H3K9me3 maintenance on a human artificial chromosome is required for segregation but not centromere epigenetic memory.

Most eukaryotic centromeres are located within heterochromatic regions. Paradoxically, heterochromatin can also antagonize de novo centromere formation, and some centromeres lack it altogether. In order to investigate the importance of heterochromatin at centromeres, we used epigenetic engineering of a synthetic alphoidtetO human artificial chromosome (HAC), to which chimeric proteins can be targeted. By tethering the JMJD2D demethylase (also known as KDM4D), we removed heterochromatin mark H3K9me3 (histone 3 lysine 9 trimethylation) specifically from the HAC centromere. This caused no short-term defects, but long-term tethering reduced HAC centromere protein levels and triggered HAC mis-segregation. However, centromeric CENP-A was maintained at a reduced level. Furthermore, HAC centromere function was compatible with an alternative low-H3K9me3, high-H3K27me3 chromatin signature, as long as residual levels of H3K9me3 remained. When JMJD2D was released from the HAC, H3K9me3 levels recovered over several days back to initial levels along with CENP-A and CENP-C centromere levels, and mitotic segregation fidelity. Our results suggest that a minimal level of heterochromatin is required to stabilize mitotic centromere function but not for maintaining centromere epigenetic memory, and that a homeostatic pathway maintains heterochromatin at centromeres.This article has an associated First Person interview with the first authors of the paper.

Effects of laparoscopic myotomy on the esophageal motility pattern of esophageal achalasia as measured by high-resolution manometry.

BACKGROUND: Esophageal achalasia can be classified on the grounds of three distinct manometric patterns that correlate well with final outcome after laparoscopic Heller-Dor myotomy (LHM). No analytical data are available, however, on the postoperative picture and its possible correlation with final outcome. The aims of this study were: (a) to investigate whether manometric patterns change after LHM for achalasia; (b) to ascertain whether postoperative patterns and/or changes can predict final outcome; and (c) to test the hypothesis that the three known patterns represent different stages in the evolution of the disease. METHODS: During the study period, we prospectively enlisted 206 consecutive achalasia patients who were assessed using high-resolution manometry (HRM) before undergoing LHM. Symptoms were scored using a detailed questionnaire. Barium swallow, endoscopy and HRM were performed, before and again 6 months after surgery. RESULTS: Preoperative HRM revealed the three known patterns with statistically different esophageal diameters (pattern I having the largest), and patients with pattern I had the highest symptom scores. The surgical treatment failed in 10 cases (4.9%). The only predictor of final outcome was the preoperative manometric pattern (p = 0.01). All patients with pattern I preoperatively had the same pattern afterward, whereas nearly 50% of patients with pattern III before LHM had patterns I or II after surgery. There were no cases showing the opposite trend. CONCLUSIONS: Neither a change of manometric pattern after surgery nor a patient's postoperative pattern was a predictor of final outcome, whereas preoperative pattern confirmed its prognostic significance. The three manometric patterns distinguishable in achalasia may represent different stages in the disease's evolution, pattern III and pattern I coinciding with the early and final stages of the disease, respectively.

Preoperative dexmedetomidine and intraoperative bradycardia in laparoscopic cholecystectomy: a meta-analysis with trial sequential analysis.

BACKGROUND: While laparoscopic surgical procedures have various advantages over traditional open techniques, artificial pneumoperitoneum is associated with severe bradycardia and cardiac arrest. Dexmedetomidine, an imidazole derivative that selectively binds to α2-receptors and has sedative and analgesic properties, can cause hypotension and bradycardia. Our primary aim was to assess the association between dexmedetomidine use and intraoperative bradycardia during laparoscopic cholecystectomy. METHODS: We performed a systematic review with a meta-analysis and trial sequential analysis using the following PICOS: adult patients undergoing endotracheal intubation for laparoscopic cholecystectomy (P); intravenous dexmedetomidine before tracheal intubation (I); no intervention or placebo administration (C); intraoperative bradycardia (primary outcome), intraoperative hypotension, hemodynamics at intubation (systolic blood pressure, mean arterial pressure, heart rate), dose needed for induction of anesthesia, total anesthesia requirements (both hypnotics and opioids) throughout the procedure, and percentage of patients requiring postoperative analgesics and experiencing postoperative nausea and vomiting and/or shivering (O); randomized controlled trials (S). RESULTS: Fifteen studies were included in the meta-analysis (980 patients). Compared to patients that did not receive dexmedetomidine, those who did had a higher risk of developing intraoperative bradycardia (RR: 2.81, 95% CI [1.34, 5.91]) and hypotension (1.66 [0.92,2.98]); however, they required a lower dose of intraoperative anesthetics and had a lower incidence of postoperative nausea and vomiting. In the trial sequential analysis for bradycardia, the cumulative z-score crossed the monitoring boundary for harm at the tenth trial. CONCLUSIONS: Patients undergoing laparoscopic cholecystectomy who receive dexmedetomidine during tracheal intubation are more likely to develop intraoperative bradycardia and hypotension.

