Patients with severe acute‐on‐chronic liver failure are disadvantaged by model for end‐stage liver disease‐based organ allocation policy

Background: Mortality for patients with acute‐on‐chronic liver failure (ACLF) may be underestimated by the model for end‐stage liver disease‐sodium (MELD‐Na) score. / Aim: To assess waitlist outcomes across varying grades of ACLF among a cohort of patients listed with a MELD‐Na score ≥35, and therefore having similar priority for liver transplantation. / Methods: We analysed the United Network for Organ Sharing (UNOS) database, years 2010‐2017. Waitlist outcomes were evaluated using Fine and Gray's competing risks regression. / Results: We identified 6342 candidates at listing with a MELD‐Na score ≥35, of whom 3122 had ACLF‐3. Extra‐hepatic organ failures were present primarily in patients with four to six organ failures. Competing risks regression revealed that candidates listed with ACLF‐3 had a significantly higher risk for 90‐day waitlist mortality (Sub‐hazard ratio (SHR) = 1.41; 95% confidence interval [CI] 1.12‐1.78) relative to patients with lower ACLF grades. Subgroup analysis of ACLF‐3 revealed that both the presence of three organ failures (SHR = 1.40, 95% CI 1.20‐1.63) or four to six organ failures at listing (SHR = 3.01; 95% CI 2.54‐3.58) was associated with increased waitlist death. Candidates with four to six organ failures also had the lowest likelihood of receiving liver transplantation (SHR = 0.61, 95% CI 0.54‐0.68). The Share 35 rule was associated with reduced 90‐day waitlist mortality among the full cohort of patients listed with ACLF‐3 and MELD‐Na score ≥35 (SHR = 0.59; 95% CI 0.49‐0.70). However, Share 35 rule implementation was not associated with reduced waitlist mortality among patients with four to six organ failures (SHR = 0.76; 95% CI 0.58‐1.02). / Conclusion: The MELD‐Na score disadvantages patients with ACLF‐3, both with and without extra‐hepatic organ failures. Incorporation of organ failures into allocation policy warrants further exploration

A Novel Antiviral Strategy against MERS-CoV and HCoV-229E Using Binase to Target Viral Genome Replication

© 2016, Springer Science+Business Media New York.RNA viruses cause most of the dangerous communicable diseases. Due to their high mutation rates, RNA viruses quickly evade selective pressures and can adapt to a new host. Therefore, new antiviral approaches are urgently needed, which target more than one specific virus variant and which would optimally prevent development of viral resistance. Among the family of coronaviruses (CoV), several human pathogenic strains (HCoV) are known to cause respiratory diseases and are implied in enteric diseases. While most strains contribute to common cold-like illnesses, others lead to severe infections. One of these viruses is the newly emerged (2012), highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) of zoonotic origin. MERS-CoV causes a severe respiratory infection with a high mortality rate of 35 %. There is no specific treatment or infection prevention available. Here, we show that the bacterial ribonuclease Binase is able to inhibit the replication of MERS-CoV and of the low-pathogenic human coronavirus 229E (HCoV-229E) in cell culture. We demonstrate that at non-toxic concentrations, Binase decreased the titers of MERS-CoV and HCoV-229E. On a molecular level, Binase treatment reduced (i) the viral subgenomic RNAs and (ii) the viral nucleocapsidprotein (N) and non-structural protein 13 (nsp13) accumulation. Furthermore, we show that the quantity of the replication/transcription complexes within the infected cells is diminished. Thus, the data obtained might allow further development of new anti-coronaviral approaches affecting viral replication, independent of the specific virus strain

Improving Bacillus Altitudinis B-388 Genome Scaffolding Using Mate-Pair Next-Generation Sequencing

