Integration of pan-omics technologies and three-dimensional in vitro tumor models: an approach toward drug discovery and precision medicine.

Despite advancements in treatment protocols, cancer is one of the leading cause of deaths worldwide. Therefore, there is a need to identify newer and personalized therapeutic targets along with screening technologies to combat cancer. With the advent of pan-omics technologies, such as genomics, transcriptomics, proteomics, metabolomics, and lipidomics, the scientific community has witnessed an improved molecular and metabolomic understanding of various diseases, including cancer. In addition, three-dimensional (3-D) disease models have been efficiently utilized for understanding disease pathophysiology and as screening tools in drug discovery. An integrated approach utilizing pan-omics technologies and 3-D in vitro tumor models has led to improved understanding of the intricate network encompassing various signalling pathways and molecular cross-talk in solid tumors. In the present review, we underscore the current trends in omics technologies and highlight their role in understanding genotypic-phenotypic co-relation in cancer with respect to 3-D in vitro tumor models. We further discuss the challenges associated with omics technologies and provide our outlook on the future applications of these technologies in drug discovery and precision medicine for improved management of cancer.

Evaluation of Xpert MTB/RIF Assay on Stool Samples for the Diagnosis of Pulmonary Tuberculosis among the Pediatric Population.

Objective Microbiological confirmation of tuberculosis (TB) in pediatric cases is challenging due to its paucibacillary nature and difficulty in specimen collection. This study aimed to validate stool as an alternative sample for the diagnosis of pediatric pulmonary TB via Xpert MTB/RIF (Xpert) assay. Materials and Methods This cross-sectional study included 75 pediatric patients up to 10 years of age with signs and symptoms suggestive of TB. From each recruited patient, pulmonary and stool samples were collected in a sterile container. The collected samples were subjected to Ziehl-Neelsen staining, BACTEC MGIT 960 culture (MGIT), Xpert, and in-house multiplex polymerase chain reaction for TB diagnosis. Results About 13.33% (10/75) of the pulmonary samples and, of them, 50% (5/75) of the stool samples were positive by Xpert assay. The sensitivity and specificity of Xpert assay with stool and pulmonary samples were 50 (95% confidence interval [CI]: 18.71-81.29%) and 100% (95% CI: 94.48-100%), respectively. Conclusion The Xpert assay on stool samples showed limited sensitivity and good specificity in the diagnosis of pulmonary TB. Therefore, it can be proposed as an alternative screening sample to diagnose TB in pediatric cases for which getting a respiratory sample is extremely difficult. However, further studies with greater number of samples and multiple baseline variables are required to support our findings. Strategies to optimize stool Xpert assay should be performed to enhance the sensitivity of this method to detect Mycobacterium tuberculosis in children.

Molecular Identification of Non-tuberculous Mycobacteria in Suspected Tuberculosis Cases in Central India.

Introduction Mycobacterium tuberculosis complex (MTBC), the primary cause of tuberculosis (TB), must be accurately identified to implement effective patient management and control strategies. Non-tuberculous mycobacteria (NTM) in suspected TB cases can result in erroneous diagnoses and needless treatment. Objective The study aimed to identify NTM in patients suspected of TB at a tertiary care hospital in central India using molecular methods. Methods This prospective study enrolled 400 suspected pulmonary and extra-pulmonary TB patients. Patients between the age of two to 90 years, of either gender, new and previously treated cases, Culture positive, patients with immune-compromised status, patients not responding to ATT, HIV positive and negative, and willing to give consent were included in the study. Liquid culture via the Mycobacterial growth indicator tube (MGIT) system was used to culture mycobacteria from clinical samples. The SD Bioline Ag MPT64 Test (Standard Diagnostics, South Korea) and in-house multiplex-PCR (mPCR) were used to differentiate between Mycobacterium tuberculosis complex and NTM species for the molecular identification of NTM GenoType® Mycobacterium Common Mycobacteria (CM) assay kit (HAIN Life Science, Nehren, Germany) was used following the manufacturer's protocol. Results Only 59/400 (14.7%) of the samples produced a positive result in MGIT culture, indicating the presence of mycobacteria, and 85.25% of the remaining 341 samples were negative for mycobacterial growth. Further investigation of these 59 cultures with mPCR and SD Bioline Ag MPT64 test showed that 12 (20.33%) cultures were determined to be NTM, while the remaining 47 (79.67%) were identified as MTBC. Genotype characterization with GenoType® mycobacterium CM assay kit revealed that five of the 12 NTM isolates (41.67%) showed patterns that were consistent with Mycobacterium (M.) fortuitum, three (25%) showed patterns that were consistent with M. abscessus, and four (33.33%) showed patterns that were consistent with M. tuberculosis. Conclusion These results emphasize the value of molecular methods for precisely identifying mycobacterial species, particularly in suspected TB cases. The high prevalence of NTM in positive cultures emphasizes the significance of differentiating between MTBC and NTM to prevent misdiagnosis and ensure proper care. Understanding the epidemiology and clinical significance of these organisms in central India is made possible by the identification of particular NTM species.

