Proteomic Biomarkers Associated with Streptococcus agalactiae Invasive Genogroups

Group B streptococcus (GBS, Streptococcus agalactiae) is a leading cause of meningitis and sepsis in newborns and an etiological agent of meningitis, endocarditis, osteoarticular and soft tissue infections in adults. GBS isolates are routinely clustered in serotypes and in genotypes. At present one GBS sequence type (i.e. ST17) is considered to be closely associated with bacterial invasiveness and novel proteomic biomarkers could make a valuable contribution to currently available GBS typing data. For that purpose we analyzed the protein profiles of 170 genotyped GBS isolates by Surface-Enhanced Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (SELDI). Univariate statistical analysis of the SELDI profiles identified four protein biomarkers significantly discriminating ST17 isolates from those of the other sequence types. Two of these biomarkers (MW of 7878 Da and 12200 Da) were overexpressed and the other two (MW of 6258 Da and 10463 Da) were underexpressed in ST17. The four proteins were isolated by mass spectrometry-assisted purification and their tryptic peptides analyzed by LC-MS/MS. They were thereby identified as the small subunit of exodeoxyribonuclease VII, the 50S ribosomal protein L7/L12, a CsbD-like protein and thioredoxin, respectively. In conclusion, we identified four candidate biomarkers of ST17 by SELDI for high-throughput screening. These markers may serve as a basis for further studies on the pathophysiology of GBS infection, and for the development of novel vaccines.     

Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling

Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.     

Influence of manufacturing processes on cell surface properties of probiotic strain <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> Lcr35<Superscript>®</Superscript>

The influence of the industrial process on the properties of probiotics, administered as complex manufactured products, has been poorly investigated. In the present study, we comparatively assessed the cell wall characteristics of the probiotic strain Lactobacillus rhamnosus Lcr35® together with three of its commercial formulations with intestinal applications. Putative secreted and transmembrane-protein-encoding genes were initially searched in silico in the genome of L. rhamnosus Lcr35®. A total of 369 candidate genes were identified which expressions were followed using a custom Lactobacillus DNA chip. Among them, 60 or 67 genes had their expression either upregulated or downregulated in the Lcr Restituo® packet or capsule formulations, compared to the native Lcr35® strain. Moreover, our data showed that the probiotic formulations (Lcr Lenio®, Lcr restituo® capsule and packet) showed a better capacity to adhere to intestinal epithelial Caco-2 cells than the native Lcr35® strain. Microbial (MATS) tests showed that the probiotic was an electron donor and that they were more hydrophilic than the native strain. The enhanced adhesion capacity of the active pharmaceutical ingredients (APIs) to epithelial Caco-2 cells and their antipathogen effect could be due to this greater surface hydrophilic character. These findings suggest that the manufacturing process influences the protein composition and the chemical properties of the cell wall. It is therefore likely that the antipathogen effect of the formulation is modulated by the industrial process. Screening of the manufactured products’ properties would therefore represent an essential step in evaluating the effects of probiotic strains.     

Matrilin-3 switches from anti- to pro-anabolic upon integration to the extracellular matrix

The extracellular matrix (ECM) has long been viewed primarily as an organized network of solid-phase ligands for integrin receptors. During degenerative processes, such as osteoarthritis, the ECM undergoes deterioration, resulting in its remodeling and in the release of some of its components. Matrilin-3 (MATN3) is an almost cartilage specific, pericellular protein acting in the assembly of the ECM of chondrocytes. In the past, MATN3 was found required for cartilage homeostasis, but also involved in osteoarthritis-related pro-catabolic functions. Here, to better understand the pathological and physiological functions of MATN3, its concentration as a circulating protein in articular fluids of human osteoarthritic patients was determined and its functions as a recombinant protein produced in human cells were investigated with particular emphasis on the physical state under which it is presented to chondrocytes. MATN3 down-regulated cartilage extracellular matrix (ECM) synthesis and up-regulated catabolism when administered as a soluble protein. When artificially immobilized, however, MATN3 induced chondrocyte adhesion via a α5β1 integrin-dependent mechanism, AKT activation and favored survival and ECM synthesis. Furthermore, MATN3 bound directly to isolated α5β1 integrin in vitro. TGFβ1 stimulation of chondrocytes allowed integration of exogenous MATN3 into their ECM and ECM-integrated MATN3 induced AKT phosphorylation and improved ECM synthesis and accumulation. In conclusion, the integration of MATN3 to the pericellular matrix of chondrocytes critically determines the direction toward which MATN3 regulates cartilage metabolism. These data explain how MATN3 plays either beneficial or detrimental functions in cartilage and highlight the important role played by the physical state of ECM molecules.     

