Fragment-based screening targeting an open form of the SARS-CoV-2 main protease binding pocket.

To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography.

The New Youngest Case of Grange Syndrome with a Novel Biallelic Pathogenic Variant in YY1AP1.

Introduction: Grange syndrome (OMIM 602531) is characterized by a constellation of symptoms of hypertension, stenosis, or occlusion of different arteries (including the cerebral, renal, abdominal, and coronary vessels) with a variable occurrence of brachysyndactyly, bone fragility, and congenital heart defects. Learning disabilities were also reported in some cases. Biallelic pathogenic variants in YY1AP1 are associated with the syndrome. Only 14 individuals with this ultra-rare syndrome (12 of them were molecularly confirmed) have hitherto been reported in the literature. Case Presentation: We herein describe a 11/2-year-old additional female case of Grange syndrome with hypertension, patent ductus arteriosus, and brachysyndactyly who was subsequently confirmed to carry a novel homozygous frameshift variant (c.2291del; p.Pro764Leufs*12) in the YY1AP1 gene through whole-exome sequencing. Conclusion: This report extends the allelic spectrum in Grange syndrome and helps provide insight into the potential role of YY1AP1 in the regulation of cellular processes.

SDU - software for high-throughput automated data collection at the Swiss Light Source.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

Anterior and Posterior Pulmonary Cusp Augmentation in the Repair of Tetralogy of Fallot.

BACKGROUND: In this retrospective study, we compared the results from 2 pulmonary valve augmentation techniques in patients undergoing surgical repair of tetralogy of Fallot. METHODS: Between 2015 and 2018, 18 patients had anterior pulmonary valve repair at our institution, and 26 patients had both anterior and posterior pulmonary valve repair. RESULTS: Patients ranged from 6 months to 30 years of age. The median follow-up period was 8 months in the anterior augmentation group and 5 months in the anterior and posterior augmentation group. Postoperative echocardiograms indicated that only 2 patients (11%) in the anterior augmentation group had moderate or severe pulmonary insufficiency, compared with no patients in the anterior and posterior augmentation group. At follow-up, pulmonary insufficiency was seen in 3 patients (17%) in the anterior augmentation group and no patients in the anterior and posterior augmentation group. CONCLUSION: Reconstruction of the native pulmonary valve accompanied by pulmonary cusp augmentation can decrease or even circumvent postoperative pulmonary insufficiency. Both anterior augmentation and anterior and posterior augmentation techniques are easily applied; however, we believe that the anterior and posterior augmentation technique is superior in terms of early postoperative and follow-up pulmonary insufficiency outcomes.

Probing ligand binding of endothiapepsin by `temperature-resolved' macromolecular crystallography.

Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery.

Fast fragment- and compound-screening pipeline at the Swiss Light Source.

Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported.

Asymptomatic cardiac tumour with premature ventricular contraction in an athlete: case report.

Cardiac fibromas are the second most common benign primary tumour of the heart in the children; the clinical features include chest pain, arrhythmia, low cardiac output due to outflow tract obstruction, and sudden cardiac death. Sports are associated with an increased risk for sudden death in athletes who are affected by cardiovascular conditions predisposing to life-threatening arrhythmias. We present a case report of 10-year-old asymptomatic boy who was referred to the paediatric cardiology department by his general practitioner for cardiac examination before participation in competitive sports. The electrocardiogram showed premature ventricular contractions originated from inferior of left ventricle. A mass was detected by 2D transthoracic echocardiography, and it was found to be compatible with fibroma on MRI. In some cases, cardiac tumours are asymptomatic as in our patient. Electrocardiogram abnormalities require detailed cardiac imaging with echocardiogram, and if necessary CT/MRI. In this article, we emphasise that detailed cardiac examination of individuals before participating in competitive sports is vital.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

Differential recognition of oligomannose isomers by glycan-binding proteins involved in innate and adaptive immunity.

