Leucyl-tRNA Synthetase Contributes to Muscle Weakness through Mammalian Target of Rapamycin Complex 1 Activation and Autophagy Suppression in a Mouse Model of Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD), caused by loss-of-function mutations in the dystrophin gene, results in progressive muscle weakness and early fatality. Impaired autophagy is one of the cellular hallmarks of DMD, contributing to the disease progression. Molecular mechanisms underlying the inhibition of autophagy in DMD are not well understood. In the current study, the DMD mouse model mdx is used for the investigation of signaling pathways leading to suppression of autophagy. Mammalian target of rapamycin complex 1 (mTORC1) is found to be hyperactive in the DMD muscles, accompanying muscle weakness and autophagy impairment. Surprisingly, Akt, a well-known upstream regulator of mTORC1, is not responsible for mTORC1 activation or the dystrophic muscle phenotypes. Instead, leucyl-tRNA synthetase (LeuRS) is found to be overexpressed in mdx muscles compared with the wild type. LeuRS is known to activate mTORC1 in a noncanonical mechanism that involves interaction with RagD, an activator of mTORC1. Disrupting LeuRS interaction with RagD by the small-molecule inhibitor BC-LI-0186 reduces mTORC1 activity, restores autophagy, and ameliorates myofiber damage in the mdx muscles. Furthermore, inhibition of LeuRS by BC-LI-0186 improves dystrophic muscle strength in an autophagy-dependent manner. Taken together, our findings uncover a noncanonical function of the housekeeping protein LeuRS as a potential therapeutic target in the treatment of DMD.

Patient Attitudes and Barriers to Endoscopy During the COVID-19 Pandemic.

BACKGROUND: After COVID-19 restrictions on nonessential procedures were lifted and safety protocols established, utilization rates of endoscopic procedures remained reduced. AIMS: This study assessed patient attitudes and barriers to scheduling endoscopy during the pandemic. METHODS: A survey was administered to patients with ordered procedures at a hospital-based setting (7/21/2020-2/19/2021) collecting demographic data, body mass index, COVID-19 relevant comorbidities, level of procedural urgency (defined by recommended scheduling window), scheduling and attendance, concerns, and awareness of safety measures. RESULTS: The average respondent was female (63.8%), age 57.6 ± 14, White (72.3%), married (76.7%), insured (99.3%), affluent English speakers (92.3%) and highly educated (at least college 90.2%). Most reported moderate to excellent COVID-19 knowledge (96.6%). Of 1039 procedures scheduled, emergent cases accounted for 5.1%, urgent 55.3% and elective 39.4%. Respondents identified appointment convenience (48.53%) as the most frequent factor impacting scheduling, also noting concern for results (28.4%). Age (p = .022), native language (p = .04), education (p = .007), self-reported COVID knowledge (p = .002), and a desire to be COVID tested pre-procedure (p = .023) were associated with arrival, more commonly in an ambulatory surgical center than hospital (p = .008). Diabetes mellitus (p = .004) and an immunocompromised state (p = .009) were adversely related to attendance. Attitudes towards safety protocols did not affect scheduling. Multivariate analysis demonstrated age, education and COVID knowledgeability were associated with procedure completion. CONCLUSIONS: Safety protocols and urgency levels were not associated with procedure completion. Pre-pandemic barriers to endoscopy persisted as dominant factors amid pandemic concerns.

Glomerular hyperfiltration is an independent predictor of postoperative outcomes: A NSQIP multi-specialty surgical cohort analysis.

