Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated.

BACKGROUND: DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. RESULTS: Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. CONCLUSIONS: Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Structural Modeling and Biochemical Characterization of Recombinant KPN_02809, a Zinc-Dependent Metalloprotease from Klebsiella pneumoniae MGH 78578

Klebsiella pneumoniae is a Gram-negative, cylindrical rod shaped opportunistic pathogen that is found in the environment as well as existing as a normal flora in mammalian mucosal surfaces such as the mouth, skin, and intestines. Clinically it is the most important member of the family of Enterobacteriaceae that causes neonatal sepsis and nosocomial infections. In this work, a combination of protein sequence analysis, structural modeling and molecular docking simulation approaches were employed to provide an understanding of the possible functions and characteristics of a hypothetical protein (KPN_02809) from K. pneumoniae MGH 78578. The computational analyses showed that this protein was a metalloprotease with zinc binding motif, HEXXH. To verify this result, a ypfJ gene which encodes for this hypothetical protein was cloned from K. pneumoniae MGH 78578 and the protein was overexpressed in Escherichia coli BL21 (DE3). The purified protein was about 32 kDa and showed maximum protease activity at 30 °C and pH 8.0. The enzyme activity was inhibited by metalloprotease inhibitors such as EDTA, 1,10-phenanthroline and reducing agent, 1,4-dithiothreitol (DTT). Each molecule of KPN_02809 protein was also shown to bind one zinc ion. Hence, for the first time, we experimentally confirmed that KPN_02809 is an active enzyme with zinc metalloprotease activity

Performance of Detecting IgM Antibodies against Enterovirus 71 for Early Diagnosis

Enterovirus 71 (EV71) infection is more likely to induce severe complications and mortality than other enteroviruses. Methods for detection of IgM antibody against EV71 had been established for years, however, the performance of the methods in the very early diagnosis of EV71 infection had not been fully evaluated, which is especially meaningful because of the short incubation period of EV71 infection. In this report, the performance of an IgM anti-EV71 assay was evaluated using acute sera collected from 165 EV71 infected patients, 165 patients infected with other enteroviruses, and more than 2,000 sera from healthy children or children with other infected diseases. The results showed a 90% sensitivity in 20 patients who were in their first illness day, and similar sensitivity remained till 4 days after onset. After then the sensitivity increased to 95% to 100% for more than one month. The specificity of the assay in non-HFMD children is 99.1% (95% CI: 98.6–99.4), similar as the 99.9% specificity in healthy adults. The cross-reaction rate in patients infected with other non-EV71 enteroviruses was 11.4%. In conclusion, the data here presented show that the detection of IgM anti-EV71 by ELISA affords a reliable, convenient, and prompt diagnosis of EV71 infection

The overmethylated genes in Helicobacter pylori-infected gastric mucosa are demethylated in gastric cancers

<p>Abstract</p> <p>Background</p> <p>The transitional-CpG sites between weakly methylated genes and densely methylated retroelements are overmethylated in the gastric mucosa infected with <it>Helicobacter pylori </it>(<it>H. pylori</it>) and they are undermethylated in the gastric cancers depending on the level of loss of heterozygosity (LOH) events. This study delineated the transitional-CpG methylation patterns of CpG-island-containing and -lacking genes in view of the retroelements.</p> <p>Methods</p> <p>The transitional-CpG sites of eight CpG-island-containing genes and six CpG-island-lacking genes were semi-quantitatively examined by performing radioisotope-labelling methylation-specific PCR under stringent conditions. The level of LOH in the gastric cancers was estimated using the 40 microsatellite markers on eight cancer-associated chromosomes. Each gene was scored as overmethylated or undermethylated based on an intermediate level of transitional-CpG methylation common in the <it>H. pylori</it>-negative gastric mucosa.</p> <p>Results</p> <p>The eight CpG-island genes examined were overmethylated depending on the proximity to the nearest retroelement in the <it>H. pylori</it>-positive gastric mucosa. The six CpG-island-lacking genes were similarly methylated in the <it>H. pylori</it>-positive and -negative gastric mucosa. In the gastric cancers, long transitional-CpG segments of the CpG-island genes distant from the retroelements remained overmethylated, whereas the overmethylation of short transitional-CpG segments close to the retroelements was not significant. Both the CpG-island-containing and -lacking genes tended to be decreasingly methylated in a LOH-level-dependent manner.</p> <p>Conclusions</p> <p>The overmethylated genes under the influence of retroelement methylation in the <it>H. pylori</it>-infected stomach are demethylated in the gastric cancers influenced by LOH.</p

Policy and prevention approaches for disordered and hazardous gaming and internet use: an international perspective