The recognition of oligomannose-type glycans in innate and adaptive immunity is elusive due to multiple closely related isomeric glycan structures. To explore the functions of oligomannoses, we developed a multifaceted approach combining mass spectrometry assignments of oligomannose substructures and the development of a comprehensive oligomannose microarray. This defined microarray encompasses both linear and branched glycans, varying in linkages, branching patterns, and phosphorylation status. With this resource, we identified unique recognition of oligomannose motifs by innate immune receptors, including DC-SIGN, L-SIGN, Dectin-2, and Langerin, broadly neutralizing antibodies against HIV gp120, N-acetylglucosamine-1-phosphotransferase, and the bacterial adhesin FimH. The results demonstrate that each protein exhibits a unique specificity to oligomannose motifs and suggest the potential to rationally design inhibitors to selectively block these protein-glycan interactions.

Identification of a novel pathogenic variant in CKAP2L and literature review in a child with Filippi syndrome and congenital talipes equinovarus.

Filippi syndrome (MIM #272440), one of the craniodigital syndromes, is a rare genetic entity with autosomal recessive inheritance and characterized by pre- and postnatal growth retardation, microcephaly, distinctive facial appearance, developmental delay/intellectual disability, and variable syndactylies of the fingers and toes. In this report, a further female patient of Filippi syndrome who additionally had a unilateral congenital talipes equinovarus (CTEV), a feature not previously recorded, is described. Genetic testing revealed a novel homozygous frameshift pathogenic variant (c.552_555delCAAA, p.Asn184Lysfs*8) in CKAP2L and thus confirmed the diagnosis of Filippi syndrome. We hope that the newly recognized feature (CTEV) will contribute to expand the clinical spectrum of this extremely rare condition. In view of the paucity of reported cases, the full spectrum of clinical findings of Filippi syndrome necessitates obviously further affected individuals/pedigrees delineation in order to elucidate the etiological and phenotypic aspects of this orphan disease appropriately.

Aortopulmonary window with anomalous origin of the right coronary artery from the pulmonary trunk.

Anomalous origin of coronary artery originating from the pulmonary artery in conjunction with the aorticopulmonary window (APW) is a rare but a significant anomaly in the era of congenital cardiac diseases. The occurrence of anomalous origin of the right coronary artery from the pulmonary artery among the associated anomalies is less than 5%. The severity of the clinical condition of these patients depends on the degree of left-right shunt and compromise of the pulmonary blood flow. We report surgical management of a case of a 45-day-old infant with APW, ventricular septal defect, and anomalous origin of coronary artery originated from the pulmonary artery.

Antibodies from Lampreys as Smart Anti-Glycan Reagents (SAGRs): Perspectives on Their Specificity, Structure, and Glyco-genomics.

The repertoire of glycans expressed by individual cells and tissues is enormous, and various estimates indicate that thousands of different glycans and "glycan determinants" are critical for functional recognition by glycan-binding proteins. Defining the steady-state expression and functional impacts of the human glycome will require a concerted worldwide effort, along with the development of new immunological, genetic, chemical, and biochemical technologies. Here, we describe the generation of smart anti-glycan reagents (SAGRs), recombinant antibodies that recognize novel glycan determinants. The antibodies are generated by the sea lamprey (Petromyzon marinus), through immunization with glycoconjugates, cells, and even tissues. SAGRs represent a versatile immunological tool for defining the expression of glycans in cells and tissues. We also present a comparison of lamprey-derived anti-carbohydrate antibodies that have been characterized to date. Finally, we explore the unique glyco-genome of the lamprey itself as it compares to those of humans and mice and how it may relate to the lamprey's inherent capacity to produce antibodies to mammalian glycans.

Tp-e interval and Tp-e/QT ratio in clinically stable pediatric heart transplant recipients.

OBJECTIVE: A study of the T wave peak-to-end (Tp-e) interval, the Tp-e/QT ratio, and the Tp-e/QTc ratio in pediatric heart transplant recipients (HTRs), a group which has a higher risk of sudden cardiac death than the normal population, has not previously been reported. The aim of this study was to assess alterations in ventricular repolarization using the Tp-e interval, Tp-e/ QT ratio, and Tp-e/QTc ratio in clinically stable pediatric HTRs. METHODS: A total of 13 clinically stable HTRs, 13 patients who had undergone cardiac surgery (CS) under cardiopulmonary bypass, and 16 healthy controls under 18 years of age were retrospectively evaluated. RESULTS: No significant differences were observed between the HTR, CS, and control groups in terms of QTc, JTc interval, and T wave amplitude (p>0.05). The Tp-e interval (p=0.001), Tp-e/QT ratio (p<0.001), and Tp-e/QTc ratio (p=0.001) were significantly higher in the HTR group compared with the CS and normal control participants. CONCLUSION: The Tp-e interval, Tp-e/QT ratio, and Tp-e/QTc ratio were elevated in stable HTRs compared with the normal and CS groups.