AIM: While high estimated glomerular filtration rate (eGFR) has been associated with increased overall mortality, its effect on postoperative outcomes is relatively understudied. We sought to investigate the association between high eGFR and 30-day postoperative outcomes using a multi-specialty surgical cohort. METHODS: Using the National Surgical Quality Improvement Program database, we selected adult for whom eGFR could be calculated using the 2021 Chronic Kidney Disease Epidemiology Collaboration equation. Based on sex-specific distributions of eGFR stratified by age quintiles, we classified patients into low (<5th percentile), normal (5-95th percentile) and high eGFR (>95th percentile). The primary outcome was a composite of any 30-day major adverse outcomes, including: death, reoperation, cardiac arrest, myocardial infarction and stroke. Secondary outcomes included 30-day infectious complications, venous thromboembolism (VTE), bleeding requiring transfusion, prolonged length of stay and unplanned readmission. After matching for demographic differences, comorbidity burden and operative characteristics, logistic regression models were used to evaluate the association between extremes of eGFR and the outcomes of interest. RESULTS: Of 1 668 447 patients, 84 115 (5.07%) had a high eGFR. High eGFR was not associated with major adverse outcomes (odds ratio [OR] 1.00 [95% confidence interval (CI): 0.97, 1.03]); however, it was associated with reoperation (OR 1.04 [95% CI: 1.00,1.08]), infectious complications (OR 1.14 [95% CI: 1.11, 1.16]), VTE (OR 1.15 [95% CI: 1.09, 1.22]) and prolonged length of stay (OR 1.19 [95% CI: 1.16, 1.21]). CONCLUSION: Our findings support an association between high eGFR and adverse 30-day postoperative outcomes.

A non-translational role of threonyl-tRNA synthetase in regulating JNK signaling during myogenic differentiation.

Aminoacyl-tRNA synthetases (aaRSs) are house-keeping enzymes that are essential for protein synthesis. However, it has become increasingly evident that some aaRSs also have non-translational functions. Here we report the identification of a non-translational function of threonyl-tRNA synthetase (ThrRS) in myogenic differentiation. We find that ThrRS negatively regulates myoblast differentiation in vitro and injury-induced skeletal muscle regeneration in vivo. This function is independent of amino acid binding or aminoacylation activity of ThrRS, and knockdown of ThrRS leads to enhanced differentiation without affecting the global protein synthesis rate. Furthermore, we show that the non-catalytic new domains (UNE-T and TGS) of ThrRS are both necessary and sufficient for the myogenic function. In searching for a molecular mechanism of this new function, we find the kinase JNK to be a downstream target of ThrRS. Our data further reveal MEKK4 and MKK4 as upstream regulators of JNK in myogenesis and the MEKK4-MKK4-JNK pathway to be a mediator of the myogenic function of ThrRS. Finally, we show that ThrRS physically interacts with Axin1, disrupts Axin1-MEKK4 interaction and consequently inhibits JNK signaling. In conclusion, we uncover a non-translational function for ThrRS in the maintenance of homeostasis of skeletal myogenesis and identify the Axin1-MEKK4-MKK4-JNK signaling axis to be an immediate target of ThrRS action.

Effects of rapamycin on growth hormone receptor knockout mice.

It is well documented that inhibition of mTORC1 (defined by Raptor), a complex of mechanistic target of rapamycin (mTOR), extends life span, but less is known about the mechanisms by which mTORC2 (defined by Rictor) impacts longevity. Here, rapamycin (an inhibitor of mTOR) was used in GHR-KO (growth hormone receptor knockout) mice, which have suppressed mTORC1 and up-regulated mTORC2 signaling, to determine the effect of concurrently decreased mTORC1 and mTORC2 signaling on life span. We found that rapamycin extended life span in control normal (N) mice, whereas it had the opposite effect in GHR-KO mice. In the rapamycin-treated GHR-KO mice, mTORC2 signaling was reduced without further inhibition of mTORC1 in the liver, muscle, and s.c. fat. Glucose and lipid homeostasis were impaired, and old GHR-KO mice treated with rapamycin lost functional immune cells and had increased inflammation. In GHR-KO MEF cells, knockdown of Rictor, but not Raptor, decreased mTORC2 signaling. We conclude that drastic reduction of mTORC2 plays important roles in impaired longevity in GHR-KO mice via disruption of whole-body homeostasis.

Lanthionine synthetase C-like protein 2 (LanCL2) is important for adipogenic differentiation.