Diagnosis of tuberculous pleural effusion in a tertiary care hospital of central India: The role of xpert Mycobacterium tuberculosis/rifampicin.

Background: In India, 15%-20% of tuberculosis (TB) cases are categorized as extra-pulmonary TB, and tuberculous pleural effusion (TPE) is the second-most common type after tuberculous lymphadenitis. However, the paucibacillary nature of TPE makes its diagnosis challenging. As a result, relying on empirical anti-TB treatment (ATT) based on clinical diagnosis becomes necessary for achieving the best possible diagnostic outcome. The study aims to determine the diagnostic utility of Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) for the detection of TB in TPE in high incidence setting of Central India. Methods: The study enrolled 321 patients who had exudative pleural effusion detected through radiological testing and were suspected of having TB. The medical procedure of thoracentesis was conducted to collect the pleural fluid, which was then subjected to both the Ziehl-Neelsen staining and Xpert MTB/RIF test. The patients who showed improvement after receiving anti-tuberculosis treatment (ATT) were considered the composite reference standard. Results: The sensitivity of smear microscopy was found to be 10.19%, while that of the Xpert MTB/RIF method was 25.93% when compared to the composite reference standard. The accuracy of clinical diagnosis was measured using receiver operating characteristics based on clinical symptoms, and it was found to be 0.858 (area under the curve). Conclusions: The study shows that Xpert MTB/RIF has significant value in diagnosing TPE, despite its low sensitivity of 25.93%. Clinical diagnosis based on symptoms was relatively accurate, but relying on symptoms alone is not enough. Using multiple diagnostic tools, including Xpert MTB/RIF, is crucial for accurate diagnosis. Xpert MTB/RIF has excellent specificity and can detect RIF resistance. Its quick results make it useful in situations where a rapid diagnosis is necessary. While it should not be the only diagnostic tool, it has a valuable role in diagnosing TPE.

A comparative analysis of molecular genotypes of Mycobacterium tuberculosis isolates from HIV-positive and HIV-negative patients.

