RIG-I has guts： Identification of a role for RIG-I in colitis ：levelopment

Countries in North America and Europe have the highest incidence of inflammatory disorders of the gastrointestinal tract. The prevalence of inflammatory bowel diseases （IBD）, which comprise Crohn＇s disease （CD） and ulcerative colitis （UC）, now ranges from 10-200 cases per 100 000 individuals [ 1 ]. Although new therapeutic approaches have been developed to improve current treatments, the etiology of this disorder remains elusive. Crohn＇s disease and ulcerative colitis are known to show similar clinical and pathological characteristics; however it is now believed that these two forms of IBD are entirely different. Epidemio- logical studies have revealed that these differences might be explained by the fact that environmental and genetic factors play important roles in the pathogenesis and susceptibility to IBD [ 1 ].     

13C Incorporation into Signature Fatty Acids as an Assay for Carbon Allocation in Arbuscular Mycorrhiza

The ubiquitous arbuscular mycorrhizal fungi consume significant amounts of plant assimilated C, but this C flow has been difficult to quantify. The neutral lipid fatty acid 16:1ω5 is a quantitative signature for most arbuscular mycorrhizal fungi in roots and soil. We measured carbon transfer from four plant species to the arbuscular mycorrhizal fungus Glomus intraradices by estimating ¹³C enrichment of 16:1ω5 and compared it with ¹³C enrichment of total root and mycelial C. Carbon allocation to mycelia was detected within 1 day in monoxenic arbuscular mycorrhizal root cultures labeled with [¹³C]glucose. The ¹³C enrichment of neutral lipid fatty acid 16:1ω5 extracted from roots increased from 0.14% 1 day after labeling to 2.2% 7 days after labeling. The colonized roots usually were more enriched for ¹³C in the arbuscular mycorrhizal fungal neutral lipid fatty acid 16:1ω5 than for the root specific neutral lipid fatty acid 18:2ω6,9. We labeled plant assimilates by using ¹³CO₂ in whole-plant experiments. The extraradical mycelium often was more enriched for ¹³C than was the intraradical mycelium, suggesting rapid translocation of carbon to and more active growth by the extraradical mycelium. Since there was a good correlation between ¹³C enrichment in neutral lipid fatty acid 16:1ω5 and total ¹³C in extraradical mycelia in different systems (r² = 0.94), we propose that the total amount of labeled C in intraradical and extraradical mycelium can be calculated from the ¹³C enrichment of 16:1ω5. The method described enables evaluation of C flow from plants to arbuscular mycorrhizal fungi to be made without extraction, purification and identification of fungal mycelia.     

The Vacuolar ATPase from <Emphasis Type="Italic">Entamoeba histolytica</Emphasis>: Molecular cloning of the gene encoding for the B subunit and subcellular localization of the protein

Background  

Entamoeba histolytica is a professional phagocytic cell where the vacuolar ATPase plays a key role. This enzyme is a multisubunit complex that regulates pH in many subcellular compartments, even in those that are not measurably acidic. It participates in a wide variety of cellular processes such as endocytosis, intracellular transport and membrane fusion. The presence of a vacuolar type H⁺-ATPase in E. histolytica trophozoites has been inferred previously from inhibition assays of its activity, the isolation of the Ehvma1 and Ehvma3 genes, and by proteomic analysis of purified phagosomes.     

Prediction of the in planta Phakopsora pachyrhizi secretome and potential effector families

Asian soybean rust (ASR), caused by the obligate biotrophic fungus Phakopsora pachyrhizi, can cause losses greater than 80%. Despite its economic importance, there is no soybean cultivar with durable ASR resistance. In addition, the P. pachyrhizi genome is not yet available. However, the availability of other rust genomes, as well as the development of sample enrichment strategies and bioinformatics tools, has improved our knowledge of the ASR secretome and its potential effectors. In this context, we used a combination of laser capture microdissection (LCM), RNAseq and a bioinformatics pipeline to identify a total of 36 350 P. pachyrhizi contigs expressed in planta and a predicted secretome of 851 proteins. Some of the predicted secreted proteins had characteristics of candidate effectors: small size, cysteine rich, do not contain PFAM domains (except those associated with pathogenicity) and strongly expressed in planta. A comparative analysis of the predicted secreted proteins present in Pucciniales species identified new members of soybean rust and new Pucciniales‐ or P. pachyrhizi‐specific families (tribes). Members of some families were strongly up‐regulated during early infection, starting with initial infection through haustorium formation. Effector candidates selected from two of these families were able to suppress immunity in transient assays, and were localized in the plant cytoplasm and nuclei. These experiments support our bioinformatics predictions and show that these families contain members that have functions consistent with P. pachyrhizi effectors.     

