New approaches to the analysis of enzymatically hydrolyzed methyl cellulose. Part 2. Comparison of various enzyme preparations

In this part of our studies, dealing with new approaches to the analysis of enzymatically hydrolyzed methyl cellulose, five different enzymes or enzyme preparations containing endoglucanases (from Bacillus agaradhaerens Cel 5A, Trichoderma reesei, Trichoderma viride, and two obtained from Trichoderma longibrachiatum) were used to hydrolyze six different methyl celluloses (MCs). The main goal was to investigate whether enzymes could be used for determination of the heterogeneity of the substituent distribution along the cellulose chain. To obtain information about the heterogeneity, it was necessary to gather information on how the enzymes affect hydrolysis. Size exclusion chromatography with multi-angle light scattering and refractive index detection (SEC-MALS/RI) was used to estimate the molar mass distribution of the MCs before and after hydrolysis. A novel internal standard addition method in combination with electrospray ionization ion trap mass spectrometry (ESI-ITMS) was used to determine the amount of formed oligomers. Two MCs, one with a degree of substitution (DS) of 1.8 and one with DS 1.3, were hydrolyzed with all of the five enzymes. The yield of summarized di- and trisaccharides was approximately 2% of the hydrolysis products for the MC with DS 1.8, whereas the product mixture, obtained from a MC with a DS of 1.3, contained 7-16% di- and trisaccharides. By a novel sample preparation method in combination with ESI-IT tandem MS, outlined in part 1 of this work, it was shown that the enzymes produced oligomers with the reducing end bearing no or only one substituent. Comparison of the methyl pattern at the nonreducing ends of the dimers and trimers indicated that the -2 subsite of the active complex is less tolerant than subsites -3 and +1. All enzymes had similar general selectivity toward the methyl substituents but also showed some differences. From both SEC-MALS/RI and ESI-ITMS, differences with respect to substituent distribution of MCs could be recognized but not for each enzyme used. Basic considerations for enzymatic hydrolysis and analysis of methyl cellulose were listed as a consequence of the results from the work.     

PfAlbas constitute a new eukaryotic DNA/RNA-binding protein family in malaria parasites

In Plasmodium falciparum, perinuclear subtelomeric chromatin conveys monoallelic expression of virulence genes. However, proteins that directly bind to chromosome ends are poorly described. Here we identify a novel DNA/RNA-binding protein family that bears homology to the archaeal protein Alba (Acetylation lowers binding affinity). We isolated three of the four PfAlba paralogs as part of a molecular complex that is associated with the P. falciparum-specific TARE6 (Telomere-Associated Repetitive Elements 6) subtelomeric region and showed in electromobility shift assays (EMSAs) that the PfAlbas bind to TARE6 repeats. In early blood stages, the PfAlba proteins were enriched at the nuclear periphery and partially co-localized with PfSir2, a TARE6-associated histone deacetylase linked to the process of antigenic variation. The nuclear location changed at the onset of parasite proliferation (trophozoite-schizont), where the PfAlba proteins were also detectable in the cytoplasm in a punctate pattern. Using single-stranded RNA (ssRNA) probes in EMSAs, we found that PfAlbas bind to ssRNA, albeit with different binding preferences. We demonstrate for the first time in eukaryotes that Alba-like proteins bind to both DNA and RNA and that their intracellular location is developmentally regulated. Discovery of the PfAlbas may provide a link between the previously described subtelomeric non-coding RNA and the regulation of antigenic variation.     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

Synthesis and SAR of selective small molecule neuropeptide Y Y2 receptor antagonists

Highly potent and selective small molecule neuropeptide Y Y2 receptor antagonists are reported. The systematic SAR exploration of a hit molecule N-(4-ethoxyphenyl)-4-[hydroxy(diphenyl)methyl]piperidine-1-carbothioamide, identified from HTS, led to the discovery of highly potent NPY Y2 antagonists 16 (CYM 9484) and 54 (CYM 9552) with IC(50) values of 19 nM and 12 nM respectively.     

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.