Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases

Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography–tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin‐like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20°C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity

Beta decay and the origin of biological chirality: New experimental results

The proposed connection between the parity-violating handedness of beta particles in radioactive decay and the sign (L) of biological chirality (the Vester-Ulbricht [V-U] hypothesis) is being investigated by measuring the theoretically predicted asymmetry in the formation of triplet positronium in amino acid enantiomers by low energy positrons under reversal of the helicity of the positrons. We find the asymmetry in leucine to be (0.8±1.0)×10 −4 , i.e. consistent with the theoretical, prediction of 10 −6 to 10 −7 . The apparatus is now sensitive enough to test the predicted asymmetry in optically active molecules which have heavy atoms at their chiral centers. The connection between these results and asymmetry in radiolysis by beta-decay electrons is made, and the implications of our limits for the V-U hypothesis discussed. Although the above limits are 10 6 times lower than direct measurements of radiolysis, they are still not small enough to allow us to rule out the V-U hypothesis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/43347/1/11084_2004_Article_BF00933685.pd

Dutch guideline for clinical foetal-neonatal and paediatric post-mortem radiology, including a review of literature

Clinical post-mortem radiology is a relatively new field of expertise and not common practice in most hospitals yet. With the declining numbers of autopsies and increasing demand for quality control of clinical care, post-mortem radiology can offer a solution, or at least be complementary. A working group consisting of radiologists, pathologists and other clinical medical specialists reviewed and evaluated the literature on the diagnostic value of post-mortem conventional radiography (CR), ultrasonography, computed tomography (PMCT), magnetic resonance imaging (PMMRI), and minimally invasive autopsy (MIA). Evidence tables were built and subsequently a Dutch national evidence-based guideline for post-mortem radiology was developed. We present this evaluation of the radiological modalities in a clinical post-mortem setting, including MIA, as well as the recently published Dutch guidelines for post-mortem radiology in foetuses, neonates, and children. In general, for post-mortem radiology modalities, PMMRI is the modality of choice in foetuses, neonates, and infants, whereas PMCT is advised in older children. There is a limited role for post-mortem CR and ultrasonography. In most cases, conventional autopsy will remain the diagnostic method of choice. CONCLUSION: Based on a literature review and clinical expertise, an evidence-based guideline was developed for post-mortem radiology of foetal, neonatal, and paediatric patients. What is Known: * Post-mortem investigations serve as a quality check for the provided health care and are important for reliable epidemiological registration. * Post-mortem radiology, sometimes combined with minimally invasive techniques, is considered as an adjunct or alternative to autopsy. What is New: * We present the Dutch guidelines for post-mortem radiology in foetuses, neonates and children. * Autopsy remains the reference standard, however minimal invasive autopsy with a skeletal survey, post-mortem computed tomography, or post-mortem magnetic resonance imaging can be complementary thereof

Determinants of Initiation Codon Selection during Translation in Mammalian Cells

Factors affecting translation of mRNA contribute to the complexity of eukaryotic proteomes. In some cases, translation of a particular mRNA can generate multiple proteins. However, the factors that determine whether ribosomes initiate translation from the first AUG codon in the transcript, from a downstream codon, or from multiple sites are not completely understood. Various mRNA properties, including AUG codon-accessibility and 5′ leader length have been proposed as potential determinants that affect where ribosomes initiate translation. To explore this issue, we performed studies using synthetic mRNAs with two in-frame AUG codons−both in excellent context. Open reading frames initiating at AUG1 and AUG2 encode large and small isoforms of a reporter protein, respectively. Translation of such an mRNA in COS-7 cells was shown to be 5′ cap-dependent and to occur efficiently from both AUG codons. AUG codon-accessibility was modified by using two different elements: an antisense locked nucleic acid oligonucleotide and an exon-junction complex. When either element was used to mask AUG1, the ratio of the proteins synthesized changed, favoring the smaller (AUG2-initiated) protein. In addition, we observed that increased leader length by itself changed the ratio of the proteins and favored initiation at AUG1. These observations demonstrate that initiation codon selection is affected by various factors, including AUG codon-accessibility and 5′ leader length, and is not necessarily determined by the order of AUG codons (5′→3′). The modulation of AUG codon accessibility may provide a powerful means of translation regulation in eukaryotic cells

Co-existence of physiologically similar sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single organic electron donor