Electroanatomic mapping-guided pediatric catheter ablation with limited/zero fluoroscopy.

OBJECTIVE: The use of fluoroscopy in pediatric catheter ablation has decreased because of mapping systems. In this study, we present the efficiency and reliability of the electroanatomic mapping system in nonfluoroscopic pediatric catheter ablation. METHODS: The medical records of patients aged <18 years who underwent ablation between November 2016 and April 2018 were evaluated. Fluoroscopy was not used in cases involving ablation of right sided-arrhythmia foci. Fluoroscopy was used only for trans-septal puncture or retroaortic approach/coronary angiography. RESULTS: A total of 76 patients underwent catheter ablation for 78 supraventricular and ventricular tachyarrhythmia substrates under the guidance of EnSite Velocity system. Fluoroscopy was used in only 14 (18.4%) of these substrates. The mean fluoroscopy duration in these 14 procedures was 5.4±3.15 min. No complications were noted, except a temporary right bundle branch block in one patient and pericardial effusion in another following cryoablation. The acute success rate in achieving complete elimination of arrhythmia substrates was 97.4% (76/78). The recurrence rate was 5.1% (4/78) at follow-up. CONCLUSION: Fluoroscopy can be completely eliminated in most pediatric catheter ablation procedures with the use of mapping systems by achieving high acute success rates and acceptable low complication rates.

Heart rate variability parameters in children with ventricular preexcitation.

INTRODUCTION: The autonomic nervous system has a regulatory effect on cardiac electrophysiology and arrhythmogenesis. We aimed to assess cardiac autonomic status using heart rate variability (HRV) parameters in children with ventricular preexcitation. METHODS: The electrocardiography, Holter monitoring, transesophageal electrophysiological study (TEEPS), and invasive electrophysiological study (EPS) results of ventricular preexcitation patients obtained over a 7-year period in our clinic were evaluated. According to the TEEPS results, patients' accessory pathway conduction was classified as adverse (n = 40) or nonadverse (n = 25). The HRV parameters of patients were compared according to tachycardia inducibility that assessed by TEEPS and EPS. Also, HRV parameters were compared in patients with adverse and nonadverse pathway conduction. Further, the HRV parameters of preexcitation patients were compared with those of healthy controls. RESULTS: LF/HF, the best measure of sympathovagal balance, was statistically higher in patients with adverse conduction than in patients without adverse conduction and controls (P  =  0.001). The LF/HF ratio was higher in ventricular preexcitation patients with inducible tachycardia than those without in EPS (P  =  0.001). In addition, the LF/HF ratio was higher in symptomatic ventricular preexcitation patients than asymptomatic ones (P  =  0.001). No difference in HRV parameters was found between preexcitation patients and controls. CONCLUSION: Autonomic tonus in patients with ventricular preexcitation may affect accessory pathway conduction properties, tachycardia inducibility, and symptomology. The indicator of sympathovagal balance, LF/HF ratio, increased in ventricular preexcitation patients with inducible tachycardia and those that were symptomatic.

Histopathological evaluation of aortic coarctation after conventional balloon angioplasty in neonates.