Rho-ROCK-dependent ezrin-radixin-moesin phosphorylation regulates Fas-mediated apoptosis in Jurkat cells

Upon engagement by its ligand, the Fas receptor (CD95/APO-1) is oligomerized in a manner dependent on F-actin. It has been shown that ezrin, a member of the ERM (ezrin-radixin-moesin) protein family can link Fas to the actin cytoskeleton. We show herein that in Jurkat cells, not only ezrin but also moesin can associate with Fas. The same observation was made in activated human peripheral blood T cells. Fas/ezrin or moesin (E/M) association increases in Jurkat cells following Fas triggering and occurs concomitantly with the formation of SDS- and 2-ME-stable high molecular mass Fas aggregates. Ezrin and moesin have to be present together for the formation of Fas aggregates since down-regulation of either ezrin or moesin expression with small interfering RNAs completely inhibits Fas aggregate formation. Although FADD (Fas-associated death domain protein) and caspase-8 associate with Fas in the absence of E/M, subsequent events such as caspase-8 activation and sensitivity to apoptosis are decreased. During the course of Fas stimulation, ezrin and moesin become phosphorylated, respectively, on T567 and on T558. This phosphorylation is mediated by the kinase ROCK (Rho-associated coiled coil-containing protein kinase) I subsequently to Rho activation. Indeed, inhibition of either Rho or ROCK prevents ezrin and moesin phosphorylation, abrogates the formation of Fas aggregates, and interferes with caspase-8 activation. Thus, phosphorylation of E/M by ROCK is involved in the early steps of apoptotic signaling following Fas triggering and regulates apoptosis induction.     

Mouse heat-shock factor 1 (HSF1) is involved in testicular response to genotoxic stress induced by doxorubicin

Heat-shock factor 1 (HSF1) protects cells and organisms against various types of stress, either by triggering a complex response that promotes cell survival or by triggering cell death when stress-induced alterations cannot be rescued. Although this dual role of HSF1 was observed in spermatogenesis exposed to heat shock or proteotoxic stress, HSF1 was also reported to contribute to cell resistance against genotoxic stress, such as that caused by doxorubicin, an anticancer drug in common clinical use. To better understand the stress/cell-dependent functions of HSF1, we used wild-type and Hsf1(tm1Ijb)/Hsf1(tm1Ijb) males to determine the role of HSF1 in the genotoxic stress response elicited in spermatogenic cells. Within 2 days after a single intraperitoneal injection of doxorubicin (DOXO; 5 mg/kg), proliferation of Hsf1+/+ but not Hsf1-/- spermatogenic cells was significantly reduced, whereas cell death was increased in mitotic germ cells and metaphase I spermatocytes. By 21 days, meiotic cells were depleted in all treated Hsf1+/+ testes but not in Hsf1-/- ones. Nevertheless, after 3 mo, spermatogenesis showed better signs of recovery in Hsf1+/+ than in Hsf1-/- males. Taken together, these data indicate that acute response to genotoxic stress in the testis involves HSF1-dependent mechanisms that induce apoptotic cell death in a TRP53-independent manner, but also intervene on a longer term to restore seminiferous tubules.     

Length–weight relationships of eight Asian freshwater fish species in Nam Theun 2 Reservoir (Laos PDR)

Length–weight relationships (LWRs) were determined for eight Asian freshwater fish species that were not yet recorded in any peer‐reviewed journal indexed in the Web of Science or the FishBase database. Fish specimens from Nam Theun 2 reservoir in Lao PDR, were collected from April 2012 to August 2016 using several methods to cover a large size‐spectrum for each species. For LWRs using the general equation from Beckman (1948), the parameter b (slope value on the growth pattern) ranges from 2.908 (Puntius brevis) to 3.429 (Rasbora paviana). This study provides an update in maximum lengths for two species and first LWRs for eight common freshwater species found mainly in reservoirs, and thus represents a means for further population dynamic evolution analyses and local fisheries management.     

Impaired Antibody Response Causes Persistence of Prototypic T Cell–Contained Virus

CD8 T cells are recognized key players in control of persistent virus infections, but increasing evidence suggests that assistance from other immune mediators is also needed. Here, we investigated whether specific antibody responses contribute to control of lymphocytic choriomeningitis virus (LCMV), a prototypic mouse model of systemic persistent infection. Mice expressing transgenic B cell receptors of LCMV-unrelated specificity, and mice unable to produce soluble immunoglobulin M (IgM) exhibited protracted viremia or failed to resolve LCMV. Virus control depended on immunoglobulin class switch, but neither on complement cascades nor on Fc receptor γ chain or Fc γ receptor IIB. Cessation of viremia concurred with the emergence of viral envelope-specific antibodies, rather than with neutralizing serum activity, and even early nonneutralizing IgM impeded viral persistence. This important role for virus-specific antibodies may be similarly underappreciated in other primarily T cell–controlled infections such as HIV and hepatitis C virus, and we suggest this contribution of antibodies be given consideration in future strategies for vaccination and immunotherapy.     

Analysis of glycated hemoglobin A1c by capillary electrophoresis and capillary isoelectric focusing

Two capillary electrophoretic methods were developed and evaluated for measurement of glycated hemoglobin A1c (HbA1c). First, a capillary electrophoresis analysis is performed with a sodium tetraborate buffer (pH 9.3) as background electrolyte in a neutrally coated capillary. HbA1c is separated from HbA0 due to specific interactions of borate anions with the cis–diol pattern in the saccharide moiety of glycohemoglobin. Second, a capillary isoelectric focusing method, which exploits a difference in pI values of HbA0 and HbA1c, is performed with Servalyt pH 6–8 or alternatively with Biolyte pH 6–8 carrier ampholytes spiked with a narrow pH cut of pH 7.2 prepared by preparative fractionation of Servalyt pH 4–9 carrier ampholytes. Both methods reflect recent developments in the methodology of capillary electrophoresis. They allow quantifying HbA1c in generic capillary electrophoresis analyzer with specificity that is consistent with previously reported electrophoretic assays in slab gels and capillaries.