The pore-forming toxin listeriolysin O mediates a novel entry pathway of L. monocytogenes into human hepatocytes

Intracellular pathogens have evolved diverse strategies to invade and survive within host cells. Among the most studied facultative intracellular pathogens, Listeria monocytogenes is known to express two invasins-InlA and InlB-that induce bacterial internalization into nonphagocytic cells. The pore-forming toxin listeriolysin O (LLO) facilitates bacterial escape from the internalization vesicle into the cytoplasm, where bacteria divide and undergo cell-to-cell spreading via actin-based motility. In the present study we demonstrate that in addition to InlA and InlB, LLO is required for efficient internalization of L. monocytogenes into human hepatocytes (HepG2). Surprisingly, LLO is an invasion factor sufficient to induce the internalization of noninvasive Listeria innocua or polystyrene beads into host cells in a dose-dependent fashion and at the concentrations produced by L. monocytogenes. To elucidate the mechanisms underlying LLO-induced bacterial entry, we constructed novel LLO derivatives locked at different stages of the toxin assembly on host membranes. We found that LLO-induced bacterial or bead entry only occurs upon LLO pore formation. Scanning electron and fluorescence microscopy studies show that LLO-coated beads stimulate the formation of membrane extensions that ingest the beads into an early endosomal compartment. This LLO-induced internalization pathway is dynamin-and F-actin-dependent, and clathrin-independent. Interestingly, further linking pore formation to bacteria/bead uptake, LLO induces F-actin polymerization in a tyrosine kinase-and pore-dependent fashion. In conclusion, we demonstrate for the first time that a bacterial pathogen perforates the host cell plasma membrane as a strategy to activate the endocytic machinery and gain entry into the host cell.     

Moving instead of asking? Performance-based tests and BASFI-questionnaire measure different aspects of physical function in ankylosing spondylitis

Introduction

Ankylosing Spondylitis (AS) is characterised by limitations in physical function. The Bath Ankylosing Spondylitis Functional Index (BASFI) is considered to be the gold-standard to assess physical function in AS patients. However, the BASFI questionnaire is a self-reported outcome measure and susceptible to subjective interpretation (under- or over-estimation). More objective outcome measures, like performance-based tests, could provide an objective outcome measurement for the evaluation of limitations in physical function. Therefore, the primary aim of this study was to determine the association between performance-based measures and the BASFI questionnaire.

Methods

In this cross-sectional study 126 AS patients completed the BASFI questionnaire and eight performance-based tests based on BASFI-items. Each test received three scores: one for performance (time or points) and a score for exertion and pain experienced during performance (using modified Borg-scale and VAS 0-100 mm, respectively). Linear regression analyses were used to assess the associations between the BASFI questionnaire and performance-based tests.

Results

The univariable association between performance and BASFI-score was moderate with a R-square of 0.31 and Beta of 0.56 (p's < 0.05). In a multivariable analysis, the association between performance, exertion and pain on the one hand and BASFI-score on the other was assessed; R-square increased to 0.54: the Beta's for exertion and pain during performance were 0.38 and 0.26, respectively; the Beta for performance decreased to 0.19 (p's < 0.05).

Conclusions

This study demonstrates that alongside actual performance, patients seem to incorporate exertion and pain in their assessment of perceived physical function on the BASFI questionnaire. Performance-based tests could provide an objective outcome measurement for the evaluation of physical function and give relevant new information in addition to the BASFI questionnaire.     

Design and applicability of DNA arrays and DNA barcodes in biodiversity monitoring

Background  

The rapid and accurate identification of species is a critical component of large-scale biodiversity monitoring programs. DNA arrays (micro and macro) and DNA barcodes are two molecular approaches that have recently garnered much attention. Here, we compare these two platforms for identification of an important group, the mammals.     

Effect of Synbiotic and Probiotic Supplementation on Serum Levels of Endothelial Cell Adhesion Molecules in Hemodialysis Patients: a Randomized Control Study

Probiotics and Antimicrobial Proteins - The aim of this study was to investigate the effect of synbiotic and probiotic supplementation on serum vascular dysfunction and necrosis markers in...     

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.     

Glycine transport in mouse eggs and preimplantation conceptuses

At least two Na+-dependent systems for glycine transport became detectable, while another became undetectable during preimplantation development of mouse conceptuses. Glycine was taken up by a process in eggs and cleavage-stage conceptuses which closely resembles system Gly. Mediated transport at these stages was more rapid at higher Cl- concentrations, sigmoidally related to the exogenous Na+ concentration, and strongly inhibited by sarcosine but not by amino acids with larger side chains. Moreover, neither Li+ nor choline could substitute for Na+ in stimulating glycine transport. System Gly was the only mediated process detected for glycine uptake in unfertilized and fertilized eggs and two-cell conceptuses, but two, less conspicuous, sarcosine-resistant, Na+-dependent components of transport also appeared to be present in eight-cell conceptuses. One of the latter components seemed to remain relatively inconspicuous when conceptuses formed blastocysts, while system Gly became undetectable. In contrast, the other less conspicuous component in eight-cell conceptuses appeared to become the most conspicuous transport process in blastocysts. The latter process, previously designated system B0,+, was shown here also to interact strongly with a broad scope of zwitterionic and cationic amino acid structures. Moreover, transport of glycine via system B0,+ was more rapid at higher Cl- concentrations, and this Na+-dependent process as well as Na+-independent leucine uptake were inhibited by choline. Furthermore, Na+-dependent amino acid transport in two-cell conceptuses and blastocysts was inhibited by 1.0 or 10 mM ouabain, but the inhibition was incomplete at both concentrations. Since Na+/K+-ATPase has not been detected in two-cell conceptuses, inhibition of amino acid transport by ouabain may not have been due solely to an effect on this enzyme. The level of system Gly activity decreased during the development of eight-cell conceptuses from eggs, and this decrease could contribute to an associated decline in intracellular glycine. Since other amino acids begin to compete strongly with glycine for transport when system B0,+ replaces system Gly in conceptuses, this qualitative change in transport activity may help account for a further decrease in the glycine content of conceptuses, reported elsewhere to occur after they form blastocysts.     

Patterns of ribosomal RNA evolution in salamanders

Sequence comparisons are presented for four segments of the large subunit of ribosomal RNA, including divergent domains D7a and D7b, portions of the large divergent domains D2, D3, and D8, and evolutionarily conservative sequences flanking divergent domains. These results resolve phylogenetic relationships among exemplars of seven families of salamanders and the three amphibian orders. Phylogenetic analysis confirms the prediction that divergent domains feature the highest relative rates of base substitution and length variation within the ribosome, but the divergent domains evolve more slowly than nuclear noncoding DNA and the silent sites of structural genes. Base substitutions demonstrate approximately twice as many transitions as transversions and an uneven distribution among sites within the divergent domains but no apparent bias in base composition. Length mutations are primarily small insertions and deletions, with deletions predominating. The divergent domains appear to be a good source of phylogenetic information for evolutionary events occurring approximately 100-200 million years ago.