The APE2 nuclease is essential for DNA double-strand break repair by microhomology-mediated end joining.

Microhomology-mediated end joining (MMEJ) is an intrinsically mutagenic pathway of DNA double-strand break (DSB) repair essential for proliferation of homologous recombination (HR)-deficient tumors. Although targeting MMEJ has emerged as a powerful strategy to eliminate HR-deficient (HRD) cancers, this is limited by an incomplete understanding of the mechanism and factors required for MMEJ repair. Here, we identify the APE2 nuclease as an MMEJ effector. We show that loss of APE2 inhibits MMEJ at deprotected telomeres and at intra-chromosomal DSBs and is epistatic with Pol Theta for MMEJ activity. Mechanistically, we demonstrate that APE2 possesses intrinsic flap-cleaving activity, that its MMEJ function in cells depends on its nuclease activity, and further identify an uncharacterized domain required for its recruitment to DSBs. We conclude that this previously unappreciated role of APE2 in MMEJ contributes to the addiction of HRD cells to APE2, which could be exploited in the treatment of cancer.

Obesity, metabolic syndrome, and inflammation: An update for anaesthetists caring for patients with obesity.

Our understanding of chronic inflammation in obesity is evolving. Suggested mechanisms include hypoxia of adipose tissue and a subsequent increase in circulating cytokines. It is now known that adipose tissue, far from being an inert tissue, produces and secretes multiple peptides that influence inflammation and metabolism, including substrates of the renin-angiotensin-aldosterone system (RAAS). RAAS blocking antihypertensive medication and cholesterol-lowering agents are now being evaluated for their metabolic and inflammation-modulating effects. Surgery also has pro-inflammatory effects, which may be exacerbated in patients with obesity. This narrative review will summarise the recent literature surrounding obesity, metabolic syndrome, inflammation, and interplay with the RAAS, with evidence-based recommendations for the optimisation of patients with obesity, prior to surgery and anaesthesia.

Revisional bariatric surgery to single-anastomosis gastric bypass: a large multi-institutional series.

BACKGROUND: Reoperation is often required after bariatric procedures. Single-anastomosis gastric bypass (SAGB) is increasingly utilized as a primary bariatric procedure. Few series document SAGB as a revisional bariatric procedure. OBJECTIVES: To describe our short-term experience with revisional SAGB, focusing on weight loss and reflux symptom outcomes. SETTING: Three hospitals in Australia with both private and public (government funded) patients. METHODS: We reviewed all revisional SAGB cases from 2012 to 2019 at. Complications were considered significant if they were Clavien-Dindo grade 3a or higher. A phone survey was conducted to assess weight loss outcomes, patient satisfaction, reflux symptoms, and other complications. RESULTS: We identified 254 patients who had a revisional bariatric procedure to SAGB (21 previous sleeve gastrectomies and 233 previous adjustable bands), with a mean follow-up of 22 ± 15.6 months (range, 1-55 mo). The mean percentage of excess weight loss was 77% (183 patients, 72%), and the number of patients with follow-ups at 1 and 4 years was 184 (73%) and 35 patients (14%). Within 30 days, there were 29 patients (11%) who required reinterventions (21 endoscopies, 1 interventional radiology procedure, and 7 reoperations) with no deaths. Beyond 30 days, 27 patients (11%) required rerevision to Roux-en-Y gastric bypass for reflux symptoms and 10 (4%) required a laparotomy or laparoscopy for another reason (e.g., bowel obstruction). At a median follow-up of 36.6 months, 87 patients (34%) completed a phone survey, 45 (52%) of whom were taking proton pump inhibitors and 66 patients (76%) of whom were satisfied with their experience. CONCLUSION: In our series, revision to SAGB was safe, with low short-term morbidity and favorable weight loss outcomes. However, beyond 1 year, a large proportion of patients experienced severe reflux symptoms and required rerevision.

ClearSight™ finger cuff versus invasive arterial pressure measurement in patients with body mass index above 45 kg/m2.