Setting: Tuberculosis Research Laboratory, Division of Clinical Microbiology and Molecular Medicine, Department of Laboratory Medicine, All India Institute of Medical Sciences, and the National Institute of Tuberculosis and Respiratory Diseases (NITRD), both situated in New Delhi. Objectives: We aimed to identify the distribution of various genotypes of M. tuberculosis among HIV-positive and HIV-negative patients suspected of having Tuberculosis, seen at the National Institute of Tuberculosis and Respiratory Diseases, New Delhi, which is a tertiary care dedicated TB hospital. Patients and methods: Genotyping by Spoligotyping and 24 loci MIRU-VNTR was performed and analyzed using SITVITWEB and MIRU-VNTRplus. Drug susceptibility patterns were also analyzed. Results: A total of 503 subjects who were PTB/EPTB suspected were recruited and 287 were culture positive. Among them, 276 had growth of Mycobacterium tuberculosis (MTB) and in 11 patients non-tuberculous mycobacteria (NTM) were grown. The isolation rate of NTM was predominantly from HIV positive [10 of 130 (7.6%)] patients. Of the total isolates of MTB, 156 (56.5%) were from HIV negative patients and 120 (43.5%) were from HIV positive patients. All 276 M. tuberculosis isolates were genotyped and tested for drug susceptibility patterns. The CAS genotype was most predominant [153 (55.4%)], followed by Beijing lineage [44 (15.9%)], East African India [25 (9.1%)] and others [54 (19.6%)]. Beijing genotype was significantly more common in HIV positive patients (22.5%) than in HIV negative patients (10.9%). In MIRU-VNTR analysis, clustering was found to be more frequent in CAS strains irrespective of HIV status. In the HIV positive group, spoligotyping could differentiate various genotypes in 90% of isolates and MIRU-VNTR analysis in 84.2% of isolates. The clustering of various MTB strains was more associated with drug resistance. Conclusion: The Beijing lineage was predominant in HIV-TB coinfected cases, even though the Central Asian Strain (CAS) was overall more predominant in the region.

Organ Involvement in COVID-19: A Molecular Investigation of Autopsied Patients.

Precise reasons for severe manifestation of SARS-CoV-2 remain unanswered, and efforts have been focused on respiratory system management. Demonstration of unequivocal presence of SARS-CoV-2 in vital body organs by cadaver autopsy was the only way to prove multi-organ involvement. Hence, the primary objective of the study was to determine presence of the SARS-CoV-2 in various organs of patients succumbing to SARS-CoV-2 infection. A total of 246 samples from different organs of 21 patients who died due to severe COVID-19 illness were investigated by qRT-PCR, and SARS-CoV-2 was detected in 181 (73.57%) samples and highest positivity of SARS-CoV-2 being (expectedly) found in nasopharynx (90.4%) followed by bilateral lungs (87.30%), peritoneal fluid (80%), pancreas (72.72%), bilateral kidneys (68.42%), liver (65%) and even in brain (47.2%). The deceased patients were categorized to three subgroups based upon the extent of organs in which SARS-CoV-2 was detected by qRT-PCR (high intensity ≥80%, intermediate intensity = 65-80% and low intensity ≤65% organs involvement). It was conclusively established that SARS-CoV-2 has the property of invasion beyond lungs and even crosses the blood-brain barrier, resulting in multi-system disease; this is probably the reason behind cytokine storm, though it is not clear whether organ damage is due to direct injury caused by the virus or result of inflammatory assault. Significant inverse correlation was found between the Ct value of lung samples and number of organs involved, implying that higher viral load in lungs is directly proportionate to involvement of extrapulmonary organs and patients with higher viral load in respiratory secretions should be monitored more closely for any warning signs and the treatment strategies should also address involvement of other organs for better outcome, because lungs, though the primary site of infection, are not the only organ system responsible for pathogenesis of systemic illness.

Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India.

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the "bridging variant" between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively.

A Parametric Targetability Evaluation Approach for Vitiligo Proteome Extracted through Integration of Gene Ontologies and Protein Interaction Topologies.

Vitiligo is a well-known skin disorder with complex etiology. Vitiligo pathogenesis is multifaceted with many ramifications. A computational systemic path was designed to first propose candidate disease proteins by merging properties from protein interaction networks and gene ontology terms. All in all, 109 proteins were identified and suggested to be involved in the onset of disease or its progression. Later, a composite approach was employed to prioritize vitiligo disease proteins by comparing and benchmarking the properties against standard target identification criteria. This includes sequence-based, structural, functional, essentiality, protein-protein interaction, vulnerability, secretability, assayability, and druggability information. The existing information was seamlessly integrated into efficient pipelines to propose a novel protocol for assessment of targetability of disease proteins. Using the online data resources and the scripting, an illustrative list of 68 potential drug targets was generated for vitiligo. While this list is broadly consistent with the research community's current interest in certain specific proteins, and suggests novel target candidates that may merit further study, it can still be modified to correspond to a user-specific environment, either by adjusting the weights for chosen criteria (i.e., a quantitative approach) or by changing the considered criteria (i.e., a qualitative approach).