Adipogenic differentiation is a highly regulated process that is necessary for metabolic homeostasis and nutrient sensing. The expression of PPARγ and the subsequent activation of adipogenic genes is critical for the process. In this study, we identified lanthionine synthetase C-like protein 2 (LanCL2) as a positive regulator of adipogenesis in 3T3-L1 cells. Knockdown of LanCL2, but not LanCL1, inhibited adipogenic differentiation, and this effect was not mediated through cAMP or Akt signaling pathways. The expression of early adipogenic markers CCAAT enhancer binding protein ß (C/EBPß) and C/EBPδ remained intact in LanCL2 knockdown cells, but levels of late adipogenic markers PPARγ and C/EBPα were suppressed. The addition of the naturally occurring PPARγ activator 15-deoxy-Δ12,14-prostaglandin J2 or conditioned medium from differentiating cells did not restore differentiation, implying that LanCL2 may not be involved in the production of a secreted endogenous PPARγ ligand. Pulldown assays demonstrated a direct physical interaction between LanCL2 and PPARγ. Consistent with a regulatory role of LanCL2, luciferase reporter assays revealed that full transcriptional activation by PPARγ was dependent on LanCL2. Taken together, our study reveals a novel role of LanCL2 in adipogenesis, specifically involved in PPARγ-mediated transactivation of downstream adipogenic genes.

hiPSC hepatocyte model demonstrates the role of unfolded protein response and inflammatory networks in α1-antitrypsin deficiency.

BACKGROUND & AIMS: α1-Antitrypsin deficiency (A1ATD) is an autosomal recessive disorder caused by mutations in the SERPINA1 gene. Individuals with the Z variant (Gly342Lys) retain polymerised protein in the endoplasmic reticulum (ER) of their hepatocytes, predisposing them to liver disease. The concomitant lack of circulating A1AT also causes lung emphysema. Greater insight into the mechanisms that link protein misfolding to liver injury will facilitate the design of novel therapies. METHODS: Human-induced pluripotent stem cell (hiPSC)-derived hepatocytes provide a novel approach to interrogate the molecular mechanisms of A1ATD because of their patient-specific genetic architecture and reflection of human physiology. To that end, we utilised patient-specific hiPSC hepatocyte-like cells (ZZ-HLCs) derived from an A1ATD (ZZ) patient, which faithfully recapitulated key aspects of the disease at the molecular and cellular level. Subsequent functional and "omics" comparisons of these cells with their genetically corrected isogenic-line (RR-HLCs) and primary hepatocytes/human tissue enabled identification of new molecular markers and disease signatures. RESULTS: Our studies showed that abnormal A1AT polymer processing (immobilised ER components, reduced luminal protein mobility and disrupted ER cisternae) occurred heterogeneously within hepatocyte populations and was associated with disrupted mitochondrial structure, presence of the oncogenic protein AKR1B10 and two upregulated molecular clusters centred on members of inflammatory (IL-18 and Caspase-4) and unfolded protein response (Calnexin and Calreticulin) pathways. These results were validated in a second patient-specific hiPSC line. CONCLUSIONS: Our data identified novel pathways that potentially link the expression of Z A1AT polymers to liver disease. These findings could help pave the way towards identification of new therapeutic targets for the treatment of A1ATD. LAY SUMMARY: This study compared the gene expression and protein profiles of healthy liver cells and those affected by the inherited disease α1-antitrypsin deficiency. This approach identified specific factors primarily present in diseased samples which could provide new targets for drug development. This study also demonstrates the interest of using hepatic cells generated from human-induced pluripotent stem cells to model liver disease in vitro for uncovering new mechanisms with clinical relevance.

The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

α1-Antitrypsin is a serine protease inhibitor produced in the liver that is responsible for the regulation of pulmonary inflammation. The commonest pathogenic gene mutation yields Z-α1-antitrypsin, which has a propensity to self-associate forming polymers that become trapped in inclusions of endoplasmic reticulum (ER). It is unclear whether these inclusions are connected to the main ER network in Z-α1-antitrypsin-expressing cells. Using live cell imaging, we found that despite inclusions containing an immobile matrix of polymeric α1-antitrypsin, small ER resident proteins can diffuse freely within them. Inclusions have many features to suggest they represent fragmented ER, and some are physically separated from the tubular ER network, yet we observed cargo to be transported between them in a cytosol-dependent fashion that is sensitive to N-ethylmaleimide and dependent on Sar1 and sec22B. We conclude that protein recycling occurs between ER inclusions despite their physical separation.-Dickens, J. A., Ordóñez, A., Chambers, J. E., Beckett, A. J., Patel, V., Malzer, E., Dominicus, C. S., Bradley, J., Peden, A. A., Prior, I. A., Lomas, D. A., Marciniak, S. J. The endoplasmic reticulum remains functionally connected by vesicular transport after its fragmentation in cells expressing Z-α1-antitrypsin.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

An antibody that prevents serpin polymerisation acts by inducing a novel allosteric behaviour.

