An α-ketoglutarate conformational switch controls iron accessibility, activation, and substrate selection of the human FTO protein.

The fat mass and obesity-associated fatso (FTO) protein is a member of the Alkb family of dioxygenases and catalyzes oxidative demethylation of N6-methyladenosine (m6A), N1-methyladenosine (m1A), 3-methylthymine (m3T), and 3-methyluracil (m3U) in single-stranded nucleic acids. It is well established that the catalytic activity of FTO proceeds via two coupled reactions. The first reaction involves decarboxylation of alpha-ketoglutarate (αKG) and formation of an oxyferryl species. In the second reaction, the oxyferryl intermediate oxidizes the methylated nucleic acid to reestablish Fe(II) and the canonical base. However, it remains unclear how binding of the nucleic acid activates the αKG decarboxylation reaction and why FTO demethylates different methyl modifications at different rates. Here, we investigate the interaction of FTO with 5-mer DNA oligos incorporating the m6A, m1A, or m3T modifications using solution NMR, molecular dynamics (MD) simulations, and enzymatic assays. We show that binding of the nucleic acid to FTO activates a two-state conformational equilibrium in the αKG cosubstrate that modulates the O2 accessibility of the Fe(II) catalyst. Notably, the substrates that provide better stabilization to the αKG conformation in which Fe(II) is exposed to O2 are demethylated more efficiently by FTO. These results indicate that i) binding of the methylated nucleic acid is required to expose the catalytic metal to O2 and activate the αKG decarboxylation reaction, and ii) the measured turnover of the demethylation reaction (which is an ensemble average over the entire sample) depends on the ability of the methylated base to favor the Fe(II) state accessible to O2.

Temperature-sensitive contacts in disordered loops tune enzyme I activity.

Homologous enzymes with identical folds often exhibit different thermal and kinetic behaviors. Understanding how an enzyme sequence encodes catalytic activity at functionally optimal temperatures is a fundamental problem in biophysics. Recently it was shown that the residues that tune catalytic activities of thermophilic/mesophilic variants of the C-terminal domain of bacterial enzyme I (EIC) are largely localized within disordered loops, offering a model system with which to investigate this phenomenon. In this work, we use molecular dynamics simulations and mutagenesis experiments to reveal a mechanism of sequence-dependent activity tuning of EIC homologs. We find that a network of contacts in the catalytic loops is particularly sensitive to changes in temperature, with some contacts exhibiting distinct linear or nonlinear temperature-dependent trends. Moreover, these trends define structurally clustered dynamical modes and can distinguish regions that tend toward order or disorder at higher temperatures. Assaying several thermophilic EIC mutants, we show that complementary mesophilic mutations to the most temperature-sensitive positions exhibit the most enhanced activity, while mutations to relatively temperature insensitive positions exhibit the least enhanced activities. These results provide a mechanistic explanation of sequence-dependent temperature tuning and offer a computational method for rational enzyme modification.

Temperature sensitive contact modes allosterically gate TRPV3.

TRPV Ion channels are sophisticated molecular sensors designed to respond to distinct temperature thresholds. The recent surge in cryo-EM structures has provided numerous insights into the structural rearrangements accompanying their opening and closing; however, the molecular mechanisms by which TRPV channels establish precise and robust temperature sensing remain elusive. In this work we employ molecular simulations, multi-ensemble contact analysis, graph theory, and machine learning techniques to reveal the temperature-sensitive residue-residue interactions driving allostery in TRPV3. We find that groups of residues exhibiting similar temperature-dependent contact frequency profiles cluster at specific regions of the channel. The dominant mode clusters on the ankyrin repeat domain and displays a linear melting trend while others display non-linear trends. These modes describe the residue-level temperature response patterns that underlie the channel's functional dynamics. With network analysis, we find that the community structure of the channel changes with temperature. And that a network of high centrality contacts connects distant regions of the protomer to the gate, serving as a means for the temperature-sensitive contact modes to allosterically regulate channel gating. Using a random forest model, we show that the contact states of specific temperature-sensitive modes are indeed predictive of the channel gate's state. Supporting the physical validity of these modes and networks are several residues identified with our analyses that are reported in literature to be functionally critical. Our results offer high resolution insight into thermo-TRP channel function and demonstrate the utility of temperature-sensitive contact analysis.

