Physiological characterization of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from agro-industrial and environmental origins with possible probiotic function

Probiotics are defined as live microorganisms, which when administered in adequate amount confer a health benefit to the host. Most of studied or commercialized probiotics contain bacteria and very few of them present yeast in its composition. In this last case, the microorganisms almost always belong to Saccharomyces genus. In the present study, it was of interest to screen among 103 non-Saccharomyces yeasts a candidate for probiotic by using in vitro and in vivo criteria. In vitro assays included growth at 37°C and production of antagonistic compounds against enteropathogenic indicators, and the in vivo assays evaluated the colonization ability of mouse gastrointestinal tract without pathologic consequences and the protective ability in mice experimentally challenged with Clostridium difficile. In conclusion, Pichia kluyveri strain 898 showed to be a potential candidate for probiotic use, based on the criteria cited above, particularly as demonstrated by its protective effect against experimental infection in mice. Interestingly, an in vivo inhibition against C. difficile observed in the animal models did not correlate with the results obtained with the in vitro assays.     

Production of biodiesel from acid waste lard

The objective of the present work was: (i) to enable biodiesel production from acid waste lard; (ii) to study the esterification reaction as possible pre-treatment at different temperatures, catalyst amount and reaction times; (iii) to evaluate biodiesel quality according to EN 14214 after basic transesterification of the pre-treated fat; and (iv) to predict the impact of using such waste as raw material in mixture with soybean oil. Temperature and catalyst amount were the most important reaction conditions which mostly affected biodiesel quality, namely viscosity and purity. The selected pre-treatment conditions were 65 °C, 2.0 wt% H₂SO₄ and 5 h, which allowed obtaining a product with a viscosity of 4.81 mm² s⁻¹ and a purity of 99.6 wt%. The proposed pre-treatment was effective to enable acid wastes as single raw materials for biodiesel production with acceptable quality; however, low yields were obtained (65 wt%). Alkali transesterification of a mixture of waste lard and soybean oil resulted in a product with a purity of 99.8 wt% and a yield of 77.8 wt%, showing that blending might be an interesting alternative to recycle such wastes. Also, because in addition to using conventional and relatively economical processes, some biodiesel properties depending on the raw material composition (such as the iodine value) might even be improved.     

Cell communication with the neural plate is required for induction of neural markers by BMP inhibition: evidence for homeogenetic induction and implications for Xenopus animal cap and chick explant assays

In Xenopus, the animal cap is very sensitive to BMP antagonists, which result in neuralization. In chick, however, only cells at the border of the neural plate can be neuralized by BMP inhibition. Here we compare the two systems. BMP antagonists can induce neural plate border markers in both ventral Xenopus epidermis and non-neural chick epiblast. However, BMP antagonism can only neuralize ectodermal cells when the BMP-inhibited cells form a continuous trail connecting them to the neural plate or its border, suggesting that homeogenetic neuralizing factors can only travel between BMP-inhibited cells. Xenopus animal cap explants contain cells fated to contribute to the neural plate border and even to the anterior neural plate, explaining why they are so easily neuralized by BMP-inhibition. Furthermore, chick explants isolated from embryonic epiblast behave like Xenopus animal caps and express border markers. We propose that the animal cap assay in Xenopus and explant assays in the chick are unsuitable for studying instructive signals in neural induction.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Detrimental role of IL-33/ST2 pathway sustaining a chronic eosinophil-dependent Th2 inflammatory response,tissue damage and parasite burden during Toxocara canis infection in mice

Toxocariasis is a neglected disease that affects people around the world. Humans become infected by accidental ingestion of eggs containing Toxocara canis infective larvae, which upon reaching the intestine, hatch, penetrate the mucosa and migrate to various tissues such as liver, lungs and brain. Studies have indicated that Th2 response is the main immune defense mechanism against toxocariasis, however, there are still few studies related to this response, mainly the IL-33/ST2 pathway. Some studies have reported an increase in IL-33 during helminth infections, including T. canis. By binding to its ST2 receptor, IL-33 stimulating the Th2 polarized immune cell and cytokine responses. Thus, we aimed to investigate the role of the IL-33/ST2 pathway in the context of T. canis larval migration and the immunological and pathophysiological aspects of the infection in the liver, lungs and brain from Wild-Type (WT) BALB/c background and genetically deficient mice for the ST2 receptor (ST2^-/-). The most important findings revealed that the IL-33/ST2 pathway is involved in eosinophilia, hepatic and cerebral parasitic burden, and induces the formation of granulomas related to tissue damage and pulmonary dysfunction. However, ST2^-/- mice, the immune response was skewed to Th1/Th17 type than Th2, that enhanced the control of parasite burden related to IgG2a levels, tissue macrophages infiltration and reduced lung dysfunction. Collectively, our results demonstrate that the Th2 immune response triggered by IL-33/ST2 pathway mediates susceptibility to T. canis, related to parasitic burden, eosinophilia and granuloma formation in which consequently contributes to tissue inflammation and injury.     

