Browsing by Author "Prashant Ranjan"

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An in vitro and computational validation of a novel loss-of-functional mutation in PAX9 associated with non-syndromic tooth agenesis
(Springer Science and Business Media Deutschland GmbH, 2023) Tanmoy Sarkar; Prashant Ranjan; Smitha Kanathur; Ankush Gupta; Parimal Das
Congenital tooth agenesis (CTA) is one of the most common craniofacial anomalies. Its frequency varies among different population depending upon the genetic heterogeneity. CTA could be of familial or sporadic and syndromic or non-syndromic. Five major genes are found to be associated with non-syndromic CTA, namely PAX9, MSX1, EDA1, AXIN2, and WNT10A. Very few studies have been carried out so far on CTA on this Indian population making this study unique and important. This study was initiated to identify potential pathogenic variant associated with congenital tooth agenesis in an India family with molar tooth agenesis. CTA was investigated and a novel c.336C > G variation was identified in the exon 3 of PAX9, leading to substitution of evolutionary conserved Cys with Trp at 112th amino acid position located at the functionally significant DNA-binding paired domain region. Functional analysis revealed that p.Cys112Trp mutation did not prevent the nuclear localization although mutant protein had higher cytoplasmic retention. EMSA using e5 probe revealed that mutant protein was unable to bind with the paired-domain-binding site. Subsequently, GST pull-down assay revealed lower binding activity of the mutant protein with its known interactor MSX1. These in vitro results were consistent with the computational results. The in vitro and computational observations altogether suggest that c.336C > G (p.Cys112Trp) variation leads to loss of function of PAX9 leading to CTA in this family. Graphical abstract: [Figure not available: see fulltext.]. © 2022, The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.
An inclusive study of deleterious missense PAX9 variants using user-friendly tools reveals structural, functional alterations, as well as potential therapeutic targets.
(Elsevier B.V., 2023) Prashant Ranjan; Parimal Das
Mutations in the PAX9 are responsible for non-syndromic tooth agenesis in humans, although their structural and functional consequences on protein phenotype, stability, and posttranslational modifications (PTMs) have not yet been adequately investigated. This in silico study focuses on retrieving the six most deleterious mutations (L21P, R26W, R28P, G51S, I87F, and K91E) of PAX9 that has been linked to severe oligodontia. Several computational algorithm methods were used to determine the deleterious effects of PAX9 mutations. Analysis of gene ontology, protein interactions, and PTMs indicated significant functional changes caused by PAX9 mutations. The structural superimposition of the wild-type and mutant PAX9 variants revealed structural changes in locations that were present in the structures of all six variations. The conserved domain analysis revealed that the areas shared by all six variations contained unique sections that lacked DNA binding or protein-protein interaction sites, suggesting prospective drug target sites for functional restoration. The protein-protein interaction network showed KDM5B as PAX9's strongest interacting partner similar to MSX1. The PAX9 protein's structural conformations, compactness, stiffness, and function may all be impacted by changes, according to MD simulations. In addition, research on cell lines and animal models may be valuable in establishing their specific roles in functional annotations. © 2023 Elsevier B.V.
An integrative analysis of functional consequences of PKD2 missense variants on RNA and protein structures: a computational approach
(Springer Nature, 2024) Chandra Devi; Prashant Ranjan; Parimal Das
Background: The PKD2, encoding polycystin-2 (PC2) protein, is second major genetic determinant of autosomal dominant polycystic kidney disease (ADPKD) after PKD1. However, the structural and functional consequences of genetic variants in PKD2 remain poorly understood. Given the complexity and heterogeneous nature of ADPKD, understanding its pathogenesis at cellular and molecular levels is vital for deciphering genotype–phenotype correlations and disease severity, thus informing patient-centered treatments. We analyzed missense variants of PKD2 to assess their impact on RNA structure using computational tools and explored associated protein structure dynamics through MD simulation. Results: Our findings reveal distinct structural alterations and dynamic behaviors associated with specific missense variants. The variants such as c.1789C > A (p.L597M), c.1109G > A (p.S370N), c.1849C > A (p.L617I), and c.646 T > C (p.Y216H) induced major changes not only in RNA structure and accessibility profile but also in protein structure dynamics. In contrast, variants such as c.915C > A (p.N305K), c.1354A > G (p.I452V), and c.568G > A (p.A190T) resulted in minor alterations in RNA structure but exhibited noticeable effects on certain parameters of protein structure dynamics. Conclusion: This study suggests the multifaceted impact of these missense variants on both RNA and protein levels. It lays the groundwork in identifying high-impact variants in terms of pathogenicity and prioritizing these for further implications in understanding disease heterogeneity and eventually contributing to the development of targeted therapeutic interventions for ADPKD. Graphical Abstract: (Figure presented.) Study Highlights : PKD2 missense variants analyzed for impact on RNA and protein Structures Alterations in RNA Structures and protein dynamics observed Computational integration of analyses aids in prioritizing variants for further study Provide insights into disease heterogeneity and potential therapeutic targets © The Author(s) 2024.
