Rapid changes in genome organisation during exit from pluripotency and the role of the nuclear envelope in maintaining the pluripotent state

The majority of Nuclear Envelope Transmembrane Proteins (NETs) are tissue specific and many of these facilitate tissue-specific genome organization. Genome organization changes dramatically during differentiation and these NETs impact this process: muscle-specific genome-organizing NETs NET39, WFS1 and TMEM38A are important for myogenesis (Robson et al, 2016) while fat-specific genome-organizing NETs TMEM120A and B are important for adipogenesis (Batrakou et al, 2015). Although during lineage specification of mouse embryonic stem cells (Peric-Hupkes et al, 2010), we do not yet understand the temporal dynamics of these changes nor the components of the nuclear envelope that orchestrate these changes during early stages of exit from pluripotency. In this thesis, I investigate the temporal dynamics of genome organization changes during pluripotency exit stimulated by LIF withdrawal. Using Fluorescence in-situ Hybridization (FISH) to label DNA, I demonstrate that some of the earliest changes in genome organization occur within the first hour of exit from pluripotency with the relocation of a locus containing three genes Triml1, Triml2 and Zfp42 (that encodes REX1, a well-known marker of pluripotency) from the nuclear interior to the nuclear periphery. The RNA and protein levels of these genes persist for several hours post exit, suggesting that reorganisation of the genome is among the very first of events occurring during lineage specification and is perhaps a higher order mechanism controlling differentiation as a change in genome organisation could affect the transcriptional profile of these cells. To try and identify the proteins involved in tethering the locus and the mechanism of release I also investigated the changes in the nuclear envelope composition as cells undergo an exit from pluripotency. I show that while certain proteins undergo post translational modifications such as phosphorylation, other new proteins are synthesised during the first two hours of exit. Using phospho-null mutants for LBR and LAP2α, I show that these play a role in the relocation of this genomic locus. Finally, I introduced tissue-specific genome-organizing NETs such as NET39 (muscle), TAPBPL (blood) and TMEM120A(fat) into embryonic stem cells and found that their introduction causes a forced exit from pluripotency. Interestingly, these NETs show specificity in their ability to affect the position of genomic loci encoding pluripotency factors like Rex1 and Nanog, strengthening the idea that these tissue specific NETs act as tethers to very specific genomic regions in order to maintain a tissue specific genome organization. The results discussed here present for the first time, a temporal view of the changes in genome organisation during such early stages of in vitro differentiation. While Rex1 repositioning has been studied in greater detail in this thesis, a more comprehensive study over the early stages of exit might reveal additional genomic loci that reposition during this phase. The rapid reorganisation of the genome following LIF withdrawal highlights the importance of tightly controlling and maintaining appropriate culture conditions for the study of pluripotency using embryonic stem cells as a model system. The study leads to conceptual advancement in stem cell biology by describing early events following exit from pluripotency and in the field of nuclear biology by identifying the NE composition in ES cells. Collectively the results demonstrate the role of the nuclear envelope in the maintenance of pluripotency and in orchestrating genome organisation changes during exit

Constrained release of lamina-associated enhancers and genes from the nuclear envelope during T-cell activation facilitates their association in chromosome compartments

The 3D organization of the genome changes concomitantly with expression changes during hematopoiesis and immune activation. Studies have focused either on lamina-associated domains (LADs) or on topologically associated domains (TADs), defined by preferential local chromatin interactions, and chromosome compartments, defined as higher-order interactions between TADs sharing functionally similar states. However, few studies have investigated how these affect one another. To address this, we mapped LADs using Lamin B1-DamID during Jurkat T-cell activation, finding significant genome reorganization at the nuclear periphery dominated by release of loci frequently important for T-cell function. To assess how these changes at the nuclear periphery influence wider genome organization, our DamID data sets were contrasted with TADs and compartments. Features of specific repositioning events were then tested by fluorescence in situ hybridization during T-cell activation. First, considerable overlap between TADs and LADs was observed with the TAD repositioning as a unit. Second, A1 and A2 subcompartments are segregated in 3D space through differences in proximity to LADs along chromosomes. Third, genes and a putative enhancer in LADs that were released from the periphery during T-cell activation became preferentially associated with A2 subcompartments and were constrained to the relative proximity of the lamina. Thus, lamina associations influence internal nuclear organization, and changes in LADs during T-cell activation may provide an important additional mode of gene regulation

