Wirkung der Enzymkombination Trypsin-Chymotrypsin-Papain auf enterohämolyierende E. coli und Salmonellen

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Plaque assay for human coronavirus NL63 using human colon carcinoma cells

<p>Abstract</p> <p>Background</p> <p>Coronaviruses cause a broad range of diseases in animals and humans. Human coronavirus (hCoV) NL63 is associated with up to 10% of common colds. Viral plaque assays enable the characterization of virus infectivity and allow for purifying virus stock solutions. They are essential for drug screening. Hitherto used cell cultures for hCoV-NL63 show low levels of virus replication and weak and diffuse cytopathogenic effects. It has not yet been possible to establish practicable plaque assays for this important human pathogen.</p> <p>Results</p> <p>12 different cell cultures were tested for susceptibility to hCoV-NL63 infection. Human colon carcinoma cells (CaCo-2) replicated virus more than 100 fold more efficiently than commonly used African green monkey kidney cells (LLC-MK2). CaCo-2 cells showed cytopathogenic effects 4 days post infection. Avicel, agarose and carboxymethyl-cellulose overlays proved suitable for plaque assays. Best results were achieved with Avicel, which produced large and clear plaques from the 4^thday of infection. The utility of plaque assays with agrose overlay was demonstrated for purifying virus, thereby increasing viral infectivity by 1 log 10 PFU/mL.</p> <p>Conclusion</p> <p>CaCo-2 cells support hCoV-NL63 better than LLC-MK2 cells and enable cytopathogenic plaque assays. Avicel overlay is favourable for plaque quantification, and agarose overlay is preferred for plaque purification. HCoV-NL63 virus stock of increased infectivity will be beneficial in antiviral screening, animal modelling of disease, and other experimental tasks.</p

Structure of the VipA/B Type VI Secretion Complex Suggests a Contraction-State-Specific Recycling Mechanism

The bacterial type VI secretion system is a multicomponent molecular machine directed against eukaryotic host cells and competing bacteria. An intracellular contractile tubular structure that bears functional homology with bacteriophage tails is pivotal for ejection of pathogenic effectors. Here, we present the 6 A cryoelectron microscopy structure of the contracted Vibrio cholerae tubule consisting of the proteins VipA and VipB. We localized VipA and VipB in the protomer and identified structural homology between the C-terminal segment of VipB and the tail-sheath protein of T4 phages. We propose that homologous segments in VipB and T4 phages mediate tubule contraction. We show that in type VI secretion, contraction leads to exposure of the ClpV recognition motif, which is embedded in the type VI-specific four-helix-bundle N-domain of VipB. Disaggregation of the tubules by the AAA+ protein ClpV and recycling of the VipA/B subunits are thereby limited to the contracted state

Differential impact of dietary branched chain and aromatic amino acids on chronic kidney disease progression in rats

The metabolism of dietary proteins generates waste products that are excreted by the kidney, in particular nitrogen-containing urea, uric acid, ammonia, creatinine, and other metabolites such as phosphates, sulfates, and protons. Kidney adaptation includes an increase in renal plasma flow (RPF) and glomerular filtration rate (GFR) and represents a burden for diseased kidneys increasing the progression rate of CKD. The present study aimed at identifying potential differences between amino acid (AA) groups constituting dietary proteins regarding their impact on RPF, GFR, and CKD progression. We utilized the well-established 5/6 nephrectomy (5/6 Nx) CKD model in rats and submitted the animals for 5 weeks to either the control diet (18% casein protein) or to diets containing 8% casein supplemented with 10% of a mix of free amino acids, representing all or only a subset of the amino acids contained in casein. Whereas the RPF and GFR measured in free moving animals remained stable during the course of the diet in rats receiving the control mix, these parameters decreased in animals receiving the branched chain amino acid (BCAA) supplementation and increased in the ones receiving the aromatic amino acids (AAAs). In animals receiving essential amino acids (EAAs) containing both BCAAs and AAAs, there was only a small increase in RPF. The kidneys of the 5/6 Nx rats receiving the BCAA diet showed the strongest increase in smooth muscle actin and collagen mRNA expression as a result of higher level of inflammation and fibrosis. These animals receiving BCAAs also showed an increase in plasma free fatty acids pointing to a problem at the level of energy metabolism. In contrast, the animals under AAA diet showed an activation of AMPK and STAT3. Taken together, our results demonstrate that subsets of EAAs contained in dietary proteins, specifically BCAAs and AAAs, exert contrasting effects on kidney functional parameters and CKD progression

Proteasome assembly from 15S precursors involves major conformational changes and recycling of the Pba1-Pba2 chaperone

The chaperones Ump1 and Pba1-Pba2 promote efficient biogenesis of 20S proteasome core particles from its subunits via 15S intermediates containing alpha and beta subunits, except beta7. Here we elucidate the structural role of these chaperones in late steps of core particle biogenesis using biochemical, electron microscopy, cross-linking and mass spectrometry analyses. In 15S precursor complexes, Ump1 is largely unstructured, lining the inner cavity of the complex along the interface between alpha and beta subunits. The alpha and beta subunits form loosely packed rings with a wider alpha ring opening than in the 20S core particle, allowing for the Pba1-Pba2 heterodimer to be partially embedded in the central alpha ring cavity. During biogenesis, the heterodimer is expelled from the alpha ring by a restructuring event that organizes the beta ring and leads to tightening of the alpha ring opening. In this way, the Pba1-Pba2 chaperone is recycled for a new round of proteasome assembly

PHENOquad: A new multi sensor platform for field phenotyping and screening of yield relevant characteristics within grapevine breeding research

Balanced and stable yield is a major trait in grapevine breeding and breeding research. Grapevine yield hereby is a complex quantitative trait, as it is influenced by multiple plant parameters, like berry size, number of berries per bunch, number of bunches per shoot, management, and environmental factors. In the current breeding process, the complexity of this trait has shown that a classification according to descriptive factors for marker development is only possible to a limited extent. Precise field phenotyping of yield-related traits is the basic prerequisite to be able to measure such quantitative traits. This, however, is the major bottleneck due to labor, time and constrains of plant material in the breeding process. For this reason, one of our main goals with the newly developed phenotyping platform PHENOquad with its multisensor system PHENOboxx is to improve phenotyping efficiency of grapevine yield to overcome the phenotyping bottleneck. The newly developed embedded vision system PHENOboxx is mounted on an "all-terrain vehicle (ATV)". This allows a fast data acquisition on a large number of individual vines. In order to evaluate the yield potential of breeding material in comparison to established grapevine cultivars, various yield-related parameters of the vines are quantified directly in the field with high spatial and temporal resolution. As key parameters for yield-related phenotyping, the number of shoots, bunches, berries and the weight of dormant pruning wood was identified. The image data acquired are annotated to train the artificial intelligence (AI). Within the process, the image analysis results are compared to annotated ground truth data and correlated with the field reference data. We expect to increase the precision, target specificity and throughput of screening grapevine material without reducing its accuracy over time by using the PHENOquad. In addition, a weighting of yield-relevant parameters would be possible. This opens up new possibilities for efficient plant evaluation in the scope of grapevine breeding. Also new application possibilities for precision viticulture are conceivable

The SARS-coronavirus-host interactome

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock