Purse-string skin closure versus linear skin closure techniques in stoma closure: a comprehensive meta-analysis with trial sequential analysis of randomised trials.

OBJECTIVES: To compare purse-string skin closure (PSC) and linear skin closure (LSC) techniques in patients undergoing stoma closure METHODS: We conducted a systematic review of literature and meta-analysis of outcomes according to PRISMA statement standards to compare PSC and LSC techniques in stoma closure. Trial sequential analysis (TSA) was performed to assess the possibility of type I or II error and compute the information size required for conclusive meta-analysis. RESULTS: We identified six randomised controlled trials (RCTs) and eight observational studies, enrolling a total of 1102 patients. The included populations in the PSC and LSC groups were comparable in terms of baseline characteristics. The risk of surgical site infection (SSI) was significantly lower in the PSC group (OR 0.10; 95% CI 0.06, 0.18; P < 0.00001). There was no difference between the two groups in terms of operative time (MD 1.80; 95% CI - 1.35, 4.96; P = 0.26), anastomotic leak (OR 0.73; 95% CI 0.21, 2.48; P = 0.61), incisional hernia (OR 0.59; 95% CI 0.25, 1.37; P = 0.22), small bowel obstruction (OR 0.96, 95% CI 0.50, 1.86; P = 0.91), and length of hospital stay (MD - 0.04; 95% CI - 0.51, 0.42; P = 0.86). Patient satisfaction was higher in the PSC group. TSA showed that the risk of type 1 error was minimal and meta-analysis was conclusive. CONCLUSIONS: PSC is associated with significantly lower risk of SSI and better patient satisfaction compared with LSC in closure of stomas and should be the closure technique of choice. The current available evidence is robust and conclusive highlighting that the results of the current study should be incorporated into clinical practice without a need for further trial data.

RNA recognition by the DNA end-binding Ku heterodimer.

Most nucleic acid-binding proteins selectively bind either DNA or RNA, but not both nucleic acids. The Saccharomyces cerevisiae Ku heterodimer is unusual in that it has two very different biologically relevant binding modes: (1) Ku is a sequence-nonspecific double-stranded DNA end-binding protein with prominent roles in nonhomologous end-joining and telomeric capping, and (2) Ku associates with a specific stem-loop of TLC1, the RNA subunit of budding yeast telomerase, and is necessary for proper nuclear localization of this ribonucleoprotein enzyme. TLC1 RNA-binding and dsDNA-binding are mutually exclusive, so they may be mediated by the same site on Ku. Although dsDNA binding by Ku is well studied, much less is known about what features of an RNA hairpin enable specific recognition by Ku. To address this question, we localized the Ku-binding site of the TLC1 hairpin with single-nucleotide resolution using phosphorothioate footprinting, used chemical modification to identify an unpredicted motif within the hairpin secondary structure, and carried out mutagenesis of the stem-loop to ascertain the critical elements within the RNA that permit Ku binding. Finally, we provide evidence that the Ku-binding site is present in additional budding yeast telomerase RNAs and discuss the possibility that RNA binding is a conserved function of the Ku heterodimer.

microRNA 1307 Is a Potential Target for SARS-CoV-2 Infection: An in Vitro Model.

microRNAs (miRs) are proposed as critical molecular targets in SARS-CoV-2 infection. Our recent in silico studies identified seven SARS-CoV-2 specific miR-like sequences, which are highly conserved with humans, including miR-1307-3p, with critical roles in COVID-19. In this current study, Vero cells were infected with SARS-CoV-2, and miR expression profiles were thereafter confirmed by qRT-PCR. miR-1307-3p was the most highly expressed miR in the infected cells; we, therefore, transiently inhibited its expression in both infected and uninfected cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay assessed cell viability following SARS-CoV-2 infection, identifying that miR-1307 expression is inversely correlated with cell viability. Lastly, changes in miR-1307-dependent pathways were analyzed through a detailed miRNOME and associated in silico analysis. In addition to our previously identified miRs, including miR-1307-3p, the upregulation of miR-193a-5p, miR-5100, and miR-23a-5p and downregulation of miR-130b-5p, miR34a-5p, miR-505-3p, miR181a-2-3p, miR-1271-5p, miR-598-3p, miR-34c-3p, and miR-129-5p were also established in Vero cells related to general lung disease-related genes following SARS-CoV-2 infection. Targeted anti-miR-1307-3p treatment rescued cell viability in infection when compared to SARS CoV-2 mediated cell cytotoxicity only. We furthermore identified by in silico analysis that miR-1307-3p is conserved in all SARS-CoV-2 sequences/strains, except in the BA.2 variant, possibly contributing to the lower disease severity of this variant, which warrants further investigation. Small RNA seq analysis was next used to evaluate alterations in the miRNOME, following miR-1307-3p manipulation, identifying critical pathobiological pathways linked to SARS-CoV-2 infection-mediated upregulation of this miR. On the basis of our findings, miRNAs like miR-1307-3p play a critical role in SARS-CoV-2 infection, including via effects on disease progression and severity.

Bacterial Adaptation to Venom in Snakes and Arachnida.

Animal venoms are considered sterile sources of antimicrobial compounds with strong membrane-disrupting activity against multidrug-resistant bacteria. However, venomous bite wound infections are common in developing nations. Investigating the envenomation organ and venom microbiota of five snake and two spider species, we observed venom community structures that depend on the host venomous animal species and evidenced recovery of viable microorganisms from black-necked spitting cobra (Naja nigricollis) and Indian ornamental tarantula (Poecilotheria regalis) venoms. Among the bacterial isolates recovered from N. nigricollis, we identified two venom-resistant, novel sequence types of Enterococcus faecalis whose genomes feature 16 virulence genes, indicating infectious potential, and 45 additional genes, nearly half of which improve bacterial membrane integrity. Our findings challenge the dogma of venom sterility and indicate an increased primary infection risk in the clinical management of venomous animal bite wounds. IMPORTANCE Notwithstanding their 3 to 5% mortality, the 2.7 million envenomation-related injuries occurring annually-predominantly across Africa, Asia, and Latin America-are also major causes of morbidity. Venom toxin-damaged tissue will develop infections in some 75% of envenomation victims, with E. faecalis being a common culprit of disease; however, such infections are generally considered to be independent of envenomation. Here, we provide evidence on venom microbiota across snakes and arachnida and report on the convergent evolution mechanisms that can facilitate adaptation to black-necked cobra venom in two independent E. faecalis strains, easily misidentified by biochemical diagnostics. Therefore, since inoculation with viable and virulence gene-harboring bacteria can occur during envenomation, acute infection risk management following envenomation is warranted, particularly for immunocompromised and malnourished victims in resource-limited settings. These results shed light on how bacteria evolve for survival in one of the most extreme environments on Earth and how venomous bites must be also treated for infections.

Identification of Schistosoma mansoni microRNAs.

BACKGROUND: MicroRNAs (miRNAs) constitute a class of single-stranded RNAs which play a crucial role in regulating development and controlling gene expression by targeting mRNAs and triggering either translation repression or messenger RNA (mRNA) degradation. miRNAs are widespread in eukaryotes and to date over 14,000 miRNAs have been identified by computational and experimental approaches. Several miRNAs are highly conserved across species. In Schistosoma, the full set of miRNAs and their expression patterns during development remain poorly understood. Here we report on the development and implementation of a homology-based detection strategy to search for miRNA genes in Schistosoma mansoni. In addition, we report results on the experimental detection of miRNAs by means of cDNA cloning and sequencing of size-fractionated RNA samples. RESULTS: Homology search using the high-throughput pipeline was performed with all known miRNAs in miRBase. A total of 6,211 mature miRNAs were used as reference sequences and 110 unique S. mansoni sequences were returned by BLASTn analysis. The existing mature miRNAs that produced these hits are reported, as well as the locations of the homologous sequences in the S. mansoni genome. All BLAST hits aligned with at least 95% of the miRNA sequence, resulting in alignment lengths of 19-24 nt. Following several filtering steps, 15 potential miRNA candidates were identified using this approach. By sequencing small RNA cDNA libraries from adult worm pairs, we identified 211 novel miRNA candidates in the S. mansoni genome. Northern blot analysis was used to detect the expression of the 30 most frequent sequenced miRNAs and to compare the expression level of these miRNAs between the lung stage schistosomula and adult worm stages. Expression of 11 novel miRNAs was confirmed by northern blot analysis and some presented a stage-regulated expression pattern. Three miRNAs previously identified from S. japonicum were also present in S. mansoni. CONCLUSION: Evidence for the presence of miRNAs in S. mansoni is presented. The number of miRNAs detected by homology-based computational methods in S. mansoni is limited due to the lack of close relatives in the miRNA repository. In spite of this, the computational approach described here can likely be applied to the identification of pre-miRNA hairpins in other organisms. Construction and analysis of a small RNA library led to the experimental identification of 14 novel miRNAs from S. mansoni through a combination of molecular cloning, DNA sequencing and expression studies. Our results significantly expand the set of known miRNAs in multicellular parasites and provide a basis for understanding the structural and functional evolution of miRNAs in these metazoan parasites.