Implications of protein conformations to modifying novel inhibitor Oseltamivir for 2009 H1N1 influenza A virus by simulation and docking studies.

Recently three FDA approved existing drugs, namely-Oseltamivir, Peramivir and Zanamivir, used against Neuraminidase (NA) for the inhibitory effect on the process of viral progeny release to inhibit infection. All NA subtypes has been divided into two groups (Group 1 and Group 2) based on phylogenetic study. Oseltamivir and Zanamivir drugs are designed for Group 2 NA but are also used against 2009 H1N1 NA that lies in Group 1. There is no specific drug available for H1N1 and, consequently, there is an urgent requirement for the same. The structure-based drug design and fragment-based drug design methods are used for building more effective and economic drug molecules. In this work, the fragment-based drug development followed by fragment evolution on the basis of protein conformations after every 10 ns of 100 ns simulation. There are two analogs of Oseltamivir acid drug discovered in this study. Only analog 1, along with Oseltamivir acid, were then docked with the native protein. The analog 1 (benzoic acid inhibitor 11) exhibited higher binding affinity value of - 10.70 kcal/mol in comparison to its predecessor. The concept of conformations and protein-ligand interactions can be useful in designing new drugs for H1N1 with high specific binding.

Construction of a Comprehensive Protein-Protein Interaction Map for Vitiligo Disease to Identify Key Regulatory Elements: A Systemic Approach.

Vitiligo is an idiopathic disorder characterized by depigmented patches on the skin due to progressive loss of melanocytes. Several genetic, immunological, and pathophysiological investigations have established vitiligo as a polygenetic disorder with multifactorial etiology. However, no definite model explaining the interplay between these causative factors has been established hitherto. Therefore, we studied the disorder at the system level to identify the key proteins involved by exploring their molecular connectivity in terms of topological parameters. The existing research data helped us in collating 215 proteins involved in vitiligo onset or progression. Interaction study of these proteins leads to a comprehensive vitiligo map with 4845 protein nodes linked with 107,416 edges. Based on centrality measures, a backbone network with 500 nodes has been derived. This has presented a clear overview of the proteins and processes involved and the crosstalk between them. Clustering backbone proteins revealed densely connected regions inferring major molecular interaction modules essential for vitiligo. Finally, a list of top order proteins that play a key role in the disease pathomechanism has been formulated. This includes SUMO2, ESR1, COPS5, MYC, SMAD3, and Cullin proteins. While this list is in fair agreement with the available literature, it also introduces new candidate proteins that can be further explored. A subnetwork of 64 vitiligo core proteins was built by analyzing the backbone and seed protein networks. Our finding suggests that the topology, along with functional clustering, provides a deep insight into the behavior of proteins. This in turn aids in the illustration of disease condition and discovery of significant proteins involved in vitiligo.

Computational model for pathway reconstruction to unravel the evolutionary significance of melanin synthesis.

Melanogenesis is a complex multistep process of high molecular weight melanins production by hydroxylation and polymerization of polyphenols. Melanins have a wide range of applications other than being a sun - protection pigment. Melanogenesis pathway exists from prokaryotes to eukaryotes. It has evolved over years owing to the fact that the melanin pigment has different roles in diverse taxa of organisms. Melanin plays a pivotal role in the existence of certain bacteria and fungi whereas in higher organisms it is a measure of protection against the harmful radiation. We have done a detailed study on various pathways known for melanin synthesis across species. It was divulged that melanin production is not restricted to tyrosine but there are other secondary metabolites that synthesize melanin in lower organisms. Furthermore the phylogenetic study of these paths was done to understand their molecular and cellular development. It has revealed that the melanin synthesis paths have co-evolved in several groups of organisms. In this study, we also introduce a method for the comparative analysis of a metabolic pathway to study its evolution based on similarity between enzymatic reactions.