Problems related to high levels of gaming and Internet usage are increasingly recognized as a potential public health burden across the developed world. The aim of this review was to present an international perspective on prevention strategies for Internet gaming disorder and related health conditions (e.g., Internet addiction), as well as hazardous gaming and Internet use. A systematic review of quantitative research evidence was conducted, followed by a search of governmental reports, policy and position statements, and health guidelines in the last decade. The regional scope included the United States, United Kingdom, Australia, China, Germany, Japan, and South Korea. Prevention studies have mainly involved school-based programs to train healthier Internet use habits in adolescents. The efficacy of selective prevention is promising but warrants further empirical attention. On an international scale, the formal recognition of gaming or Internet use as a disorder or as having quantifiable harms at certain levels of usage has been foundational to developing structured prevention responses. The South Korean model, in particular, is an exemplar of a coordinated response to a public health threat, with extensive government initiatives and long-term strategic plans at all three levels of prevention (i.e., universal, selective, and indicated). Western regions, by comparison, are dominated by prevention approaches led by non-profit organizations and private enterprise. The future of prevention of gaming and Internet problems ultimately relies upon all stakeholders working collaboratively in the public interest, confronting the reality of the evidence base and developing practical, ethical, and sustainable countermeasures

Functional Expression of the Extracellular Calcium Sensing Receptor (CaSR) in Equine Umbilical Cord Matrix Size-Sieved Stem Cells

The present study investigates the effects of high external calcium concentration ([Ca(2+)](o)) and the calcimimetic NPS R-467, a known calcium-sensing receptor (CaSR) agonist, on growth/proliferation of two equine size-sieved umbilical cord matrix mesenchymal stem cell (eUCM-MSC) lines. The involvement of CaSR on observed cell response was analyzed at both the mRNA and protein level.A large (>8 µm in diameter) and a small (<8 µm) cell line were cultured in medium containing: 1) low [Ca(2+)](o) (0.37 mM); 2) high [Ca(2+)](o) (2.87 mM); 3) NPS R-467 (3 µM) in presence of high [Ca(2+)](o) and 4) the CaSR antagonist NPS 2390 (10 µM for 30 min.) followed by incubation in presence of NPS R-467 in medium with high [Ca(2+)](o). Growth/proliferation rates were compared between groups. In large cells, the addition of NPS R-467 significantly increased cell growth whereas increasing [Ca(2+)](o) was not effective in this cell line. In small cells, both higher [Ca(2+)](o) and NPS R-467 increased cell growth. In both cell lines, preincubation with the CaSR antagonist NPS 2390 significantly inhibited the agonistic effect of NPS R-467. In both cell lines, increased [Ca(2+)](o) and/or NPS R-467 reduced doubling time values.Treatment with NPS R-467 down-regulated CaSR mRNA expression in both cell lines. In large cells, NPS R-467 reduced CaSR labeling in the cytosol and increased it at cortical level.In conclusion, calcium and the calcimimetic NPS R-467 reduce CaSR mRNA expression and stimulate cell growth/proliferation in eUCM-MSC. Their use as components of media for eUCM-MSC culture could be beneficial to obtain enough cells for down-stream purposes

Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome

<p>Abstract</p> <p>Background</p> <p>Flax (<it>Linum usitatissimum </it>L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome.</p> <p>Results</p> <p>The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding.</p> <p>Conclusion</p> <p>The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly.</p

Global overview of the management of acute cholecystitis during the COVID-19 pandemic (CHOLECOVID study)

Background: This study provides a global overview of the management of patients with acute cholecystitis during the initial phase of the COVID-19 pandemic. Methods: CHOLECOVID is an international, multicentre, observational comparative study of patients admitted to hospital with acute cholecystitis during the COVID-19 pandemic. Data on management were collected for a 2-month study interval coincident with the WHO declaration of the SARS-CoV-2 pandemic and compared with an equivalent pre-pandemic time interval. Mediation analysis examined the influence of SARS-COV-2 infection on 30-day mortality. Results: This study collected data on 9783 patients with acute cholecystitis admitted to 247 hospitals across the world. The pandemic was associated with reduced availability of surgical workforce and operating facilities globally, a significant shift to worse severity of disease, and increased use of conservative management. There was a reduction (both absolute and proportionate) in the number of patients undergoing cholecystectomy from 3095 patients (56.2 per cent) pre-pandemic to 1998 patients (46.2 per cent) during the pandemic but there was no difference in 30-day all-cause mortality after cholecystectomy comparing the pre-pandemic interval with the pandemic (13 patients (0.4 per cent) pre-pandemic to 13 patients (0.6 per cent) pandemic; P = 0.355). In mediation analysis, an admission with acute cholecystitis during the pandemic was associated with a non-significant increased risk of death (OR 1.29, 95 per cent c.i. 0.93 to 1.79, P = 0.121). Conclusion: CHOLECOVID provides a unique overview of the treatment of patients with cholecystitis across the globe during the first months of the SARS-CoV-2 pandemic. The study highlights the need for system resilience in retention of elective surgical activity. Cholecystectomy was associated with a low risk of mortality and deferral of treatment results in an increase in avoidable morbidity that represents the non-COVID cost of this pandemic