Structural determinants of ivabradine block of the open pore of HCN4.

HCN1-4 channels are the molecular determinants of the If/Ih current that crucially regulates cardiac and neuronal cell excitability. HCN dysfunctions lead to sinoatrial block (HCN4), epilepsy (HCN1), and chronic pain (HCN2), widespread medical conditions awaiting subtype-specific treatments. Here, we address the problem by solving the cryo-EM structure of HCN4 in complex with ivabradine, to date the only HCN-specific drug on the market. Our data show ivabradine bound inside the open pore at 3 Å resolution. The structure unambiguously proves that Y507 and I511 on S6 are the molecular determinants of ivabradine binding to the inner cavity, while F510, pointing outside the pore, indirectly contributes to the block by controlling Y507. Cysteine 479, unique to the HCN selectivity filter (SF), accelerates the kinetics of block. Molecular dynamics simulations further reveal that ivabradine blocks the permeating ion inside the SF by electrostatic repulsion, a mechanism previously proposed for quaternary ammonium ions.

Rare and Common Genetic Variation Underlying Atrial Fibrillation Risk.

Importance: Atrial fibrillation (AF) has a substantial genetic component. The importance of polygenic risk is well established, while the contribution of rare variants to disease risk warrants characterization in large cohorts. Objective: To identify rare predicted loss-of-function (pLOF) variants associated with AF and elucidate their role in risk of AF, cardiomyopathy (CM), and heart failure (HF) in combination with a polygenic risk score (PRS). Design, Setting, and Participants: This was a genetic association and nested case-control study. The impact of rare pLOF variants was evaluated on the risk of incident AF. HF and CM were assessed in cause-specific Cox regressions. End of follow-up was July 1, 2022. Data were analyzed from January to October 2023. The UK Biobank enrolled 502 480 individuals aged 40 to 69 years at inclusion in the United Kingdom between March 13, 2006, and October 1, 2010. UK residents of European ancestry were included. Individuals with prior diagnosis of AF were excluded from analyses of incident AF. Exposures: Rare pLOF variants and an AF PRS. Main Outcomes and Measures: Risk of AF and incident HF or CM prior to and subsequent to AF diagnosis. Results: A total of 403 990 individuals (218 489 [54.1%] female) with a median (IQR) age of 58 (51-63) years were included; 24 447 were diagnosed with incident AF over a median (IQR) follow-up period of 13.3 (12.4-14.0) years. Rare pLOF variants in 6 genes (TTN, RPL3L, PKP2, CTNNA3, KDM5B, and C10orf71) were associated with AF. Of these, TTN, RPL3L, PKP2, CTNNA3, and KDM5B replicated in an external cohort. Combined with high PRS, rare pLOF variants conferred an odds ratio of 7.08 (95% CI, 6.03-8.28) for AF. Carriers with high PRS also had a substantial 10-year risk of AF (16% in female individuals and 24% in male individuals older than 60 years). Rare pLOF variants were associated with increased risk of CM both prior to AF (hazard ratio [HR], 3.13; 95% CI, 2.24-4.36) and subsequent to AF (HR, 2.98; 95% CI, 1.89-4.69). Conclusions and Relevance: Rare and common genetic variation were associated with an increased risk of AF. The findings provide insights into the genetic underpinnings of AF and may aid in future genetic risk stratification.

Ion channel inhibition by targeted recruitment of NEDD4-2 with divalent nanobodies.

Targeted recruitment of E3 ubiquitin ligases to degrade traditionally undruggable proteins is a disruptive paradigm for developing new therapeutics. Two salient limitations are that <2% of the ~600 E3 ligases in the human genome have been exploited to produce proteolysis targeting chimeras (PROTACs), and the efficacy of the approach has not been demonstrated for a vital class of complex multi-subunit membrane proteins- ion channels. NEDD4-1 and NEDD4-2 are physiological regulators of myriad ion channels, and belong to the 28-member HECT (homologous to E6AP C-terminus) family of E3 ligases with widespread roles in cell/developmental biology and diverse diseases including various cancers, immunological and neurological disorders, and chronic pain. The potential efficacy of HECT E3 ligases for targeted protein degradation is unexplored, constrained by a lack of appropriate binders, and uncertain due to their complex regulation by layered intra-molecular and posttranslational mechanisms. Here, we identified a nanobody that binds with high affinity and specificity to a unique site on the N-lobe of the NEDD4-2 HECT domain at a location physically separate from sites critical for catalysis- the E2 binding site, the catalytic cysteine, and the ubiquitin exosite- as revealed by a 3.1 Å cryo-electron microscopy reconstruction. Recruiting endogenous NEDD4-2 to diverse ion channel proteins (KCNQ1, ENaC, and CaV2.2) using a divalent (DiVa) nanobody format strongly reduced their functional expression with minimal off-target effects as assessed by global proteomics, compared to simple NEDD4-2 overexpression. The results establish utility of a HECT E3 ligase for targeted protein downregulation, validate a class of complex multi-subunit membrane proteins as susceptible to this modality, and introduce endogenous E3 ligase recruitment with DiVa nanobodies as a general method to generate novel genetically-encoded ion channel inhibitors.

