Manipulation of the Tn5 transpososome assembly pathway and its effect on reaction dynamics

Transposable elements are an almost ubiquitous feature in all three domains of life, and are a significant driver of genomic evolution. These discrete genetic elements are capable of mobilisation and amplification, which allows them to multiply. A subset of these elements, the cut-and-paste transposons, excise themselves entirely from the surrounding DNA and integrate themselves elsewhere in the genome. Hsmar1, a cut-and-paste element of the mariner transposon family, was recently investigated to examine the kinetics of the complex formation pathway. This transposase forms a dimer in solution before binding to first one transposon end, then recruiting the other. This pathway, Synapsis by Naked End Capture (S-NEC), is found in both prokaryotes and eukaryotes. Tn5, of the IS4 family, is an example of the alternative pathway, Synapsis by Protein Dimerisation (S-PD). In this pathway, the transposase is monomeric in solution. Each monomer binds to a transposon end independently, before combining to form a synaptic complex. This pathway has been seen widely in prokaryotes, but no eukaryotic elements tested to date employ it. To understand what limitations the complex formation pathway might place upon an S-PD transposase in a eukaryotic genome, the Tn5 transposase was manipulated. Forming the transposase into a single-chain dimer allowed it to mimic S-NEC, an alteration that made it hyperactive in prokaryotes and eukaryotes. As the eukaryotic genome contains chromatin, adapting the Tn5 transposase to this might overcome some of the low activity seen. The fusion of a chromatin binding domain to the transposase was not able to enhance the frequency of transposition, which may suggest that the presence of chromatin is not causing significant inhibition. The results presented in this work suggest two significant conclusions. The first is that the S-NEC reaction dynamics are significantly preferable to S-PD within eukaryotes. The second is that S-PD itself may be a system of regulation of transposition, to prevent activity levels that could be detrimental to the host

Pooled analysis of WHO Surgical Safety Checklist use and mortality after emergency laparotomy

Background The World Health Organization (WHO) Surgical Safety Checklist has fostered safe practice for 10 years, yet its place in emergency surgery has not been assessed on a global scale. The aim of this study was to evaluate reported checklist use in emergency settings and examine the relationship with perioperative mortality in patients who had emergency laparotomy. Methods In two multinational cohort studies, adults undergoing emergency laparotomy were compared with those having elective gastrointestinal surgery. Relationships between reported checklist use and mortality were determined using multivariable logistic regression and bootstrapped simulation. Results Of 12 296 patients included from 76 countries, 4843 underwent emergency laparotomy. After adjusting for patient and disease factors, checklist use before emergency laparotomy was more common in countries with a high Human Development Index (HDI) (2455 of 2741, 89.6 per cent) compared with that in countries with a middle (753 of 1242, 60.6 per cent; odds ratio (OR) 0.17, 95 per cent c.i. 0.14 to 0.21, P <0001) or low (363 of 860, 422 per cent; OR 008, 007 to 010, P <0.001) HDI. Checklist use was less common in elective surgery than for emergency laparotomy in high-HDI countries (risk difference -94 (95 per cent c.i. -11.9 to -6.9) per cent; P <0001), but the relationship was reversed in low-HDI countries (+121 (+7.0 to +173) per cent; P <0001). In multivariable models, checklist use was associated with a lower 30-day perioperative mortality (OR 0.60, 0.50 to 073; P <0.001). The greatest absolute benefit was seen for emergency surgery in low- and middle-HDI countries. Conclusion Checklist use in emergency laparotomy was associated with a significantly lower perioperative mortality rate. Checklist use in low-HDI countries was half that in high-HDI countries.Peer reviewe

Global variation in anastomosis and end colostomy formation following left-sided colorectal resection