Genomic loci mispositioning in Tmem120a knockout mice yields latent lipodystrophy

Little is known about how the observed fat-specific pattern of 3D-spatial genome organisation is established. Here we report that adipocyte-specific knockout of the gene encoding nuclear envelope transmembrane protein Tmem120a disrupts fat genome organisation, thus causing a lipodystrophy syndrome. Tmem120a deficiency broadly suppresses lipid metabolism pathway gene expression and induces myogenic gene expression by repositioning genes, enhancers and miRNA-encoding loci between the nuclear periphery and interior. Tmem120a(−/−) mice, particularly females, exhibit a lipodystrophy syndrome similar to human familial partial lipodystrophy FPLD2, with profound insulin resistance and metabolic defects that manifest upon exposure to an obesogenic diet. Interestingly, similar genome organisation defects occurred in cells from FPLD2 patients that harbour nuclear envelope protein encoding LMNA mutations. Our data indicate TMEM120A genome organisation functions affect many adipose functions and its loss may yield adiposity spectrum disorders, including a miRNA-based mechanism that could explain muscle hypertrophy in human lipodystrophy

Face mask usage during Covid-19 pandemic: prevalence and factors associated in Malaysian Medical School

Coronavirus disease 2019 (COVID-19) is a contagious disease caused by a newly discovered coronavirus strain. Anxiety, anticipation, and lack of knowledge in the previous experience on severe acute respiratory syndrome (SARS) and influenza A virus subtype H1N1 (H1N1) pandemic have a substantial impact on significant public health behaviours including wearing face mask in public spaces. We investigate the prevalence of face mask usage and its associated factors during COVID19 amongst students and staff of the Faculty of Medicine and Health Sciences (FMHS), Universiti Putra Malaysia (UPM). Using a cross-sectional study, 220 respondents participated in an electronic version of a self-administered questionnaire. Analyses were performed using the Chi-square test, IBM SPSS Statistics 25, with statistical significance at p<0.05. Out of the 220 respondents, 197 (89.5%) reported that they wore face masks all the time. Most of the respondents (90.3%) had a high level of precautionary measures against COVID-19, 89.6% selected the internet as their source of information and 90.9% had a good attitude level towards measures against COVID-19. There were significant associations between hygienic practices (p=0.001), source of information (internet) (p=0.025), and attitude towards COVID-19 (p=0.001) with face mask usage. This study demonstrated a high prevalence (89.5%) of face mask usage during the COVID-19 outbreak

Chemical interrogation of nuclear size identifies compounds with cancer cell line specific effects on migration and invasion

[Image: see text] Background: Lower survival rates for many cancer types correlate with changes in nuclear size/scaling in a tumor-type/tissue-specific manner. Hypothesizing that such changes might confer an advantage to tumor cells, we aimed at the identification of commercially available compounds to guide further mechanistic studies. We therefore screened for Food and Drug Administration (FDA)/European Medicines Agency (EMA)-approved compounds that reverse the direction of characteristic tumor nuclear size changes in PC3, HCT116, and H1299 cell lines reflecting, respectively, prostate adenocarcinoma, colonic adenocarcinoma, and small-cell squamous lung cancer. Results: We found distinct, largely nonoverlapping sets of compounds that rectify nuclear size changes for each tumor cell line. Several classes of compounds including, e.g., serotonin uptake inhibitors, cyclo-oxygenase inhibitors, β-adrenergic receptor agonists, and Na(+)/K(+) ATPase inhibitors, displayed coherent nuclear size phenotypes focused on a particular cell line or across cell lines and treatment conditions. Several compounds from classes far afield from current chemotherapy regimens were also identified. Seven nuclear size-rectifying compounds selected for further investigation all inhibited cell migration and/or invasion. Conclusions: Our study provides (a) proof of concept that nuclear size might be a valuable target to reduce cell migration/invasion in cancer treatment and (b) the most thorough collection of tool compounds to date reversing nuclear size changes specific to individual cancer-type cell lines. Although these compounds still need to be tested in primary cancer cells, the cell line-specific nuclear size and migration/invasion responses to particular drug classes suggest that cancer type-specific nuclear size rectifiers may help reduce metastatic spread

