The angiotensin II type 2 receptor for pain control.

All well-known deleterious effects of angiotensin (Ang) II, including vasoconstriction, inflammation, water and salt retention, and vascular remodeling, are mediated via its type 1 (AT1) receptor. This explains why AT1 receptor blockers (ARBs) and inhibitors of Ang II synthesis, such as ACE inhibitors and renin inhibitors, are beneficial for cardiovascular disease. Yet, Ang II has a second receptor, the Ang II type 2 (AT2) receptor, the function of which, even after over 20 years of research, remains largely unknown. In this issue, Marion et al. provide a new chapter to the AT2 receptor story.

High diversity in Delta variant across countries revealed by genome-wide analysis of SARS-CoV-2 beyond the Spike protein.

The highly contagious Delta variant of SARS-CoV-2 has become a prevalent strain globally and poses a public health challenge around the world. While there has been extensive focus on understanding the amino acid mutations in the Delta variant's Spike protein, the mutational landscape of the rest of the SARS-CoV-2 proteome (25 proteins) remains poorly understood. To this end, we performed a systematic analysis of mutations in all the SARS-CoV-2 proteins from nearly 2 million SARS-CoV-2 genomes from 176 countries/territories. Six highly prevalent missense mutations in the viral life cycle-associated Membrane (I82T), Nucleocapsid (R203M, D377Y), NS3 (S26L), and NS7a (V82A, T120I) proteins are almost exclusive to the Delta variant compared to other variants of concern (mean prevalence across genomes: Delta = 99.74%, Alpha = 0.06%, Beta = 0.09%, and Gamma = 0.22%). Furthermore, we find that the Delta variant harbors a more diverse repertoire of mutations across countries compared to the previously dominant Alpha variant. Overall, our study underscores the high diversity of the Delta variant between countries and identifies a list of amino acid mutations in the Delta variant's proteome for probing the mechanistic basis of pathogenic features such as high viral loads, high transmissibility, and reduced susceptibility against neutralization by vaccines.

Single-cell RNA-seq reveals developmental plasticity with coexisting oncogenic states and immune evasion programs in ETP-ALL.

Lineage plasticity and stemness have been invoked as causes of therapy resistance in cancer, because these flexible states allow cancer cells to dedifferentiate and alter their dependencies. We investigated such resistance mechanisms in relapsed/refractory early T-cell progenitor acute lymphoblastic leukemia (ETP-ALL) carrying activating NOTCH1 mutations via full-length single-cell RNA sequencing (scRNA-seq) of malignant and microenvironmental cells. We identified 2 highly distinct stem-like states that critically differed with regard to cell cycle and oncogenic signaling. Fast-cycling stem-like leukemia cells demonstrated Notch activation and were effectively eliminated in patients by Notch inhibition, whereas slow-cycling stem-like cells were Notch independent and rather relied on PI3K signaling, likely explaining the poor efficacy of Notch inhibition in this disease. Remarkably, we found that both stem-like states could differentiate into a more mature leukemia state with prominent immunomodulatory functions, including high expression of the LGALS9 checkpoint molecule. These cells promoted an immunosuppressive leukemia ecosystem with clonal accumulation of dysfunctional CD8+ T cells that expressed HAVCR2, the cognate receptor for LGALS9. Our study identified complex interactions between signaling programs, cellular plasticity, and immune programs that characterize ETP-ALL, illustrating the multidimensionality of tumor heterogeneity. In this scenario, combination therapies targeting diverse oncogenic states and the immune ecosystem seem most promising to successfully eliminate tumor cells that escape treatment through coexisting transcriptional programs.

Terpenes in Cannabis sativa Inhibit Capsaicin Responses in Rat DRG Neurons via Na+/K+ ATPase Activation.

Terpenes in Cannabis sativa exert analgesic effects, but the mechanisms are uncertain. We examined the effects of 10 terpenes on capsaicin responses in an established model of neuronal hypersensitivity. Adult rat DRG neurons cultured with neurotrophic factors NGF and GDNF were loaded with Fura2AM for calcium imaging, and treated with individual terpenes or vehicle for 5 min, followed by 1 µMol capsaicin. In vehicle treated control experiments, capsaicin elicited immediate and sustained calcium influx. Most neurons treated with terpenes responded to capsaicin after 6-8 min. Few neurons showed immediate capsaicin responses that were transient or normal. The delayed responses were found to be due to calcium released from the endoplasmic reticulum, as they were maintained in calcium/magnesium free media, but not after thapsigargin pre-treatment. Terpene inhibition of calcium influx was reversed after washout of medium, in the absence of terpenes, and in the presence of the Na+/K+ ATPase inhibitor ouabain, but not CB1 or CB2 receptor antagonists. Thus, terpenes inhibit capsaicin evoked calcium influx by Na+/K+ ATPase activation. Immunofluorescence showed TRPV1 co-expression with α1ß1 Na+/K+ ATPase in most neurons while others were either TRPV1 or α1ß1 Na+/K+ ATPase positive.

