The Matricellular Protein Periostin Is Required for Sost Inhibition and the Anabolic Response to Mechanical Loading and Physical Activity

Periostin (gene Postn) is a secreted extracellular matrix protein involved in cell recruitment and adhesion and plays an important role in odontogenesis. In bone, periostin is preferentially expressed in the periosteum, but its functional significance remains unclear. We investigated Postn^−/− mice and their wild type littermates to elucidate the role of periostin in the skeletal response to moderate physical activity and direct axial compression of the tibia. Furthermore, we administered a sclerostin-blocking antibody to these mice in order to demonstrate the influence of sustained Sost expression in their altered bone phenotypes. Cancellous and cortical bone microarchitecture as well as bending strength were altered in Postn^−/− compared with Postn^+/+ mice. Exercise and axial compression both significantly increased bone mineral density and trabecular and cortical microarchitecture as well as biomechanical properties of the long bones in Postn^+/+ mice by increasing the bone formation activity, particularly at the periosteum. These changes correlated with an increase of periostin expression and a consecutive decrease of Sost in the stimulated bones. In contrast, mechanical stimuli had no effect on the skeletal properties of Postn^−/− mice, where base-line expression of Sost levels were higher than Postn^+/+ and remained unchanged following axial compression. In turn, the concomitant injection of sclerostin-blocking antibody rescued the bone biomechanical response in Postn^−/− mice. Taken together, these results indicate that the matricellular periostin protein is required for Sost inhibition and thereby plays an important role in the determination of bone mass and microstructural in response to loading.     

Multilocus sequence typing supports the hypothesis that Ochrobactrum anthropi displays a human-associated subpopulation

Background  

Ochrobactrum anthropi is a versatile bacterial species with strains living in very diverse habitats. It is increasingly recognized as opportunistic pathogen in hospitalized patients. The population biology of the species particularly with regard to the characteristics of the human isolates is being investigated. To address this issue, we proposed a polyphasic approach consisting in Multi-Locus Sequence Typing (MLST), multi-locus phylogeny, genomic-based fingerprinting by pulsed-field gel electrophoresis (PFGE) and antibiotyping.     

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

Background

Hyperactivity of the epithelial sodium (Na⁺) channel (ENaC) and increased Na⁺ absorption by airway epithelial cells leading to airway surface liquid dehydration and impaired mucociliary clearance are thought to play an important role in the pathogenesis of cystic fibrosis (CF) pulmonary disease. In airway epithelial cells, ENaC is constitutively activated by endogenous trypsin-like serine proteases such as Channel-Activating Proteases (CAPs). It was recently reported that ENaC activity could also be stimulated by apical treatment with human neutrophil elastase (hNE) in a human airway epithelial cell line, suggesting that hNE inhibition could represent a novel therapeutic approach for CF lung disease. However, whether hNE can also activate Na⁺ reabsorption in primary human nasal epithelial cells (HNEC) from control or CF patients is currently unknown.

Methods

We evaluated by short-circuit current (I_sc) measurements the effects of hNE and EPI-hNE4, a specific hNE inhibitor, on ENaC activity in primary cultures of HNEC obtained from control (9) and CF (4) patients.

Results

Neither hNE nor EPI-hNE4 treatments did modify I_sc in control and CF HNEC. Incubation with aprotinin, a Kunitz-type serine protease inhibitor that blocks the activity of endogenous CAPs, decreased I_sc by 27.6% and 54% in control and CF HNEC, respectively. In control and CF HNEC pretreated with aprotinin, hNE did significantly stimulate I_sc, an effect which was blocked by EPI-hNE4.

Conclusions

These results indicate that hNE does activate ENaC and transepithelial Na⁺ transport in both normal and CF HNEC, on condition that the activity of endogenous CAPs is first inhibited. The potent inhibitory effect of EPI-hNE4 on hNE-mediated ENaC activation observed in our experiments highlights that the use of EPI-hNE4 could be of interest to reduce ENaC hyperactivity in CF airways.     

The chloroplast lumen and stromal proteomes of Arabidopsis thaliana show differential sensitivity to short- and long-term exposure to low temperature

Cold acclimation and over-wintering by herbaceous plants are energetically expensive and are dependent on functional plastid metabolism. To understand how the stroma and the lumen proteomes adapt to low temperatures, we have taken a proteomic approach (difference gel electrophoresis) to identify proteins that changed in abundance in Arabidopsis chloroplasts during cold shock (1 day), and short- (10 days) and long-term (40 days) acclimation to 5 degrees C. We show that cold shock (1 day) results in minimal change in the plastid proteomes, while short-term (10 days) acclimation results in major changes in the stromal but few changes in the lumen proteome. Long-term acclimation (40 days) results in modulation of the proteomes of both compartments, with new proteins appearing in the lumen and further modulations in protein abundance occurring in the stroma. We identify 43 differentially displayed proteins that participate in photosynthesis, other plastid metabolic functions, hormone biosynthesis and stress sensing and signal transduction. These findings not only provide new insights into the cold response and acclimation of Arabidopsis, but also demonstrate the importance of studying changes in protein abundance within the relevant cellular compartment.     

