Serial Lift-Out: sampling the molecular anatomy of whole organisms.

Cryo-focused ion beam milling of frozen-hydrated cells and subsequent cryo-electron tomography (cryo-ET) has enabled the structural elucidation of macromolecular complexes directly inside cells. Application of the technique to multicellular organisms and tissues, however, is still limited by sample preparation. While high-pressure freezing enables the vitrification of thicker samples, it prolongs subsequent preparation due to increased thinning times and the need for extraction procedures. Additionally, thinning removes large portions of the specimen, restricting the imageable volume to the thickness of the final lamella, typically <300 nm. Here we introduce Serial Lift-Out, an enhanced lift-out technique that increases throughput and obtainable contextual information by preparing multiple sections from single transfers. We apply Serial Lift-Out to Caenorhabditis elegans L1 larvae, yielding a cryo-ET dataset sampling the worm's anterior-posterior axis, and resolve its ribosome structure to 7 Å and a subregion of the 11-protofilament microtubule to 13 Å, illustrating how Serial Lift-Out enables the study of multicellular molecular anatomy.

Enhanced Mitochondria-SR Tethering Triggers Adaptive Cardiac Muscle Remodeling.

BACKGROUND: Cardiac contractile function requires high energy from mitochondria, and Ca2+ from the sarcoplasmic reticulum (SR). Via local Ca2+ transfer at close mitochondria-SR contacts, cardiac excitation feedforward regulates mitochondrial ATP production to match surges in demand (excitation-bioenergetics coupling). However, pathological stresses may cause mitochondrial Ca2+ overload, excessive reactive oxygen species production and permeability transition, risking homeostatic collapse and myocyte loss. Excitation-bioenergetics coupling involves mitochondria-SR tethers but the role of tethering in cardiac physiology/pathology is debated. Endogenous tether proteins are multifunctional; therefore, nonselective targets to scrutinize interorganelle linkage. Here, we assessed the physiological/pathological relevance of selective chronic enhancement of cardiac mitochondria-SR tethering. METHODS: We introduced to mice a cardiac muscle-specific engineered tether (linker) transgene with a fluorescent protein core and deployed 2D/3D electron microscopy, biochemical approaches, fluorescence imaging, in vivo and ex vivo cardiac performance monitoring and stress challenges to characterize the linker phenotype. RESULTS: Expressed in the mature cardiomyocytes, the linker expanded and tightened individual mitochondria-junctional SR contacts; but also evoked a marked remodeling with large dense mitochondrial clusters that excluded dyads. Yet, excitation-bioenergetics coupling remained well-preserved, likely due to more longitudinal mitochondria-dyad contacts and nanotunnelling between mitochondria exposed to junctional SR and those sealed away from junctional SR. Remarkably, the linker decreased female vulnerability to acute massive ß-adrenergic stress. It also reduced myocyte death and mitochondrial calcium-overload-associated myocardial impairment in ex vivo ischemia/reperfusion injury. CONCLUSIONS: We propose that mitochondria-SR/endoplasmic reticulum contacts operate at a structural optimum. Although acute changes in tethering may cause dysfunction, upon chronic enhancement of contacts from early life, adaptive remodeling of the organelles shifts the system to a new, stable structural optimum. This remodeling balances the individually enhanced mitochondrion-junctional SR crosstalk and excitation-bioenergetics coupling, by increasing the connected mitochondrial pool and, presumably, Ca2+/reactive oxygen species capacity, which then improves the resilience to stresses associated with dysregulated hyperactive Ca2+ signaling.

Expanded clinical phenotype spectrum correlates with variant function in SCN2A-related disorders.

SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel Nav1.2 are rare, with clinically heterogeneous expressions that include epilepsy, autism and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relationship to channel function, 81 patients (36 female, 44%, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their Nav1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal onset, n = 27; infant onset, n = 18; and later onset n = 24; and autism without seizures, n = 12) was strongly correlated with a non-seizure severity index (P = 0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (P = 0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland Adaptive Behavior composite score of 49.5 (>3 standard deviations below the norm-referenced mean of the test). Epileptic spasms were significantly more common in infant-onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (P = 0.007). Primary phenotype was also strongly correlated with variant function (P < 0.0001); gain-of-function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy and to severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups, representing: (i) primarily later-onset epilepsy with moderate loss-of-function variants and low severity indices; (ii) mostly infant-onset epilepsy with moderate loss-of-function variants but higher severity indices; and (iii) late-onset and autism only, with the lowest severity indices (mostly zero) and severe/complete loss-of-function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relationship between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on Nav1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance towards clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.

Developmental constraints enforce altruism and avert the tragedy of the commons in a social microbe.

Organisms often cooperate through the production of freely available public goods. This can greatly benefit the group but is vulnerable to the "tragedy of the commons" if individuals lack the motivation to make the necessary investment into public goods production. Relatedness to groupmates can motivate individual investment because group success ultimately benefits their genes' own self-interests. However, systems often lack mechanisms that can reliably ensure that relatedness is high enough to promote cooperation. Consequently, groups face a persistent threat from the tragedy unless they have a mechanism to enforce investment when relatedness fails to provide adequate motivation. To understand the real threat posed by the tragedy and whether groups can avert its impact, we determine how the social amoeba Dictyostelium discoideum responds as relatedness decreases to levels that should induce the tragedy. We find that, while investment in public goods declines as overall within-group relatedness declines, groups avert the expected catastrophic collapse of the commons by continuing to invest, even when relatedness should be too low to incentivize any contribution. We show that this is due to a developmental buffering system that generates enforcement because insufficient cooperation perturbs the balance of a negative feedback system controlling multicellular development. This developmental constraint enforces investment under the conditions expected to be most tragic, allowing groups to avert a collapse in cooperation. These results help explain how mechanisms that suppress selfishness and enforce cooperation can arise inadvertently as a by-product of constraints imposed by selection on different traits.

Altered neurological and neurobehavioral phenotypes in a mouse model of the recurrent KCNB1-p.R306C voltage-sensor variant.

Pathogenic variants in KCNB1 are associated with a neurodevelopmental disorder spectrum that includes global developmental delays, cognitive impairment, abnormal electroencephalogram (EEG) patterns, and epilepsy with variable age of onset and severity. Additionally, there are prominent behavioral disturbances, including hyperactivity, aggression, and features of autism spectrum disorder. The most frequently identified recurrent variant is KCNB1-p.R306C, a missense variant located within the S4 voltage-sensing transmembrane domain. Individuals with the R306C variant exhibit mild to severe developmental delays, behavioral disorders, and a diverse spectrum of seizures. Previous in vitro characterization of R306C described altered sensitivity and cooperativity of the voltage sensor and impaired capacity for repetitive firing of neurons. Existing Kcnb1 mouse models include dominant negative missense variants, as well as knockout and frameshifts alleles. While all models recapitulate key features of KCNB1 encephalopathy, mice with dominant negative alleles were more severely affected. In contrast to existing loss-of-function and dominant-negative variants, KCNB1-p.R306C does not affect channel expression, but rather affects voltage-sensing. Thus, modeling R306C in mice provides a novel opportunity to explore impacts of a voltage-sensing mutation in Kcnb1. Using CRISPR/Cas9 genome editing, we generated the Kcnb1R306C mouse model and characterized the molecular and phenotypic effects. Consistent with the in vitro studies, neurons from Kcnb1R306C mice showed altered excitability. Heterozygous and homozygous R306C mice exhibited hyperactivity, altered susceptibility to chemoconvulsant-induced seizures, and frequent, long runs of slow spike wave discharges on EEG, reminiscent of the slow spike and wave activity characteristic of Lennox Gastaut syndrome. This novel model of channel dysfunction in Kcnb1 provides an additional, valuable tool to study KCNB1 encephalopathies. Furthermore, this allelic series of Kcnb1 mouse models will provide a unique platform to evaluate targeted therapies.

Cellular and behavioral effects of altered NaV1.2 sodium channel ion permeability in Scn2aK1422E mice.