BACKGROUND: Measuring blood pressure in patients with obesity is challenging. The ClearSight™ finger cuff (FC) uses the vascular unloading technique to provide continuous non-invasive blood pressure measurements. We aimed to test the agreement of the FC with invasive radial arterial monitoring (INV) in patients with obesity. METHODS: Participants had a body mass index (BMI) ≥45 kg/m2 and underwent laparoscopic bariatric surgery. FC and INV measurements were obtained simultaneously every 5 min on each patient, following induction of anesthesia. Agreement over time was assessed using modified Bland-Altman plots and error grid analysis permitted clinical interpretation of the results. Four-quadrant plots allowed assessment of concordance in blood pressure changes. RESULTS: The 30 participants had a median (IQR) BMI of 50.2 kg/m2 (IQR 48.3-55.3). The observed bias (SD, 95% limits of agreement) for systolic blood pressure (SBP) was 14.3 mmHg (14.1, -13.4 - 42.0), 5.2 mmHg (10.9, -16.0 - 26.5) for mean arterial pressure (MAP) and 2.6 mmHg (10.8, -18.6 - 23.8) for diastolic blood pressure (DBP). Error grid analysis showed that the proportion of readings in risk zones A-E were 90.8, 6.5, 2.7, 0 and 0% for SBP and 91.4, 4.3, 4.3, 0 and 0% for MAP, respectively. Discordance occurred in ≤8% of pairs for consecutive change in SBP, MAP and DBP. CONCLUSIONS: The vascular unloading technique was not adequately in agreement with radial arterial monitoring. Evaluation in a larger sample is required before recommending this technique for intraoperative monitoring of patients with BMI ≥45 kg/m2.

Improving Access to Bariatric Surgery for Rural and Remote Patients: Experiences from a State-Wide Bariatric Telehealth Service in Australia.

BACKGROUND: The state of Queensland, Australia, is large (1.85 million km2). The provision of bariatric care across the state is difficult as most major hospitals are concentrated in the capital city of Brisbane. We implemented a state-wide telehealth service to improve access for rural patients in a public bariatric service. We report our early experiences with this service. METHODS: We reviewed all patients seen in the Bariatric Telehealth Clinic from 2017 to 2019. Patients underwent consultation through video-link software at their local hospital with the multidisciplinary team in Brisbane (surgeon, dietician and clinical nurse). Distances from Brisbane and number of visits were calculated. Telehealth patients were contacted by phone to complete a survey regarding their experiences. This was a 17-question Likert-style survey with scores from 1 (strongly disagree) to 5 (strongly agree). RESULTS: A total of 85 new patients underwent their initial consultation via telehealth. Each patient had a variable number of in-person as well as telehealth consultations both before and after surgery. Mean distance from telehealth consultation site to Brisbane was 614 km (range 149-2472 km). In total, 41 (48%) completed the survey. With regard to telehealth saving time and money, improving access to bariatric care, and desire to use telehealth again, the mean score was 4 out of 5 or higher for all questions (i.e., agree or strongly agree). There was no identifiable post-operative complication that was caused or exacerbated by telehealth. CONCLUSION: Bariatric surgical telehealth appointments are feasible and preferred by most patients residing in rural and remote locations. Each consultation avoids significant travel time and cost for the patient and health service, with no obvious adverse outcomes. Telehealth improves equity and access to specialist services for rural and remote patients.

Perioperative Blood Pressure Monitoring in Patients With Obesity.

Blood pressure monitoring plays a key part in the preoperative, intraoperative, and postoperative care of all patients. In patients with obesity, hypertension indicates increased metabolic and surgical risk and may signal the presence of significant medical comorbidities, including obstructive sleep apnea. Avoidance of hypertension postoperatively is necessary to minimize bleeding risk after surgery. Hypotension in the postoperative period may be the first sign of significant complications that require urgent management. With the problem of being overweight or obese now affecting two-thirds of adults in Western countries, the proportion of patients with obesity in perioperative environments is similarly increasing. Detection of aberrations in blood pressure is contingent on the accuracy of blood pressure measurement methods. Patients with obesity tend to have a large arm circumference and "cone-shaped arms." Standard blood pressure cuffs fit such patients poorly, and this compromises the accuracy of measurements. Alternatives to arm blood pressure cuffs, some made specifically for individuals with obesity, have been evaluated but are not widely available to clinicians. This focused narrative review will discuss the relevance of hypertension management in the care of patients with obesity, highlight the currently available methods for perioperative monitoring of blood pressure, and explore the opportunities that exist to improve the perioperative blood pressure care in patients with obesity undergoing surgical procedures.