Deriving and validating a risk prediction model for long COVID-19: protocol for an observational cohort study using linked Scottish data.

INTRODUCTION: COVID-19 is commonly experienced as an acute illness, yet some people continue to have symptoms that persist for weeks, or months (commonly referred to as 'long-COVID'). It remains unclear which patients are at highest risk of developing long-COVID. In this protocol, we describe plans to develop a prediction model to identify individuals at risk of developing long-COVID. METHODS AND ANALYSIS: We will use the national Early Pandemic Evaluation and Enhanced Surveillance of COVID-19 (EAVE II) platform, a population-level linked dataset of routine electronic healthcare data from 5.4 million individuals in Scotland. We will identify potential indicators for long-COVID by identifying patterns in primary care data linked to information from out-of-hours general practitioner encounters, accident and emergency visits, hospital admissions, outpatient visits, medication prescribing/dispensing and mortality. We will investigate the potential indicators of long-COVID by performing a matched analysis between those with a positive reverse transcriptase PCR (RT-PCR) test for SARS-CoV-2 infection and two control groups: (1) individuals with at least one negative RT-PCR test and never tested positive; (2) the general population (everyone who did not test positive) of Scotland. Cluster analysis will then be used to determine the final definition of the outcome measure for long-COVID. We will then derive, internally and externally validate a prediction model to identify the epidemiological risk factors associated with long-COVID. ETHICS AND DISSEMINATION: The EAVE II study has obtained approvals from the Research Ethics Committee (reference: 12/SS/0201), and the Public Benefit and Privacy Panel for Health and Social Care (reference: 1920-0279). Study findings will be published in peer-reviewed journals and presented at conferences. Understanding the predictors for long-COVID and identifying the patient groups at greatest risk of persisting symptoms will inform future treatments and preventative strategies for long-COVID.

Effect of dexmedetomidine on hemodynamic responses to tracheal intubation: A meta-analysis with meta-regression and trial sequential analysis.

STUDY OBJECTIVE: An uncontrolled adrenergic response during tracheal intubation may lead to life-threatening complications. Dexmedetomidine binds to α2-receptors and may attenuate this response. The primary aim of our meta-analysis is to investigate dexmedetomidine efficacy in attenuating sympathetic response to tracheal intubation, compared with placebo or no dexmedetomidine, in terms of heart rate and blood pressure at intubation. DESIGN: Meta-analysis with meta-regression and trial sequential analysis. SETTING: Systematic search from inception until December 1, 2020 in the following databases: Pubmed, Scopus, the Cochrane Central Register of Controlled Trials, EMBASE and Google Scholar. INTERVENTIONS: All randomized controlled trials investigating intravenous dexmedetomidine as premedication in adult patients undergoing tracheal intubation were included in our study. Studies were included without any language or publication date restriction. A trial sequential analysis and a post-hoc meta-regression were performed on the main outcomes. MEASUREMENTS: Hemodynamic parameters and heart rate at tracheal intubation, dose of anesthetic needed for induction of anesthesia, total anesthetic requirement throughout the operative procedure, postoperative pain and percentage of patients requiring analgesics at 24 postoperative hours, postoperative nausea and vomiting, intraoperative and postoperative bradycardia, hypotension, dizziness, shivering and/or respiratory depression. MAIN RESULTS: Ninety-nine included studies randomized 6833 patients. During laryngoscopy, all hemodynamic parameters were significantly greater in the no dexmedetomidine group. In particular, in the dexmedetomidine group, systolic blood pressure differed by -21.8 mm Hg (95% CI -26.6 to -17.1, p-value < 0.001, I2 97%), mean arterial pressure by -12.8 mm Hg (95% CI -15.6 to -10.0, p-value < 0.001, I2 98%), and heart rate by -16.9 bpm (95% CI -19.8 to -13.9, p-value < 0.001, I2 98%). CONCLUSIONS: Patients receiving premedication with dexmedetomidine for tracheal intubation, compared with no dexmedetomidine, have a lower blood pressure and heart rate, however, the risk of bradycardia and hypotension is relevant and its use during daily practice should be cautiously evaluated for each patient.