Solution Structure Ensembles of the Open and Closed Forms of the ∼130 kDa Enzyme I via AlphaFold Modeling, Coarse Grained Simulations, and NMR.

Large-scale interdomain rearrangements are essential to protein function, governing the activity of large enzymes and molecular machineries. Yet, obtaining an atomic-resolution understanding of how the relative domain positioning is affected by external stimuli is a hard task in modern structural biology. Here, we show that combining structural modeling by AlphaFold2 with coarse-grained molecular dynamics simulations and NMR residual dipolar coupling data is sufficient to characterize the spatial domain organization of bacterial enzyme I (EI), a ∼130 kDa multidomain oligomeric protein that undergoes large-scale conformational changes during its catalytic cycle. In particular, we solve conformational ensembles for EI at two different experimental temperatures and demonstrate that a lower temperature favors sampling of the catalytically competent closed state of the enzyme. These results suggest a role for conformational entropy in the activation of EI and demonstrate the ability of our protocol to detect and characterize the effect of external stimuli (such as mutations, ligand binding, and post-translational modifications) on the interdomain organization of multidomain proteins. We expect the ensemble refinement protocol described here to be easily transferrable to the investigation of the structure and dynamics of other uncharted multidomain systems and have assembled a Google Colab page (https://potoyangroup.github.io/Seq2Ensemble/) to facilitate implementation of the presented methodology elsewhere.

Serratus anterior and pectoralis plane blocks for robotically assisted mitral valve repair: a randomised clinical trial.

BACKGROUND: Minimally invasive cardiac surgery provokes substantial pain and therefore analgesic consumption. The effect of fascial plane blocks on analgesic efficacy and overall patient satisfaction remains unclear. We therefore tested the primary hypothesis that fascial plane blocks improve overall benefit analgesia score (OBAS) during the initial 3 days after robotically assisted mitral valve repair. Secondarily, we tested the hypotheses that blocks reduce opioid consumption and improve respiratory mechanics. METHODS: Adults scheduled for robotically assisted mitral valve repairs were randomised to combined pectoralis II and serratus anterior plane blocks or to routine analgesia. The blocks were ultrasound-guided and used a mixture of plain and liposomal bupivacaine. OBAS was measured daily on postoperative Days 1-3 and were analysed with linear mixed effects modelling. Opioid consumption was assessed with a simple linear regression model and respiratory mechanics with a linear mixed model. RESULTS: As planned, we enrolled 194 patients, with 98 assigned to blocks and 96 to routine analgesic management. There was neither time-by-treatment interaction (P=0.67) nor treatment effect on total OBAS over postoperative Days 1-3 with a median difference of 0.08 (95% confidence interval [CI]: -0.50 to 0.67; P=0.69) and an estimated ratio of geometric means of 0.98 (95% CI: 0.85-1.13; P=0.75). There was no evidence of a treatment effect on cumulative opioid consumption or respiratory mechanics. Average pain scores on each postoperative day were similarly low in both groups. CONCLUSIONS: Serratus anterior and pectoralis plane blocks did not improve postoperative analgesia, cumulative opioid consumption, or respiratory mechanics during the initial 3 days after robotically assisted mitral valve repair. CLINICAL TRIAL REGISTRATION: NCT03743194.

Two-year outcomes after transcatheter aortic valve-in-valve implantation in degenerated surgical valves.

BACKGROUND: Transcatheter aortic valve-in-valve implantation (ViV-TAVI) has emerged in recent years as a safe alternative to redo surgery in high-risk patients. Although early results are encouraging, data beyond short-term outcomes are lacking. Herein, we aimed to assess the 2-year outcomes after ViV-TAVI. METHODS: Patients undergoing ViV-TAVI for degenerated surgical valves between 2013 and 2019 at the Cleveland Clinic were reviewed. The coprimary endpoints were all-cause mortality and congestive heart failure (CHF) hospitalizations. We used time-to-event analyses to assess the primary outcomes. Further, we measured the changes in transvalvular gradients and the incidence of structural valve deterioration (SVD). RESULTS: One hundred and eighty-eight patients were studied (mean age = 76 years; 65% males). At 2 years of follow-up, all-cause mortality and CHF hospitalizations occurred in 15 (8%) and 28 (14.9%) patients, respectively. On multivariable analysis, the postprocedural length of stay was a significant predictor for both all-cause mortality (hazard ratio [HR] = 1.1; 95% confidence interval [CI]: 1.01, 1.19) and CHF hospitalization (HR = 1.16; 95% CI: 1.07, 1.27). However, the internal diameter of the surgical valve was not associated with significant differences in both primary endpoints. For hemodynamic outcomes, nine patients (4.8%) developed SVD. The mean and peak transvalvular pressure gradients remained stable over the follow-up period. CONCLUSION: ViV-TAVI for degenerated surgical valves was associated with favorable 2-year clinical and hemodynamic outcomes. Further studies are needed to better understand the role of ViV-TAVI as a treatment option in the life management of aortic valve disease.