Calcimycin mediates apoptosis in breast and cervical cancer cell lines by inducing intracellular calcium levels in a P2RX4-dependent manner
(Elsevier B.V., 2024) Neha; Prashant Ranjan; Parimal Das
Background: Calcimycin (A23187) is a polyether antibiotic and divalent cation ionophore, extracted from Streptomyces chartrecensis. With wide variety of antimicrobial activities, it also exhibits cytotoxicity of tumor cells. Calcimycin exhibit therapeutic potential against tumor cell growth; however, the molecular mechanism remains to be fully elucidated. Present study explores the mechanism of calcimycin-induced apoptosis cancer cell lines. Methods: Apoptotic induction in a dose-dependent manner were recorded with MTT assays, Phase contrast imaging, wound healing assay, fluorescence imaging by DAPI and AO/EB staining and FACS using cell line model. Mitochondrial potential was analyzed by TMRM assay as Ca2+ signaling is well known to be influenced and synchronized by mitochondria also. Results: Calcimycin induces apoptosis in dose dependent manner, also accompanied by increased intracellular calcium-level and expression of purinergic receptor-P2RX4, a ligand-gated ion channel. Conclusion: Calcimycin tends to increase the intracellular calcium level, mRNA expression of ATP receptor P2RX4, and phosphorylation of p38. Blocking of either intracellular calcium by BAPTA-AM, P2RX4 expression by antagonist 5-BDBD, and phospho-p38 by SB203580, abrogated the apoptotic activity of calcimycin. General significance: Taken together, these results show that calcimycin induces apoptosis in P2RX4 and ATP mediated intracellular Ca2+ and p38 MAPK mediated pathway in both the cancer cell lines. This study explored a new mode of action for calcimycin in cancer that could be potentially employed in future studies for cancer therapeutic research. This study disentangles that the calcimycin-induced apoptotic cell death is P2RX4 and ATP involved, intracellular Ca2+ and p38 MAPK mediated pathway. © 2023
Computational exploration of protein structure dynamics and RNA structural consequences of PKD1 missense variants: implications in ADPKD pathogenesis
(Springer Science and Business Media Deutschland GmbH, 2024) Chandra Devi; Prashant Ranjan; Sonam Raj; Parimal Das
We analyzed the impact of nine previously identified missense PKD1 variants from our studies, including c.6928G > A p.G2310R, c.8809G > A p.E2937K, c.2899 T > C p.W967R, c.6284A > G p.D2095G, c.6644G > A p.R2215Q, c.7810G > A p.D2604N, c.11249G > C p.R3750P, c.1001C > T p.T334M, and c.3101A > G p.N1034S on RNA structures and PC1 protein structure dynamics utilizing computational tools. RNA structure analysis was done using short RNA snippets of 41 nucleotides with the variant position at the 21st nucleotide, ensuring 20 bases on both sides. The secondary structures of these RNA snippets were predicted using RNAstructure. Structural changes of the mutants compared to the wild type were analyzed using the MutaRNA webserver. Molecular dynamics (MD) simulation of PC1 wild-type and mutant protein regions were performed using GROMACS 2018 (GROMOS96 54a7 force field). Findings revealed that five variants including c.8809G > A (p.E2937K), c.11249G > C (p.R3750P), c.3101A > G (p.N1034S), c.6928G > A (p.G2310R), c.6644G > A (p.R2215Q) exhibited major alterations in RNA structures and thereby their interactions with other proteins or RNAs affecting protein structure dynamics. While certain variants have minimal impact on RNA conformations, their observed alterations in MD simulations indicate impact on protein structure dynamics highlighting the importance of evaluating the functional consequences of genetic variants by considering both RNA and protein levels. The study also emphasizes that each missense variant exerts a unique impact on RNA stability, and protein structure dynamics, potentially contributing to the heterogeneous clinical manifestations and progression observed in Autosomal Dominant Polycystic Kidney Disease (ADPKD) patients offering a novel perspective in this direction. Thus, the utility of studying the structure dynamics through computational tools can help in prioritizing the variants for their functional implications, understanding the molecular mechanisms underlying variability in ADPKD presentation and developing targeted therapeutic interventions. © King Abdulaziz City for Science and Technology 2024.