Cost-effective HLA typing with tagging SNPs predicts celiac disease risk haplotypes in the Finnish, Hungarian, and Italian populations

Human leukocyte antigen (HLA) genes, located on chromosome 6p21.3, have a crucial role in susceptibility to various autoimmune and inflammatory diseases, such as celiac disease and type 1 diabetes. Certain HLA heterodimers, namely DQ2 (encoded by the DQA1*05 and DQB1*02 alleles) and DQ8 (DQA1*03 and DQB1*0302), are necessary for the development of celiac disease. Traditional genotyping of HLA genes is laborious, time-consuming, and expensive. A novel HLA-genotyping method, using six HLA-tagging single-nucleotide polymorphisms (SNPs) and suitable for high-throughput approaches, was described recently. Our aim was to validate this method in the Finnish, Hungarian, and Italian populations. The six previously reported HLA-tagging SNPs were genotyped in patients with celiac disease and in healthy individuals from Finland, Hungary, and two distinct regions of Italy. The potential of this method was evaluated in analyzing how well the tag SNP results correlate with the HLA genotypes previously determined using traditional HLA-typing methods. Using the tagging SNP method, it is possible to determine the celiac disease risk haplotypes accurately in Finnish, Hungarian, and Italian populations, with specificity and sensitivity ranging from 95% to 100%. In addition, it predicts homozygosity and heterozygosity for a risk haplotype, allowing studies on genotypic risk effects. The method is transferable between populations and therefore suited for large-scale research studies and screening of celiac disease among high-risk individuals or at the population level. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Lotta Koskinen and Jihane Romanos are authors with equal contribution.     

Lateral Distribution of the Transmembrane Domain of Influenza Virus Hemagglutinin Revealed by Time-resolved Fluorescence Imaging

Influenza virus hemagglutinin (HA) has been suggested to be enriched in liquid-ordered lipid domains named rafts, which represent an important step in virus assembly. We employed Förster resonance energy transfer (FRET) via fluorescence lifetime imaging microscopy to study the interaction of the cytoplasmic and transmembrane domain (TMD) of HA with agly co sylphos pha tidyl ino si tol (GPI)-anchored peptide, an established marker for rafts in the exoplasmic leaflet of living mammalian plasma membranes. Cyan fluorescent protein (CFP) was fused to GPI, whereas the HA sequence was tagged with yellow fluorescent protein (YFP) on its exoplasmic site (TMD-HA-YFP), avoiding any interference of fluorescent proteins with the proposed role of the cytoplasmic domain in lateral organization of HA. Constructs were expressed in Chinese hamster ovary cells (CHO-K1) for which cholesterol-sensitive lipid nanodomains and their dimension in the plasma membrane have been described (Sharma, P., Varma, R., Sarasij, R. C., Ira, Gousset, K., Krishnamoorthy, G., Rao, M., and Mayor, S. (2004) Cell 116, 577–589). Upon transfection in CHO-K1 cells, TMD-HA-YFP is partially expressed as a dimer. Only dimers are targeted to the plasma membrane. Clustering of TMD-HA-YFP with GPI-CFP was observed and shown to be reduced upon cholesterol depletion, a treatment known to disrupt rafts. No indication for association of TMD-HA-YFP with GPI-CFP was found when palmitoylation, an important determinant of raft targeting, was suppressed. Clustering of TMD-HA-YFP and GPI-CFP was also observed in purified plasma membrane suspensions by homoFRET. We concluded that the pal mit oy lated TMD-HA alone is sufficient to recruit HA to cholesterol-sensitive nanodomains. The corresponding construct of the spike protein E2 of Semliki Forest virus did not partition preferentially in such domains.Assembly of enveloped viruses requires the selective recruitment of viral components at distinct sites of the host cell membranes from which viruses bud. One of the most intensely studied enveloped viruses with respect to assembly is the influenza virus, in which budding takes place at the plasma membrane of epithelial cells. Three membrane proteins are embedded in the influenza virus envelope: hemagglutinin (HA),3 which mediates binding of the virus to the host cell and fusion with cell target membrane (1); neuraminidase; and the proton channel M2. The inner viral membrane leaflet is covered by the matrix protein M1, which is supposed to mediate binding of the eight viral RNA-nucleoprotein complexes harboring the genetic information of the virus. Several studies support a role of lipid domains as a platform for enrichment of viral components. HA, the most abundant envelope protein of the influenza virus, has been found to be enriched in detergent-resistant membrane (DRM) fractions (2–4). Typical lipid components of those fractions are saturated phospholipids, glycosphingolipids, and cholesterol, which are known to form liquid-ordered domains (5). This has led to the idea that so-called lipid rafts, which resemble liquid-ordered domains, could function as assembly sites. Support for this hypothesis was given by the observation that the lipid composition of the influenza virus envelope is more similar to that of a raft than to the overall plasma membrane (2, 6).As it has been shown that DRM fractions may not represent the native state of lipid domains, in particular of rafts (7, 8), subsequent efforts have focused on other techniques to assess the lateral organization of HA. Electron microscopy studies using immunogold labeling (4, 9), Förster resonance energy transfer (FRET) measurements between fluorescent HA antibodies in fixed cells (9), and investigations on photoactivatable HA in living cells (10) have revealed cholesterol-sensitive clustering of HA in the plasma membrane of mammalian cells at lengths between 20 and 900 nm.A specific problem encountered in studying the lateral organization of proteins in the plasma membrane is that lipid domains as rafts are typically organized at a submicroscopic level. Indeed, several attempts to image raft domains in biological membranes have suggested that rafts are very small and highly dynamic (11, 12). A guiding study in the characterization of lipid domains in biological membranes has been performed by Mayor and colleagues (13) on the plasma membrane of CHO-K1 cells. Based on homoFRET measurements they have shown that about 20–40% of GFP-tagged glycosylphosphatidylinositol (GPI) (for review see Brown and Rose (14)) and other GPI-anchored proteins are organized with about three to four copies in small cholesterol-sensitive clusters. Mathematical modeling of those experimental data is consistent with a domain diameter of about 5 nm (13).In the present study we investigated the lateral organization of the C terminus of HA, corresponding to the transmembrane domain (TMD), and the cytoplasmic tail (CT) of the protein in the plasma membrane of CHO-K1 cells, taking advantage of the well characterized spatial arrangement of the raft marker GPI in those cells (see above). Lateral organization was studied essentially by fluorescence lifetime imaging microscopy (FLIM)-based FRET between CFP (donor) and YFP (acceptor). For this purpose, we replaced the ectodomain of HA by YFP and studied FRET between this construct and GPI-CFP. As a complementary approach, we performed ensemble measurements in suspensions of plasma membranes purified from cells expressing fluorescent GPI and HA constructs. We measured homoFRET by time-resolved fluorescence anisotropy, providing information on the aggregation/clustering state of the fluorescent constructs, which is important in rationalizing the FLIM-FRET data.     

