Genetic and biochemical characterization of the cell wall hydrolase activity of the major secreted protein of Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG (LGG) produces two major secreted proteins, designated here Msp1 (LGG_00324 or p75) and Msp2 (LGG_00031 or p40), which have been reported to promote the survival and growth of intestinal epithelial cells. Intriguingly, although each of these proteins shares homology with cell wall hydrolases, a physiological function that correlates with such an enzymatic activity remained to be substantiated in LGG. To investigate the bacterial function, we constructed knock-out mutants in the corresponding genes aiming to establish a genotype to phenotype relation. Microscopic examination of the msp1 mutant showed the presence of rather long and overly extended cell chains, which suggests that normal daughter cell separation is hampered. Subsequent observation of the LGG wild-type cells by immunofluorescence microscopy revealed that the Msp1 protein accumulates at the septum of exponential-phase cells. The cell wall hydrolyzing activity of the Msp1 protein was confirmed by zymogram analysis. Subsequent analysis by RP-HPLC and mass spectrometry of the digestion products of LGG peptidoglycan (PG) by Msp1 indicated that the Msp1 protein has D-glutamyl-L-lysyl endopeptidase activity. Immunofluorescence microscopy and the failure to construct a knock-out mutant suggest an indispensable role for Msp2 in priming septum formation in LGG.     

Design of a medical record review study on the incidence and preventability of adverse events requiring a higher level of care in Belgian hospitals

ABSTRACT: BACKGROUND: Adverse events are unintended patient injuries that arise from healthcare management resulting in disability, prolonged hospital stay or death. Adverse events that require intensive care admission imply a considerable financial burden to the healthcare system. The epidemiology of adverse events in Belgian hospitals has never been assessed systematically. FINDINGS: A multistage retrospective review study of patients requiring a transfer to a higher level of care will be conducted in six hospitals in the province of Limburg. Patient records are reviewed starting from January 2012 by a clinical team consisting of a research nurse, a physician and a clinical pharmacist. Besides the incidence and the level of causation and preventability, also the type of adverse events and their consequences (patient harm, mortality and length of stay) will be assessed. Moreover, the adequacy of the patient records and quality/usefulness of the method of medical record review will be evaluated. DISCUSSION: This paper describes the rationale for a retrospective review study of adverse events that necessitate a higher level of care. More specifically, we are particularly interested in increasing our understanding in the preventability and root causes of these events in order to implement improvement strategies. Attention is paid to the strengths and limitations of the study design.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Modulation of neuronal pentraxin 1 expression in rat pancreatic β-cells submitted to chronic glucotoxic stress

Insulin secretory granules are β-cell vesicles dedicated to insulin processing, storage, and release. The secretion of insulin secretory granule content in response to an acute increase of glucose concentration is a highly regulated process allowing normal glycemic homeostasis. Type 2 diabetes is a metabolic disease characterized by chronic hyperglycemia. The consequent prolonged glucose exposure is known to exert deleterious effects on the function of various organs, notably impairment of insulin secretion by pancreatic β-cells and induction of apoptosis. It has also been described as modifying gene and protein expression in β-cells. Therefore, we hypothesized that a modulation of insulin secretory granule protein expression induced by chronic hyperglycemia may partially explain β-cell dysfunction. To identify the potential early molecular mechanisms underlying β-cell dysfunction during chronic hyperglycemia, we performed SILAC and mass spectrometry experiments to monitor changes in the insulin secretory granule proteome from INS-1E rat insulinoma β-cells cultivated either with 11 or 30 mm of glucose for 24 h. Fourteen proteins were found to be differentially expressed between these two conditions, and several of these proteins were not described before to be present in β-cells. Among them, neuronal pentraxin 1 was only described in neurons so far. Here we investigated its expression and intracellular localization in INS-1E cells. Furthermore, its overexpression in glucotoxic conditions was confirmed at the mRNA and protein levels. According to its role in hypoxia-ischemia-induced apoptosis described in neurons, this suggests that neuronal pentraxin 1 might be a new β-cell mediator in the AKT/GSK3 apoptotic pathway. In conclusion, the modification of specific β-cell pathways such as apoptosis and oxidative stress may partially explain the impairment of insulin secretion and β-cell failure, observed after prolonged exposure to high glucose concentrations.     

A strand specific high resolution normalization method for chip-sequencing data employing multiple experimental control measurements

Background  

High-throughput sequencing is becoming the standard tool for investigating protein-DNA interactions or epigenetic modifications. However, the data generated will always contain noise due to e.g. repetitive regions or non-specific antibody interactions. The noise will appear in the form of a background distribution of reads that must be taken into account in the downstream analysis, for example when detecting enriched regions (peak-calling). Several reported peak-callers can take experimental measurements of background tag distribution into account when analysing a data set. Unfortunately, the background is only used to adjust peak calling and not as a pre-processing step that aims at discerning the signal from the background noise. A normalization procedure that extracts the signal of interest would be of universal use when investigating genomic patterns.     

Hsp110 is a nucleotide-activated exchange factor for Hsp70

Hsp110 proteins constitute a subfamily of the Hsp70 chaperones and are potent nucleotide exchange factors (NEFs) for canonical Hsp70s of the eukaryotic cytosol. Here, we show that the NEF activity of the yeast Hsp110 homologue Sse1 itself is controlled by nucleotide. Nucleotide binding results in formation of a stabilized conformation of Sse1 that is required for association with the yeast Hsp70 Ssa1. The interaction triggers release of bound ADP from Ssa1, but nucleotide persists bound to Sse1 in the complex. Surprisingly, removal of this nucleotide does not affect the integrity of the complex. Instead, rebinding of ATP to the Hsp70 prompts the dissociation of the complex. Our data demonstrate that in contrast to previously characterized NEFs for Hsp70 chaperones, the NEF activity of Sse1 requires nucleotide binding and let us propose a new model for Hsp110 function.