BACKGROUND: Optimal management strategy for native aortic coarctation in neonates and young infants is still a matter of debate. The surgical procedure, histopathologic research, and clinical outcome in 15 neonates who underwent surgery after successful balloon angioplasty is the basis of this study. METHOD: Between 01 October, 2014 and 01 August, 2017, we enrolled 15 patients with native aortic coarctation for this study. These patients had complications regarding recoarctation, following balloon angioplasty intervention at our institute and other centres. Surgically extracted parts were examined histopathologically and patient's data were collected retrospectively.ResultThe reasons for recurrence of recoarctation after balloon angioplasty are as follows: patients with higher preoperative echocardiographic gradients had recoarctation earlier, neointimal proliferation, aortic intimal fibrosis at the region of ductal insertion, and ductal residual tissue debris after balloon angioplasty. No repeat intervention was required in the 15 patients who underwent surgery followed by balloon angioplasty. Early mortality was seen in one patient after surgery. Postoperative complication in the surgical group occurred in the form of chylothorax in one patient. CONCLUSION: In centres in which the neonatal ICU is inexperienced, balloon angioplasty is particularly recommended. In developing neonatal clinics, balloon angioplasty, when performed on patients at their earliest possible age, delays actual corrective operation to a later date, which in turn provides less risky surgical outcomes in infants who are gaining weight, growing, and do not have any haemodynamic complaints.

Conformational switch of the bacterial adhesin FimH in the absence of the regulatory domain: Engineering a minimalistic allosteric system.

For many biological processes such as ligand binding, enzymatic catalysis, or protein folding, allosteric regulation of protein conformation and dynamics is fundamentally important. One example is the bacterial adhesin FimH, where the C-terminal pilin domain exerts negative allosteric control over binding of the N-terminal lectin domain to mannosylated ligands on host cells. When the lectin and pilin domains are separated under shear stress, the FimH-ligand interaction switches in a so-called catch-bond mechanism from the low- to high-affinity state. So far, it has been assumed that the pilin domain is essential for the allosteric propagation within the lectin domain that would otherwise be conformationally rigid. To test this hypothesis, we generated mutants of the isolated FimH lectin domain and characterized their thermodynamic, kinetic, and structural properties using isothermal titration calorimetry, surface plasmon resonance, nuclear magnetic resonance, and X-ray techniques. Intriguingly, some of the mutants mimicked the conformational and kinetic behaviors of the full-length protein and, even in absence of the pilin domain, conducted the cross-talk between allosteric sites and the mannoside-binding pocket. Thus, these mutants represent a minimalistic allosteric system of FimH, useful for further mechanistic studies and antagonist design.

What contributes to an effective mannose recognition domain?

In general, carbohydrate-lectin interactions are characterized by high specificity but also low affinity. The main reason for the low affinities are desolvation costs, due to the numerous hydroxy groups present on the ligand, together with the typically polar surface of the binding sites. Nonetheless, nature has evolved strategies to overcome this hurdle, most prominently in relation to carbohydrate-lectin interactions of the innate immune system but also in bacterial adhesion, a process key for the bacterium's survival. In an effort to better understand the particular characteristics, which contribute to a successful carbohydrate recognition domain, the mannose-binding sites of six C-type lectins and of three bacterial adhesins were analyzed. One important finding is that the high enthalpic penalties caused by desolvation can only be compensated for by the number and quality of hydrogen bonds formed by each of the polar hydroxy groups engaged in the binding process. In addition, since mammalian mannose-binding sites are in general flat and solvent exposed, the half-lives of carbohydrate-lectin complexes are rather short since water molecules can easily access and displace the ligand from the binding site. In contrast, the bacterial lectin FimH benefits from a deep mannose-binding site, leading to a substantial improvement in the off-rate. Together with both a catch-bond mechanism (i.e., improvement of affinity under shear stress) and multivalency, two methods commonly utilized by pathogens, the affinity of the carbohydrate-FimH interaction can be further improved. Including those just described, the various approaches explored by nature to optimize selectivity and affinity of carbohydrate-lectin interactions offer interesting therapeutic perspectives for the development of carbohydrate-based drugs.

Two cases of Kawasaki disease presented with acute febrile jaundice.

Kawasaki disease is an acute, systemic vasculitis of unknown etiology. Although gastrointestinal involvement does not belong to the classic diagnostic criteria; diarrhea, abdominal pain, hepatic dysfunction, hydrops of gallbladder, and acute febrile cholestatic jaundice are reported in patients with Kawasaki disease. We describe here two cases presented with fever, and acute jaundice as initial features of Kawasaki disease.