A combination of culture-dependent and independent methods was used to study the co-existence of different sulfate-reducing bacteria (SRB) in an upflow anaerobic sludge bed reactor treating sulfate-rich wastewater. The wastewater was fed with ethanol as an external electron donor. Twenty six strains of SRB were randomly picked and isolated from the highest serial dilution that showed growth (i.e. 108). Repetitive enterobacterial palindromic polymerase chain reaction and whole cell protein profiling revealed a low genetic diversity, with only two genotypes among the 26 strains obtained in the pure culture. The low genetic diversity suggests the absence of micro-niches within the reactor, which might be due to a low spatial and temporal micro-heterogeneity. The total 16S rDNA sequencing of two representative strains L3 and L7 indicated a close relatedness to the genus Desulfovibrio. The two strains differed in as many as five physiological traits, which might allow them to occupy distinct niches and thus co-exist within the same habitat. Whole cell hybridisation with fluorescently labeled oligonucleotide probes was performed to characterise the SRB community in the reactor. The isolated strains Desulfovibrio L3 and Desulfovibrio L7 were the most dominant SRB, representing 30–35% and 25–35%, respectively, of the total SRB community. Desulfobulbus-like bacteria contributed for 20–25%, and the Desulfobacca acetoxidans-specific probe targeted approximately 15–20% of the total SRB. The whole cell hybridisation results thus revealed a consortium of four different species of SRB that can be enriched and maintained on a single energy source in a full-scale sulfidogenic reactor

Prebiotic potential of a maize-based soluble fibre and impact of dose on the human gut microbiota

Dietary management of the human gut microbiota towards a more beneficial composition is one approach that may improve host health. To date, a large number of human intervention studies have demonstrated that dietary consumption of certain food products can result in significant changes in the composition of the gut microbiota i.e. the prebiotic concept. Thus the prebiotic effect is now established as a dietary approach to increase beneficial gut bacteria and it has been associated with modulation of health biomarkers and modulation of the immune system. Promitor™ Soluble Corn Fibre (SCF) is a well-known maize-derived source of dietary fibre with potential selective fermentation properties. Our aim was to determine the optimum prebiotic dose of tolerance, desired changes to microbiota and fermentation of SCF in healthy adult subjects. A double-blind, randomised, parallel study was completed where volunteers (n = 8/treatment group) consumed 8, 14 or 21 g from SCF (6, 12 and 18 g/fibre delivered respectively) over 14-d. Over the range of doses studied, SCF was well tolerated Numbers of bifidobacteria were significantly higher for the 6 g/fibre/day compared to 12g and 18g/fibre delivered/day (mean 9.25 and 9.73 Log10 cells/g fresh faeces in the pre-treatment and treatment periods respectively). Such a numerical change of 0.5 Log10 bifidobacteria/g fresh faeces is consistent with those changes observed for inulin-type fructans, which are recognised prebiotics. A possible prebiotic effect of SCF was therefore demonstrated by its stimulation of bifidobacteria numbers in the overall gut microbiota during a short-term intervention

Gene Expression Profiles of Colonic Mucosa in Healthy Young Adult and Senior Dogs

Background: We have previously reported the effects of age and diet on nutrient digestibility, intestinal morphology, and large intestinal fermentation patterns in healthy young adult and senior dogs. However, a genome-wide molecular analysis of colonic mucosa as a function of age and diet has not yet been performed in dogs. Methodology/Principal Findings: Colonic mucosa samples were collected from six senior (12-year old) and six young adult (1-year old) female beagles fed one of two diets (animal protein-based vs. plant protein-based) for 12 months. Total RNA in colonic mucosa was extracted and hybridized to Affymetrix GeneChipH Canine Genome Arrays. Results indicated that the majority of gene expression changes were due to age (212 genes) rather than diet (66 genes). In particular, the colonic mucosa of senior dogs had increased expression of genes associated with cell proliferation, inflammation, stress response, and cellular metabolism, whereas the expression of genes associated with apoptosis and defensive mechanisms were decreased in senior vs. young adult dogs. No consistent diet-induced alterations in gene expression existed in both age groups, with the effects of diet being more pronounced in senior dogs than in young adult dogs. Conclusion: Our results provide molecular insight pertaining to the aged canine colon and its predisposition to dysfunction and disease. Therefore, our data may aid in future research pertaining to age-associated gastrointestinal physiologica

Quantitative Subcellular Proteome and Secretome Profiling of Influenza A Virus-Infected Human Primary Macrophages

Influenza A viruses are important pathogens that cause acute respiratory diseases and annual epidemics in humans. Macrophages recognize influenza A virus infection with their pattern recognition receptors, and are involved in the activation of proper innate immune response. Here, we have used high-throughput subcellular proteomics combined with bioinformatics to provide a global view of host cellular events that are activated in response to influenza A virus infection in human primary macrophages. We show that viral infection regulates the expression and/or subcellular localization of more than one thousand host proteins at early phases of infection. Our data reveals that there are dramatic changes in mitochondrial and nuclear proteomes in response to infection. We show that a rapid cytoplasmic leakage of lysosomal proteins, including cathepsins, followed by their secretion, contributes to inflammasome activation and apoptosis seen in the infected macrophages. Also, our results demonstrate that P2X7 receptor and src tyrosine kinase activity are essential for inflammasome activation during influenza A virus infection. Finally, we show that influenza A virus infection is associated with robust secretion of different danger-associated molecular patterns (DAMPs) suggesting an important role for DAMPs in host response to influenza A virus infection. In conclusion, our high-throughput quantitative proteomics study provides important new insight into host-response against influenza A virus infection in human primary macrophages