GPIomics: global analysis of glycosylphosphatidylinositol‐anchored molecules of Trypanosoma cruzi

Glycosylphosphatidylinositol (GPI) anchoring is a common, relevant posttranslational modification of eukaryotic surface proteins. Here, we developed a fast, simple, and highly sensitive (high attomole‐low femtomole range) method that uses liquid chromatography‐tandem mass spectrometry (LC‐MSⁿ) for the first large‐scale analysis of GPI‐anchored molecules (i.e., the GPIome) of a eukaryote, Trypanosoma cruzi, the etiologic agent of Chagas disease. Our genome‐wise prediction analysis revealed that approximately 12% of T. cruzi genes possibly encode GPI‐anchored proteins. By analyzing the GPIome of T. cruzi insect‐dwelling epimastigote stage using LC‐MSⁿ, we identified 90 GPI species, of which 79 were novel. Moreover, we determined that mucins coded by the T. cruzi small mucin‐like gene (TcSMUG S) family are the major GPI‐anchored proteins expressed on the epimastigote cell surface. TcSMUG S mucin mature sequences are short (56–85 amino acids) and highly O‐glycosylated, and contain few proteolytic sites, therefore, less likely susceptible to proteases of the midgut of the insect vector. We propose that our approach could be used for the high throughput GPIomic analysis of other lower and higher eukaryotes.     

Redox thiol status plays a central role in the mobilization and metabolism of nitric oxide in human red blood cells

We assessed the redox thiol status influence on nitric oxide (NO) metabolism and efflux in erythrocytes stimulated with acetylcholinesterase substrate (acetylcholine, ACh) and inhibitor (velnacrine maleate, VM). Erythrocyte suspensions from healthy donors were incubated with increasing concentrations of dithiothreitol (1-50 μM), in the presence and absence of acetylcholine/velnacrine (10 μM). Levels of NO, nitrite/nitrate, S-nitrosohemoglobin, peroxynitrite and S-nitrosoglutathione were determined by spectrofluorimetric and spectrophotometric methods.Dithiothreitol significantly mobilized NO toward nitrite/nitrate and S-nitrosoglutathione, and decreased the amount of NO efflux. Both ACh/VM induce changes on the levels of erythrocyte nitrite/nitrate dependent on the DTT concentration. Higher levels of peroxynitrite and S-nitrosoglutathione were seen with velnacrine in presence of DTT 1 and 50 μM.We concluded that dithiothreitol-induced activation of erythrocyte thiol status decreases NO efflux and allows greater intracellular NO mobilization onto different derivative molecules, both in the absence and presence of acetylcholinesterase substrate and inhibitor.