Early mortality and neurologic outcomes following mitral valve surgery in the very elderly.

OBJECTIVES: Valve repair is the gold standard for treatment of degenerative mitral valve disease. As the population ages, patients undergoing valve degeneration and therefore considered for mitral valve surgery will naturally be getting older. We sought to evaluate whether mitral repair retained a survival advantage over replacement in patients ≥80 years old. METHODS: A retrospective cohort study was performed using data acquired from the United Kingdom National Adult Cardiac Surgery Audit for the outcomes of in-hospital mortality and postoperative cerebrovascular event (CVA). Individual multivariable logistic regression models were created to investigate adjusted associations between these outcomes and type of mitral valve operation, repair or replacement. Additionally, associations between the individual model parameters and in-hospital mortality and CVA were investigated. RESULTS: A total of 1140 patients underwent mitral repair (66.4%, median age 82.3), and 577 patients underwent mitral replacement (33.6%, median age 82.1). The overall age range was 80-92. The incidence of in-hospital mortality favored the repair group (4.4% vs. 8.3%, p = .001). Multivariable logistic regression modeling demonstrated an increased adjusted odds of in-hospital mortality for mitral valve replacement (MVR) (odd ratio [OR]: 2.01, 1.15-3.50, p = .01). The only other parameter associated with an increased adjusted odds of in-hospital mortality was postoperative dialysis (OR: 14.2, 7.67-26.5, p < .001). There was not a demonstrated association between MVR and perioperative CVA (OR: 1.11, 0.49-2.4, p = .8). CONCLUSIONS: In patients ≥80 years old, mitral valve repair (MVr) was shown to be associated with a decreased adjusted odds of mortality, with a null association with CVA. These results suggest that, if feasible, MVr should remain the preferred management strategy, even in the very elderly.

Incidence and short-term outcomes of surgical bailout after transcatheter mitral valve repair with the MitraClip system.

OBJECTIVES: This study sought to investigate the incidence and outcomes of surgical bailout (SB) after transcatheter mitral valve repair (TMVr) with MitraClip. BACKGROUND: TMVr poses a risk of serious procedural complications, possibly requiring urgent open surgery for SB. However, little is known about the risk of SB cases after TMVr. METHODS: We retrospectively identified patients who underwent TMVr using the Nationwide Readmissions Database 2014-2017. SB was defined as open thoracotomy for heart and aorta during the same hospitalization. Annual hospital volume was defined as the annual number of TMVr cases in each hospital in each year. RESULTS: Among 15,032 eligible patients, SB was required in 214 (1.42%), of whom 134 (62.6%) underwent mitral valve surgery (113 replacements; 21 repairs). The incidence of SB was decreasing significantly over the 4 years (5.26% in 2014; 0.43% in 2017; ptrend < .001). There was a significant nonlinear, inverse association of annual hospital volume with the incidence of SB. In-hospital death (15.0 vs. 2.1%; p < .001) and other in-hospital adverse events were significantly more frequent in patients with than without SB, whereas the 30-day readmission rate was similar (13.2 vs. 15.1%; p = .572). After adjustment for patient and hospital characteristics, SB was significantly associated with higher in-hospital mortality (odds ratio = 6.67, 95% confidence interval = 4.35, 10.23, p < .001). CONCLUSIONS: This study suggests that although the incidence of SB after TMVr is decreasing, SB is required more frequently in lower-volume hospitals and carries high in-hospital mortality. Further efforts are needed to understand the reasons for SB and improve outcomes in patients needing SB.