Global importance analysis: An interpretability method to quantify importance of genomic features in deep neural networks.

Deep neural networks have demonstrated improved performance at predicting the sequence specificities of DNA- and RNA-binding proteins compared to previous methods that rely on k-mers and position weight matrices. To gain insights into why a DNN makes a given prediction, model interpretability methods, such as attribution methods, can be employed to identify motif-like representations along a given sequence. Because explanations are given on an individual sequence basis and can vary substantially across sequences, deducing generalizable trends across the dataset and quantifying their effect size remains a challenge. Here we introduce global importance analysis (GIA), a model interpretability method that quantifies the population-level effect size that putative patterns have on model predictions. GIA provides an avenue to quantitatively test hypotheses of putative patterns and their interactions with other patterns, as well as map out specific functions the network has learned. As a case study, we demonstrate the utility of GIA on the computational task of predicting RNA-protein interactions from sequence. We first introduce a convolutional network, we call ResidualBind, and benchmark its performance against previous methods on RNAcompete data. Using GIA, we then demonstrate that in addition to sequence motifs, ResidualBind learns a model that considers the number of motifs, their spacing, and sequence context, such as RNA secondary structure and GC-bias.

Assessment of prevailing practices for identification of tuberculosis in children admitted in nutritional rehabilitation centres: A multicentre study.

Background & objectives: To eliminate tuberculosis (TB), the National TB Elimination Programme (NTEP) has given an algorithm for diagnosis and treatment of TB in children. This study was carried out to assess the prevailing practices to identify TB in severely malnourished children admitted to various nutrition rehabilitation centres (NRCs). Methods: A retrospective chart review of 41 NRCs across five States having a maximum number of NRCs was carried out. Details of children admitted to the NRCs and the investigations carried out in the form of obtaining X-ray film, tuberculin skin test (TST) and gastric aspirate for cartridge-based nucleic acid amplification test (CBNAAT) for diagnosis of TB over three months were collected. Results: A total of 2121 children with severe acute malnourished (SAM) across 41 NRCs (Bhopal, Jodhpur, Patna, Kolkata, Lucknow and Ratlam) were evaluated. X-ray of the chest was done in 473 (22%), TST was done in 135 (6%) and gastric aspirate was collected in 56 (3%) children. CBNAAT was done in only 56 SAM children. Ten children among those screened were confirmed to have pulmonary TB and were linked to treatment as per the National TB guidelines. Interpretation & conclusions: Although NTEP suggests universal screening of all malnourished children admitted in the NRCs, but the prevailing practices indicate that the NRCs are grossly under-utilizing molecular diagnostic tests for diagnosis of TB. There is a gap between prevailing practices and suggested guidelines for the identification of TB in children admitted in the NRCs.

Mycobacterium tuberculosis strain lineage in mixed tribal population across India and Andaman Nicobar Island.

In India, the tribal population constitutes almost 8.6% of the nation's total population. Despite their large presence, there are only a few reports available on Mycobacterium tuberculosis (M. tb) strain prevalence in Indian tribal communities considering the mobile nature of this population and also the influence of the mainstream populations they coexist within many areas for their livelihood. This study attempts to provide critical information pertaining to the TB strain diversity, its public health implications, and distribution among the tribal population in eleven Indian states and Andaman & Nicobar (A&N) Island. The study employed a population-based molecular approach. Clinical isolates were received from 66 villages (10 states and Island) and these villages were selected by implying situation analysis. A total of 78 M. tb clinical isolates were received from 10 different states and A&N Island. Among these, 16 different strains were observed by spoligotyping technique. The major M. tb strains spoligotype belong to the Beijing, CAS1_DELHI, and EAI5 family of M. tb strains followed by EAI1_SOM, EAI6_BGD1, LAM3, LAM6, LAM9, T1, T2, U strains. Drug-susceptibility testing (DST) results showed almost 15.4% of clinical isolates found to be resistant to isoniazid (INH) or rifampicin (RMP) + INH. Predominant multidrug-resistant (MDR-TB) isolates seem to be Beijing strain. Beijing, CAS1_DELHI, EAI3_IND, and EAI5 were the principal strains infecting mixed tribal populations across India. Despite the small sample size, this study has demonstrated higher diversity among the TB strains with significant MDR-TB findings. Prevalence of Beijing MDR-TB strains in Central, Southern, Eastern India and A&N Island indicates the transmission of the TB strains.