© 2016, Springer Science+Business Media New York.Bacillus species, generally regarded as soil microorganisms, are present in human gastrointestinal tract (GIT) in quantities, which cannot be explained by their entrance with food only. They are capable of growing in GIT and interacting with intestinal microbiota and host organism by excretion of enzymes and low-molecular weight compounds, which exert digestion-facilitating, antagonistic, immunomodulating, antiviral, anticancer properties or mediate cell communication. For better understanding of its probiotic potential, we have sequenced genome of Bacillus altitudinis B-388 using mate-pair technology. It allowed us to improve quality of the genome sequence. The number of contigs decreased from 59 to 8. N50 contig length increased by four times. The number of identified genes raised from 3730 to 3774 (3645 proteins and 73 RNAs) with the reduction of frameshifted genes. The calculated size of B. altitudinis B-388 genome is 3,743,699 bp, with a G + C content of 41.17 mol%. Additional incomplete prophage sequence in genome of B. altitudinis B-388 was revealed. It was found that cryptic plasmid encodes SoxR, an oxidative stress response regulator. To date, the reported sequence is the most thorough presentation of B. altitudinis genome among four whole-genome sequences of this species deposited in GenBank

Antiviral Activity of Bacterial Extracellular Ribonuclease Against Single-, Double-Stranded RNA and DNA Containing Viruses in Cell Cultures

© 2016, Springer Science+Business Media New York.Binase, a small guanyl-preferring extracellular ribonuclease of Gram-positive non-pathogenic soil bacteria Bacillus pumilus. Binase is a well-known bacterial ribonuclease, and the most essential properties of the enzyme were characterized. Binase has demonstrated antiviral activity in various virus-infected animal models. Most experiments associated with binase treatment of virus-infected animals were performed using single stranded RNA viruses. It is still unclear, whether binase is able to inactivate the double stranded RNA virus. Moreover, the phenomenon of the antiviral activity of binase against the DNA containing virus in animal model is not explained. Here, we presented the experimental results reflecting binase effect towards eukaryotic cells infected with viruses containing different types of nucleic acids. The obtained data revealed the bacterial ribonuclease binase mode of action against single stranded RNA influenza A virus, double stranded RNA reovirus and DNA containing herpes virus to prove future application of new antiviral tool with a broad range of activity

A Set of Genetic Constructs for Binase and Barstar Overproduction

© 2016, Springer Science+Business Media New York.Low molecular weight guanyl-preferring ribonuclease (RNase) secreted by Bacillus pumilus is referred to as binase and regarded as a potential agent to target oncogenic diseases and viral infections. Depending on research purposes, native binase or tagged and mutated forms are required for application. Specific inhibition of binase activity is achieved by binding to RNase inhibitor barstar. Native and mutant forms of binase are routinely obtained by ion-exchange chromatography, while tagged versions of binase, which can be purified faster and less tedious than non-tagged, are currently unavailable. Here, recombinant DNA techniques were employed to improve binase overexpression in bacterial host cells, its purification, and subsequent detection in downstream assays using affinity tag. Here, recombinant DNA techniques were employed to improve binase overexpression in bacterial host cells. Binase-barstar genetic cassette was modified to obtain strong two-gene operon with the possibility of rapid exchange of binase and/or barstar genes with homologous or mutated ones as well as the generation of C-terminally His-tagged binase and/or barstar. The binase-barstar operon was introduced into pET26b vector as well as into pET26b-pET15b hybrid vector that was created in this study. As a result, plasmids allowed the induced production of extracellular non-tagged, N- or C-terminal His-tagged binase and synchronous intracellular production of non-tagged or C-His-tagged barstar. The constructs can be further sub-cloned in gram-positive bacterial hosts to ensure native production conditions

Ribonuclease from bacillus acts as an antiviral agent against negative- and positive-sense single stranded human respiratory RNA viruses

Copyright © 2017 Raihan Shah Mahmud et al.Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses

Three-step procedure for preparation of pure Bacillus altitudinis ribonuclease

© 2015 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.Ribonucleases are considered as promising tools for anticancer treatment due to their selective cytotoxicity against tumor cells. We investigated a new RNase from Bacillus altitudinis termed BALNASE (B. altitudinisRNase). Balnase is a close homolog of the well-known cytotoxic binase, differing by only one amino acid residue: nonpolar hydrophobic alanine at position 106 in the balnase molecule is replaced by a polar uncharged threonine in binase. The most exciting question is how the physico-chemical properties and biological effects of RNase might be changed by A106T substitution. Here, we have developed a chromatography-based rapid and modern technique for the purification of this new RNase which allowed us to get a protein sample of high quality with specific activity of 1.2 × 106 units in preparative amounts, suitable for further investigation of its biological properties

