Mapping interactions of calmodulin and neuronal NO synthase by crosslinking and mass spectrometry.

Neuronal nitric oxide synthase (nNOS) is a homodimeric cytochrome P450-like enzyme that catalyzes the conversion of L-arginine to nitric oxide in the presence of NADPH and molecular oxygen. The binding of calmodulin (CaM) to a linker region between the FAD/FMN-containing reductase domain, and the heme-containing oxygenase domain is needed for electron transfer reactions, reduction of the heme, and NO synthesis. Due to the dynamic nature of the reductase domain and low resolution of available full-length structures, the exact conformation of the CaM-bound active complex during heme reduction is still unresolved. Interestingly, hydrogen-deuterium exchange and mass spectrometry studies revealed interactions of the FMN domain and CaM with the oxygenase domain for iNOS, but not nNOS. This finding prompted us to utilize covalent crosslinking and mass spectrometry to clarify interactions of CaM with nNOS. Specifically, MS-cleavable bifunctional crosslinker disuccinimidyl dibutyric urea was used to identify thirteen unique crosslinks between CaM and nNOS as well as 61 crosslinks within the nNOS. The crosslinks provided evidence for CaM interaction with the oxygenase and reductase domain residues as well as interactions of the FMN domain with the oxygenase dimer. Cryo-EM studies, which gave a high-resolution model of the oxygenase domain, along with crosslink-guided docking provided a model of nNOS that brings the FMN within 15 Å of the heme in support for a more compact conformation than previously observed. These studies also point to the utility of covalent crosslinking and mass spectrometry in capturing transient dynamic conformations that may not be captured by hydrogen-deuterium exchange and mass spectrometry experiments.

Aortic dilatation: Value of echocardiography in the systematic assessment of elite rugby players in the French National Rugby League (LNR).

The value of echocardiography in the screening of athletes in addition to the electrocardiogram is debated and still unclear. 336 rugby players in French professional divisions (Top 14, Pro D2) were prospectively assessed with electrocardiogram and echocardiography. 75% were Caucasian, 16.4% Pacific Islanders, and 8.6% Afro-Caribbean. Six (1.8%) players had electrocardiogram abnormalities, exclusively negative T waves. Twenty-one (6.25%) of them had abnormal echocardiography findings: one possible early hypertrophic cardiomyopathy, one anomalous origin of coronary artery, two left ventricular dilatations, one isolated bicuspid aortic valve, two aortic regurgitations, and 14 ascending aortic dilatations. The median aortic diameter was modestly correlated with age: 32 mm [23-48] in players aged ≤25 years vs 33.5 mm [24-50] in those aged >25 years (P = 0.02, correlation coefficient -.01). This tendency increased with cumulative hours of weight training: 34 mm [24-50] in forwards vs 32 mm [25-44] in backs (P = 0.01); and ethnicity, with Pacific Islanders having higher values in both raw data and body surface area or height-indexed data than Afro-Caribbeans and Caucasians: 34 [25-50] vs 32 [27-48] and 33 [23-49] mm (P = 0.017); 15 [12.2-21] vs 14.8 [11-19.9] and 14.8 [10-20.9] mm/m2 (P < 0.0001); 18.5 [14-25] mm/m vs 17.4 [14.8-25] mm/m and 17.6 [12.2-25.3] mm/m (P = 0.0125). In a population of professional rugby players, echocardiography was contributive. The main anomaly was aortic dilatation (14/336, 4.2%). While this is proportionally much higher than in other sports, the cutoffs need to be defined more precisely by including the criterion of ethnicity, as is already the case for electrocardiography.

Characterization of repolarization in Brugada syndrome patients during exercise testing: Dynamic angle evaluation.

BACKGROUND: A new ECG criterion has been studied in Brugada syndrome (BrS) at rest to differentiate type 2 and incomplete right bundle branch block (IRBBB). METHODS: We assessed this criterion during exercise comparing BrS (46 patients) and IRBBB (17 patients). A beta angle was measured from lead V1 between the upslope of S-wave and the downslope of the r'-wave. RESULTS: Beta angle was significantly larger in BrS at rest (58±24° vs 25±15°, p<0.001), exercise (47±26° vs 15±11°, p<0.001), and recovery (46±24° vs 21±12°, p<0.001) with a reduction in angle at exercise compared to rest. There was a significant rebound in angle at recovery in the control group to (p<0.001); no such rebound was observed in the BrS group (p=NS). CONCLUSION: Beta angle study at rest and its evolution at exercise could help discriminate BrS patients from healthy subjects.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor-binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with stabilized Spike ectodomain. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high-affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high-affinity (0.53-4.2 nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron and Delta pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

Computational pipeline provides mechanistic understanding of Omicron variant of concern neutralizing engineered ACE2 receptor traps.