Prognostic model to predict postoperative acute kidney injury in patients undergoing major gastrointestinal surgery based on a national prospective observational cohort study.

Background: Acute illness, existing co-morbidities and surgical stress response can all contribute to postoperative acute kidney injury (AKI) in patients undergoing major gastrointestinal surgery. The aim of this study was prospectively to develop a pragmatic prognostic model to stratify patients according to risk of developing AKI after major gastrointestinal surgery. Methods: This prospective multicentre cohort study included consecutive adults undergoing elective or emergency gastrointestinal resection, liver resection or stoma reversal in 2-week blocks over a continuous 3-month period. The primary outcome was the rate of AKI within 7 days of surgery. Bootstrap stability was used to select clinically plausible risk factors into the model. Internal model validation was carried out by bootstrap validation. Results: A total of 4544 patients were included across 173 centres in the UK and Ireland. The overall rate of AKI was 14·2 per cent (646 of 4544) and the 30-day mortality rate was 1·8 per cent (84 of 4544). Stage 1 AKI was significantly associated with 30-day mortality (unadjusted odds ratio 7·61, 95 per cent c.i. 4·49 to 12·90; P < 0·001), with increasing odds of death with each AKI stage. Six variables were selected for inclusion in the prognostic model: age, sex, ASA grade, preoperative estimated glomerular filtration rate, planned open surgery and preoperative use of either an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Internal validation demonstrated good model discrimination (c-statistic 0·65). Discussion: Following major gastrointestinal surgery, AKI occurred in one in seven patients. This preoperative prognostic model identified patients at high risk of postoperative AKI. Validation in an independent data set is required to ensure generalizability

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

PANC Study (Pancreatitis: A National Cohort Study): national cohort study examining the first 30 days from presentation of acute pancreatitis in the UK

Abstract Background Acute pancreatitis is a common, yet complex, emergency surgical presentation. Multiple guidelines exist and management can vary significantly. The aim of this first UK, multicentre, prospective cohort study was to assess the variation in management of acute pancreatitis to guide resource planning and optimize treatment. Methods All patients aged greater than or equal to 18 years presenting with acute pancreatitis, as per the Atlanta criteria, from March to April 2021 were eligible for inclusion and followed up for 30 days. Anonymized data were uploaded to a secure electronic database in line with local governance approvals. Results A total of 113 hospitals contributed data on 2580 patients, with an equal sex distribution and a mean age of 57 years. The aetiology was gallstones in 50.6 per cent, with idiopathic the next most common (22.4 per cent). In addition to the 7.6 per cent with a diagnosis of chronic pancreatitis, 20.1 per cent of patients had a previous episode of acute pancreatitis. One in 20 patients were classed as having severe pancreatitis, as per the Atlanta criteria. The overall mortality rate was 2.3 per cent at 30 days, but rose to one in three in the severe group. Predictors of death included male sex, increased age, and frailty; previous acute pancreatitis and gallstones as aetiologies were protective. Smoking status and body mass index did not affect death. Conclusion Most patients presenting with acute pancreatitis have a mild, self-limiting disease. Rates of patients with idiopathic pancreatitis are high. Recurrent attacks of pancreatitis are common, but are likely to have reduced risk of death on subsequent admissions. </jats:sec