Serpins are important regulators of proteolytic pathways with an antiprotease activity that involves a conformational transition from a metastable to a hyperstable state. Certain mutations permit the transition to occur in the absence of a protease; when associated with an intermolecular interaction, this yields linear polymers of hyperstable serpin molecules, which accumulate at the site of synthesis. This is the basis of many pathologies termed the serpinopathies. We have previously identified a monoclonal antibody (mAb4B12) that, in single-chain form, blocks α1-antitrypsin (α1-AT) polymerisation in cells. Here, we describe the structural basis for this activity. The mAb4B12 epitope was found to encompass residues Glu32, Glu39 and His43 on helix A and Leu306 on helix I. This is not a region typically associated with the serpin mechanism of conformational change, and correspondingly the epitope was present in all tested structural forms of the protein. Antibody binding rendered ß-sheet A - on the opposite face of the molecule - more liable to adopt an 'open' state, mediated by changes distal to the breach region and proximal to helix F. The allosteric propagation of induced changes through the molecule was evidenced by an increased rate of peptide incorporation and destabilisation of a preformed serpin-enzyme complex following mAb4B12 binding. These data suggest that prematurely shifting the ß-sheet A equilibrium towards the 'open' state out of sequence with other changes suppresses polymer formation. This work identifies a region potentially exploitable for a rational design of ligands that is able to dynamically influence α1-AT polymerisation.

Increased ERK signalling promotes inflammatory signalling in primary airway epithelial cells expressing Z α1-antitrypsin.

Overexpression of Z α1-antitrypsin is known to induce polymer formation, prime the cells for endoplasmic reticulum stress and initiate nuclear factor kappa B (NF-κB) signalling. However, whether endogenous expression in primary bronchial epithelial cells has similar consequences remains unclear. Moreover, the mechanism of NF-κB activation has not yet been elucidated. Here, we report excessive NF-κB signalling in resting primary bronchial epithelial cells from ZZ patients compared with wild-type (MM) controls, and this appears to be mediated by mitogen-activated protein/extracellular signal-regulated kinase, EGF receptor and ADAM17 activity. Moreover, we show that rather than being a response to protein polymers, NF-κB signalling in airway-derived cells represents a loss of anti-inflammatory signalling by M α1-antitrypsin. Treatment of ZZ primary bronchial epithelial cells with purified plasma M α1-antitrypsin attenuates this inflammatory response, opening up new therapeutic options to modulate airway inflammation in the lung.

A single-chain variable fragment intrabody prevents intracellular polymerization of Z α1-antitrypsin while allowing its antiproteinase activity.

Mutant Z α1-antitrypsin (E342K) accumulates as polymers within the endoplasmic reticulum (ER) of hepatocytes predisposing to liver disease, whereas low levels of circulating Z α1-antitrypsin lead to emphysema by loss of inhibition of neutrophil elastase. The ideal therapy should prevent polymer formation while preserving inhibitory activity. Here we used mAb technology to identify interactors with Z α1-antitrypsin that comply with both requirements. We report the generation of an mAb (4B12) that blocked α1-antitrypsin polymerization in vitro at a 1:1 molar ratio, causing a small increase of the stoichiometry of inhibition for neutrophil elastase. A single-chain variable fragment (scFv) intrabody was generated based on the sequence of mAb4B12. The expression of scFv4B12 within the ER (scFv4B12KDEL) and along the secretory pathway (scFv4B12) reduced the intracellular polymerization of Z α1-antitrypsin by 60%. The scFv4B12 intrabody also increased the secretion of Z α1-antitrypsin that retained inhibitory activity against neutrophil elastase. MAb4B12 recognized a discontinuous epitope probably located in the region of helices A/C/G/H/I and seems to act by altering protein dynamics rather than binding preferentially to the native state. This novel approach could reveal new target sites for small-molecule intervention that may block the transition to aberrant polymers without compromising the inhibitory activity of Z α1-antitrypsin.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress.

