DNA translocases and origin region segregation in B. subtilis

Specialized mechanisms involved in chromosome segregation, septum placement, and chromosome dimer resolution contribute to the maintenance of chromosome integrity throughout the cell cycle. The first part of this work focuses on the investigation of DNA translocases in the Gram positive model organism Bacillus subtilis, which move the chromosomes away from the division plane by directed DNA transport. SpoIIIE is a membrane-integral translocase that also acts during sporulation, while SftA is associated with the division septum by an unknown mechanism and was reported to be a soluble protein in vitro. The solubility of SftA in vivo was proven with cell fractionation experiments, and the part of the protein that serves for septal targeting was determined through the localization of different truncations of fluorescently labeled SftA, which helped narrow down the stretch of amino acids responsible for targeting the protein to the septum. Expression in a Eukaryotic heterologous system revealed an interaction between SftA and FtsA, but not FtsZ. Further evidence was provided by single molecule tracking experiments whereby the fraction of soluble SftA molecules increased in an FtsA depletion background as compared to a wild type background. Concomitant investigation on the single molecule level of SpoIIIE, a membrane associated DNA translocase in B. subtilis, and PfkA, a soluble phosphofructokinase, revealed a different behavior of the two translocases (SftA and SpoIIIE): SftA has a septal bound fraction, and a small soluble fast moving fraction,comparable in diffusion coefficient to the exclusively soluble PfkA tracked under the same conditions. SpoIIIE is much slower in comparison, and even its “fast” moving fraction is much slower than that of SftA. It seems to move slowly along the membrane with no specific enrichment at the septum, even after Mitomycin C (MMC) treatment. The second part of this work focused on the count of nucleoids, origin, and terminus of replication regions under conditions of fast and slow growth, with or without the induction of double strand breaks with MMC. B. subtilis seemed to be predominantly diploid, with a fraction of polyploid and monoploid cells which changed depending on the growth conditions. Replication of the origin proximal regions increased after DNA damage induction, as was shown by the increase in the number of origins during the time of DNA repair, while the number of termini remained constant. Time lapse experiments of the segregation of the tagged origin regions revealed that the movement is best described as directed diffusion, but seems to be quite robust and continues, though slower, after MMC treatment or treatment with Ciprofloxacin which blocks the topoisomerase I

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

New genetic loci link adipose and insulin biology to body fat distribution.

Body fat distribution is a heritable trait and a well-established predictor of adverse metabolic outcomes, independent of overall adiposity. To increase our understanding of the genetic basis of body fat distribution and its molecular links to cardiometabolic traits, here we conduct genome-wide association meta-analyses of traits related to waist and hip circumferences in up to 224,459 individuals. We identify 49 loci (33 new) associated with waist-to-hip ratio adjusted for body mass index (BMI), and an additional 19 loci newly associated with related waist and hip circumference measures (P < 5 × 10(-8)). In total, 20 of the 49 waist-to-hip ratio adjusted for BMI loci show significant sexual dimorphism, 19 of which display a stronger effect in women. The identified loci were enriched for genes expressed in adipose tissue and for putative regulatory elements in adipocytes. Pathway analyses implicated adipogenesis, angiogenesis, transcriptional regulation and insulin resistance as processes affecting fat distribution, providing insight into potential pathophysiological mechanisms

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Chromosome segregation in B. subtilis is highly heterogeneous

Objective!#!The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level.!##!Results!#!B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation

DNA translocases and origin region segregation in B. subtilis

Specialized mechanisms involved in chromosome segregation, septum placement, and chromosome dimer resolution contribute to the maintenance of chromosome integrity throughout the cell cycle. The first part of this work focuses on the investigation of DNA translocases in the Gram positive model organism Bacillus subtilis, which move the chromosomes away from the division plane by directed DNA transport. SpoIIIE is a membrane-integral translocase that also acts during sporulation, while SftA is associated with the division septum by an unknown mechanism and was reported to be a soluble protein in vitro. The solubility of SftA in vivo was proven with cell fractionation experiments, and the part of the protein that serves for septal targeting was determined through the localization of different truncations of fluorescently labeled SftA, which helped narrow down the stretch of amino acids responsible for targeting the protein to the septum. Expression in a Eukaryotic heterologous system revealed an interaction between SftA and FtsA, but not FtsZ. Further evidence was provided by single molecule tracking experiments whereby the fraction of soluble SftA molecules increased in an FtsA depletion background as compared to a wild type background. Concomitant investigation on the single molecule level of SpoIIIE, a membrane associated DNA translocase in B. subtilis, and PfkA, a soluble phosphofructokinase, revealed a different behavior of the two translocases (SftA and SpoIIIE): SftA has a septal bound fraction, and a small soluble fast moving fraction,comparable in diffusion coefficient to the exclusively soluble PfkA tracked under the same conditions. SpoIIIE is much slower in comparison, and even its “fast” moving fraction is much slower than that of SftA. It seems to move slowly along the membrane with no specific enrichment at the septum, even after Mitomycin C (MMC) treatment. The second part of this work focused on the count of nucleoids, origin, and terminus of replication regions under conditions of fast and slow growth, with or without the induction of double strand breaks with MMC. B. subtilis seemed to be predominantly diploid, with a fraction of polyploid and monoploid cells which changed depending on the growth conditions. Replication of the origin proximal regions increased after DNA damage induction, as was shown by the increase in the number of origins during the time of DNA repair, while the number of termini remained constant. Time lapse experiments of the segregation of the tagged origin regions revealed that the movement is best described as directed diffusion, but seems to be quite robust and continues, though slower, after MMC treatment or treatment with Ciprofloxacin which blocks the topoisomerase I