Revealing biomolecular structure and motion with neural ab initio cryo-EM reconstruction.

Proteins and other biomolecules form dynamic macromolecular machines that are tightly orchestrated to move, bind, and perform chemistry. Cryo-electron microscopy (cryo-EM) can access the intrinsic heterogeneity of these complexes and is therefore a key tool for understanding mechanism and function. However, 3D reconstruction of the resulting imaging data presents a challenging computational problem, especially without any starting information, a setting termed ab initio reconstruction. Here, we introduce a method, DRGN-AI, for ab initio heterogeneous cryo-EM reconstruction. With a two-step hybrid approach combining search and gradient-based optimization, DRGN-AI can reconstruct dynamic protein complexes from scratch without input poses or initial models. Using DRGN-AI, we reconstruct the compositional and conformational variability contained in a variety of benchmark datasets, process an unfiltered dataset of the DSL1/SNARE complex fully ab initio, and reveal a new "supercomplex" state of the human erythrocyte ankyrin-1 complex. With this expressive and scalable model for structure determination, we hope to unlock the full potential of cryo-EM as a high-throughput tool for structural biology and discovery.

Structural and molecular basis of choline uptake into the brain by FLVCR2.

Choline is an essential nutrient that the human body needs in vast quantities for cell membrane synthesis, epigenetic modification and neurotransmission. The brain has a particularly high demand for choline, but how it enters the brain remains unknown1-3. The major facilitator superfamily transporter FLVCR1 (also known as MFSD7B or SLC49A1) was recently determined to be a choline transporter but is not highly expressed at the blood-brain barrier, whereas the related protein FLVCR2 (also known as MFSD7C or SLC49A2) is expressed in endothelial cells at the blood-brain barrier4-7. Previous studies have shown that mutations in human Flvcr2 cause cerebral vascular abnormalities, hydrocephalus and embryonic lethality, but the physiological role of FLVCR2 is unknown4,5. Here we demonstrate both in vivo and in vitro that FLVCR2 is a BBB choline transporter and is responsible for the majority of choline uptake into the brain. We also determine the structures of choline-bound FLVCR2 in both inward-facing and outward-facing states using cryo-electron microscopy. These results reveal how the brain obtains choline and provide molecular-level insights into how FLVCR2 binds choline in an aromatic cage and mediates its uptake. Our work could provide a novel framework for the targeted delivery of therapeutic agents into the brain.

Post-transcriptional regulation and subcellular localization of G-protein γ7 subunit: implications for striatal function and behavioral responses to cocaine.

The striatal D1 dopamine receptor (D1R) and A2a adenosine receptor (A2aR) signaling pathways play important roles in drug-related behaviors. These receptors activate the Golf protein comprised of a specific combination of αolfß2γ7 subunits. During assembly, the γ7 subunit sets the cellular level of the Golf protein. In turn, the amount of Golf protein determines the collective output from both D1R and A2aR signaling pathways. This study shows the Gng7 gene encodes multiple γ7 transcripts differing only in their non-coding regions. In striatum, Transcript 1 is the predominant isoform. Preferentially expressed in the neuropil, Transcript 1 is localized in dendrites where it undergoes post-transcriptional regulation mediated by regulatory elements in its 3' untranslated region that contribute to translational suppression of the γ7 protein. Earlier studies on gene-targeted mice demonstrated loss of γ7 protein disrupts assembly of the Golf protein. In the current study, morphological analysis reveals the loss of the Golf protein is associated with altered dendritic morphology of medium spiny neurons. Finally, behavioral analysis of conditional knockout mice with cell-specific deletion of the γ7 protein in distinct populations of medium spiny neurons reveals differential roles of the Golf protein in mediating behavioral responses to cocaine. Altogether, these findings provide a better understanding of the regulation of γ7 protein expression, its impact on Golf function, and point to a new potential target and mechanisms for treating addiction and related disorders.