Background End colostomy rates following colorectal resection vary across institutions in high-income settings, being influenced by patient, disease, surgeon and system factors. This study aimed to assess global variation in end colostomy rates after left-sided colorectal resection. Methods This study comprised an analysis of GlobalSurg-1 and -2 international, prospective, observational cohort studies (2014, 2016), including consecutive adult patients undergoing elective or emergency left-sided colorectal resection within discrete 2-week windows. Countries were grouped into high-, middle- and low-income tertiles according to the United Nations Human Development Index (HDI). Factors associated with colostomy formation versus primary anastomosis were explored using a multilevel, multivariable logistic regression model. Results In total, 1635 patients from 242 hospitals in 57 countries undergoing left-sided colorectal resection were included: 113 (6·9 per cent) from low-HDI, 254 (15·5 per cent) from middle-HDI and 1268 (77·6 per cent) from high-HDI countries. There was a higher proportion of patients with perforated disease (57·5, 40·9 and 35·4 per cent; P < 0·001) and subsequent use of end colostomy (52·2, 24·8 and 18·9 per cent; P < 0·001) in low- compared with middle- and high-HDI settings. The association with colostomy use in low-HDI settings persisted (odds ratio (OR) 3·20, 95 per cent c.i. 1·35 to 7·57; P = 0·008) after risk adjustment for malignant disease (OR 2·34, 1·65 to 3·32; P < 0·001), emergency surgery (OR 4·08, 2·73 to 6·10; P < 0·001), time to operation at least 48 h (OR 1·99, 1·28 to 3·09; P = 0·002) and disease perforation (OR 4·00, 2·81 to 5·69; P < 0·001). Conclusion Global differences existed in the proportion of patients receiving end stomas after left-sided colorectal resection based on income, which went beyond case mix alone

Manipulation of the Tn5 transpososome assembly pathway and its effect on reaction dynamics

Transposable elements are an almost ubiquitous feature in all three domains of life, and are a significant driver of genomic evolution. These discrete genetic elements are capable of mobilisation and amplification, which allows them to multiply. A subset of these elements, the cut-and-paste transposons, excise themselves entirely from the surrounding DNA and integrate themselves elsewhere in the genome. Hsmar1, a cut-and-paste element of the mariner transposon family, was recently investigated to examine the kinetics of the complex formation pathway. This transposase forms a dimer in solution before binding to first one transposon end, then recruiting the other. This pathway, Synapsis by Naked End Capture (S-NEC), is found in both prokaryotes and eukaryotes. Tn5, of the IS4 family, is an example of the alternative pathway, Synapsis by Protein Dimerisation (S-PD). In this pathway, the transposase is monomeric in solution. Each monomer binds to a transposon end independently, before combining to form a synaptic complex. This pathway has been seen widely in prokaryotes, but no eukaryotic elements tested to date employ it. To understand what limitations the complex formation pathway might place upon an S-PD transposase in a eukaryotic genome, the Tn5 transposase was manipulated. Forming the transposase into a single-chain dimer allowed it to mimic S-NEC, an alteration that made it hyperactive in prokaryotes and eukaryotes. As the eukaryotic genome contains chromatin, adapting the Tn5 transposase to this might overcome some of the low activity seen. The fusion of a chromatin binding domain to the transposase was not able to enhance the frequency of transposition, which may suggest that the presence of chromatin is not causing significant inhibition. The results presented in this work suggest two significant conclusions. The first is that the S-NEC reaction dynamics are significantly preferable to S-PD within eukaryotes. The second is that S-PD itself may be a system of regulation of transposition, to prevent activity levels that could be detrimental to the host

Isolation and Characterisation of Bacteriophage Selective for Key Acinetobacter baumannii Capsule Chemotypes.

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (-20 to 60 °C) and pH (5.0-9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38-39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections

Thigh-length compression stockings and DVT after stroke

Controversy exists as to whether neoadjuvant chemotherapy improves survival in patients with invasive bladder cancer, despite randomised controlled trials of more than 3000 patients. We undertook a systematic review and meta-analysis to assess the effect of such treatment on survival in patients with this disease

Evolutionary and functional history of the escherichia coli K1 capsule

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage