Targeting prolyl-tRNA synthetase via a series of ATP-mimetics to accelerate drug discovery against toxoplasmosis.

The prolyl-tRNA synthetase (PRS) is a validated drug target for febrifugine and its synthetic analog halofuginone (HFG) against multiple apicomplexan parasites including Plasmodium falciparum and Toxoplasma gondii. Here, a novel ATP-mimetic centered on 1-(pyridin-4-yl) pyrrolidin-2-one (PPL) scaffold has been validated to bind to Toxoplasma gondii PRS and kill toxoplasma parasites. PPL series exhibited potent inhibition at the cellular (T. gondii parasites) and enzymatic (TgPRS) levels compared to the human counterparts. Cell-based chemical mutagenesis was employed to determine the mechanism of action via a forward genetic screen. Tg-resistant parasites were analyzed with wild-type strain by RNA-seq to identify mutations in the coding sequence conferring drug resistance by computational analysis of variants. DNA sequencing established two mutations, T477A and T592S, proximal to terminals of the PPL scaffold and not directly in the ATP, tRNA, or L-pro sites, as supported by the structural data from high-resolution crystal structures of drug-bound enzyme complexes. These data provide an avenue for structure-based activity enhancement of this chemical series as anti-infectives.

Manganese-dependent microRNA trimming by 3'→5' exonucleases generates 14-nucleotide or shorter tiny RNAs.

MicroRNAs (miRNAs) are about 22-nucleotide (nt) noncoding RNAs forming the effector complexes with Argonaute (AGO) proteins to repress gene expression. Although tiny RNAs (tyRNAs) shorter than 19 nt have been found to bind to plant and vertebrate AGOs, their biogenesis remains a long-standing question. Here, our in vivo and in vitro studies show several 3'→5' exonucleases, such as interferon-stimulated gene 20 kDa (ISG20), three prime repair exonuclease 1 (TREX1), and ERI1 (enhanced RNAi, also known as 3'hExo), capable of trimming AGO-associated full-length miRNAs to 14-nt or shorter tyRNAs. Their guide trimming occurs in a manganese-dependent manner but independently of the guide sequence and the loaded four human AGO paralogs. We also show that ISG20-mediated guide trimming makes Argonaute3 (AGO3) a slicer. Given the high Mn2+ concentrations in stressed cells, virus-infected cells, and neurodegeneration, our study sheds light on the roles of the Mn2+-dependent exonucleases in remodeling gene silencing.

Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant.

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function in vivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel in vivo animal model. At baseline, young AnkBp.E1458G+/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G+/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G+/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G+/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G+/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in in vivo cardiac function as well as the impact of single AnkB loss-of-function variants in vivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity.

Double drugging of prolyl-tRNA synthetase provides a new paradigm for anti-infective drug development.

Toxoplasmosis is caused by Toxoplasma gondii and in immunocompromised patients it may lead to seizures, encephalitis or death. The conserved enzyme prolyl-tRNA synthetase (PRS) is a validated druggable target in Toxoplasma gondii but the traditional 'single target-single drug' approach has its caveats. Here, we describe two potent inhibitors namely halofuginone (HFG) and a novel ATP mimetic (L95) that bind to Toxoplasma gondii PRS simultaneously at different neighbouring sites to cover all three of the enzyme substrate subsites. HFG and L95 act as one triple-site inhibitor in tandem and form an unusual ternary complex wherein HFG occupies the 3'-end of tRNA and the L-proline (L-pro) binding sites while L95 occupies the ATP pocket. These inhibitors exhibit nanomolar IC50 and EC50 values independently, and when given together reveal an additive mode of action in parasite inhibition assays. This work validates a novel approach and lays a structural framework for further drug development based on simultaneous targeting of multiple pockets to inhibit druggable proteins.

Diagnostic accuracy of daytime polysomnography: a reappraisal during the COVID-19 era.

Level I conventional polysomnography (PSG), the gold standard for diagnosing obstructive sleep apnea (OSA), requires an overnight stay. This study evaluated the role of daytime PSG as an alternative diagnostic tool. A prospective cohort study was undertaken with consecutive patients with suspected OSA at a tertiary care sleep center. The primary objective was to evaluate the sensitivity and diagnostic accuracy of daytime PSG for diagnosing OSA. The secondary objective was to find out the factors associated with a falsely negative daytime PSG result. All individuals were subjected to level I daytime PSG, done in the sleep lab in the presence of an experienced sleep technician during the daytime from 12 PM to 4 PM. Out of 162 patients, 105 underwent daytime PSG. OSA was diagnosed on daytime PSG in 86.7 out of the 19 remaining patients refused a repeat PSG study. Out of the 12 individuals who underwent the nighttime PSG for confirmatory diagnosis, 10 were diagnosed as OSA (false negatives), and 2 were confirmed as not-OSA (true negatives). The sensitivity, diagnostic accuracy, and negative predictive value of daytime PSG were 89.58%, 89.80%, and 16.67%, respectively. The false negatives had a higher prevalence of mild OSA. Daytime PSG is sensitive in diagnosing OSA and can be considered in individuals with severe symptoms at centers with a high patient load or when the individual wishes to avoid a nighttime study. A negative result in daytime PSG must be followed by conventional overnight PSG for confirmatory diagnosis.

Relevance of KCNJ5 in Pathologies of Heart Disease.

Abnormalities in G-protein-gated inwardly rectifying potassium (GIRK) channels have been implicated in diseased states of the cardiovascular system; however, the role of GIRK4 (Kir3.4) in cardiac physiology and pathophysiology has yet to be completely understood. Within the heart, the KACh channel, consisting of two GIRK1 and two GIRK4 subunits, plays a major role in modulating the parasympathetic nervous system's influence on cardiac physiology. Being that GIRK4 is necessary for the functional KACh channel, KCNJ5, which encodes GIRK4, it presents as a therapeutic target for cardiovascular pathology. Human variants in KCNJ5 have been identified in familial hyperaldosteronism type III, long QT syndrome, atrial fibrillation, and sinus node dysfunction. Here, we explore the relevance of KCNJ5 in each of these diseases. Further, we address the limitations and complexities of discerning the role of KCNJ5 in cardiovascular pathophysiology, as identical human variants of KCNJ5 have been identified in several diseases with overlapping pathophysiology.

Management of bronchial asthma in 2021.

To The Editor, The Global Initiative for Asthma (GINA) 2021 update was published on the 28th of April, 2021. There are significant changes, including treatment of mild asthma, the role of azithromycin, treatment of asthma in COVID-19 times, and role of biologics...

GINA 2020: what's new and why?

The Global initiative against asthma (GINA) 2020 strategy has been released with some changes and updates. GINA recommends the continuation of medications, avoidance of nebulization and spirometry, and ensuring a written asthma action plan in COVID-19 times. GINA 2020 specifies which step of management is to be followed according to the patient's symptoms in an easy flowchart. Clinicians need to be aware of the changes and the evidence behind them.

COVID-19 in intensive care. Some necessary steps for health care workers.

Due to the nature of their profession, health care personnel (HCP) have always been easy targets for transmission of communicable diseases like COVID-19. Shielding HCPs is of consequential significance in ensuring continued health care for the whole population in addition to reducing further spread. Close contact, repeated contact and prolonged contact are unavoidable in the intensive care (IC) environment. It is not uncommon for IC-HCPs to get carried away during an emergent situation, such as that posed by a suddenly deteriorating patient, and forgo the protective barriers that protect them from contracting a communicable infection. Some notable precautionary measures are mentioned below. This is by no means an exhaustive list.

Evaluation of the clinical profile, laboratory parameters and outcome of two hundred COVID-19 patients from a tertiary centre in India.

COVID-19 is a pandemic with over 5 million cases worldwide. The disease has imposed a huge burden on health resources. Evaluation of clinical and epidemiological profiles of such patients can help in understanding and managing the outbreak more efficiently. This study was a prospective observational analysis of 200 diagnosed COVID-19 patients admitted to a tertiary care center from 20th march to 8th May 2020. All these patients were positive for COVID-19 by an oro-nasopharyngeal swab-rtPCR based testing. Analyses of demographic factors, clinical characteristics, comorbidities, laboratory parameters, and the outcomes were performed. The mean age of the population was 40 years with a slight male predominance (116 patients out of 200, 58%). A majority of the patients (147, 73.5 %) were symptomatic, with fever being the most common symptom (109, 54.5%), followed by cough (91, 45.5%). An older age, presence of symptoms and their duration, leukocytosis, a high quick SOFA score, a high modified SOFA score, need for ventilator support, an AST level more than 3 times the upper limit of normal (ULN), and a serum creatinine level of 2 mg/dl or greater were at a significantly higher risk of ICU admission and mortality. Presence of diabetes mellitus, AST > three times ULN, serum creatinine 2 mg/dl or higher, and a qSOFA score of 1 or higher were all associated with significantly greater odds of critical care requirement. Triage and severity assessment helps in deciding the requirement for a hospital stay and ICU admission for COVID-19 which can easily be done using clinical and laboratory parameters. A mild, moderate and severe category approach with defined criteria and treatment guidelines will help in judicious utilization of health-care resources, especially for developing countries like India.   *Other members of the Safdarjung Hospital COVID-19 working group: Balvinder Singh (Microbiology), MK Sen (Pulmonary Medicine), Shibdas Chakrabarti (Pulmonary Medicine), NK Gupta (Pulmonary medicine), AJ Mahendran (Pulmonary Medicine), Ramesh Meena (Medicine), G Usha (Anaesthesiology), Santvana Kohli (Anaesthesiology), Sahil Diwan (Anaesthesiology), Rushika Saksena (Microbiology), Vikramjeet Dutta (Microbiology), Anupam Kr Anveshi (Microbiology).

Follow-up study of COVID-19 sequelae (FOSCO study).

INTRODUCTION: We undertook the first study from India to evaluate the long-term health effects of coronavirus disease 2019 (COVID-19). METHODS: The patients enrolled in our post-COVID-19 clinic were followed up for assessment at 1, 3, 6 and 12 months after recovery from acute disease prospectively. RESULTS: 200 patients with mean age of 50.72 years and 57.5% males were analysed. 42.5% had severe and 17% had moderate disease at the time of diagnosis. The persistence of symptoms beyond 1 month of diagnosis was seen in 72.5% (145/200) patients. 8% (16/200) of the patients had post-COVID-19 complications that required rehospitalisation after discharge or recovery from acute COVID-19. The complications included respiratory failure (2%), lung cavities (3.5%), fungal infection, pericardial effusion, pneumothorax and death. The symptoms were persistent beyond 3 months in 51% (102/200) and beyond 6 months in 17.5% (35/200) of cases. The patients with persistent symptoms beyond 3 months and 6 months had significantly higher intensive care unit (ICU) admission during acute COVID-19, severe disease during acute COVID-19, and higher prevalence of comorbidities compared to the recovered patients. The clinical recovery was attained in 95.5% (91/200) patients, and the radiological recovery was attained in 97.92% patients at 1 year. The mean duration to clinical recovery was 174.2 days. CONCLUSIONS: COVID-19 recovery takes longer time. However, clinico-radiological recovery is attained in >95% cases by one year.

A study on the prevalence of RLS in OSA and the consequences of co-occurrence.

Background: Restless leg syndrome (RLS) is common among patients with obstructive sleep apnoea (OSA) but the prognostic importance of this is not studied. We have called OSA and RLS coexistence as ComOSAR. Materials and Methods: A prospective observational study was done on patients referred for polysomnography (PSG) with the aims to evaluate 1) the prevalence of RLS in OSA and comparing it with RLS in non-OSA, 2) the prevalence of insomnia, psychiatric, metabolic and cognitive disorders in ComOSAR versus OSA alone, 3) chronic obstructive airway disease (COAD) in ComOSAR versus OSA alone. OSA, RLS and insomnia were diagnosed as per respective guidelines. They were evaluated for psychiatric disorders, metabolic disorders, cognitive disorders and COAD. Results: Of 326 patients enrolled, 249 were OSA and 77 were non-OSA. 61/249 OSA patients, i.e. 24.4% had comorbid RLS, i.e. ComOSAR. RLS in non-OSA patients was similar (22/77, i.e. 28.5%); P = 0.41. ComOSAR had a significantly higher prevalence of insomnia (26% versus 10.1%; P = 0.016), psychiatric disorders (73.7% versus 48.4%; P = 0.00026) and cognitive deficits (72.1% versus 54.7%, P = 0.016) compared to OSA alone. Metabolic disorders like metabolic syndrome, diabetes mellitus, hypertension and coronary artery disease were also observed in a significantly higher number of patients with ComOSAR versus OSA alone (57% versus 34%; P = 0.0015). COAD was also seen in a significantly higher number of patients with ComOSAR compared to OSA alone (49% versus 19% respectively; P = 0.00001). Conclusion: It is essential to look for RLS in patients with OSA as it leads to a significantly higher prevalence of insomnia, and cognitive, metabolic and psychiatric disorders. COAD is also more common in ComOSAR compared to OSA alone.