Genetic variants in SCN2A, encoding the NaV1.2 voltage-gated sodium channel, are associated with a range of neurodevelopmental disorders with overlapping phenotypes. Some variants fit into a framework wherein gain-of-function missense variants that increase neuronal excitability lead to developmental and epileptic encephalopathy, while loss-of-function variants that reduce neuronal excitability lead to intellectual disability and/or autism spectrum disorder (ASD) with or without co-morbid seizures. One unique case less easily classified using this framework is the de novo missense variant SCN2A-p.K1422E, associated with infant-onset developmental delay, infantile spasms and features of ASD. Prior structure-function studies demonstrated that K1422E substitution alters ion selectivity of NaV1.2, conferring Ca2+ permeability, lowering overall conductance and conferring resistance to tetrodotoxin (TTX). Based on heterologous expression of K1422E, we developed a compartmental neuron model incorporating variant channels that predicted reductions in peak action potential (AP) speed. We generated Scn2aK1422E mice and characterized effects on neurons and neurological/neurobehavioral phenotypes. Cultured cortical neurons from heterozygous Scn2aK1422E/+ mice exhibited lower current density with a TTX-resistant component and reversal potential consistent with mixed ion permeation. Recordings from Scn2aK1442E/+ cortical slices demonstrated impaired AP initiation and larger Ca2+ transients at the axon initial segment during the rising phase of the AP, suggesting complex effects on channel function. Scn2aK1422E/+ mice exhibited rare spontaneous seizures, interictal electroencephalogram abnormalities, altered induced seizure thresholds, reduced anxiety-like behavior and alterations in olfactory-guided social behavior. Overall, Scn2aK1422E/+ mice present with phenotypes similar yet distinct from other Scn2a models, consistent with complex effects of K1422E on NaV1.2 channel function.

A comprehensive analysis of POLE/POLD1 genomic alterations in colorectal cancer.

INTRODUCTION: Pathogenic mutations in POLE/POLD1 lead to decreased fidelity of DNA replication, resulting in a high tumor mutational burden (TMB-H), defined as TMB ≥ 10 mut/Mb, independent of deficient mismatch repair (dMMR) and microsatellite instability high (MSI-H) status. METHODS: De-identified records of patients with colorectal cancer (CRC) profiled with the Tempus xT assay (DNA-seq of 595-648 genes at 500×) were identified from the Tempus Database. RESULTS: Among 9136 CRC samples profiled, the frequency of POLE/POLD1 genomic alterations was 2.4% (n = 217). Copy number loss was the most common genomic alteration (64%, n = 138) of POLE/POLD1, followed by copy number amplifications (18%, n = 40) and short variant mutations (18%, n = 39). The POLE/POLD1 mutated group presented with a higher frequency of TMB-H phenotype relative to wild type (WT; 22% vs. 9%, P < .001), with a median TMB of 127 mut/Mb in the TMB-H POLE/POLD1 subset. The TMB showed a dramatic contrast between POLE/POLD1 short variant mutations as compared to the group with copy number alterations, with a TMB of 159 mut/Mb vs 15 mut/Mb, respectively. Thus, the short variant mutations represented the so-called ultra-hypermutated phenotype. The POLE/POLD1 mutated group, as compared to WT, exhibited a higher rate of coexisting mutations, including APC, ALK, ATM, BRCA2, and RET mutations. CONCLUSION: Patients with POLE/POLD1 mutations exhibited significant differences across immunological markers (ie, TMB, MMR, and MSI-H) and molecular co-alterations. Those with short variant mutations represented 18% of the POLE/POLD1 cohort and 0.4% of the total cohort examined. This group of patients had a median TMB of 159 mut/Mb (range 34-488), representing the ultra-hypermutated phenotype. This group of patients is important to identify given the potential for exceptional response to immune checkpoint inhibitors.

AGA Rapid Clinical Practice Update on the Management of Patients Taking GLP-1 Receptor Agonists Prior to Endoscopy: Communication.

