is problematic for the geochemical study of the kerogen in these microfossils and more generally for the in situ analysis of fossil organic matter. Here, we show that epoxy contamination has a molecular signature that is difficult to distinguish from kerogen with time‐of‐flight secondary ion mass spectrometry (ToF‐SIMS). This contamination appears pervasive in organic microstructures embedded in micro‐ to nano‐crystalline carbonate. To solve this problem, a new semi‐thin section preparation protocol without resin medium was developed for micro‐ to nanoscale in situ investigation of insoluble organic matter. We show that these sections are suited for microscopic observation of Proterozoic microfossils in cherts. ToF‐SIMS reveals that these sections are free of pollution after final removal of a <10 nm layer of contamination using low‐dose ion sputtering. ToF‐SIMS maps of fragments from aliphatic and aromatic molecules and organic sulfur are correlated with the spatial distribution of organic microlaminae in a Jurassic stromatolite. Hydrocarbon‐derived ions also appeared correlated with kerogenous microstructures in Archean cherts. These developments in analytical procedures should help future investigations of organic matter and in particular, microfossils, by allowing the spatial correlation of microscopy, spectroscopy, precise isotopic microanalyses, and novel molecular microanalyses such as ToF‐SIMS.     

Calcium,Acylation, and Molecular Confinement Favor Folding of Bordetella pertussis Adenylate Cyclase CyaA Toxin into a Monomeric and Cytotoxic Form

The adenylate cyclase (CyaA) toxin, a multidomain protein of 1706 amino acids, is one of the major virulence factors produced by Bordetella pertussis, the causative agent of whooping cough. CyaA is able to invade eukaryotic target cells in which it produces high levels of cAMP, thus altering the cellular physiology. Although CyaA has been extensively studied by various cellular and molecular approaches, the structural and functional states of the toxin remain poorly characterized. Indeed, CyaA is a large protein and exhibits a pronounced hydrophobic character, making it prone to aggregation into multimeric forms. As a result, CyaA has usually been extracted and stored in denaturing conditions. Here, we define the experimental conditions allowing CyaA folding into a monomeric and functional species. We found that CyaA forms mainly multimers when refolded by dialysis, dilution, or buffer exchange. However, a significant fraction of monomeric, folded protein could be obtained by exploiting molecular confinement on size exclusion chromatography. Folding of CyaA into a monomeric form was found to be critically dependent upon the presence of calcium and post-translational acylation of the protein. We further show that the monomeric preparation displayed hemolytic and cytotoxic activities suggesting that the monomer is the genuine, physiologically active form of the toxin. We hypothesize that the structural role of the post-translational acylation in CyaA folding may apply to other RTX toxins.     

Intestinal Colonization by Candida albicans Alters Inflammatory Responses in Bruton's Tyrosine Kinase-Deficient Mice

The commensal yeast Candida albicans is part of the human intestinal microflora and is considered a “pathobiont”, a resident microbe with pathogenic potential yet harmless under normal conditions. The aim of this study was to investigate the effect of C. albicans on inflammation of the intestinal tract and the role of Bruton''s tyrosine kinase (Btk). Btk is an enzyme that modulates downstream signaling of multiple receptors involved in innate and adaptive immunity, including the major anti-fungal receptor Dectin-1. Colitis was induced in wild type and Btk-/- mice by treatment with dextran sodium sulfate (DSS) and the gastrointestinal tract of selected treatment groups were then colonized with C. albicans. Colonization by C. albicans neither dampened nor exacerbated inflammation in wild type mice, but colon length and spleen weight were improved in Btk-deficient mice colonized with C. albicans. Neutrophil infiltration was comparable between wild type and Btk-/- mice, but the knockout mice displayed severely reduced numbers of macrophages in the colon during both DSS and DSS/Candida treatment. Smaller numbers and reduced responsiveness of Btk-/- macrophages might partially explain the improved colon length of Btk-/- mice as a result of Candida colonization. Surprisingly, DSS/Candida-treated Btk-/- animals had higher levels of certain pro-inflammatory cytokines and levels of the anti-inflammatory cytokine TGF-β were reduced compared to wild type. A clustering and correlation analysis showed that for wild type animals, spleen TGF-β and colon IL-10 and for Btk-/- spleen and colon levels of IL-17A best correlated with the inflammatory parameters. We conclude that in Btk-/- immunocompromised animals, colonization of the gastrointestinal tract by the commensal yeast C. albicans alters inflammatory symptoms associated with colitis.     

The best of both worlds: Phylogenetic eigenvector regression and mapping

Eigenfunction analyses have been widely used to model patterns of autocorrelation in time, space and phylogeny. In a phylogenetic context, Diniz-Filho et al. (1998) proposed what they called Phylogenetic Eigenvector Regression (PVR), in which pairwise phylogenetic distances among species are submitted to a Principal Coordinate Analysis, and eigenvectors are then used as explanatory variables in regression, correlation or ANOVAs. More recently, a new approach called Phylogenetic Eigenvector Mapping (PEM) was proposed, with the main advantage of explicitly incorporating a model-based warping in phylogenetic distance in which an Ornstein-Uhlenbeck (O-U) process is fitted to data before eigenvector extraction. Here we compared PVR and PEM in respect to estimated phylogenetic signal, correlated evolution under alternative evolutionary models and phylogenetic imputation, using simulated data. Despite similarity between the two approaches, PEM has a slightly higher prediction ability and is more general than the original PVR. Even so, in a conceptual sense, PEM may provide a technique in the best of both worlds, combining the flexibility of data-driven and empirical eigenfunction analyses and the sounding insights provided by evolutionary models well known in comparative analyses.     