Clinical outcomes associated with retrograde arterial perfusion in minimally invasive mitral valve surgery: a systematic review.

INTRODUCTION: Given several reports of an increased neurologic risk with retrograde arterial perfusion in minimally invasive mitral valve surgery, we sought to identify and synthesize the best available evidence on the influence of perfusion strategy on post-operative clinical outcomes in this population. METHODS: A systematic search of PubMed, EMBASE, MEDLINE, and Cochrane library databases was performed to identify publications comparing clinical outcomes associated with antegrade and retrograde arterial perfusion in minimally invasive mitral valve surgery. Pre-specified outcomes of interest were neurologic events, mortality, and renal failure. The search was performed by two independent reviewers, with data abstraction following. RESULTS: Seven observational studies were included in this review, with a total patient population of 5,385. Six were retrospective cohort in design, with a single small prospective cohort study identified. When available, adjusted publication-specific risk estimates were abstracted and included preferentially over unadjusted or reviewer-derived risk estimates. Meta-analysis was felt to be heavily flawed in the context of few small studies identified and was not performed. In adjusted estimates, there appeared to be an increased risk of neurologic complications with retrograde arterial perfusion. There was a null pattern apparent between arterial perfusion strategy and each of 30-day mortality and renal failure. CONCLUSION: Retrograde arterial perfusion in minimally invasive mitral valve surgery may be associated with an increased risk of neurologic events, without affecting the risk of 30-day mortality or renal failure. Although these patterns were identified, an overall paucity of evidence justifies further study.

Techniques for Mitral Valve Re-repair.

The emergence of mitral valve repair as the preferred treatment for severe mitral regurgitation (MR) caused by degenerative disease has been accompanied by an increasing number of valve repair failures seen by surgeons. Consequently, the feasibility of valve re-repair vs valve replacement at the time of reoperation has become a valid clinical consideration. In this report we explore the mechanisms of mitral valve repair failure as well as factors that meaningfully influence the likelihood of a successful re-repair. We provide illustrations of techniques for re-repair that we have used with reliable success, informed by the mechanism of repair failure. Lastly, we share our outcomes for mitral valve re-repair over the last 5 years and discuss our experience using the techniques illustrated in this report.

Long-term neural regeneration following injury to the peroneal branch of the sciatic nerve in sheep.

Peripheral nerves (PNs) are frequently injured as a result of trauma or disease. Development of therapies to regenerate PNs requires the use of animal models, typically beginning in rodents and progressing to larger species. There are several large animal models of PN regeneration that each has their benefits and drawbacks. Sheep have been used in PN studies due to their similarities in body weight to humans and the ease and lesser expense in their care and housing relative to other species. We have investigated the use of sheep for studies of PN regeneration and have developed and tested an injury model in the peroneal branch of the sciatic nerve. Three experimental groups were tested on mature sheep: a bisection; a 5-cm reverse autograft; and sham surgery. Protocols were developed for the post-operative care for animals with this injury, and regeneration was tracked for extended time points via compound muscle action potentials (CMAPs) and endpoint assessments of nerve morphometry, muscle mass and muscle fibrosis. Results indicate the practical viability of this PN injury model and show distinctions in the degree and rate of regeneration between bisection and reverse autograft that persisted 14 months. This long-term study shows bisections lead to significantly improved CMAPS and muscle mass and lesser muscle fibrosis as compared to reverse autograft. The persistence of these discernable changes between two relatively similar experimental groups out to extended time points is an indication of the sensitivity of this nerve section and its potential applicability for comparative studies.

Comparison of acute recoil after valve deployment and after post-dilation in patients undergoing transfemoral-transcatheter aortic valve replacement with SAPIEN-3 valve.