SH3BGRL3 binds to myosin 1c in a calcium dependent manner and modulates migration in the MDA-MB-231 cell line.

BACKGROUND: The human SH3 domain Binding Glutamic acid Rich Like 3 (SH3BGRL3) gene is highly conserved in phylogeny and widely expressed in human tissues. However, its function is largely undetermined. The protein was found to be overexpressed in several tumors, and recent work suggested a possible relationship with EGFR family members. We aimed at further highlighting on these issues and investigated SH3BGRL3 molecular interactions and its role in cellular migration ability. RESULTS: We first engineered the ErbB2-overexpressing SKBR3 cells to express exogenous SH3BGRL3, as well as wild type Myo1c or different deletion mutants. Confocal microscopy analysis indicated that SH3BGRL3 co-localized with Myo1c and ErbB2 at plasma membranes. However, co-immunoprecipitation assays and mass spectrometry demonstrated that SH3BGRL3 did not directly bind ErbB2, but specifically recognized Myo1c, on its IQ-bearing neck region. Importantly, the interaction with Myo1c was Ca2+-dependent. A role for SH3BGRL3 in cell migration was also assessed, as RNA interference of SH3BGRL3 in MDA-MB-231 cells, used as a classical migration model, remarkably impaired the migration ability of these cells. On the other side, its over-expression increased cell motility. CONCLUSION: The results of this study provide insights for the formulation of novel hypotheses on the putative role of SH3BGRL3 protein in the regulation of myosin-cytoskeleton dialog and in cell migration. It could be envisaged the SH3BGRL3-Myo1c interaction as a regulation mechanism for cytoskeleton dynamics. It is well known that, at low Ca2+ concentrations, the IQ domains of Myo1c are bound by calmodulin. Here we found that binding of Myo1c to SH3BGRL3 requires instead the presence of Ca2+. Thus, it could be hypothesized that Myo1c conformation may be modulated by Ca2+-driven mechanisms that involve alternative binding by calmodulin or SH3BGRL3, for the regulation of cytoskeletal activity.

Are thromboelastometric and thromboelastographic parameters associated with mortality in septic patients? A systematic review and meta-analysis.

BACKGROUND: Thromboelastometry/elastography (ROTEM/TEG) showed promising results for diagnosis of sepsis-induced coagulopathy, but their association with the outcome is unclear. Our aim was to assess any difference in ROTEM/TEG measurements between septic survivors and non-survivors. METHODS: Pubmed, Web of Science, Embase and Cochrane Library databases were investigated. The research aimed to include any randomized or observational study: i) on septic adult patients admitted to Intensive Care Unit (ICU) or Emergency Department (ED); ii) including ROTEM/TEG; iii) assessing mortality. RESULTS: Seven prospective and four retrospective observational studies (952 patients) were included. According to the INTEM/kaolin-assay, clotting time (CT)/R (standardized mean difference(SMD) -0.29, 95% CI -0.49 to -0.09, p = 0.004) and clot formation time (CFT)/K (SMD -0.42, 95% CI -0.78 to -0.06, p = 0.02) were shorter in survivors. According to the EXTEM-assay, CT was shorter (MD -11.66 s, 95% CI -22.59 to -0.73, p = 0.04), while MCF was higher (MD 3.49 mm, 95% CI 0.43 to 6.55, p = 0.03) in survivors. A hypocoagulable profile was more frequent in non-survivors (OR 0.31, 95%CI 0.18 to 0.55, p < 0.0001). Overall, the risk of bias of the included studies was moderate and the quality of evidence low. CONCLUSIONS: Hypocoagulability and lower MCF in EXTEM may be associated with higher mortality in sepsis.

Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation.

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies.

It is generally accepted that chromatin containing the histone H3 variant CENP-A is an epigenetic mark maintaining centromere identity. However, the pathways leading to the formation and maintenance of centromere chromatin remain poorly characterized due to difficulties of analysis of centromeric repeats in native chromosomes. To address this problem, in our previous studies we generated a human artificial chromosome (HAC) whose centromere contains a synthetic alpha-satellite (alphoid) DNA array containing the tetracycline operator, the alphoidtetO-HAC. The presence of tetO sequences allows the specific targeting of the centromeric region in the HAC with different chromatin modifiers fused to the tetracycline repressor. The alphoidtetO-HAC has been extensively used to investigate protein interactions within the kinetochore and to define the epigenetic signature of centromeric chromatin to maintain a functional kinetochore. In this study, we developed a novel synthetic HAC containing two alphoid DNA arrays with different targeting sequences, tetO, lacO and gal4, the alphoidhybrid-HAC. This new HAC can be used for detailed epigenetic engineering studies because its kinetochore can be simultaneously or independently targeted by different chromatin modifiers and other fusion proteins.

Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies.

Since their description in the late 1990s, Human Artificial Chromosomes (HACs) bearing functional kinetochores have been considered as promising systems for gene delivery and expression. More recently a HAC assembled from a synthetic alphoid DNA array has been exploited in studies of centromeric chromatin and in assessing the impact of different epigenetic modifications on kinetochore structure and function in human cells. This HAC was termed the alphoidtetO-HAC, as the synthetic monomers each contained a tetO sequence in place of the CENP-B box that can be targeted specifically with tetR-fusion proteins. Studies in which the kinetochore chromatin of the alphoidtetO-HAC was specifically modified, revealed that heterochromatin is incompatible with centromere function and that centromeric transcription is important for centromere assembly and maintenance. In addition, the alphoidtetO-HAC was modified to carry large gene inserts that are expressed in target cells under conditions that recapitulate the physiological regulation of endogenous loci. Importantly, the phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. AlphoidtetO-HAC-based technology has also been used to develop new drug screening and assessment strategies to manipulate the CIN phenotype in cancer cells. In summary, the alphoidtetO-HAC is proving to be a versatile tool for studying human chromosome transactions and structure as well as for genome and cancer studies.

The natural history of achalasia: Evidence of a continuum-"The evolutive pattern theory".

BACKGROUND: It is currently unclear if the three manometric patterns of esophageal achalasia represent distinct entities or part of a disease continuum. The study's aims were: a) to test the hypothesis that the three patterns represent different stages in the evolution of achalasia; b) to investigate whether manometric patterns change after Laparoscopic-Heller-Dor (LHD). METHODS: We assessed the patients diagnosed with achalasia who underwent LHD as their first treatment from 1992 to 2016. Their symptoms were scored using a detailed questionnaire for dysphagia, food-regurgitation, and chest pain. Barium-swallow, endoscopy, and esophageal-manometry were performed before and 6 months after surgery. RESULTS: The study population consisted of 511 patients (M:F=283:228). Patients' demographic and clinical data showed that those with pattern III had a shorter history of symptoms, a higher incidence of chest pain, and a less dilated gullet (p<0.001). All patients with a sigmoid-shaped mega-esophagus had pattern I achalasia. One patient with a diagnosis of pattern III achalasia developed pattern II at a follow-up manometry before surgery. At a median follow-up of 30 months (IQR 12-56), the outcome of surgery was positive in 479 patients (91.7%). All patients with pattern I preoperatively had the same pattern after LHD, whereas more than 50% of patients with pattern III before treatment showed pattern I or II after surgery. CONCLUSIONS: This study supports the hypothesis/theory that the different manometric patterns represent different stages in the evolution of the disease-where pattern III is the earliest stage, pattern II an intermediate stage, and pattern I the final stage.

Postoperative Gastroesophageal Reflux After Laparoscopic Heller-Dor for Achalasia: True Incidence with an Objective Evaluation.

INTRODUCTION: The most common complication after laparoscopic Heller-Dor (LHD) is gastroesophageal reflux disease (GERD). The present study aimed (a) to analyze the true incidence of postoperative reflux by objectively assessing a large group of LHD patients and (b) to see whether the presence of typical GERD symptoms correlates with the real incidence of postoperative reflux. METHODS: After LHD, patients were assessed by means of a symptom score, endoscopy, esophageal manometry, and 24-h pH monitoring. Patients were assigned to three groups: those did not accept to perform 24-h pH monitoring (group NP); those with normal postoperative pH findings (group A); and those with pathological postoperative acid exposure (group B). RESULTS: Four hundred sixty-three of the 806 LHD patients agreed to undergo follow-up 24-h pH monitoring. Normal pH findings were seen in 423 patients (group A, 91.4 %), while 40 (8.6 %) had a pathological acid exposure (group B). The median symptom scores were similar: 3.0 (IQR 0-8) in group A and 6.0 (IQR 0-10) in group B (p = 0.29). At endoscopy, the percentage of esophagitis was also similar (11 % in group A, 19 % in group B; p = 0.28). CONCLUSIONS: This study demonstrated that, after LHD was performed by experienced surgeons, the true incidence of postoperative GERD is very low. The incidence of this possible complication should be assessed by pH monitoring because endoscopic findings and symptoms may be misleading.