Live Cell Imaging Reveals pH Oscillations in Saccharomyces cerevisiae During Metabolic Transitions.

Addition of glucose to starved Saccharomyces cerevisiae initiates collective NADH dynamics termed glycolytic oscillations. Numerous questions remain about the extent to which single cells can oscillate, if oscillations occur in natural conditions, and potential physiological consequences of oscillations. In this paper, we report sustained glycolytic oscillations in single cells without the need for cyanide. Glucose addition to immobilized cells induced pH oscillations that could be imaged with fluorescent sensors. A population of cells had oscillations that were heterogeneous in frequency, start time, stop time, duration and amplitude. These changes in cytoplasmic pH were necessary and sufficient to drive changes in NADH. Oscillators had lower mitochondrial membrane potentials and budded more slowly than non-oscillators. We also uncovered a new type of oscillation during recovery from H2O2 challenge. Our data show that pH in S. cerevisiae changes over several time scales, and that imaging pH offers a new way to measure glycolytic oscillations on individual cells.

Voltage-gated calcium flux mediates Escherichia coli mechanosensation.

Electrically excitable cells harness voltage-coupled calcium influx to transmit intracellular signals, typically studied in neurons and cardiomyocytes. Despite intense study in higher organisms, investigations of voltage and calcium signaling in bacteria have lagged due to their small size and a lack of sensitive tools. Only recently were bacteria shown to modulate their membrane potential on the timescale of seconds, and little is known about the downstream effects from this modulation. In this paper, we report on the effects of electrophysiology in individual bacteria. A genetically encoded calcium sensor expressed in Escherichia coli revealed calcium transients in single cells. A fusion sensor that simultaneously reports voltage and calcium indicated that calcium influx is induced by voltage depolarizations, similar to metazoan action potentials. Cytoplasmic calcium levels and transients increased upon mechanical stimulation with a hydrogel, and single cells altered protein concentrations dependent on the mechanical environment. Blocking voltage and calcium flux altered mechanically induced changes in protein concentration, while inducing calcium flux reproduced these changes. Thus, voltage and calcium relay a bacterial sense of touch and alter cellular lifestyle. Although the calcium effectors remain unknown, these data open a host of new questions about E. coli, including the identity of the underlying molecular players, as well as other signals conveyed by voltage and calcium. These data also provide evidence that dynamic voltage and calcium exists as a signaling modality in the oldest domain of life, and therefore studying electrophysiology beyond canonical electrically excitable cells could yield exciting new findings.

Flavivirus sfRNA suppresses antiviral RNA interference in cultured cells and mosquitoes and directly interacts with the RNAi machinery.

Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3' UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes.

XRN1 stalling in the 5' UTR of Hepatitis C virus and Bovine Viral Diarrhea virus is associated with dysregulated host mRNA stability.

We demonstrate that both Hepatitis C virus (HCV) and Bovine Viral Diarrhea virus (BVDV) contain regions in their 5' UTRs that stall and repress the enzymatic activity of the cellular 5'-3' exoribonuclease XRN1, resulting in dramatic changes in the stability of cellular mRNAs. We used biochemical assays, virus infections, and transfection of the HCV and BVDV 5' untranslated regions in the absence of other viral gene products to directly demonstrate the existence and mechanism of this novel host-virus interaction. In the context of HCV infection, we observed globally increased stability of mRNAs resulting in significant increases in abundance of normally short-lived mRNAs encoding a variety of relevant oncogenes and angiogenesis factors. These findings suggest that non-coding regions from multiple genera of the Flaviviridae interfere with XRN1 and impact post-transcriptional processes, causing global dysregulation of cellular gene expression which may promote cell growth and pathogenesis.