Requirements for septal localization and chromosome segregation activity of the DNA translocase SftA from Bacillus subtilis\textit {Bacillus subtilis}

$\textit {Bacillus subtilis}$ possesses 2 DNA translocases that affect late stages of chromosome segregation: SftA separates nonsegregated DNA prior to septum closure, while SpoIIIE rescues septum-entrapped DNA. We provide evidence that SftA is associated with the division machinery via a stretch of 47 amino acids within its N-terminus, suggesting that SftA is recruited by protein-protein interactions with a component of the division machinery. SftA was also recruited to mid-cell in the absence of its first 20 amino acids, which are proposed to contain a membrane-binding motif. Cell fractionation experiments showed that SftA can be found in the cytosolic fraction, and to a minor degree in the membrane fraction, showing that it is a soluble protein in vivo. The expression of truncated SftA constructs led to a dominant $\it sftA$ deletion phenotype, even at very low induction rates of the truncated proteins, indicating that the incorporation of nonfunctional monomers into SftA hexamers abolishes functionality. Mobility shift experiments and surface plasmon binding studies showed that SftA binds to DNA in a cooperative manner, and demonstrated low ATPase activity when binding to short nucleotides rather than to long stretches of DNA

Global variation in postoperative mortality and complications after cancer surgery: a multicentre, prospective cohort study in 82 countries

Background: 80% of individuals with cancer will require a surgical procedure, yet little comparative data exist on early outcomes in low-income and middle-income countries (LMICs). We compared postoperative outcomes in breast, colorectal, and gastric cancer surgery in hospitals worldwide, focusing on the effect of disease stage and complications on postoperative mortality. Methods: This was a multicentre, international prospective cohort study of consecutive adult patients undergoing surgery for primary breast, colorectal, or gastric cancer requiring a skin incision done under general or neuraxial anaesthesia. The primary outcome was death or major complication within 30 days of surgery. Multilevel logistic regression determined relationships within three-level nested models of patients within hospitals and countries. Hospital-level infrastructure effects were explored with three-way mediation analyses. This study was registered with ClinicalTrials.gov, NCT03471494. Findings: Between April 1, 2018, and Jan 31, 2019, we enrolled 15 958 patients from 428 hospitals in 82 countries (high income 9106 patients, 31 countries; upper-middle income 2721 patients, 23 countries; or lower-middle income 4131 patients, 28 countries). Patients in LMICs presented with more advanced disease compared with patients in high-income countries. 30-day mortality was higher for gastric cancer in low-income or lower-middle-income countries (adjusted odds ratio 3·72, 95% CI 1·70–8·16) and for colorectal cancer in low-income or lower-middle-income countries (4·59, 2·39–8·80) and upper-middle-income countries (2·06, 1·11–3·83). No difference in 30-day mortality was seen in breast cancer. The proportion of patients who died after a major complication was greatest in low-income or lower-middle-income countries (6·15, 3·26–11·59) and upper-middle-income countries (3·89, 2·08–7·29). Postoperative death after complications was partly explained by patient factors (60%) and partly by hospital or country (40%). The absence of consistently available postoperative care facilities was associated with seven to 10 more deaths per 100 major complications in LMICs. Cancer stage alone explained little of the early variation in mortality or postoperative complications. Interpretation: Higher levels of mortality after cancer surgery in LMICs was not fully explained by later presentation of disease. The capacity to rescue patients from surgical complications is a tangible opportunity for meaningful intervention. Early death after cancer surgery might be reduced by policies focusing on strengthening perioperative care systems to detect and intervene in common complications. Funding: National Institute for Health Research Global Health Research Unit