Field assessment in Cameroon of a reader of POC-CCA lateral flow strips for the quantification of Schistosoma mansoni circulating cathodic antigen in urine

BackgroundDetermining Schistosoma mansoni infection rate and intensity is challenging due to the low sensitivity of the Kato-Katz (KK) test that underestimates the true disease prevalence. Circulating cathodic antigen (CCA) excreted in urine is constantly produced by adult worms and has been used as the basis of a simple, non-invasive point of care test (POC-CCA) for Schistosoma mansoni infections. Although the abundance of CCA in urine is proportional to worm burden, the POC-CCA test is marketed as a qualitative test, making it difficult to investigate the wide range of infection intensities. This study was designed to compare the prevalence and intensity of S. mansoni by KK and POC-CCA and quantify, on fresh and frozen (<-20°C) urine samples, CCA using the visual scores and the ESEquant LR3 reader.MethodologyStool and urine samples were collected from 759 school-aged children. The prevalence and intensity of S. mansoni were determined using KK and POC-CCA. The degree of the positivity of POC-CCA was estimated by quantifying CCA on fresh and frozen urine samples using visual scores and strip reader. The prevalence, the infection intensity as well the relative amounts of CCA were compared.ResultsThe S. mansoni infection rates inferred from POC-CCA and KK were 40.7% and 9.4% respectively. Good correlations were observed between infection intensities recorded by; i) the reader and visual scoring system on fresh (Rho = 0.89) and frozen samples (Rho = 0.97), ii) the reader on fresh urine samples and KK (epg) (Rho = 0.44). Nevertheless, 238 POC-CCA positive children were negative for KK, and sixteen of them had high levels of CCA. The correlation between results from the reader on fresh and frozen samples was good (Rho = 0.85). On frozen samples, CCA was not detected in 55 samples that were positive in fresh urine samples.ConclusionThis study confirmed the low sensitivity of KK and the high capacity of POC-CCA to provide reliable data on the prevalence and intensity of S. mansoni infections. The lateral flow reader enabled accurate quantification of CCA under field conditions on fresh and frozen urine samples with less time and effort than KK.     

Proprotein convertase PC7 enhances the activation of the EGF receptor pathway through processing of the EGF precursor

Although the processing profile of the membrane-bound epidermal growth factor precursor (pro-EGF) is tissue-specific, it has not been investigated at the cellular level nor have the cognate proteinases been defined. Among the proprotein convertases (PCs), only the membrane-bound PC7, the most ancient and conserved basic amino acid-specific PC family member, induces the processing of pro-EGF into an ～115-kDa transmembrane form (EGF-115) at an unusual VHPR(290)↓A motif. Because site-directed mutagenesis revealed that Arg(290) is not critical, the generation of EGF-115 by PC7 is likely indirect. This was confirmed by testing a wide range of protease inhibitors, which revealed that the production of EGF-115 is most probably achieved via the activation by PC7 of a latent serine and/or cysteine protease(s). EGF-115 is more abundant at the cell surface than pro-EGF and is associated with a stronger EGF receptor (EGFR) activation, as evidenced by higher levels of phosphorylated ERK1/2. This suggests that the generation of EGF-115 represents a regulatory mechanism of juxtacrine EGFR activation. Thus, PC7 is distinct from the other PCs in its ability to enhance the activation of the cell surface EGFR.     

Identification of genetic markers for differentiation of Shiga toxin-producing, enteropathogenic, and avirulent strains of Escherichia coli O26

Shiga toxin-producing Escherichia coli (STEC) O26 is one of the top five enterohemorrhagic E. coli (EHEC) O groups most often associated with hemorrhagic colitis and hemolytic uremic syndrome (HUS) worldwide. STEC O26 is considered to have evolved from enteropathogenic (EPEC) O26 strains through the acquisition of Shiga toxin (Stx)-encoding genes. Our PCR data identified several STEC-like strains expressing all features of STEC except Stx production and carrying remnants of Stx phages that were probably derivatives of EHEC O26. EHEC and EPEC O26 strains phenotypically resemble O26 EHEC-like and apathogenic E. coli O26 strains and are therefore undistinguishable by cultural methods. A clear discrimination between the different O26 groups is required for diagnostics in patients and for control of food safety. To develop an assay for specific detection of EHEC and EHEC-like O26 strains, we used a high-throughput PCR approach for selection of discriminative genetic markers among 33 tested genes mostly encoding type III secretion system effector proteins. The genes ECs1822, nleH1-2, nleA, nleC, nleH1-1, nleG, nleG2, nleG6-1, nleG6-2, espJ, espM2, nleG8-2, espG, ent (or espL2), nleB, nleE, efa1, and espB were detected at different frequencies in O26 EHEC, EHEC-like, and EPEC strains, indicating the possible role of these genes in virulence of human pathogenic O26 strains. The espK and espN genes were detected only in EHEC and EHEC-like O26 strains. espK was present in 99.14% of EHEC and 91.14% of EHEC-like O26 strains and was hence the best candidate as a genetic marker for characterizing these pathogroups. These data were corroborated by a genotyping real-time PCR test based on allelic discrimination of the arcA (aerobic respiratory control protein A) gene. The results indicate that a combination of molecular detection tools for O26 wzx (wzx(O26)), eae-beta, stx, espK, and arcA genotyping is highly discriminative for clear identification of EHEC and EHEC-like E. coli O26 strains. This simple diagnostic test might be applicable in hospital service laboratories or public health laboratories to test strains isolated from stools of patients suffering from diarrhea.