Structural perspective on the activation of RNAse P RNA by protein.

Ribonucleoprotein particles are central to numerous cellular pathways, but their study in vitro is often complicated by heterogeneity and aggregation. We describe a new technique to characterize these complexes trapped as homogeneous species in a nondenaturing gel. Using this technique, in conjunction with phosphorothioate footprinting analysis, we identify the protein-binding site and RNA folding states of ribonuclease P (RNase P), an RNA-based enzyme that, in vivo, requires a protein cofactor to catalyze the 5' maturation of precursor transfer RNA (pre-tRNA). Our results show that the protein binds to a patch of conserved RNA structure adjacent to the active site and influences the conformation of the RNA near the tRNA-binding site. The data are consistent with a role of the protein in substrate recognition and support a new model of the holoenzyme that is based on a recently solved crystal structure of RNase P RNA.

Cosmopolitan heterotrophic microeukaryotes are active bacterial grazers in experimental oil-polluted systems.

We investigated the population dynamics and prevailing 18S rDNA phylotypes of microeukaryotes (

COPASAAR--a database for proteomic analysis of single amino acid repeats.

BACKGROUND: Single amino acid repeats make up a significant proportion in all of the proteomes that have currently been determined. They have been shown to be functionally and medically significant, and are associated with cancers and neuro-degenerative diseases such as Huntington's Chorea, where a poly-glutamine repeat is responsible for causing the disease. The COPASAAR database is a new tool to facilitate the rapid analysis of single amino acid repeats at a proteome level. The database aims to simplify the comparison of repeat distributions between proteomes in order to provide a better understanding of their function and evolution. RESULTS: A comparative analysis of all proteomes in the database (currently 244) shows that single amino acid repeats account for about 12-14% of the proteome of any given species. They are more common in eukaryotes (14%) than in either archaea or bacteria (both 13%). Individual analyses of proteomes show that long single amino acid repeats (6+ residues) are much more common in the Eukaryotes and that longer repeats are usually made up of hydrophilic amino acids such as glutamine, glutamic acid, asparagine, aspartic acid and serine. CONCLUSION: COPASAAR is a useful tool for comparative proteomics that provides rapid access to amino acid repeat data that can be readily data-mined. The COPASAAR database can be queried at the kingdom, proteome or individual protein level. As the amount of available proteome data increases this will be increasingly important in order to automate proteome comparison. The insights gained from these studies will give a better insight into the evolution of protein sequence and function.

One gene, two diseases and three conformations: molecular dynamics simulations of mutants of human prion protein at room temperature and elevated temperatures.

Fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD) are associated to the same mutation at codon 178 but differentiate into clinicopathologically distinct diseases determined by this mutation and a naturally occurring methionine-valine polymorphism at codon 129 of the prion protein gene. It has been suggested that the clinical and pathological difference between FFI and CJD is caused by different conformations of the prion protein. Using molecular dynamics (MD), we investigated the effect of the mutation at codon 178 and the polymorphism at codon 129 on prion protein dynamics and conformation at normal and elevated temperatures. Four model structures were examined with a focus on their dynamics and conformational changes. The results showed differences in stability and dynamics between polymorphic variants. Methionine variants demonstrated a higher stability than valine variants. Elongation of existing beta-sheets and formation of new beta-sheets was found to occur more readily in valine polymorphic variants. We also discovered the inhibitory effect of proline residue on existing beta-sheet elongation.

Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation.

Molecular dynamics simulations have been used extensively to model the folding and unfolding of proteins. The rates of folding and unfolding should follow the Arrhenius equation over a limited range of temperatures. This study shows that molecular dynamic simulations of the unfolding of crambin between 500K and 560K do follow the Arrhenius equation. They also show that while there is a large amount of variation between the simulations the average values for the rate show a very high degree of correlation.

The European and Japanese outbreaks of H5N8 derive from a single source population providing evidence for the dispersal along the long distance bird migratory flyways.

The origin of recent parallel outbreaks of the high pathogenicity H5N8 avian flu virus in Europe and in Japan can be traced to a single source population, which has most likely been spread by migratory birds. By using Bayesian coalescent methods to analyze the DNA sequences of the virus to find the times for divergence and combining this sequence data with bird migration data we can show the most likely locations and migratory pathways involved in the origin of the current outbreak. This population was most likely located in the Siberian summer breeding grounds of long-range migratory birds. These breeding grounds provide a connection between different migratory flyways and explain the current outbreaks in remote locations. By combining genetic methods and epidemiological data we can rapidly identify the sources and the dispersion pathways for novel avian influenza outbreaks.