Molecular Dissection of Mycobacterium tuberculosis Integration Host Factor Reveals Novel Insights into the Mode of DNA Binding and Nucleoid Compaction.

The annotated whole-genome sequence of Mycobacterium tuberculosis indicated that Rv1388 (Mtihf) likely encodes a putative 20 kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or organization of mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF-duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg170, Arg171, and Arg173, which might be involved in DNA binding, and a conserved proline (P150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methylmethanesulfonate could be complemented with the wild-type Mtihf, but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, bind with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαß. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes compaction of DNA into nucleoid-like or higher-order filamentous structures. We hence propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Molecular dissection of Mycobacterium tuberculosis integration host factor reveals novel insights into the mode of DNA binding and nucleoid compaction.

The annotated whole-genome sequence of Mycobacterium tuberculosis revealed that Rv1388 (Mtihf) is likely to encode for a putative 20-kDa integration host factor (mIHF). However, very little is known about the functional properties of mIHF or the organization of the mycobacterial nucleoid. Molecular modeling of the mIHF three-dimensional structure, based on the cocrystal structure of Streptomyces coelicolor IHF duplex DNA, a bona fide relative of mIHF, revealed the presence of Arg-170, Arg-171, and Arg-173, which might be involved in DNA binding, and a conserved proline (Pro-150) in the tight turn. The phenotypic sensitivity of Escherichia coli ΔihfA and ΔihfB strains to UV and methyl methanesulfonate could be complemented with the wild-type Mtihf but not its alleles bearing mutations in the DNA-binding residues. Protein-DNA interaction assays revealed that wild-type mIHF, but not its DNA-binding variants, binds with high affinity to fragments containing attB and attP sites and curved DNA. Strikingly, the functionally important amino acid residues of mIHF and the mechanism(s) underlying its binding to DNA, DNA bending, and site-specific recombination are fundamentally different from that of E. coli IHFαß. Furthermore, we reveal novel insights into IHF-mediated DNA compaction depending on the placement of its preferred binding sites; mIHF promotes DNA compaction into nucleoid-like or higher order filamentous structures. We therefore propose that mIHF is a distinct member of a subfamily of proteins that serve as essential cofactors in site-specific recombination and nucleoid organization and that these findings represent a significant advance in our understanding of the role(s) of nucleoid-associated proteins.

Mechanisms underlying clinical efficacy of Angiotensin II type 2 receptor (AT2R) antagonist EMA401 in neuropathic pain: clinical tissue and in vitro studies.

BACKGROUND: The clinical efficacy of the Angiotensin II (AngII) receptor AT2R antagonist EMA401, a novel peripherally-restricted analgesic, was reported recently in post-herpetic neuralgia. While previous studies have shown that AT2R is expressed by nociceptors in human DRG (hDRG), and that EMA401 inhibits capsaicin responses in cultured hDRG neurons, the expression and levels of its endogenous ligands AngII and AngIII in clinical neuropathic pain tissues, and their signalling pathways, require investigation. We have immunostained AngII, AT2R and the capsaicin receptor TRPV1 in control post-mortem and avulsion injured hDRG, control and injured human nerves, and in cultured hDRG neurons. AngII, AngIII, and Ang-(1-7) levels were quantified by ELISA. The in vitro effects of AngII, AT2R agonist C21, and Nerve growth factor (NGF) were measured on neurite lengths; AngII, NGF and EMA401 effects on expression of p38 and p42/44 MAPK were measured using quantitative immunofluorescence, and on capsaicin responses using calcium imaging. RESULTS: AngII immunostaining was observed in approximately 75% of small/medium diameter neurons in control (n = 5) and avulsion injured (n = 8) hDRG, but not large neurons i.e. similar to TRPV1. AngII was co-localised with AT2R and TRPV1 in hDRG and in vitro. AngII staining by image analysis showed no significant difference between control (n = 12) and injured (n = 13) human nerves. AngII levels by ELISA were also similar in control human nerves (4.09 ± 0.36 pmol/g, n = 31), injured nerves (3.99 ± 0.79 pmol/g, n = 7), and painful neuromas (3.43 ± 0.73 pmol/g, n = 12); AngIII and Ang-(1-7) levels were undetectable (<0.03 and 0.05 pmol/g respectively). Neurite lengths were significantly increased in the presence of NGF, AngII and C21 in cultured DRG neurons. AngII and, as expected, NGF significantly increased signal intensity of p38 and p42/44 MAPK, which was reversed by EMA401. AngII mediated sensitization of capsaicin responses was not observed in the presence of MAP kinase inhibitor PD98059, and the kinase inhibitor staurosporine. CONCLUSION: The major AT2R ligand in human peripheral nerves is AngII, and its levels are maintained in injured nerves. EMA401 may act on paracrine/autocrine mechanisms at peripheral nerve terminals, or intracrine mechanisms, to reduce neuropathic pain signalling in AngII/NGF/TRPV1-convergent pathways.

