Dramatic Repositioning of c-Myb to Different Promoters during the Cell Cycle Observed by Combining Cell Sorting with Chromatin Immunoprecipitation

The c-Myb transcription factor is a critical regulator of proliferation and stem cell differentiation, and mutated alleles of c-Myb are oncogenic, but little is known about changes in c-Myb activity during the cell cycle. To map the association of c-Myb with specific target genes during the cell cycle, we developed a novel Fix-Sort-ChIP approach, in which asynchronously growing cells were fixed with formaldehyde, stained with Hoechst 33342 and separated into different cell cycle fractions by flow sorting, then processed for chromatin immunoprecipitation (ChIP) assays. We found that c-Myb actively repositions, binding to some genes only in specific cell cycle phases. In addition, the specificity of c-Myb is dramatically different in small subpopulations of cells, for example cells in the G2/M phase of the cell cycle, than in the bulk population. The repositioning of c-Myb during the cell cycle is not due to changes in its expression and also occurs with ectopically expressed, epitope-tagged versions of c-Myb. The repositioning occurs in established cell lines, in primary human CD34+ hematopoietic progenitors and in primary human acute myeloid leukemia cells. The combination of fixation, sorting and ChIP analysis sheds new light on the dynamic nature of gene regulation during the cell cycle and provides a new type of tool for the analysis of gene regulation in small subsets of cells, such as cells in a specific phase of the cell cycle

Mouse mammary stem cells express prognostic markers for triple-negative breast cancer

Introduction Triple negative breast cancer (TNBC) is a heterogeneous group of tumours in which chemotherapy, the current mainstay of systemic treatment, is often initially beneficial but with a high risk of relapse and metastasis. There is currently no means of predicting which TNBC will relapse. We tested the hypothesis that the biological properties of normal stem cells are re-activated in tumour metastasis and that, therefore, the activation of normal mammary stem cell-associated gene sets in primary TNBC would be highly prognostic for relapse and metastasis. Methods Mammary basal stem and myoepithelial cells were isolated by flow cytometry and tested in low dose transplant assays. Gene expression microarrays were used to establish expression profiles of the stem and myoepithelial populations; these were compared to each other and to our previously established mammary epithelial gene expression profiles. Stem cell genes were classified by Gene Ontology (GO) analysis and the expression of a subset analysed in the stem cell population at single cell resolution. Activation of stem cell genes was interrogated across different breast cancer cohorts and within specific subtypes and tested for clinical prognostic power. Results A set of 323 genes was identified that was expressed significantly more highly in the purified basal stem cells compared to all other cells of the mammary epithelium. 109 out of 323 genes had been associated with stem cell features in at least one other study in addition to our own, providing further support for their involvement in the biology of this cell type. GO analysis demonstrated an enrichment of these genes for an association with cell migration, cytoskeletal regulation and tissue morphogenesis, consistent with a role in invasion and metastasis. Single cell resolution analysis showed that individual cells co-expressed both epithelial- and mesenchymal-associated genes/proteins. Most strikingly, we demonstrated that strong activity of this stem cell gene set in TNBCs identified those tumours most likely to rapidly progress to metastasis. Conclusions Our findings support the hypothesis that the biological properties of normal stem cells are drivers of metastasis and that these properties can be used to stratify patients with a highly heterogeneous disease such as TNBC

Long-term follow-up of IPEX syndrome patients after different therapeutic strategies : an international multicenter retrospective study

Background: Immunodysregulation polyendocrinopathy enteropathy x-linked(IPEX) syndrome is a monogenic autoimmune disease caused by FOXP3 mutations. Because it is a rare disease, the natural history and response to treatments, including allogeneic hematopoietic stem cell transplantation (HSCT) and immunosuppression (IS), have not been thoroughly examined. Objective: This analysis sought to evaluate disease onset, progression, and long-term outcome of the 2 main treatments in long-term IPEX survivors. Methods: Clinical histories of 96 patients with a genetically proven IPEX syndrome were collected from 38 institutions worldwide and retrospectively analyzed. To investigate possible factors suitable to predict the outcome, an organ involvement (OI) scoring system was developed. Results: We confirm neonatal onset with enteropathy, type 1 diabetes, and eczema. In addition, we found less common manifestations in delayed onset patients or during disease evolution. There is no correlation between the site of mutation and the disease course or outcome, and the same genotype can present with variable phenotypes. HSCT patients (n = 58) had a median follow-up of 2.7 years (range, 1 week-15 years). Patients receiving chronic IS (n 5 34) had a median follow-up of 4 years (range, 2 months-25 years). The overall survival after HSCT was 73.2% (95% CI, 59.4-83.0) and after IS was 65.1% (95% CI, 62.8-95.8). The pretreatment OI score was the only significant predictor of overall survival after transplant (P = .035) but not under IS. Conclusions: Patients receiving chronic IS were hampered by disease recurrence or complications, impacting long-term.disease-free survival. When performed in patients with a low OI score, HSCT resulted in disease resolution with better quality of life, independent of age, donor source, or conditioning regimen