Contributions of the TEL-patch amino acid cluster on TPP1 to telomeric DNA synthesis by human telomerase.

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch--a cluster of amino acids on the surface of the shelterin component TPP1--is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment--binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

Reduced bio basis function neural network for identification of protein phosphorylation sites: comparison with pattern recognition algorithms.

Protein phosphorylation is a post-translational modification performed by a group of enzymes known as the protein kinases or phosphotransferases (Enzyme Commission classification 2.7). It is essential to the correct functioning of both proteins and cells, being involved with enzyme control, cell signalling and apoptosis. The major problem when attempting prediction of these sites is the broad substrate specificity of the enzymes. This study employs back-propagation neural networks (BPNNs), the decision tree algorithm C4.5 and the reduced bio-basis function neural network (rBBFNN) to predict phosphorylation sites. The aim is to compare prediction efficiency of the three algorithms for this problem, and examine knowledge extraction capability. All three algorithms are effective for phosphorylation site prediction. Results indicate that rBBFNN is the fastest and most sensitive of the algorithms. BPNN has the highest area under the ROC curve and is therefore the most robust, and C4.5 has the highest prediction accuracy. C4.5 also reveals the amino acid 2 residues upstream from the phosporylation site is important for serine/threonine phosphorylation, whilst the amino acid 3 residues upstream is important for tyrosine phosphorylation.

Evaluation of double formalin--Lugol's fixation in assessing number and biomass of ciliates: an example of estimations at mesoscale in NE Atlantic.

Ciliated protozoa are potential grazers of primary and bacterial production and act as intermediaries between picoplankton and copepods and other large suspension feeders. Accurate determination of ciliate abundance and feeding mode is crucial in oceanic carbon budget estimations. However, the impact of different fixatives on the abundance and cell volume of ciliates has been investigated in only a few studies using either laboratory cultures or natural populations. Lugol's solution and formalin are the most commonly used fixatives for the preservation of ciliates samples. In the present study, the aim was to compare 0.4% Lugol's solution and 2% borated-formalin fixation and evaluate the need of counting duplicate samples each using a different fixative. For this, a large number of samples (n = 110) from the NE Atlantic was analyzed in the frame of POMME program (Multidisciplinary Mesoscale Ocean Program). We established a statistically significant relationship (p < 0.0001) between Lugol's and formalin fixed samples for both abundance (r2 = 0.50) and biomass (r2 = 0.76) of aloricate ciliates which showed that counts were higher in Lugol's solution by a factor of 2 and a non-taxon specific cell-loss in formalin. However, loricate ciliate abundance in our samples which were represented primarily by Tintinnus spp. did not show any difference between the two treatments. Abundance and biomass of mixotrophic ciliates (chloroplast-bearing cells) were for various reasons underestimated in both treatments. Our results show that unique fixation by formalin may severely underestimate ciliates abundance and biomass although their population may not alter. For this reason, Lugol's solution is best for the estimation of their abundance and biomass. However, for counts of mixotrophs and the evaluation of the ecological role of ciliates in carbon flux, double fixation is essential. Compromises regarding the fixatives have lead to severe underestimations of mixotrophs in studies conducted by now.

The Robustness of Pathway Analysis in Identifying Potential Drug Targets in Non-Small Cell Lung Carcinoma.

The identification of genes responsible for causing cancers from gene expression data has had varied success. Often the genes identified depend on the methods used for detecting expression patterns, or on the ways that the data had been normalized and filtered. The use of gene set enrichment analysis is one way to introduce biological information in order to improve the detection of differentially expressed genes and pathways. In this paper we show that the use of network models while still subject to the problems of normalization is a more robust method for detecting pathways that are differentially overrepresented in lung cancer data. Such differences may provide opportunities for novel therapeutics. In addition, we present evidence that non-small cell lung carcinoma is not a series of homogeneous diseases; rather that there is a heterogeny within the genotype which defies phenotype classification. This diversity helps to explain the lack of progress in developing therapies against non-small cell carcinoma and suggests that drug development may consider multiple pathways as treatment targets.

A global phylogenetic analysis in order to determine the host species and geography dependent features present in the evolution of avian H9N2 influenza hemagglutinin.