EMA401, an orally administered highly selective angiotensin II type 2 receptor antagonist, as a novel treatment for postherpetic neuralgia: a randomised, double-blind, placebo-controlled phase 2 clinical trial.

BACKGROUND: Existing treatments for postherpetic neuralgia, and for neuropathic pain in general, are limited by modest efficacy and unfavourable side-effects. The angiotensin II type 2 receptor (AT2R) is a new target for neuropathic pain. EMA401, a highly selective AT2R antagonist, is under development as a novel neuropathic pain therapeutic agent. We assessed the therapeutic potential of EMA401 in patients with postherpetic neuralgia. METHODS: In this multicentre, placebo-controlled, double-blind, randomised, phase 2 clinical trial, we enrolled patients (aged 22-89 years) with postherpetic neuralgia of at least 6 months' duration from 29 centres across six countries. We randomly allocated 183 participants to receive either oral EMA401 (100 mg twice daily) or placebo for 28 days. Randomisation was done according to a centralised randomisation schedule, blocked by study site, which was generated by an independent, unmasked statistician. Patients and staff at each site were masked to treatment assignment. We assessed the efficacy, safety, and pharmacokinetics of EMA401. The primary efficacy endpoint was change in mean pain intensity between baseline and the last week of dosing (days 22-28), measured on an 11-point numerical rating scale. The primary efficacy analysis was intention to treat. This trial is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000822987. FINDINGS: 92 patients were assigned to EMA401 and 91 were assigned to placebo. The patients given EMA401 reported significantly less pain compared with baseline values in the final week of treatment than did those given placebo (mean reductions in pain scores -2.29 [SD 1.75] vs -1.60 [1.66]; difference of adjusted least square means -0.69 [SE 0.25]; 95% CI -1.19 to -0.20; p=0.0066). No serious adverse events related to EMA401 occurred. Overall, 32 patients reported 56 treatment-emergent adverse events in the EMA401 group compared with 45 such events reported by 29 patients given placebo. INTERPRETATION: EMA401 (100 mg twice daily) provides superior relief of postherpetic neuralgia compared with placebo at the end of 28 days of treatment. EMA401 was well tolerated by patients. FUNDING: Spinifex Pharmaceuticals.

Topical TrkA Kinase Inhibitor CT327 is an Effective, Novel Therapy for the Treatment of Pruritus due to Psoriasis: Results from Experimental Studies, and Efficacy and Safety of CT327 in a Phase 2b Clinical Trial in Patients with Psoriasis.

Pruritus is an important symptom in psoriasis with no targeted treatment. Tropomyosin-receptor kinase A (TrkA) is associated with pruritus and psoriatic plaque formation. We report the efficacy of a TrkA inhibitor, CT327, on pruritus in psoriasis. A randomised, double-blind, vehicle-controlled Phase 2b clinical trial was conducted in 160 subjects. No effect was found on psoriasis severity using Investigator's Global Assessment (primary endpoint). However, clinically and statistically significant reductions in pruritus were observed in the 108 patient subset reporting at least moderate pruritus at baseline (37.1 mm visual analogue scale improvement (95% CI [-37.5, -6.2], p = 0.0067) for lowest dose; secondary endpoint). Significant modified Psoriasis Area and Severity Index reductions were found in this subset (p < 0.05). Experiments exploring capsaicin-mediated calcium influx, important in pruritus signalling, were performed in sensory neurons. CT327 inhibited capsaicin responses, indicating action at the nerve growth factor-TrkA-TRPV1 pathway. TrkA is a key target in pruritus, and CT327 has potential to become an effective and safe first-in-class treatment.