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

Endoplasmic reticulum polymers impair luminal protein mobility and sensitize to cellular stress in alpha1-antitrypsin deficiency.

UNLABELLED: Point mutants of alpha1 -antitrypsin (α1AT) form ordered polymers that are retained as inclusions within the endoplasmic reticulum (ER) of hepatocytes in association with neonatal hepatitis, cirrhosis, and hepatocellular carcinoma. These inclusions cause cell damage and predispose to ER stress in the absence of the classical unfolded protein response (UPR). The pathophysiology underlying this ER stress was explored by generating cell models that conditionally express wild-type (WT) α1AT, two mutants that cause polymer-mediated inclusions and liver disease (E342K [the Z allele] and H334D) and a truncated mutant (Null Hong Kong; NHK) that induces classical ER stress and is removed by ER-associated degradation. Expression of the polymeric mutants resulted in gross changes in the ER luminal environment that recapitulated the changes observed in liver sections from individuals with PI*ZZ α1AT deficiency. In contrast, expression of NHK α1AT caused electron lucent dilatation and expansion of the ER throughout the cell. Photobleaching microscopy in live cells demonstrated a decrease in the mobility of soluble luminal proteins in cells that express E342K and H334D α1AT, when compared to those that express WT and NHK α1AT (0.34 ± 0.05, 0.22 ± 0.03, 2.83 ± 0.30, and 2.84 ± 0.55 µm(2) /s, respectively). There was no effect on protein mobility within ER membranes, indicating that cisternal connectivity was not disrupted. Polymer expression alone was insufficient to induce the UPR, but the resulting protein overload rendered cells hypersensitive to ER stress induced by either tunicamycin or glucose depletion. CONCLUSION: Changes in protein diffusion provide an explanation for the cellular consequences of ER protein overload in mutants that cause inclusion body formation and α1AT deficiency.

Perceptions and Understanding of Transgender Patient Care: A Survey of Practicing Laryngologists.

OBJECTIVES: Transgender individuals face significant health disparities including deficiencies in physician education, knowledge, and comfort with transgender health care. As the prevalence of the transgender population increases more individuals may seek gender-affirming surgery. Herein, we present a survey study which presents data on (1) the current practice patterns, (2) the familiarity with, (3) the perception of, and (4) the future educational goals of transgender health care among laryngologists in the United States. METHODS: A cross-sectional survey study of practicing laryngologists in the United States. RESULTS: A total of 53 laryngologists participated in the study, with 50 (94.3%) coming from an academic practice. Survey response rate was 32.3% (54/167). The number of patients cared for and surgeries performed were significantly associated with self-perceived overall competence (p < 0.001 and p < 0.001), surgical competence (p = 0.013 and p < 0.001), and comfort counseling patients on gender-affirming surgeries (p < 0.001 and p < 0.001). Most obtained training through real-world experience (n = 46, 86.8%), whereas only 11 (20.7%) had formal training in residency or fellowship. Although 37 (70%) of participants felt competent caring for transgender patients, 38 (72%) want to learn more about transgender care, and 49 (93%) support incorporating transgender care into otolaryngology residency/fellowship curricula. CONCLUSION: There is a need for an increased awareness of transgender healthcare issues to address disparities experienced by this diverse population. Many laryngologists report wanting to learn more about this developing part of our field and support incorporating transgender care into training. We attempt to spotlight the degree by which practicing laryngologists are familiar, competent, and comfortable with transgender care. LEVEL OF EVIDENCE: 5 Laryngoscope, 134:3215-3219, 2024.

Comparison of keratometry using SS-OCT and dual Scheimpflug-Placido imaging in postmyopic corneal refractive surgery eyes.