The SARS-CoV-2 Omicron variant, with 15 mutations in Spike receptor binding domain (Spike-RBD), renders virtually all clinical monoclonal antibodies against WT SARS-CoV-2 ineffective. We recently engineered the SARS-CoV-2 host entry receptor, ACE2, to tightly bind WT-Spike-RBD and prevent viral entry into host cells ("receptor traps"). Here we determine cryo-EM structures of our receptor traps in complex with full length Spike. We develop a multi-model pipeline combining Rosetta protein modeling software and cryo-EM to allow interface energy calculations even at limited resolution and identify interface side chains that allow for high affinity interactions between our ACE2 receptor traps and Spike-RBD. Our structural analysis provides a mechanistic rationale for the high affinity (0.53 - 4.2nM) binding of our ACE2 receptor traps to Omicron-RBD confirmed with biolayer interferometry measurements. Finally, we show that ACE2 receptor traps potently neutralize Omicron- and Delta-pseudotyped viruses, providing alternative therapeutic routes to combat this evolving virus.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation.

Without an effective prophylactic solution, infections from SARS-CoV-2 continue to rise worldwide with devastating health and economic costs. SARS-CoV-2 gains entry into host cells via an interaction between its Spike protein and the host cell receptor angiotensin converting enzyme 2 (ACE2). Disruption of this interaction confers potent neutralization of viral entry, providing an avenue for vaccine design and for therapeutic antibodies. Here, we develop single-domain antibodies (nanobodies) that potently disrupt the interaction between the SARS-CoV-2 Spike and ACE2. By screening a yeast surface-displayed library of synthetic nanobody sequences, we identified a panel of nanobodies that bind to multiple epitopes on Spike and block ACE2 interaction via two distinct mechanisms. Cryogenic electron microscopy (cryo-EM) revealed that one exceptionally stable nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains (RBDs) locked into their inaccessible down-state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for SARS-CoV-2 Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains stability and function after aerosolization, lyophilization, and heat treatment. These properties may enable aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia, promising to yield a widely deployable, patient-friendly prophylactic and/or early infection therapeutic agent to stem the worst pandemic in a century.

An ultrapotent synthetic nanobody neutralizes SARS-CoV-2 by stabilizing inactive Spike.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus enters host cells via an interaction between its Spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2). By screening a yeast surface-displayed library of synthetic nanobody sequences, we developed nanobodies that disrupt the interaction between Spike and ACE2. Cryo-electron microscopy (cryo-EM) revealed that one nanobody, Nb6, binds Spike in a fully inactive conformation with its receptor binding domains locked into their inaccessible down state, incapable of binding ACE2. Affinity maturation and structure-guided design of multivalency yielded a trivalent nanobody, mNb6-tri, with femtomolar affinity for Spike and picomolar neutralization of SARS-CoV-2 infection. mNb6-tri retains function after aerosolization, lyophilization, and heat treatment, which enables aerosol-mediated delivery of this potent neutralizer directly to the airway epithelia.

New Technique for Surgical Epicardial Implantation of a Cardioverter-Defibrillator in Children and Adults With Congenital Heart Disease.

BACKGROUND: We assessed a surgical technique for implanting a cardioverter-defibrillator. The indications for cardioverter-defibrillator implantation in pediatric patients and adults with congenital heart disease are relatively specific and require multidisciplinary discussion regarding implantation modalities. We coupled the positioning of two coils sutured to the pericardium with an implantable cardioverter-defibrillator device inserted within a supradiaphragmatic pocket in a population of children and adults with congenital heart disease. METHODS: Thirteen consecutive patients, either children or adults with congenital heart disease, underwent the implantation of a single-chamber implantable cardioverter-defibrillator in our center. All patients were systematically followed at 3, 6, and 12 months after implantation. RESULTS: Patients were mainly male (n = 9, 69%), and mean age was 21 ± 12 years. Median follow-up was 13 months. All patients underwent surgery without acute complication. One patient needed a second surgery because of defibrillation coils fracture. Neither infectious complication nor inappropriate shock was noted. There were two appropriate shocks in 1 patient. CONCLUSIONS: This new technique for surgical epicardial implantation of a cardioverter-defibrillator in children and adults with congenital heart disease is safe and feasible. These results should be confirmed by prospective studies with long-term follow-up.

Subcutaneous implantable cardioverter defibrillators in children, young adults and patients with congenital heart disease.

The demonstration of severe complications in patients implanted with a transvenous implantable cardioverter defibrillator (ICD) has led to the development of devices equipped with a subcutaneous lead. This new technique offers numerous advantages but also certain disadvantages. Various studies or anecdotal clinical cases have specifically been conducted with this subcutaneous defibrillation system in children and/or patients with congenital heart disease. Results of these studies suggest: 1) a high feasibility despite being limited by a selection process that excludes patients requiring permanent pacing and patients declared ineligible during pre-screening; 2) good efficacy of electrical shocks in reducing induced or spontaneous ventricular arrhythmias; 3) in this specific subset of patients, 2 types of complications have been particularly described: a risk of device exteriorization and infection, and a large number of inappropriate therapies primarily related to T-wave oversensing. The subcutaneous ICD could therefore constitute the gold standard for patients with complex congenital heart disease with no venous access to the heart or with a persistent shunt increasing the risk of systemic emboli as well as in young patients with channelopathy or hypertrophic cardiomyopathy not requiring long-term pacing. Technological change (reduction in device size, better differentiation between R- and T-waves, possibility of pacing if device coupled with a leadless pacemaker) could reduce the limitations and complications and thereby increase the indications in this sub-group of patients.