PocketAnnotate: towards site-based function annotation.

A computational pipeline PocketAnnotate for functional annotation of proteins at the level of binding sites has been proposed in this study. The pipeline integrates three in-house algorithms for site-based function annotation: PocketDepth, for prediction of binding sites in protein structures; PocketMatch, for rapid comparison of binding sites and PocketAlign, to obtain detailed alignment between pair of binding sites. A novel scheme has been developed to rapidly generate a database of non-redundant binding sites. For a given input protein structure, putative ligand-binding sites are identified, matched in real time against the database and the query substructure aligned with the promising hits, to obtain a set of possible ligands that the given protein could bind to. The input can be either whole protein structures or merely the substructures corresponding to possible binding sites. Structure-based function annotation at the level of binding sites thus achieved could prove very useful for cases where no obvious functional inference can be obtained based purely on sequence or fold-level analyses. An attempt has also been made to analyse proteins of no known function from Protein Data Bank. PocketAnnotate would be a valuable tool for the scientific community and contribute towards structure-based functional inference. The web server can be freely accessed at http://proline.biochem.iisc.ernet.in/pocketannotate/.

Dual role of signaling pathways in myeloma requires cell type-specific targeting of ligand-receptor interactions.

ABSTRACT: Although most patients with multiple myeloma respond to treatment initially, therapy resistance develops almost invariably, and only a subset of patients show durable responses to immunomodulatory therapies. Although the immune microenvironment has been extensively studied in patients with myeloma, its composition is currently not used as prognostic markers in clinical routine. We hypothesized that the outcome of immune signaling pathway engagement can be highly variable, depending on which 2 cellular populations participate in this interaction. This would have important prognostic and therapeutic implications, suggesting that it is crucial for immune pathways to be targeted in a specific cellular context. To test this hypothesis, we investigated a cohort of 25 patients with newly diagnosed multiple myeloma. We examined the complex regulatory networks within the immune compartment and their impact on disease progression. Analysis of immune cell composition and expression profiles revealed significant differences in the B-cell compartment associated with treatment response. Transcriptional states in patients with short time to progression demonstrated an enrichment of pathways promoting B-cell differentiation and inflammatory responses, which may indicate immune dysfunction. Importantly, the analysis of molecular interactions within the immune microenvironment highlights the dual role of signaling pathways, which can either be associated with good or poor prognosis depending on the cell types involved. Our findings therefore argue that therapeutic strategies targeting ligand-receptor interactions should take into consideration the composition of the microenvironment and the specific cell types involved in molecular interactions.

Reduced Thalamic γ-Aminobutyric Acid (GABA) in Painless but Not Painful Diabetic Peripheral Neuropathy.

Alterations in the structure, function, and microcirculation of the thalamus, a key brain region involved in pain pathways, have previously been demonstrated in patients with painless and painful diabetic peripheral neuropathy (DPN). However, thalamic neurotransmitter levels including γ-aminobutyric acid (GABA) (inhibitory neurotransmitter) and glutamate (excitatory neurotransmitter) in different DPN phenotypes are not known. We performed a magnetic resonance spectroscopy study and quantified GABA and glutamate levels within the thalamus, in a carefully characterized cohort of participants with painless and painful DPN. Participants with DPN (painful and painless combined) had a significantly lower GABA:H2O ratio compared with those without DPN (healthy volunteers [HV] and participants with diabetes without DPN [no DPN]). Participants with painless DPN had the lowest GABA:H2O ratio, which reached significance compared with HV and no DPN, but not painful DPN. There was no difference in GABA:H2O in painful DPN compared with all other groups. A significant correlation with GABA:H2O and neuropathy severity was also seen. This study demonstrates that lower levels of thalamic GABA in participants with painless DPN may reflect neuroplasticity due to reduced afferent pain impulses, whereas partially preserved levels of GABA in painful DPN may indicate that central GABAergic pathways are involved in the mechanisms of neuropathic pain in diabetes.

Local delivery of molecules from a nanopipette for quantitative receptor mapping on live cells.