The Impact of Heller Myotomy on Integrated Relaxation Pressure in Esophageal Achalasia.

BACKGROUND: A new high-resolution manometry (HRM) parameter, the integrated relaxation pressure (IRP), has been proposed for the assessment of esophageal-gastric junction (EGJ) relaxation. Our aim was to assess the effect of Heller myotomy on IRP in achalasia patients. METHODS: We prospectively collected data on achalasia patients who underwent HRM between 2009-2014. Barium swallow was used to assess esophageal diameter and shape. Manometric diagnoses were performed by using the Chicago Classification v3. All patients with a confirmed diagnosis of achalasia were treated surgically with Heller Myotomy RESULTS: One hundred thirty-nine consecutive achalasia patients (M:F = 72:67) represented the study population. All the patients had 100% simultaneous waves but 11 had an IRP < 15 mmHg. At median follow-up of 28 months, the median of IRP was significantly lower after surgery (27.4 [IQR 20.4-35] vs 7.1 [IQR 4.4-9.8]; p < 0.001), and so were the lower esophageal sphincter (LES) resting pressure (27 [IQR 18-33] vs 6 [IQR 3-11]; p < 0.001). At univariate analysis, IRP correlated with the gender, LES resting residual pressure, and dysphagia score. CONCLUSIONS: This is the first study to have examined the role of IRP in achalasia, and how it changes after surgical treatment. An increased preoperative IRP correlated directly with a more severe dysphagia. The IRP was restored to normal by Heller myotomy.

Mucosal Perforation During Laparoscopic Heller Myotomy Has No Influence on Final Treatment Outcome.

BACKGROUND: The aims of the study were (a) to examine the final outcome in patients experiencing accidental mucosal perforation during laparoscopic Heller myotomy with Dor fundoplication (LHD) and (b) to evaluate whether perforation episodes might influence the way in which surgeons subsequently approached the LHD procedure. METHODS: We studied all consecutive patients that underwent LHD between 1992 and 2015. Patients were divided into two main groups: those who experienced an intraoperative mucosal perforation (group P) and those whose LHD was uneventful (group NP). Two additional groups were compared: group A, which consisted of patients operated by a given surgeon immediately before a perforation episode occurred, and group B, which included those operated immediately afterwards. RESULTS: Eight hundred seventy-five patients underwent LHD; a mucosal perforation was detected in 25 patients (2.9 %), which was found unrelated to patients' symptom's score and age, radiological stage, manometric pattern, or the surgeon's experience. The median postoperative symptom score was similar for the two groups as the failure rate: 92 failures in group NP (10.8 %) and 4 in group P (16 %) (p = 0.34); moreover, symptoms recurred in 2 patients of group A (10 %) and 3 patients of group B (15 %) (p = 0.9). CONCLUSIONS: Accidental perforation during LHD is infrequent and impossible to predict on the grounds of preoperative therapy or the surgeon's personal experience. Despite a longer surgical procedure and hospital stay, the outcome of LHD is much the same as for patients undergoing uneventful myotomy. A recent mucosal perforation does not influence the surgeon's subsequent performance.

Metformin inhibits cell cycle progression of B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (CLL) was believed to result from clonal accumulation of resting apoptosis-resistant malignant B lymphocytes. However, it became increasingly clear that CLL cells undergo, during their life, iterative cycles of re-activation and subsequent clonal expansion. Drugs interfering with CLL cell cycle entry would be greatly beneficial in the treatment of this disease. 1, 1-Dimethylbiguanide hydrochloride (metformin), the most widely prescribed oral hypoglycemic agent, inexpensive and well tolerated, has recently received increased attention for its potential antitumor activity. We wondered whether metformin has apoptotic and anti-proliferative activity on leukemic cells derived from CLL patients. Metformin was administered in vitro either to quiescent cells or during CLL cell activation stimuli, provided by classical co-culturing with CD40L-expressing fibroblasts. At doses that were totally ineffective on normal lymphocytes, metformin induced apoptosis of quiescent CLL cells and inhibition of cell cycle entry when CLL were stimulated by CD40-CD40L ligation. This cytostatic effect was accompanied by decreased expression of survival- and proliferation-associated proteins, inhibition of signaling pathways involved in CLL disease progression and decreased intracellular glucose available for glycolysis. In drug combination experiments, metformin lowered the apoptotic threshold and potentiated the cytotoxic effects of classical and novel antitumor molecules. Our results indicate that, while CLL cells after stimulation are in the process of building their full survival and cycling armamentarium, the presence of metformin affects this process.