DESCRIPTION: The purpose of this American Gastroenterological Association (AGA) Institute Rapid Clinical Practice Update (CPU) Communication is to review the available evidence and provide expert advice regarding the evolving management of patients taking GLP-1 receptor agonists prior to endoscopy. METHODS: This CPU was commissioned and approved by the AGA Institute Clinical Practice Updates Committee (CPUC) and the AGA Governing Board to provide timely guidance on a topic of high clinical importance to the AGA membership and underwent internal peer review by the CPUC and external peer review through standard procedures of Clinical Gastroenterology and Hepatology. This communication incorporates important and recently published studies in this field, and it reflects the experiences of the authors who are experts in bariatric medicine and/or endoscopy.

Transoral Outlet Reduction: Expert Tips, Tricks, and Troubleshooting.

Transoral outlet reduction (TORe) is an incisionless, endoscopic procedure to address weight recurrence after Roux-en-Y gastric bypass. Given the chronic, progressive nature of obesity and the minimally invasive, anatomy preserving technique of TORe, the procedure is expected to be met with high patient acceptance and widening clinical adoption. Nevertheless, the approach to TORe has been heterogeneous. As endoscopic bariatric therapies are increasingly incorporated into the multidisciplinary management of obesity, it is crucial to have a standardized, evidence-based framework for their implementation. In this review, based on the available literature and the authors' combined experience of over 1,000 TORe procedures, we present our approach to patient selection, procedural technique, troubleshooting, and patient aftercare unique to TORe.

Development of a novel endoscopic suturing simulator: validation and impact on clinical learning curve (with video).

BACKGROUND AND AIMS: Endoscopic suturing is a complex skill with broad applications. The aim of this study was to develop and validate a novel endoscopic suturing simulator and scoring system for the purse-string suture pattern. METHODS: This was a prospective study of an endoscopic suturing simulator that consists of a circular opening representing a dilated gastrojejunal anastomosis of Roux-en-Y gastric bypass with 12 small target holes around its circumference. Purse-string suturing is performed in a counterclockwise fashion. Time allotted is 5 minutes, and each successful bite is awarded 10 points. Participants were divided into the novice, intermediate, and experienced groups. Validity evidence based on content, validity evidence based on other variables, and validity evidence based on consequences of testing were assessed. RESULTS: Seventeen subjects (3 novice, 7 intermediate, and 7 experienced) participated in the study. Validity evidence based on content: The content validity index for realism, relevance, and representativeness was 0.89, 1.00, and 1.00, respectively. Validity evidence based on other variables: The novice, intermediate, and experienced groups scored 30.0 ± 8.2, 57.1 ± 28.1, and 131.2 ± 51.7 (P = .001). Validity evidence based on consequences of testing: The simulator group required 5 ± 5 transoral outlet reduction cases before being able to independently complete an entire purse-string, whereas the non-simulator group required 38 ± 11 clinical cases (P < .0001). CONCLUSIONS: This novel endoscopic suturing simulator seems realistic, relevant, and representative of the clinical suturing experience. In addition, it seems effective at objectively assessing suturing skills and shortening the clinical learning curve.

Bariatric endoscopic antral myotomy: a pilot study assessing technical feasibility, physiologic changes, and preliminary efficacy.

BACKGROUND AND AIMS: Gastric balloons and endoscopic sleeve gastroplasty appear to work by delaying gastric emptying. We hypothesized that pylorus-sparing antral myotomy would inhibit the antral pump, inducing gastric retention and similarly resulting in weight loss. METHODS: In this single-center pilot study, we assessed bariatric endoscopic antral myotomy (BEAM) using submucosal tunneling. The primary outcomes were feasibility, safety, and efficacy at 6 and 12 months, whereas the secondary outcomes were changes in the gastric-emptying rate and gastroparesis cardinal symptom index (GCSI) score. RESULTS: Six subjects underwent successful BEAM. One required needle decompression, and another developed pulmonary embolism, treated without sequela. At 6 and 12 months, patients achieved 9.1% ± 8.9% and 12.2% ± 7.1% total weight loss (P < .0005). The gastric-emptying rate was delayed by 36.6% in those with ≥10% total weight loss. The GCSI score increased significantly at 12 months, particularly regarding early satiety. CONCLUSIONS: This pilot study suggests BEAM is feasible and appears to induce delayed gastric emptying that is associated with significant weight loss, without symptoms of gastroparesis.