Bacteriophages of soil bacilli: A new multivalent phage of Bacillus altitudinis

© 2017, Allerton Press, Inc. Bacillus are soil saprophytes, facultative anaerobes developing in the temperature range of 28–37°С. 16S rRNA cataloging shows that these bacteria form a coherent class with broad variability of virulence. Bacillus phages can be extensively used for phagotyping bacteria in the process of soil, water, and food monitoring. Bacillus phages can also be used as vectors in horizontal gene transfer and potential therapeutic agents. Thus, description of the biological diversity of the Bacillus phages is useful for further development of tools used in molecular biology and biomedicine. In this work, the scheme for isolation of soil bacteriophages was unified, which allowed ten bacillus phages to be isolated from different types of soil. It was shown that the number of phages depended on the soil fertility, decreasing as the soil changed from black soil to chestnut soil to gray forest soil to uncontaminated urban soil to oil-contaminated urban soil. A new polyvalent DNA-containing bacteriophage SRT01hs of B. altitudinis (it is also able to infect B. subtilis, B. cereus, and B. pumilus, but not B. licheniformis and B. atrophaeus) was described in detail. It has a typical structure: a total length of 360 nm and an icosahedron-shaped head 100 nm in diameter. Several phages simultaneously attack a B. altitudinis cell by increasing the level of intracellular low-molecular RNA. Infection with the phage virtually eliminates the stationary growth phase of infected bacilli and leads to a permanent increase in the number of phages in cultural liquor, with the exception of the time period of high activity of the secreted ribonuclease

Fast MCMC sampling for hidden markov models to determine copy number variations

<p>Abstract</p> <p>Background</p> <p>Hidden Markov Models (HMM) are often used for analyzing Comparative Genomic Hybridization (CGH) data to identify chromosomal aberrations or copy number variations by segmenting observation sequences. For efficiency reasons the parameters of a HMM are often estimated with maximum likelihood and a segmentation is obtained with the Viterbi algorithm. This introduces considerable uncertainty in the segmentation, which can be avoided with Bayesian approaches integrating out parameters using Markov Chain Monte Carlo (MCMC) sampling. While the advantages of Bayesian approaches have been clearly demonstrated, the likelihood based approaches are still preferred in practice for their lower running times; datasets coming from high-density arrays and next generation sequencing amplify these problems.</p> <p>Results</p> <p>We propose an approximate sampling technique, inspired by compression of discrete sequences in HMM computations and by <it>kd</it>-trees to leverage spatial relations between data points in typical data sets, to speed up the MCMC sampling.</p> <p>Conclusions</p> <p>We test our approximate sampling method on simulated and biological ArrayCGH datasets and high-density SNP arrays, and demonstrate a speed-up of 10 to 60 respectively 90 while achieving competitive results with the state-of-the art Bayesian approaches.</p> <p><it>Availability: </it>An implementation of our method will be made available as part of the open source GHMM library from <url>http://ghmm.org</url>.</p

Pulmonary Tuberculosis and Drug Resistance in Dhaka Central Jail, the Largest Prison in Bangladesh

There are limited data on TB among prison inmates in Bangladesh. The aim of the study was to determine the prevalence of pulmonary tuberculosis (TB), its drug resistance and risk factors in Dhaka Central Jail, the largest prison in Bangladesh.Cross sectional survey with, active screening of a total number of 11,001 inmates over a period of 2 years. Sputum samples from TB suspects were taken for acid- fast bacilli (AFB) microscopy, culture and drug susceptibility testing. (5.37, 4.02–7.16).The study results revealed a very high prevalence of TB in the prison population in Dhaka Central Jail. Entry examinations and active symptom screening among inmates are important to control TB transmission inside the prison. Identifying undiagnosed smear-negative TB cases remains a challenge to combat this deadly disease in this difficult setting