Influence of mathematical models and correction factors on binding results of polyphenols and retinol with β-lactoglobulin measured with fluorescence quenching

β-Lactoglobulin is frequently researched as food grade nanotransporter for hydrophobic compounds like polyphenols and fatty soluble vitamins. The effects of self-fluorescence and inner filtering caused by ligands on the analysis of ligand-β-lactoglobulin-binding were evaluated using fluorescence quenching data. Without correction for self-fluorescence or inner filtering the binding constants are either overestimated or binding is erroneously assumed. Additionally, the variance between the derived numbers of binding sites resulting by using different mathematical models with the same data set is about 50 %. Thus here we suggest the use of at least two very different mathematical approaches when deriving fluorescence titration data and evaluating the underlying restrictions of the respective model thoroughly. The greatest differences were found to concern K′a or K′d, whereas n seems to be relatively stable. The Cogan plot seems to be the best choice for FQ calculations, given that it does not require an estimation of added ligand concentration and that it is insensitive to residual fluorescence.     

Ubiquitous urease affects soybean susceptibility to fungi

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.     

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.     

A Novel MicroRNA-132-Surtuin-1 Axis Underlies Aberrant B-cell Cytokine Regulation in Patients with Relapsing-Remitting Multiple Sclerosis

Clinical trial results demonstrating that B-cell depletion substantially reduces new relapses in patients with multiple sclerosis (MS) have established that B cells play a role in the pathophysiology of MS relapses. The same treatment appears not to impact antibodies directed against the central nervous system, which underscores the contribution of antibody-independent functions of B cells to disease activity. One mechanism by which B cells are now thought to contribute to MS activity is by over-activating T cells, including through aberrant expression of B cell pro-inflammatory cytokines. However, the mechanisms underlying the observed B cell cytokine dysregulation in MS remain unknown. We hypothesized that aberrant expression of particular microRNAs might be involved in the dysregulated pro-inflammatory cytokine responses of B cells of patients with MS. Through screening candidate microRNAs in activated B cells of MS patients and matched healthy subjects, we discovered that abnormally increased secretion of lymphotoxin and tumor necrosis factor α by MS B cells is associated with abnormally increased expression of miR-132. Over-expression of miR-132 in normal B cells significantly enhanced their production of lymphotoxin and tumor necrosis factor α. The over-expression of miR-132 also suppressed the miR-132 target, sirtuin-1. We confirmed that pharmacological inhibition of sirtuin-1 in normal B cells induces exaggerated lymphotoxin and tumor necrosis factor α production, while the abnormal production of these cytokines by MS B cells can be normalized by resveratrol, a sirtuin-1 activator. These results define a novel miR-132-sirtuin-1 axis that controls pro-inflammatory cytokine secretion by human B cells, and demonstrate that a dysregulation of this axis underlies abnormal pro-inflammatory B cell cytokine responses in patients with MS.     

Measurement of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in cultured rat mesencephalic neurons by high-performance liquid chromatography with electrochemical detection

An HPLC method with electrochemical detection for the simultaneous measurement of serotonin (5-hydroxytryptamine) and 5-hydroxyindoleacetic acid in primary mesencephalic cell culture is described. The serotonin and 5-hydroxyindoleacetic acid cell content was measured on different days of growth in vitro; after twelve days in culture the amounts of serotonin and 5-hydroxyindoleacetic acid detected were 916.0 ± 70.2 and 215.8 ± 15.5 pg per well, respectively. The heterogeneity of neurons in our cultures and their capacity to take up serotonin were assessed by measuring the amounts of exogenous serotonin taken up in the presence of different monoamine uptake inhibitors. This method, sensitive and reliable, can represent a valid alternative to the use of labelled compounds.