Identification and Characterization of a Bacterial Periplasmic Solute Binding Protein That Binds l-Amino Acid Amides.

Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.

Golf cart injuries have similar severity to all-terrain vehicle injuries in children: a multicenter comparison over a 5-year period.

Background: Golf carts (GCs) and all-terrain vehicles (ATVs) are popular forms of personal transport. Although ATVs are considered adventurous and dangerous, GCs are perceived to be safer. Anecdotal experience suggests increasing numbers of both GC and ATV injuries, as well as high severity of GC injuries in children. This multicenter study examined GC and ATV injuries and compared their injury patterns, resource utilization, and outcomes. Methods: Pediatric trauma centers in Florida submitted trauma registry patients age <16 years from January 2016 to June 2021. Patients with GC or ATV mechanisms were identified. Temporal trends were evaluated. Injury patterns, resource utilization, and outcomes for GCs and ATVs were compared. Intensive care unit admission and immediate surgery needs were compared using multivariable logistic regression. Results: We identified 179 GC and 496 ATV injuries from 10 trauma centers. GC and ATV injuries both increased during the study period (R2 0.4286, 0.5946, respectively). GC patients were younger (median 11 vs 12 years, p=0.003) and had more intracranial injuries (34% vs 19%, p<0.0001). Overall Injury Severity Score (5 vs 5, p=0.27), intensive care unit (ICU) admission (20% vs 16%, p=0.24), immediate surgery (11% vs 11%, p=0.96), and mortality (1.7% vs 1.4%, p=0.72) were similar for GCs and ATVs, respectively. The risk of ICU admission (OR 1.19, 95% CI 0.74 to 1.93, p=0.47) and immediate surgery (OR 1.04, 95% CI 0.58 to 1.84, p=0.90) remained similar on multivariable logistic regression. Conclusions: During the study period, GC and ATV injuries increased. Despite their innocuous perception, GCs had a similar injury burden to ATVs. Heightened safety measures for GCs should be considered. Level of evidence: III, prognostic/epidemiological.

Atrial fibrillation: age at diagnosis, incident cardiovascular events, and mortality.

BACKGROUND AND AIMS: Patients with atrial fibrillation (AF) are at increased risks of cardiovascular diseases and mortality, but risks according to age at diagnosis have not been reported. This study investigated age-specific risks of outcomes among patients with AF and the background population. METHODS: This nationwide population-based cohort study included patients with AF and controls without outcomes by the application of exposure density matching on the basis of sex, year of birth, and index date. The absolute risks and hazard rates were stratified by age groups and assessed using competing risk survival analyses and Cox regression models, respectively. The expected differences in residual life years among participants were estimated. RESULTS: The study included 216 579 AF patients from year 2000 to 2020 and 866 316 controls. The mean follow-up time was 7.9 years. Comparing AF patients with matched controls, the hazard ratios among individuals ≤50 years was 8.90 [95% confidence interval (CI), 7.17-11.0] for cardiomyopathy, 8.64 (95% CI, 7.74-9.64) for heart failure, 2.18 (95% CI, 1.89-2.52) for ischaemic stroke, and 2.74 (95% CI, 2.53-2.96) for mortality. The expected average loss of life years among individuals ≤50 years was 9.2 years (95% CI, 9.0-9.3) years. The estimates decreased with older age. CONCLUSIONS: The findings show that earlier diagnosis of AF is associated with a higher hazard ratio of subsequent myocardial disease and shorter life expectancy. Further studies are needed to determine causality and whether AF could be used as a risk marker among particularly younger patients.

Use of veterinary medicinal products in the Philippines: regulations, impact, challenges, and recommendations.