PURPOSE: To compare simulated (SimK) and total (True-K) keratometry and corneal astigmatism values between the IOLMaster 700 (IOLM) and Galilei G4 (G4) devices in postmyopic laser refractive surgery eyes. SETTING: Methodist Eye Associates, Blanton Eye Institute, Houston Methodist Hospital, Houston, Texas. DESIGN: Retrospective cohort study. METHODS: A chart review was conducted on patients with prior myopic laser-assisted in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK), undergoing phacoemulsification at a single institution from May 2019 through January 2022, who underwent imaging with both the IOLM and G4. Exclusion criteria were prior radial keratotomy, keratoectatic diseases, and inability to obtain a reliable image. Mean, flat, and steep SimK and True-K (TK from the IOLM and TCP IOL from the G4) values and astigmatism magnitude were compared. RESULTS: 50 eyes of 50 patients were included. The mean difference in SimK and True-K between devices (IOLM - G4) was -0.04 (95% CI -0.13 to 0.06; P > .05) diopters (D) and 1.14 (95% CI 1.02 to 1.25; P < .05) D, respectively. The IOLM measured steeper True-K values than the G4. There were no statistically significant differences between devices for all other SimK values, whereas for True-K there were significant differences in flat K and steep K ( P < .05), but not astigmatism magnitude. CONCLUSIONS: Despite an overall good correlation in postmyopic laser refractive surgery eyes in keratometry and astigmatism measurements, there is a significant difference in True-K, with the IOLM measuring steeper values by about 1.0 D compared with the G4, similar to prior studies on nonrefractive surgery eyes.

Morbidity and mortality of multivisceral resection with radical nephrectomy for locally advanced renal cell carcinoma: An analysis of the National Surgical Quality Improvement Program (NSQIP) database.

INTRODUCTION: Locally advanced renal cell carcinoma (RCC) can rarely invade into adjacent abdominal viscera without clinical evidence of distant metastases. The role of multivisceral resection (MVR) of involved adjacent organs at the time of radical nephrectomy (RN) remains poorly described and quantified. Using a national database, we aimed to evaluate the association between RN+MVR and 30-day postoperative complications. METHODS AND MATERIALS: We conducted a retrospective cohort study of adult patients undergoing RN for RCC with and without MVR between 2005 and 2020 using the ACS-NSQIP database. The primary outcome was a composite of any of the following 30-day major postoperative complications: mortality, reoperation, cardiac event, and neurologic event. Secondary outcomes included individual components of the composite primary outcome, as well as infectious and venous thromboembolic complications, unplanned intubation and ventilation, transfusion, readmission, and prolonged length of stay (LOS). Groups were balanced using propensity score matching. Likelihood of complications was assessed by conditional logistic regression adjusted for unbalanced total operation time. Postoperative complications were compared by Fisher's exact test among subtypes of resection. RESULTS: A total of 12,417 patients were identified: 12,193 (98.2%) undergoing RN alone and 224 (1.8%) undergoing RN+MVR. Patients undergoing RN+MVR were more likely to experience major complications (odds ratio [OR] 2.46; 95% confidence interval [CI] 1.28-4.74). However, there was no significant association between RN+MVR and postoperative mortality (OR 2.49; 95% CI 0.89-7.01). RN+MVR was associated with higher rates of reoperation (OR 7.85; 95% CI 2.38-25.8), sepsis (OR 5.45; 95% CI 1.83-16.2), surgical site infection (OR 4.41; 95% CI 2.14-9.07), blood transfusion (OR 2.24; 95% CI 1.55-3.22), readmission (OR 1.78; 95% CI 1.11-2.84), infectious complications (OR 2.62; 95% CI 1.62-4.24), and longer hospital stay (5 days [IQR 3-8] vs. 4 days [IQR 3-7]; OR 2.31 [95% CI 2.13-3.03]).  There was no heterogeneity in the association between subtype of MVR and major complication rate. CONCLUSION: Undergoing RN+MVR is associated with an increased risk of 30-day postoperative morbidity, including infectious complications, reoperation, blood transfusion, prolonged LOS, and readmission.

The infective polymerization of conformationally unstable antithrombin mutants may play a role in the clinical severity of antithrombin deficiency.