BACKGROUND: Transcatheter aortic valves are prone to acute recoil similar to the metal-based coronary stents. However, it is not clear if recoil remains a factor only after the initial valve deployment or also after post-dilation. METHODS: We conducted a retrospective observational study of patients who underwent transfemoral transcatheter aortic valve replacement (TAVR) with SAPIEN-3 valve. Acute recoil at the upper, central, and lower levels of the valve was calculated in both anteroposterior right anterior oblique (RAO) and lateral left anterior oblique (LAO) views after initial deployment as well as after post-dilation. The average recoil of the RAO and LAO views was also calculated and described as RAO/LAO. RESULTS: The acute recoil in the RAO/LAO views (mean ± SD) was 3.9 ± 1.1% after valve deployment in the whole study population (n = 257). Among the subset of patients who required post-dilation (n = 133), the mean acute recoil in the RAO/LAO views was found to be greater after initial valve deployment as compared with after post-dilation (3.8 ± 1.1% vs. 3.0 ± 0.9%; p < .001). Further, acute recoil was significantly greater in the RAO view than the LAO view and at the central level of the prosthesis as compared with the upper and lower levels. Those findings were consistent after initial deployment as well as after post-dilation. Clinical outcomes were similar between patients who required post-dilation compared to those who did not. In multivariable logistic regression analysis, only smaller valve cover index was found to be an independent predictor of 30-day mild or greater aortic regurgitation (OR 0.007; 95% CI 0.0001-0.707; p = .035). CONCLUSION: Acute elastic recoil of the SAPIEN-3 valve was significantly less after post-dilation as compared with after deployment. It was also greater when measured in the RAO view as compared with the LAO view. Furthermore, acute recoil was not homogenous across the height of the valve stent frame.

Early failure after non-resectional mitral valve repair with artificial chordae.

Severe recurrent mitral regurgitation within 1 year of mitral valve repair is usually attributed to a technical issue with the original repair procedure. However, when artificial chordae are employed to correct mitral valve prolapse, ventricular remodeling (ie, decreased ventricular size) can lead to recurrent prolapse and valve dysfunction. To illustrate this phenomenon, we present two patients who experienced early failure after undergoing mitral valve repair with artificial chordae.

Robotic trans-mitral septal myectomy and papillary muscle reorientation for HOCM combined with or without mitral valve repair: Technical aspects - How we do it.

Hypertrophic obstructive cardiomyopathy (HOCM) is one of the more common genetic disorders. The pathophysiology and natural history of the disease have been well studied. Left ventricular outflow tract obstruction (LVOTO) and systolic anterior motion (SAM) of the anterior mitral leaflet can result in sudden cardiac death, progressive heart failure and arrythmias. Surgical septal myectomy for HOCM is the standard of care and is routinely performed through a median sternotomy. Septal myectomy has also been performed using the trans-atrial, trans-mitral approach either directly or with robotic assistance. In cases with severe LVOT obstruction in the setting of only mild to moderate proximal septal hypertrophy, intrinsic problems with the mitral valve contribute. Typically, these are hypermobile papillary muscles and or excessive height of the anterior mitral leaflet. Combining septal myectomy with reorientation of hypermobile anteriorly positioned papillary muscles has shown to prevent SAM and thereby additionally decrease the subvalvular aortic outflow obstruction. Our extensive experience in both septal myectomy and robotic mitral valve repair has given us a different perspective in approaching the primary mitral regurgitation in HOCM patients where a combined septal myectomy, papillary muscle reorientation and complex mitral valve repair has been safely performed using the less invasive robotic-assisted approach.Our objective here is to discuss the technical aspects of the procedure.

Left Of Bang Interventions in Trauma: ethical implications for military medical prophylaxis.

Advances in medical capability should be accompanied by discussion of their ethical implications. In the military medical context there is a growing interest in developing prophylactic interventions that will mitigate the effects of trauma and improve survival. The ethics of this novel capability are currently unexplored. This paper describes the concept of trauma prophylaxis (Left Of Bang Interventions in Trauma) and outlines some of the ethical issues that need to be considered, including within concept development, research and implementation. Trauma prophylaxis can be divided into interventions that do not (type 1) and those that do (type 2) have medical enhancement as an unintended side effect of their prophylactic action. We conclude that type 1 interventions have much in common with established military medical prophylaxis, and the potentially enhancing qualities of type 2 interventions raise different issues. We welcome further debate on both interventions.

UK Role 4 military infectious diseases and tropical medicine cases in 2005-2013.