Tunneled or Pocket Creation Method versus Conventional Endoscopic Submucosal Dissection for Gastric Lesions - A Systematic Review and Meta-analysis.

BACKGROUND AND AIMS: Tunneled endoscopic submucosal dissection (T-ESD) and pocket creation method ESD (PCM-ESD) are considered to have technical advantages over conventional ESD (C-ESD). However, data comparing these techniques for ESD of gastric lesions is limited. METHODS: PubMed and Cochrane databases were reviewed for relevant studies from their inception to October 31, 2023. Studies comparing T-ESD or PCM-ESD (T/PCM-ESD) to C-ESD for gastric lesions were included. The primary outcomes were dissection speed and en bloc resection. Secondary outcomes were R0 resection, recurrence, perforation, and post-ESD bleeding. A random effects meta-analysis was conducted. RESULTS: Eight observational studies (359 patients - T/PCM-ESD, 670 patients - C-ESD) were included. T/PCM-ESD was associated with a significantly faster dissection speed (Mean Difference: 4.42 mm2/min, 95% CI: 2.05, 6.79, I2 = 79%). There were no significant differences between the groups in terms of en bloc resection (risk ratio (RR): 1.01, 95% confidence interval (CI): 1.00-1.03, I2 = 0%), R0 resection (RR: 1.03, 95% CI: 0.99-1.07, I2 = 0%) and recurrence (RR: 0.73, 95% CI: 0.14-3.84, I2 = 0%). While T/PCM-ESD was associated with a significantly lower risk of perforation (RR: 0.21, 95% CI: 0.06-0.80, I2 = 0%), post-ESD bleeding rates were not significantly different. CONCLUSION: T/PCM-ESD facilitates faster and safer gastric ESD than conventional ESD with comparable en bloc resection, R0 resection, and recurrence rates. A future randomized controlled control trial is required.

EUS-guided liver palpation as a screening tool for advanced fibrosis and cirrhosis in patients with suspected metabolic dysfunction-associated steatotic liver disease: a pilot study.

BACKGROUND AND AIMS: Endoscopic liver "palpation" can be performed by indenting the liver surface under EUS. Indentation depth is measured with the use of sonographic calipers. We hypothesized that fibrotic livers are more difficult to indent, and that indentation can accurately predict liver fibrosis staging. We compared EUS-guided liver palpation and conventional screening modalities in patients with suspected metabolic dysfunction-associated steatotic liver disease. METHODS: This was a cross-sectional pilot study. Consecutive patients at 3 hospitals from 2021 to 2023 underwent EUS-guided palpation with liver biopsy. Liver palpation was compared with fibrosis-4 index (FIB-4), aspartate transaminase to platelet ratio index (APRI), nonalcoholic fatty liver disease fibrosis score (NFS), and transient elastography in predicting fibrosis staging on histology. Area under the receiver operating characteristic curve analysis was performed. RESULTS: Seventy-three patients were included. Mean age was 49.1 years, and 71.2% were female. Mean body mass index was 41.1 kg/m.2 Indentation depth was negatively correlated with fibrosis stage (Kruskal-Willis test, P < .0001). EUS palpation demonstrated c-statistics of 0.79 and 0.95 in discriminating advanced fibrosis and cirrhosis, respectively. EUS liver palpation was superior to NFS in predicting advanced fibrosis (P = .0057) and superior to APRI and NFS in predicting cirrhosis (P = .0099 and P = .045, respectively). EUS palpation was not significantly different from FIB-4. EUS palpation was superior to transient elastography in predicting cirrhosis (P = .045). When optimal cutoffs were used, indentation measurement ≤3.5 mm yielded 100% predictive value for ruling in advanced fibrosis, and ≥4.0 mm yielded 100% predictive value for ruling out cirrhosis. CONCLUSIONS: EUS liver palpation is a novel, accurate, and easy-to-use screening tool for advanced fibrosis and cirrhosis in patients with metabolic dysfunction-associated steatotic liver disease.