Preoperative Factors and Three Year Weight Change in the Longitudinal Assessment of Bariatric Surgery (LABS) Consortium

BACKGROUND: Limited data guide the prediction of weight loss success or failure following bariatric surgery according to pre-surgery factors. There is significant variation in weight change following bariatric surgery and much interest in identifying pre-operative factors that may contribute to these differences. OBJECTIVE: This report evaluates the associations of a comprehensive set of baseline factors and three-year weight change. SETTING: Ten hospitals in six geographically diverse clinical centers in the United States. METHODS: PARTICIPANTS AND INTERVENTIONS: Adults undergoing a first bariatric surgical procedure as part of clinical care by participating surgeons were recruited between 2006 and 2009. Participants completed research assessments utilizing standardized and detailed data collection on over 100 preoperative and operative parameters for individuals undergoing Roux-en-Y gastric bypass (RYGB) and laparoscopic adjustable gastric banding (LAGB). Weight was measured 3 years following surgery. METHODS: MAIN OUTCOME MEASURES: Percent weight change for RYGB or LAGB from baseline to 3 years was analyzed as both a continuous and dichotomous outcome with cut points at 25% for RYGB and 10% for LAGB. Multivariable linear and logistic regression models were used to identify independent baseline predictors of the continuous and categorical outcomes, respectively. RESULTS: The median weight loss 3 years following surgery for RYGB (n=1513) participants was 31.5% (IQR: 24.6%–38.4%; range, 59.2% loss to 0.9% gain) of baseline weight and 16.0% (IQR: 8.1%–23.1%; range, 56.1% loss to 12.5% gain) for LAGB (n=509) participants. The median age was 46 years for RYGB and 48 years for LAGB; 80% of RYGB participants and 75% of LAGB participants were female; and the median baseline Body Mass Index (BMI) was 46 kg/m(2) for RYGB and 44 kg/m(2) for LAGB. For RYGB, Black participants lost 2.7% less weight compared to Whites and participants with diabetes at baseline had 3.7% less weight loss at year 3 than those without diabetes at baseline. There were small but statistically significant differences in weight change for RYGB in those with abnormal kidney function and current or recent smoking. For LAGB participants, those with a large band had 75% greater odds of experiencing less than 10% weight loss after adjusting for BMI and sex. CONCLUSIONS: Few baseline variables were associated with three year weight change and the effects were small. These results indicate that baseline variables have limited predictive value for an individual’s chance of a successful weight loss outcome following bariatric surgery. TRIAL REGISTRATION: NCT00465829, ClinicalTrials.go

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

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

<i>hmgcs1</i> regulates facial development by a cholesterol independent mechanism.

<p>(A-C) Wildtype AB embryos were treated with 0.01% DMSO (vehicle), 8uM lonafarnib (Lona) or 3 uM Ro 48 8071 (Ro-48) beginning at 5 hours post fertilization and for a period of 4 days post fertilization (dpf). Alcian blue was used to analyze the cartilage structures of the developing zebrafish head and neck. (A-C) demonstrate the dissected viscerocranium of 4 day old larvae. (DMSO n = 30, Ro-48 n = 14, and Lona n = 8) (D-F) Whole mount <i>in situ</i> hybridization (ISH) was performed at 3 days post fertilization (protruding mouth stage) with embryos treated with 0.01% DMSO (vehicle), 8uM lonafarnib (Lona) or 2.5 uM Ro 48 8071 (Ro-48) (DMSO n = 20, Ro 48 n = 27, and Lona n = 30) (G) Schematic representation of viscerocranium. The extension of the Meckel’s cartilage (mandible) was measured as a detection for the presence of a facial cleft phenotype. Distance is indicated by the red line. (G’) Average was normalized and represented relative to the vehicle control group. Asterisk denotes statistical significance of p<0.001. Statistical analysis was performed using a T-test.</p