INTRODUCTION: Infectious diseases are a frequent cause of morbidity among British troops. The aim of this paper is to describe the spectrum of infectious diseases seen when UK service personnel are evacuated for definitive care to the Role 4 Medical Treatment Facility based at Birmingham Heartlands Hospital. METHOD: A retrospective analysis of all military patients presenting with infectious diseases and treated at Birmingham Heartlands Hospital between 14 April 2005 and 31 December 2013 was undertaken. RESULTS: During this period, 502 patients were identified. Infections originated in 49 countries, most commonly Afghanistan (46% cases), the UK (10% cases) and Belize (9% of cases). The most common presentations were dermatological conditions, gastroenterological illnesses and undifferentiated fevers. CONCLUSION: UK service personnel in significant numbers continue to suffer a wide range of infectious diseases, acquired throughout the globe, which often require specialist tertiary infection services to diagnose and manage. Future prospective data collection is recommended to identify trends, which in turn will inform military training needs and future research priorities in the Defence Medical Services (DMS) and allows development of appropriate policies and clinical guidelines for management of DMS personnel with infectious diseases.

Neuropathologic features of TOMM40 '523 variant on late-life cognitive decline.

INTRODUCTION: The study investigated the role of neuropathologies in the relationship between TOMM40 '523 genotype and late-life cognitive decline. METHODS: Participants were community-dwelling older persons who had annual cognitive assessments and brain autopsies after death. Genotyping used DNA from peripheral blood or postmortem brain tissue. Linear mixed models assessed the extent to which the association of '523 genotype with cognitive decline is attributable to neuropathologies. RESULTS: Relative to ε3/ε3 homozygotes with '523-S/VL or '523-VL/VL genotype, both '523-L carriers and ε3/ε3 homozygotes with '523-S/S genotype had faster cognitive decline. The association of '523-L with cognitive decline was attenuated and no longer significant after controlling for Alzheimer's and other neuropathologies. By contrast, the association of '523-S/S was unchanged. DISCUSSION: There are two distinct TOMM40 '523 signals in relation to late-life cognitive decline. One signal primarily acts through AD and other common neuropathologies, whereas the other operates through a different mechanism.

Novel porcine model of cutaneous myiasis: a training tool for healthcare workers.

Cutaneous myiasis is a well-described problem in travellers to endemic regions including military personnel. Realistic training is important to ensure that healthcare workers have the confidence and expertise to recognise cutaneous myiasis and safely remove larvae if required. A model is described here that is simple, reproducible and realistic, and will allow for training of military healthcare workers in safe surgical removal of larvae when required.

The SSV Evaluation System: A Tool to Prioritize Short Structural Variants for Studies of Possible Regulatory and Causal Variants.

Short structural variants (SSVs) are short genomic variants (<50 bp) other than SNPs. It has been suggested that SSVs contribute to many human complex traits. However, high-throughput analysis of SSVs presents numerous technical challenges. In order to facilitate the discovery and assessment of SSVs, we have developed a prototype bioinformatics tool, "SSV evaluation system," which is a searchable, annotated database of SSVs in the human genome, with associated customizable scoring software that is used to evaluate and prioritize SSVs that are most likely to have significant biological effects and impact on disease risk. This new bioinformatics tool is a component in a larger strategy that we have been using to discover potentially important SSVs within candidate genomic regions that have been identified in genome-wide association studies, with the goal to prioritize potential functional/causal SSVs and focus the follow-up experiments on a relatively small list of strong candidate SSVs. We describe our strategy and discuss how we have used the SSV evaluation system to discover candidate causal variants related to complex neurodegenerative diseases. We present the SSV evaluation system as a powerful tool to guide genetic investigations aiming to uncover SSVs that underlie human complex diseases including neurodegenerative diseases in aging.

New Genetic Approaches to AD: Lessons from APOE-TOMM40 Phylogenetics.

Clinical trials for Alzheimer's disease are now focusing on the earliest stages of the disease with the goal of delaying dementia onset. There is great utility in using genetic variants to identify individuals at high age-dependent risk when the goal is to begin treatment before the development of any cognitive symptoms. Genetic variants identified through large-scale genome-wide association studies have not substantially improved the accuracy provided by APOE genotype to identify people at high risk of late-onset Alzheimer's disease (LOAD). We describe novel approaches, focused on molecular phylogenetics, to finding genetic variants that predict age at LOAD onset with sufficient accuracy and precision to be useful. We highlight the discovery of a polymorphism in TOMM40 that, in addition to APOE, may improve risk prediction and review how TOMM40 genetic variants may impact the develop of LOAD independently from APOE. The analysis methods described in this review may be useful for other genetically complex human diseases.