American Society for Gastrointestinal Endoscopy-European Society of Gastrointestinal Endoscopy guideline on primary endoscopic bariatric and metabolic therapies for adults with obesity.

This joint ASGE-ESGE guideline provides an evidence-based summary and recommendations regarding the role of endoscopic bariatric and metabolic therapies (EBMTs) in the management of obesity. The document was developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework. It evaluates the efficacy and safety of EBMT devices and procedures that currently have CE mark or FDA-clearance/approval, or that had been approved within five years of document development. The guideline suggests the use of EBMTs plus lifestyle modification in patients with a BMI of ≥ 30 kg/m2, or with a BMI of 27.0-29.9 kg/m2 with at least 1 obesity-related comorbidity. Furthermore, it suggests the utilization of intragastric balloons and devices for endoscopic gastric remodeling (EGR) in conjunction with lifestyle modification for this patient population.

The effect of endoscopic gastric plication on portosystemic pressure gradient in patients with nonalcoholic fatty liver disease and compensated advanced chronic liver disease.

BACKGROUND: The goals of therapy for patients with nonalcoholic fatty liver disease and compensated advanced chronic liver disease include weight loss and reduction of the portosystemic pressure gradient (PPG) to decrease the risk of hepatic decompensation. Endoscopic gastric plication (EGP) is an effective endoscopic weight loss procedure. This study aimed to assess the effect of EGP on PPG. METHODS: In this prospective pilot study, patients with nonalcoholic fatty liver disease and compensated advanced chronic liver disease underwent endoscopic ultrasound-guided PPG measurement prior to and at 6 months following EGP. Primary outcomes were the change in PPG and proportion of patients experiencing ≥ 20 % reduction in PPG at 6 months. Secondary outcomes included percent total weight loss (TWL) and changes in noninvasive tests of fibrosis. RESULTS: 20 patients were included. Baseline median body mass index and liver stiffness measurement were 40.2 kg/m2 (range 30.1-56.7) and 14.7 kPa (range 8.2-36), respectively. At 6 months, median PPG decreased from 5.4 mmHg (range 0.7-19.6) to 1.8 mmHg (range 0.4-17.6) (P = 0.002), with 79 % (11/14) experiencing ≥ 20 % reduction. Patients experienced 12.5 % (6.5 %-26.1 %) TWL (P < 0.001) at 6 months, with 89 % (17/19) achieving ≥ 7 % and 68 % (13/19) achieving ≥ 10 % TWL. There were significant improvements in noninvasive tests of fibrosis. CONCLUSION: EGP appeared to be effective at reducing PPG in patients with nonalcoholic fatty liver disease and compensated advanced chronic liver disease.

American Society for Gastrointestinal Endoscopy-European Society of Gastrointestinal Endoscopy guideline on primary endoscopic bariatric and metabolic therapies for adults with obesity.

This joint ASGE-ESGE guideline provides an evidence-based summary and recommendations regarding the role of endoscopic bariatric and metabolic therapies (EBMTs) in the management of obesity. The document was developed using the Grading of Recommendations, Assessment, Development and Evaluation (GRADE) framework. It evaluates the efficacy and safety of EBMT devices and procedures that currently have CE mark or FDA-clearance/approval, or that had been approved within five years of document development. The guideline suggests the use of EBMTs plus lifestyle modification in patients with a BMI of ≥30 kg/m2, or with a BMI of 27.0-29.9 kg/m2 with at least 1 obesity-related comorbidity. Furthermore, it suggests the utilization of intragastric balloons and devices for endoscopic gastric remodeling (EGR) in conjunction with lifestyle modification for this patient population.

Efficacy and Safety of Gastric Per-Oral Endoscopic Myotomy (G-POEM): A Systematic Review and Meta-Analysis.