Doxycycline inhibition of abdominal aortic aneurysm growth: a systematic review of the literature.

Abdominal aortic aneurysm (AAA) is a common disease and a major cause of death through rupture, the risk of which increases with aneurysm size. There is approximately a 5 year interval from when aneurysmal dilatation develops until it reaches a size where surgery is indicated. Slowing, or arresting, aneurysm growth during this period would be beneficial. Aneurysmal aortic wall degeneration is a multifactorial, chronic inflammatory process resulting via activation of matrix metalloproteinases (MMPs), in destruction of mural connective tissue. Doxycycline, a tetracycline antibiotic, is a known inhibitor of MMPs. Animal studies of doxcycline for AAA provide significant evidence of a beneficial effect. However, the human studies, comprising 6 controlled trials and 2 cohort studies, provide conflicting evidence. They are generally of poor methodological quality with small numbers (just 255 subjects analyzed), lack of adjustment for confounding variables, short term doxycycline exposure and a lack of long term follow up. Standardization of dose (per unit weight) and confirmation of compliance remain other systemic failings. The safety of long-term doxycycline use is yet to be proved. The evidence for any beneficial effect of doxycycline as a treatment for AAA, therefore, remains weak. Further studies are required and will ideally be multicentre, involve large subject numbers and be of high quality randomization and blinding with longer periods of doxycycline exposure, confirmation of compliance, standardization of confounding variables and prolonged follow up.

Persistence of host response against glochidia larvae in Micropterus salmoides.

Host fish acquire resistance to the parasitic larvae (glochidia) of freshwater mussels (Unionidae). Glochidia metamorphose into juvenile mussels while encysted on host fish. We investigated the duration of acquired resistance of largemouth bass, Micropterus salmoides (Lacepède, 1802) to glochidia of the broken rays mussel, Lampsilis reeveiana (Call, 1887). Fish received three successive priming infections with glochidia to induce an immune response. Primed fish were held at 22-23 degrees C and were challenged (re-infected) at intervals after priming. Metamorphosis success was quantified as the percent of attached glochidia that metamorphosed to the juvenile stage and were recovered alive. Metamorphosis success at 3, 7, and 12 months after priming was significantly lower on primed fish (26%, 40%, and 68% respectively) than on control fish (85%, 93%, and 92% respectively). A second group of largemouth bass was similarly primed and blood was extracted. Immunoblotting was used to detect host serum antibodies to L. reeveiana glochidia proteins. Serum antibodies were evident in primed fish, but not in naive control fish. Acquired resistance of host fish potentially affects natural reproduction and artificial propagation of unionids, many of which are of conservation concern.

Cross-resistance of largemouth bass to glochidia of unionid mussels.

We tested whether host fish that acquired resistance to glochidia of one mussel species were cross-resistant to glochidia of other species. Largemouth bass (Micropterus salmoides) were primed with 4-5 successive infections of glochidia of Lampsilis reeveiana. The percentage of attached glochidia that survived and transformed to the juvenile stage (transformation success) was compared between primed fish and naïve controls. Transformation success of L. reeveiana, Lampsilis abrupta, Villosa iris, and Utterbackia imbecillis was significantly lower on primed fish (37.8%, 43.5%, 67.0%, and 13.2%, respectively) than on control fish (89.0%, 89.7%, 90.0%, and 22.2% respectively). Immunoblotting was used to analyze the binding of serum antibodies from primed fish with glochidia proteins. Antibodies bound to glochidia proteins of similar molecular weight from L. reeveiana and L. abrupta. Bound proteins of V. iris differed in molecular weight from those of the Lampsilis species. There was no binding to specific glochidia proteins of U. imbecillis or Strophitus undulatus. Our results indicate that host-acquired resistance can extend across mussel genera and subfamilies and might involve both specific and nonspecific mechanisms. Understanding the specificity of acquired resistance of hosts to glochidia could enhance understanding of the evolutionary and ecological relationships between mussels and their host fishes.