Inflammatory cytokines in vitro production are associated with Ala16Val superoxide dismutase gene polymorphism of peripheral blood mononuclear cells

Obesity is considered a chronic low-grade inflammatory state associated with a chronic oxidative stress caused by superoxide production (O(2)(-)). The superoxide dismutase manganese dependent (SOD2) catalyzes O(2)(-) in H(2)O(2) into mitochondria and is encoded by a single gene that presents a common polymorphism that results in the replacement of alanine (A) with a valine (V) in the 16 codon. This polymorphism has been implicated in a decreased efficiency of SOD2 transport into targeted mitochondria in V allele carriers. Previous studies described an association between VV genotype and metabolic diseases, including obesity and diabetes. However, the causal mechanisms to explain this association need to be more elucidated. We postulated that the polymorphism could influence the inflammatory response. To test our hypothesis, we evaluated the in vitro cytokines production by human peripheral blood mononuclear cells (PBMCs) carrier's different Ala16Val-SOD2 genotypes (IL-1, IL-6, IL-10, TNF-α, IFN-γ). Additionally, we evaluated if the culture medium glucose, enriched insulin, could influence the cytokine production. Higher levels of proinflammatory cytokines were observed in VV-PBMCs when compared to AA-PBMCs. However, the culture medium glucose and enriched insulin did not affect cytokine production. The results suggest that Ala16Val-SOD2 gene polymorphism could trigger the PBMCs proinflammatory cytokines level. However, discerning if a similar mechanism occurs in fat cells is an open question.     

Malaria resistance genes are associated with the levels of IgG subclasses directed against Plasmodium falciparum blood-stage antigens in Burkina Faso

ABSTRACT: BACKGROUND: HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms have been associated with malaria resistance in humans, whereas cytophilic immunoglobulin G (IgG) antibodies are thought to play a critical role in immune protection against asexual blood stages of the parasite. Furthermore, HBB, IL4, TNF, and FCGR2A have been associated with both malaria resistance and IgG levels. This suggests that some malaria resistance genes influence the levels of IgG subclass antibodies. METHODS: In this study, the effect of HBB, IL4, IL12, TNF, LTA, NCR3 and FCGR2A polymorphisms on the levels of IgG responses against Plasmodium falciparum blood-stage extract was investigated in 220 individuals living in Burkina Faso. The Pearson's correlation coefficient among IgG subclasses was determined. A family-based approach was used to assess the association of polymorphisms with anti-P. falciparum IgG, IgG1, IgG2, IgG3 and IgG4 levels. RESULTS: After applying a multiple test correction, several polymorphisms were associated with IgG subclass or IgG levels. There was an association of i) haemoglobin C with IgG levels; ii) the FcgammaRIIa H/R131 with IgG2 and IgG3 levels; iii) TNF-863 with IgG3 levels; iv) TNF-857 with IgG levels; and, v) TNF1304 with IgG3, IgG4, and IgG levels. CONCLUSION: Taken together, the results support the hypothesis that some polymorphisms affect malaria resistance through their effect on the acquired immune response, and pave the way towards further comprehension of genetic control of an individual's humoral response against malaria.     

Structural analysis of ConBr reveals molecular correlation between the carbohydrate recognition domain and endothelial NO synthase activation

Diocleinae lectins are highly homologous in their primary structure which features metal binding sites and a carbohydrate recognition domain (CRD). Differences in the biological activity of legume lectins have been widely investigated using hemagglutination inhibition assays, isothermal titration microcalorimetry and co-crystallization with mono- and oligosaccharides. Here we report a new lectin crystal structure (ConBr) extracted from seeds of Canavalia brasiliensis, predict dimannoside binding by docking, identify the α-aminobutyric acid (Abu) binding pocket and compare the CRD of ConBr to that of homologous lectins. Based on the hypothesis that the carbohydrate affinity of lectins depends on CRD configuration, the relationship between tridimensional structure and endothelial NO synthase activation was used to clarify differences in biological activity. Our study established a correlation between the position of CRD amino acid side chains and the stimulation of NO release from endothelium.     

Bayesian multisensory integration and cross-modal spatial links.

Our perception of the word is the result of combining information between several senses, such as vision, audition and proprioception. These sensory modalities use widely different frames of reference to represent the properties and locations of object. Moreover, multisensory cues come with different degrees of reliability, and the reliability of a given cue can change in different contexts. The Bayesian framework--which we describe in this review--provides an optimal solution to deal with this issue of combining cues that are not equally reliable. However, this approach does not address the issue of frames of references. We show that this problem can be solved by creating cross-modal spatial links in basis function networks. Finally, we show how the basis function approach can be combined with the Bayesian framework to yield networks that can perform optimal multisensory combination. On the basis of this theory, we argue that multisensory integration is a dialogue between sensory modalities rather that the convergence of all sensory information onto a supra-modal area.