The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process

Antimicrobial resistance among migrants in Europe: a systematic review and meta-analysis

BACKGROUND: Rates of antimicrobial resistance (AMR) are rising globally and there is concern that increased migration is contributing to the burden of antibiotic resistance in Europe. However, the effect of migration on the burden of AMR in Europe has not yet been comprehensively examined. Therefore, we did a systematic review and meta-analysis to identify and synthesise data for AMR carriage or infection in migrants to Europe to examine differences in patterns of AMR across migrant groups and in different settings. METHODS: For this systematic review and meta-analysis, we searched MEDLINE, Embase, PubMed, and Scopus with no language restrictions from Jan 1, 2000, to Jan 18, 2017, for primary data from observational studies reporting antibacterial resistance in common bacterial pathogens among migrants to 21 European Union-15 and European Economic Area countries. To be eligible for inclusion, studies had to report data on carriage or infection with laboratory-confirmed antibiotic-resistant organisms in migrant populations. We extracted data from eligible studies and assessed quality using piloted, standardised forms. We did not examine drug resistance in tuberculosis and excluded articles solely reporting on this parameter. We also excluded articles in which migrant status was determined by ethnicity, country of birth of participants' parents, or was not defined, and articles in which data were not disaggregated by migrant status. Outcomes were carriage of or infection with antibiotic-resistant organisms. We used random-effects models to calculate the pooled prevalence of each outcome. The study protocol is registered with PROSPERO, number CRD42016043681. FINDINGS: We identified 2274 articles, of which 23 observational studies reporting on antibiotic resistance in 2319 migrants were included. The pooled prevalence of any AMR carriage or AMR infection in migrants was 25·4% (95% CI 19·1-31·8; I2 =98%), including meticillin-resistant Staphylococcus aureus (7·8%, 4·8-10·7; I2 =92%) and antibiotic-resistant Gram-negative bacteria (27·2%, 17·6-36·8; I2 =94%). The pooled prevalence of any AMR carriage or infection was higher in refugees and asylum seekers (33·0%, 18·3-47·6; I2 =98%) than in other migrant groups (6·6%, 1·8-11·3; I2 =92%). The pooled prevalence of antibiotic-resistant organisms was slightly higher in high-migrant community settings (33·1%, 11·1-55·1; I2 =96%) than in migrants in hospitals (24·3%, 16·1-32·6; I2 =98%). We did not find evidence of high rates of transmission of AMR from migrant to host populations. INTERPRETATION: Migrants are exposed to conditions favouring the emergence of drug resistance during transit and in host countries in Europe. Increased antibiotic resistance among refugees and asylum seekers and in high-migrant community settings (such as refugee camps and detention facilities) highlights the need for improved living conditions, access to health care, and initiatives to facilitate detection of and appropriate high-quality treatment for antibiotic-resistant infections during transit and in host countries. Protocols for the prevention and control of infection and for antibiotic surveillance need to be integrated in all aspects of health care, which should be accessible for all migrant groups, and should target determinants of AMR before, during, and after migration. FUNDING: UK National Institute for Health Research Imperial Biomedical Research Centre, Imperial College Healthcare Charity, the Wellcome Trust, and UK National Institute for Health Research Health Protection Research Unit in Healthcare-associated Infections and Antimictobial Resistance at Imperial College London

Surgical site infection after gastrointestinal surgery in high-income, middle-income, and low-income countries: a prospective, international, multicentre cohort study

Background: Surgical site infection (SSI) is one of the most common infections associated with health care, but its importance as a global health priority is not fully understood. We quantified the burden of SSI after gastrointestinal surgery in countries in all parts of the world. Methods: This international, prospective, multicentre cohort study included consecutive patients undergoing elective or emergency gastrointestinal resection within 2-week time periods at any health-care facility in any country. Countries with participating centres were stratified into high-income, middle-income, and low-income groups according to the UN's Human Development Index (HDI). Data variables from the GlobalSurg 1 study and other studies that have been found to affect the likelihood of SSI were entered into risk adjustment models. The primary outcome measure was the 30-day SSI incidence (defined by US Centers for Disease Control and Prevention criteria for superficial and deep incisional SSI). Relationships with explanatory variables were examined using Bayesian multilevel logistic regression models. This trial is registered with ClinicalTrials.gov, number NCT02662231. Findings: Between Jan 4, 2016, and July 31, 2016, 13 265 records were submitted for analysis. 12 539 patients from 343 hospitals in 66 countries were included. 7339 (58·5%) patient were from high-HDI countries (193 hospitals in 30 countries), 3918 (31·2%) patients were from middle-HDI countries (82 hospitals in 18 countries), and 1282 (10·2%) patients were from low-HDI countries (68 hospitals in 18 countries). In total, 1538 (12·3%) patients had SSI within 30 days of surgery. The incidence of SSI varied between countries with high (691 [9·4%] of 7339 patients), middle (549 [14·0%] of 3918 patients), and low (298 [23·2%] of 1282) HDI (p < 0·001). The highest SSI incidence in each HDI group was after dirty surgery (102 [17·8%] of 574 patients in high-HDI countries; 74 [31·4%] of 236 patients in middle-HDI countries; 72 [39·8%] of 181 patients in low-HDI countries). Following risk factor adjustment, patients in low-HDI countries were at greatest risk of SSI (adjusted odds ratio 1·60, 95% credible interval 1·05–2·37; p=0·030). 132 (21·6%) of 610 patients with an SSI and a microbiology culture result had an infection that was resistant to the prophylactic antibiotic used. Resistant infections were detected in 49 (16·6%) of 295 patients in high-HDI countries, in 37 (19·8%) of 187 patients in middle-HDI countries, and in 46 (35·9%) of 128 patients in low-HDI countries (p < 0·001). Interpretation: Countries with a low HDI carry a disproportionately greater burden of SSI than countries with a middle or high HDI and might have higher rates of antibiotic resistance. In view of WHO recommendations on SSI prevention that highlight the absence of high-quality interventional research, urgent, pragmatic, randomised trials based in LMICs are needed to assess measures aiming to reduce this preventable complication