Mutations affecting mobile domains of antithrombin induce conformational instability resulting in protein polymerization that associates with a severe clinical phenotype, probably by an unknown gain of function. By homology with other conformational diseases, we speculated that these variants might infect wild-type (WT) monomers reducing the anticoagulant capacity. Infective polymerization of WT polymers and different P1 mutants (p.R425del, p.R425C and p.R425H) were evaluated by using native gels and radiolabeled WT monomers and functional assays. Human embryonic kidney cells expressing the Epstein-Barr nuclear antigen 1 (HEK-EBNA) cells expressing inducible (p.R425del) or two novel constitutive (p.F271S and p.M370T) conformational variants were used to evaluate intracellular and secreted antithrombin under mild stress (pH 6.5 and 39°C for 5 h). We demonstrated the conformational sensitivity of antithrombin London (p.R425del) to form polymers under mild heating. Under these conditions purified antithrombin London recruited WT monomers into growing polymers, reducing the anticoagulant activity. This process was also observed in the plasma of patients with p.R425del, p.R425C and p.R425H mutations. Under moderate stress, coexpression of WT and conformational variants in HEK-EBNA cells increased the intracellular retention of antithrombin and the formation of disulfide-linked polymers, which correlated with impaired secretion and reduction of anticoagulant activity in the medium. Therefore, mutations inducing conformational instability in antithrombin allow its polymerization with the subsequent loss of function, which under stress could sequestrate WT monomers, resulting in a new prothrombotic gain of function, particularly relevant for intracellular antithrombin. The in vitro results suggest a temporal and severe plasma antithrombin deficiency that may contribute to the development of the thrombotic event and to the clinical severity of these mutations.

COVID-19 and communication: A sentiment analysis of US state governors' official press releases.

OBJECTIVES: This study examines the contents of official communication from United States governors' offices related to the COVID-19 pandemic to assess patterns in communication and to determine if they correlate with trends for COVID cases and deaths. METHODS: We collected text data for all COVID-19 related press releases between March 1 and December 31, 2020 from the US governors' office websites in all 50 states. An automated parsing and sentiment analyzer assessed descriptive statistics and trends in tone, including positivity and negativity. RESULTS: We included a total of 7,720 press releases in this study. We found that both positive and negative sentiments were homogenous across states at the beginning of the pandemic but became heterogeneous as the pandemic evolved. The same trend applied to the frequency and tone of press releases. Sentiments across states were overall positive with a small level of negativity. We observed a reactive official communication to the evolution of the number of COVID-19 cases rather than responsive or preventive. CONCLUSIONS: The findings of both positivity and negativity in press communications suggest that the effect of discounted importance was present in official communications. Our findings support a state-dependent optimal communication frequency and tone, agreeing with the curvilinear communication model of organizational theory and implying that feedback cycles between government officials and public response should be shortened to rapidly maximize communication efficacy during the pandemic. Future research should identify and evaluate the drivers of the large differences in communication tone across states and validate the reactive characteristics of COVID-19 official communications.

Pancreatic head vs pancreatic body/tail cancer: Are they different?

BACKGROUND: The impact of pancreatic tumor location on patient survival has been studied in large national data-based analyses which yielded controversial results. AIM: To explore if pancreatic head cancer (PHC) and pancreatic body/tail cancer (PBTC) have different overall survival (OS), molecular signature and response to chemotherapy. METHODS: We retrospectively queried patient records from July 2016 to June 2020 in our institution. Patient demographics, cancer stage on diagnosis, tumor location, somatic mutations, treatment, and survival are recorded and analyzed. A test is considered statistically significant if the P value was < 0.05. RESULTS: We reviewed 101 patients with complete records, among which 67 (66.34%) were PHC and 34 (33.66%) were PBTC. More PHC were diagnosed at younger age [61.49 vs 68.97, P = 0.010], earlier stages (P = 0.006) and underwent surgical resection (P = 0.025). There were no significant differences among all mutations and pathways studied except for TP53 mutations (37.0% in PHC vs 70.0% in PBTC, P = 0.03). OS was not statistically different between PHC and PBTC (P = 0.636) in the overall population and in subgroups according to surgical resection status or stages. In terms of response to chemotherapy, chemotherapy regimens (FOLFIRINOX-based vs gemcitabine-based) didn't impact disease free interval in those who had surgical resection in either PHC (P = 0.546) or PBTC (P = 0.654), or the duration of response to first line palliative treatment in those with advanced disease in PHC (P = 0.915) or PBTC (P = 0.524). CONCLUSION: Even though PHC and PBTC have similar poor OS and response to chemotherapy, the different presentations and molecular profiles indicate they are different diseases. Utilization of molecular profiling to develop targeted therapy for individualization of treatment is needed.