Agricultural production is a major driver of the Philippine economy. Mass production of animal products, such as livestock and poultry farming, is one of the most prominent players in the field. Filipino farmers use veterinary medicinal products (VMPs) when raising agricultural animals to improve animal growth and prevent diseases. Unfortunately, the extensive use of VMPs, particularly antibiotics, has been linked to drug resistance in animals, particularly antibiotics. Antimicrobial gene products produced in animals due to the prolonged use of VMPs can passed on to humans when they consume animal products. This paper reviews information on the use of VMPs in the Philippines, including the regulations, their impact, challenges, and potential recommendations. The Philippines has existing legislation regulating VMP use. Several agencies were tasked to regulate the use of VMPs, such as the Department of Agriculture, the Department of Health, and the Philippine National Action Plan. Unfortunately, there is a challenge to implementing these regulations, which affects consumers. The unregulated use of VMPs influences the transmission of antibiotic residues from animals to crops to humans. This challenge should be addressed, with more focus on stricter regulation.

Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Optimizing Triage Practice in Pediatric Trauma: Lessons From Under-triage and Over-triage Rates and Risk Factors.

BACKGROUND: Triage accuracy is essential for delivering effective trauma care, especially in the pediatric population where unique challenges exist. The purpose of this study was to investigate risk factors contributing to under-triage and over-triage in an urban pediatric trauma center. METHODS: This retrospective cohort study included all trauma activations at an urban level 1 trauma center between January 1, 2021, and July 31, 2023 (patients <18 years old.) Patients who were under- or over-triaged were identified based on the level of trauma activation and injury severity score. RESULTS: There were 1094 trauma activations included in this study. The rate of under-triage was 3.8% (n = 42) and over-triage was 13.6% (n = 149). Infants aged 0-1 years had the highest rate of under-triage (10.9%, n = 19, P < .001), while those aged 11-17 had the highest rate of over-triage (17.0%, n = 82, P = .003). Non-accidental trauma was the strongest risk factor for under-triage (OR 30.2 [6.4-142.8] P < .001). Penetrating mechanism was the strongest risk factor for over-triage (OR 12.2 [5.6-26.2] P < .001). DISCUSSION: This study reveals the complexity of trauma triage in the pediatric population. We identified key predictive factors, such as age, comorbidities, and mechanism of injury, that can be used to refine triage practices and improve the care of pediatric trauma patients.

Strength and Functional Outcomes Following Achilles Tendon Reconstruction With Hamstring Tendon Autograft Augmentation.

BACKGROUND: The proposed advantages of hamstring autograft reconstruction when compared to alternative procedures, such as flexor hallucis longus (FHL) transfer, V-Y lengthening, and allograft reconstruction, are improved healing and reproduction of normal tendon biomechanics and reduced morbidity within the foot and ankle. In this study, we examined the effect of Achilles tendon reconstruction using hamstring autografts on strength and functional outcomes. METHODS: Patients who underwent Achilles repair with a hamstring autograft for insertional or midsubstance tendinopathy, delayed diagnosis of rupture, or infection after primary repair were evaluated for inclusion. Forty-six patients were identified; 12 further augmented with an FHL transfer are included in the analysis. Isokinetic testing was completed with a Biodex dynamometer under supervision of a physical therapist masked to surgical side. Pre- and postoperative Foot and Ankle Outcome Scores (FAOS, before March 2016) or Patient-Reported Outcomes Measurement Information System (PROMIS, after March 2016) surveys were collected. RESULTS: For knee flexion, peak torque was not significantly different when comparing operative and nonoperative sides at 180 degrees/second (45.38 Nm vs 45.96 Nm; P = .69) nor at 300 degrees/second (44.2 Nm vs 47.02 Nm; P = .069). Knee extension absolute peak torque was only found to be significantly weaker on the operative side at the faster testing (75.5 Nm vs 79.56 Nm; P < .05). Peak ankle plantarflexion torque was significantly weaker on the operative side at both the slower speed (60 degrees/second: 39.9 Nm vs 48.76 Nm; P < .005) and the faster speed (120 degrees/second: 31.3 Nm vs 40.7 Nm; P < .001). Average power for ankle plantarflexion did not differ significantly from the operative side to the nonoperative side in the slower test (26.46 W vs 27.48 W; P = .60) but did significantly differ on the faster test (32.13 W vs 37.63 W; P = .041). At an average of 19.9 months postoperation, all physical function and pain-related patient-reported outcome scores showed clinically and statistically significant improvement. CONCLUSION: Achilles reconstruction with a hamstring autograft ± FHL transfer allowed patients with severe Achilles pathology to return to good subjective function, with modest deficits in calf strength compared with the uninjured side. Overall knee flexion strength did not appear impaired. These results suggest that hamstring autograft reconstruction is a viable method to treat these complex cases involving a lack of healthy tissue, allowing patients to return to symptom-free physical function and athletic activity. LEVEL OF EVIDENCE: Level IV, case series.