Characterization of a tumor-associated activating mutation of the p110β PI 3-kinase.

The PI3-kinase pathway is commonly activated in tumors, most often by loss of PTEN lipid phosphatase activity or the amplification or mutation of p110α. Oncogenic mutants have commonly been found in p110α, but rarely in any of the other catalytic subunits of class I PI3-kinases. We here characterize a p110β helical domain mutation, E633K, first identified in a Her2-positive breast cancer. The mutation increases basal p110β activity, but does not affect activation of p85/p110β dimers by phosphopeptides or Gβγ. Expression of the mutant causes increases in Akt and S6K1 activation, transformation, chemotaxis, proliferation and survival in low serum. E633 is conserved among class I PI3 Ks, and its mutation in p110β is also activating. Interestingly, the E633K mutant occurs near a region that interacts with membranes in activated PI 3-kinases, and its mutation abrogates the requirement for an intact Ras-binding domain in p110β-mediated transformation. We propose that the E633K mutant activates p110β by enhancing its basal association with membranes. This study presents the first analysis of an activating oncogenic mutation of p110β

Effect of p110β mutant on transformation and chemotaxis.

<p>(A) NIH 3T3 cells stably expressing wild type or E633K p110β were plated in soft agar and colonies were counted after 3 weeks. Colony counts are normalized to the number of colonies produced by cells expressing p110β alone. (B) Equal number of NIH 3T3 cells stably expressing wild type or E633K p110β were plated and left to grow to confluence for 10 days. Foci were counted and normalized to cells expressing wild-type p110β. (C) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated. Data are mean ± SEM of triplicate samples from two experiments.</p

Akt signaling, proliferation and survival of cells expressing mutant p110β.

<p>(A) Expression level of wild-type or E633K myc-p110β in stably-transfected cells. (B) Cells stably expressing wild type or E633K p10β were incubated overnight in 10%, 0.5% or 0% NCS media. Whole cell lysates were analyzed by western blotting with anti-pT308 Akt, anti-pT389 S6K, and anti-β-actin antibodies. (C-E) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in (C) 10% NCS medium, (D) 0.5% NCS medium, or (E) 0% NCS medium, and assayed using the MTT assay. (F) Cells stably expressing wild type or E633K p110β were incubated for 24 hours in 10%, 0.5%, or 0% NCS medium. Cell viability was assayed by Trypan blue staining. Dead cells are displayed as percent of total number of cells. Data are mean ± SEM of triplicate samples from two separate experiments.</p

Role of kinase activity on the increased proliferation and migration by the p110β mutant.

<p>(A) Cells stably expressing wild-type or E633K p110β were plated in 96-well plates, incubated for 24 and 48 hours in 10% NCS, with or without 200 nM TGX-221, and assayed using the MTT assay. (B) NIH 3T3 cells stably expressing wild type or E633K p110β were starved overnight and plated either in 0% or 10% NCS in transwell chambers, and incubated with media containing 0% or 10% NCS in the lower chamber and upper chambers as indicated, with or without 200 nM TGX-221 in both chambers as indicated. Data are mean ± SEM of at least duplicate samples from two separate experiments.</p

Characterization of the lipid kinase activity of the p110β mutant.

<p>(A) HEK 293T cells were transfected with p85 and wild type or E633K myc-p110β. Anti-myc immunoprecipitates were analyzed by western blotting and for lipid kinase activity. (B) Anti-myc immunoprecipitates from cells transfected as above were incubated for 2 hours with pY-peptide and assayed for lipid kinase activity. (C) Anti-myc immunoprecipitates from cells transfected as above were incubated with lipid vesicles/Gβ₁γ₂ subunits for 10 minutes and assayed for lipid kinase activity. (D) Sequence alignment of p110α, p110β, p110γ and p110δ focusing on the acidic patch containing the E633 p110β residue, highlighted in red. (E) Specific activity of wild-type and D626K p110α co-expressed with p85 in HEK 293T cells and assayed as above. All data are mean ± SEM of triplicate determination from three separate experiments.</p