Using nanopipettes to locally deliver molecules to the surface of living cells could potentially open up studies of biological processes down to the level of single molecules. However, in order to achieve precise and quantitative local delivery it is essential to be able to determine the amount and distribution of the molecules being delivered. In this work, we investigate how the size of the nanopipette, the magnitude of the applied pressure or voltage, which drives the delivery, and the distance to the underlying surface influences the number and spatial distribution of the delivered molecules. Analytical expressions describing the delivery are derived and compared with the results from finite element simulations and experiments on delivery from a 100 nm nanopipette in bulk solution and to the surface of sensory neurons. We then developed a setup for rapid and quantitative delivery to multiple subcellular areas, delivering the molecule capsaicin to stimulate opening of Transient Receptor Potential Vanilloid subfamily member 1 (TRPV1) channels, membrane receptors involved in pain sensation. Overall, precise and quantitative delivery of molecules from nanopipettes has been demonstrated, opening up many applications in biology such as locally stimulating and mapping receptors on the surface of live cells.

ResidualBind: Uncovering Sequence-Structure Preferences of RNA-Binding Proteins with Deep Neural Networks.

Deep neural networks have demonstrated improved performance at predicting sequence specificities of DNA- and RNA-binding proteins. However, it remains unclear why they perform better than previous methods that rely on k-mers and position weight matrices. Here, we highlight a recent deep learning-based software package, called ResidualBind, that analyzes RNA-protein interactions using only RNA sequence as an input feature and performs global importance analysis for model interpretability. We discuss practical considerations for model interpretability to uncover learned sequence motifs and their secondary structure preferences.

Pulmonary Tuberculosis in Severely Malnourished Children Admitted to Nutrition Rehabilitation Centers: A Multicenter Study.

OBJECTIVES: To identify prevalence of pulmonary tuberculosis (TB) in severely malnourished children admitted to nutritional rehabilitation centers. METHODS: A multicenter cross-sectional study involving 41 nutrition rehabilitation centres (NRCs) across India was carried out to document prevalence of pulmonary tuberculosis in acute severe malnourished children admitted in NRCs. After training of the NRC staff to follow algorithm provided by national tuberculosis elimination program, children admitted to NRCs were screened for pulmonary tuberculosis. RESULTS: A total of 4356 children were enrolled across all the sites. Gastric aspirate for Cartridge based nucleic acid amplification test (CBNAAT), tuberculin skin test (TST) and X-ray film of chest were done in more than 99% of enrolled subjects. A total of 189 children (4.3%) had pulmonary tuberculosis. Eighty-seven (1.99%) were microbiologically confirmed by positive CBNAAT. On multivariate analysis, only significant association was with history of contact with TB patient in family. CONCLUSIONS: The present results suggest that a significant proportion (>4%) of children admitted in NRCs suffer from pulmonary tuberculosis. It is feasible to improve diagnosis of tuberculosis as a whole and microbiologically confirmed TB.

Expanding repertoire of SARS-CoV-2 deletion mutations contributes to evolution of highly transmissible variants.

The emergence of highly transmissible SARS-CoV-2 variants and vaccine breakthrough infections globally mandated the characterization of the immuno-evasive features of SARS-CoV-2. Here, we systematically analyzed 2.13 million SARS-CoV-2 genomes from 188 countries/territories (up to June 2021) and performed whole-genome viral sequencing from 102 COVID-19 patients, including 43 vaccine breakthrough infections. We identified 92 Spike protein mutations that increased in prevalence during at least one surge in SARS-CoV-2 test positivity in any country over a 3-month window. Deletions in the Spike protein N-terminal domain were highly enriched for these 'surge-associated mutations' (Odds Ratio = 14.19, 95% CI 6.15-32.75, p value = 3.41 × 10-10). Based on a longitudinal analysis of mutational prevalence globally, we found an expanding repertoire of Spike protein deletions proximal to an antigenic supersite in the N-terminal domain that may be one of the key contributors to the evolution of highly transmissible variants. Finally, we generated clinically annotated SARS-CoV-2 whole genome sequences from 102 patients and identified 107 unique mutations, including 78 substitutions and 29 deletions. In five patients, we identified distinct deletions between residues 85-90, which reside within a linear B cell epitope. Deletions in this region arose contemporaneously on a diverse background of variants across the globe since December 2020. Overall, our findings based on genomic-epidemiology and clinical surveillance suggest that the genomic deletion of dispensable antigenic regions in SARS-CoV-2 may contribute to the evasion of immune responses and the evolution of highly transmissible variants.