BACKGROUND: Gastric per-oral endoscopic myotomy (G-POEM) is an innovative treatment that has become increasingly utilized for patients with refractory gastroparesis. The aim of this systematic review and meta-analysis was to evaluate the safety and effectiveness of G-POEM for the treatment of gastroparesis. METHODS: Individualized search strategies were developed through February 2021 in accordance with the PRISMA and MOOSE guidelines. This meta-analysis was performed by calculating pooled proportions and mean difference preprocedure and postprocedure with rates estimated using random effects models. Measured outcomes included technical success, clinical success, improvement in gastroparesis cardinal symptom index (GCSI), change in gastric emptying rate, alterations in impedance planimetry (functional lumen imaging probe [FLIP]) assessment, and adverse events. RESULTS: A total of 20 studies (n=797 patients; 67.41% female) were included. The mean age was 48.92±11.61 y, with an average duration of 4.24±1.11 y since gastroparesis diagnosis. Technical success was 98.47% [(95% CI: 97.14, 99.19);I2=0.00] with a mean myotomy length of 3.78±1.16 cm. In terms of clinical success, mean preprocedure GCSI scores were 3.38±0.37 and improved significantly postprocedure [weighted mean difference -1.56 (95% CI: -1.89 to -1.24); I2=82.53; P<0.001]. Gastric retention after 4 hours demonstrated ~50% improvement (preprocedure 43.08±9.24% versus postprocedure 22.97±10.19%; P<0.001). FLIP assessment with 40 mL and 50 mL balloons demonstrated a significant increase in diameter, distensibility index, and cross-sectional area postprocedure (all P<0.05). Procedure-associated adverse events occurred among 10.92% [(95% CI 5.09 to 19.32); I2=82.85] of patients. CONCLUSION: G-POEM appears safe and highly effective for the treatment of patients with refractory gastroparesis regardless of symptom predominance or etiology.

Obesity Is Associated with Altered Rectal Sensitivity in Chronic Constipation.

BACKGROUND: Defecation dysfunction may contribute to chronic constipation (CC), but the impact of obesity on anorectal physiology in CC remains unclear. We aimed to evaluate the relationship between obesity and anorectal function on physiologic testing in patients presenting with CC. METHODS: This was a retrospective cohort study of consecutive adults who underwent high resolution anorectal manometry (HRAM) at a tertiary center for CC. Patient demographics, clinical history, surgical/obstetric history, medications, and HRAM results were reviewed. Patients were classified into obese (BMI > 30 kg/m2) vs non-obese (BMI < 30 kg/m2) groups at the time of HRAM. Fisher-exact/student t-test for univariate analyses and general linear regression for multivariable analysis were performed. RESULTS: 383 adults (mean 50.3 years; 85.8% female) with CC were included. On HRAM, patients with obesity had lower anal sphincter resting tone (37.3 vs 48.5 mmHg, p = 0.005) and maximum squeeze pressure (104.8 mmHg vs 120.0 mmHg, p = 0.043). No significant differences in dyssynergia (61% vs 53%, p = 0.294) and failed balloon expulsion (18% vs 25%, p = 0.381) were found between obese and non-obese groups. On balloon distention testing, the maximum tolerated (163.5 vs 147.6 mL, p = 0.042) and urge sensation (113.9 vs 103.7 mL, p = 0.048) volumes were significantly increased among patients with obesity. After adjusting for potential confounders, obesity remained independently associated with increased maximum tolerated volume (ß-coefficient 13.7, p = 0.049). CONCLUSION: Obesity was independently associated with altered rectal sensitivity among patients with CC. Altered rectal sensation may play an important role in CC among patients with obesity. Anorectal physiology testing should be considered to understand the pathophysiology and guide management.

Potency Assay Variability Estimation in Practice.

During the drug development process, testing potency plays an important role in the quality assessment required for the manufacturing and marketing of biologics. Due to multiple operational and biological factors, higher variability is usually observed in bioassays compared with physicochemical methods. In this paper, we discuss different sources of bioassay variability and how this variability can be statistically estimated. In addition, we propose an algorithm to estimate the variability of reportable results associated with different numbers of runs and their corresponding OOS rates under a given specification. Numerical experiments are conducted on multiple assay formats to elucidate the empirical distribution of bioassay variability.

SARS-CoV-2 Rapidly Infects Peripheral Sensory and Autonomic Neurons, Contributing to Central Nervous System Neuroinvasion before Viremia.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.