Loss of Cardiac Splicing Regulator RBM20 Is Associated With Early-Onset Atrial Fibrillation.

We showed an association between atrial fibrillation and rare loss-of-function (LOF) variants in the cardiac splicing regulator RBM20 in 2 independent cohorts. In a rat model with loss of RBM20, we demonstrated altered splicing of sarcomere genes (NEXN, TTN, TPM1, MYOM1, and LDB3), and differential expression in key cardiac genes. We identified altered sarcomere and mitochondrial structure on electron microscopy imaging and found compromised mitochondrial function. Finally, we demonstrated that 3 novel LOF variants in RBM20, identified in patients with atrial fibrillation, lead to significantly reduced splicing activity. Our results implicate alternative splicing as a novel proarrhythmic mechanism in the atria.

New Ceramic Heads With Titanium Sleeves on Retained Femoral Components: Results of Over 500 Revision Total Hip Arthroplasties.

BACKGROUND: Ceramic heads are frequently combined with titanium sleeves in revision total hip arthroplasties (THAs), ostensibly to protect the ceramic head from existing damage to the retained trunnion. Although widely adopted, data on the performance and safety of this construct are minimal. The purpose of this study was to describe implant survivorships, radiographic results, and clinical outcomes of patients who underwent revision THA with a ceramic head and titanium sleeve on a retained femoral component. METHODS: We identified 516 revision THAs with femoral component retention (328 acetabular-only revisions and 188 bearing surface exchanges) treated with a new ceramic head and titanium sleeve between 2000 and 2020. Mean age at revision was 64 years, 56% were women, and mean body mass index was 30. The indications for revision THA were adverse local tissue reaction (25%), acetabular loosening (24%), dislocation (17%), infection (5%), and other (29%). Kaplan-Meier survivorships were analyzed, radiographs reviewed, and Harris Hip Scores evaluated. Mean follow-up was 4 years (range, 2 to 10). RESULTS: There were no reoperations or failures for ceramic head fracture, taper corrosion, or head/sleeve disengagement. The 10-year survivorship free of any re-revision was 85%. Indications for the 57 re-revisions included dislocation (33), infection (13), acetabular component loosening (7), periprosthetic fracture (2), psoas impingement (1), and sciatic nerve irritation (1). The 10-year survivorship free of any reoperation was 82%. There were an additional 14 reoperations. Radiographically, 1.9% had progressive femoral radiolucent lines, and 4.7% had progressive acetabular radiolucent lines. Mean Harris Hip Score was 81 at 2 years. CONCLUSIONS: New ceramic heads with titanium sleeves in revision THAs with retained femoral components were durable and reliable with no cases of ceramic head fracture or taper complications at mean 4-year follow-up, including those revised for adverse local tissue reaction. LEVEL OF EVIDENCE: IV.

Pulsed radiofrequency or surgery for anterior cutaneous nerve entrapment syndrome: Long-term results of a randomized controlled trial.

PURPOSE: Patients with anterior cutaneous nerve entrapment syndrome (ACNES) often require a step-up treatment strategy including abdominal wall injections, pulsed radiofrequency (PRF) or a neurectomy. Long-term success rates of PRF and surgery are largely unknown. The aim of the current study was to report on the long-term efficacy of PRF and neurectomy in ACNES patients who earlier participated in the randomized controlled PULSE trial. METHODS: Patients who completed the PULSE trial were contacted about pain status and additional treatments in the following years. Treatment success was based on numerical rating scale (NRS) following IMMPACT recommendations and Patient Global Impression of Change (PGIC) scores. RESULTS: A total of 44 of the original 60 patients were eligible for analysis (73.3%). Median follow-up was 71.5 months. One patient (4.3%) was still free of pain after a single PRF session, and five additional patients (21.7%) were free of pain by repetitive PRF treatments. By contrast, 13 patients (61.9%) in the neurectomy group were still free of pain without additional treatments. All pain recurrences and therefore primary re-interventions occurred in the first 2 years after the initial treatment. CONCLUSION: Approximately one in five ACNES patients undergoing PRF treatment reports long-term success obviating the need of surgical intervention. Surgery for ACNES is long-term effective in approximately two of three operated patients. Recurrent ACNES beyond 2 years after either intervention is rare.