A complete phylogenetic analysis of all of the H9N2 hemagglutinin sequences that were collected between 1966 and 2012 was carried out in order to build a picture of the geographical and host specific evolution of the hemagglutinin protein. To improve the quality and applicability of the output data the sequences were divided into subsets based upon location and host species. The phylogenetic analysis of hemagglutinin reveals that the protein has distinct lineages between China and the Middle East, and that wild birds in both regions retain a distinct form of the H9 molecule, from the same lineage as the ancestral hemagglutinin. The results add further evidence to the hypothesis that the current predominant H9N2 hemagglutinin lineage might have originated in Southern China. The study also shows that there are sampling problems that affect the reliability of this and any similar analysis. This raises questions about the surveillance of H9N2 and the need for wider sampling of the virus in the environment. The results of this analysis are also consistent with a model where hemagglutinin has predominantly evolved by neutral drift punctuated by occasional selection events. These selective events have produced the current pattern of distinct lineages in the Middle East, Korea and China. This interpretation is in agreement with existing studies that have shown that there is widespread intra-country sequence evolution.

Identification of human TERT elements necessary for telomerase recruitment to telomeres.

Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. In this study, we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease.

Weight loss and metabolic outcomes of bariatric surgery in men versus women - A matched comparative observational cohort study.

BACKGROUND: Despite the high prevalence of morbid obesity, the global frequency of bariatric surgery in men is significantly lower than in women. It is unclear if this is due to the perception of poorer outcomes in men. OBJECTIVES: Compare weight loss and metabolic outcomes in men vs. women after bariatric surgery. SETTING: University teaching hospital in North West England. METHODS: We performed an observational cohort analysis of 79 men matched to 79 women for baseline age (± 5 years), body mass index (BMI; ± 2 kg/m(2)), bariatric procedure (69 gastric bypass and 10 sleeve gastrectomy each), type 2 diabetes (33 each), and continuous positive airway pressure (CPAP) therapy for obstructive sleep apnoea (OSA; 40 each). RESULTS: Overall mean (95% confidence interval) reduction in BMI was 17.5 (15.7-19.4) kg/m(2) (P<0.001) at 24 months. There was no significant difference between men and women in mean percentage excess BMI loss (65.8% vs. 72.9%) at 24 months. Likewise, there were significant reductions in blood pressure, glycosylated haemoglobin and total cholesterol-to-high density lipoprotein cholesterol overall but no significant gender differences. Postoperatively, 77.5% of men and 90.0% of women with OSA discontinued CPAP therapy (non-significant). CONCLUSIONS: Weight loss and metabolic outcomes after bariatric surgery are of similar magnitude in men compared to women. The use of bariatric surgery in eligible men should be encouraged.

Analysis of gene expression data from non-small cell lung carcinoma cell lines reveals distinct sub-classes from those identified at the phenotype level.

Microarray data from cell lines of Non-Small Cell Lung Carcinoma (NSCLC) can be used to look for differences in gene expression between the cell lines derived from different tumour samples, and to investigate if these differences can be used to cluster the cell lines into distinct groups. Dividing the cell lines into classes can help to improve diagnosis and the development of screens for new drug candidates. The micro-array data is first subjected to quality control analysis and then subsequently normalised using three alternate methods to reduce the chances of differences being artefacts resulting from the normalisation process. The final clustering into sub-classes was carried out in a conservative manner such that sub-classes were consistent across all three normalisation methods. If there is structure in the cell line population it was expected that this would agree with histological classifications, but this was not found to be the case. To check the biological consistency of the sub-classes the set of most strongly differentially expressed genes was be identified for each pair of clusters to check if the genes that most strongly define sub-classes have biological functions consistent with NSCLC.

Developing stochastic models for spatial inference: bacterial chemotaxis.

BACKGROUND: Biological systems are inherently inhomogeneous and spatial effects play a significant role in processes such as pattern formation. At the cellular level proteins are often localised either through static attachment or via a dynamic equilibrium. As well as spatial heterogeneity many cellular processes exhibit stochastic fluctuations and so to make inferences about the location of molecules there is a need for spatial stochastic models. A test case for spatial models has been bacterial chemotaxis which has been studied extensively as a model of signal transduction. RESULTS: By creating specific models of a cellular system that incorporate the spatial distributions of molecules we have shown how the fit between simulated and experimental data can be used to make inferences about localisation, in the case of bacterial chemotaxis. This method allows the robust comparison of different spatial models through alternative model parameterisations. CONCLUSIONS: By using detailed statistical analysis we can reliably infer the parameters for the spatial models, and also to evaluate alternative models. The statistical methods employed in this case are particularly powerful as they reduce the need for a large number of simulation replicates. The technique is also particularly useful when only limited molecular level data is available or where molecular data is not quantitative.