Enhancing tomato growth and early blight disease resistance through green-synthesized silver nanoparticles: Insights into plant physiology
(Elsevier B.V., 2024) Jeetu Narware; Satyendra P. Singh; Jharjhari chakma; Prashant Ranjan; Lopamudra Behera; Parimal Das; Nazia Manzar; Abhijeet Shankar Kashyap
Tomato (Solanum lycopersicum) is a globally significant crop, but its cultivation is challenged by the devastating Early Blight disease caused by Alternaria solani, leading to substantial yield losses. Silver nanoparticles (AgNPs) have emerged as potential antifungal agents, garnering increasing attention. In this study, we investigated the impact of biogenic AgNPs on tomato plant defense mechanisms during a three-way interaction with plant pathogens and nanoparticles. Additionally, the study explored the integration of biochemical markers to assess plant response. Histochemical analyses confirmed the presence of oxidative stress markers (H2O2 and O−2), as well as callose and lignin deposition, supporting the involvement of defensive responses in this study. Metabolic profiling revealed that tomato plants exposed to 20 ppm AgNPs exhibited maximal accumulation levels of various secondary metabolites compared to the control group. Remarkably, the concurrent application of AgNPs and plants resulted in enhanced plant resistance against biotic stress, as evidenced by reduced stress parameters and stress enzyme activity. The antioxidant enzymes (PO, SOD, CAT, LPX) exhibited significant variations among treatments, emphasizing the influence of AgNPs on maintaining reactive oxygen species (ROS) homeostasis in plant cells. These findings highlight the potential of nanotechnology-based approaches in bolstering food supply and promoting sustainable agriculture. Our study underscores the significance of integrating biochemical markers to monitor and assess plant response during the interaction with nanoparticles and pathogens. This integration offers a promising avenue for elucidating the underlying mechanisms of plant defense and optimizing agricultural practices. In conclusion, our research elucidates the potential of silver nanoparticles in augmenting plant resistance against Early Blight disease in tomato, ultimately contributing to sustainable agriculture. The incorporation of biochemical markers further advances our understanding of the intricate interplay between nanoparticles, plant pathogens, and plant defense mechanisms. © 2024 SAAB
Functional evaluation of novel chromon derivative compounds for recognition of G-quadruplex structure
(Elsevier B.V., 2024) Neha Neha; Prashant Ranjan; Surendra Kumar; Roop Shikha Singh; Daya Shankar Pandey; Parimal Das
Currently, G-quadruplex structure targeting strategies are considered as a promising anticancer approach. The purpose of this research is to expand the options for G-quadruplex targeting ligands, particularly emphasizing fluorescent ligands. Our study contributes to the field by providing additional choices for G-quadruplex binders with a notable focus on selectivity towards G-quadruplex topology over duplex DNA. In the search of selective and potent G-quadruplex binders, here we discuss an analysis of a few chroman derivatives ligands named A and C and their respective borondifluoride complexes B and D as a quadruplex targeting compounds which found to stabilize G-quadruplex structure. To investigate the binding characteristics of these molecules with G-quadruplex vs. duplex selectivity, In vitro biophysical studies were performed by steady-state fluorescence, UV–visible titration, fluorescent TO displacement assay, CD thermal melting, circular dichroism spectroscopy, and cellular imaging by employing both telomeric and PRCC G-quadruplex forming sequences. Our investigation shows that these chromam ligands and their complexes are able to selectively bind and stabilize parallel and mixed hybrid topology of G-quadruplex both In vitro and in cellular conditions. A molecular docking and MD simulation study also suggests the binding of these compounds with G-quadruplex conformation. Collectively our study suggests these chroman complexes for the first time as a potentially useful fluorescent chemical product for G-quadruplex specific ligands and expands an option for G-quadruplex targeting ligands. © 2024
Impact of B.1.617 and RBD SARS-CoV-2 variants on vaccine efficacy: An in-silico approach
(Indian Association of Medical Microbiologists, 2022) Prashant Ranjan; Neha; Chandra Devi; Garima Jain; Chandana Basu Mallick; Parimal Das
Purpose: The existing panels of COVID-19 vaccines are based on the spike protein of an earlier SARS-CoV-2 strain that emerged in Wuhan, China. However, the evolving nature of SARS-CoV-2 has resulted in the emergence of new variants, thereby posing a greater challenge in the management of the disease. India faced a deadlier second wave of infections very recently, and genomic surveillance revealed that the B.1.617 variant and its sublineages are responsible for the majority of the cases. Hence, it's crucial to determine if the current vaccines available can be effective against these variants. Methods: To address this, we performed molecular dynamics (MD) simulation on B.1.617 along with K417G variants and other RBD variants. We studied structural alteration of the spike protein and factors affecting antibody neutralization and immune escape via In silico docking. Results: We found that in seven of the 12 variants studied, there was a structural alteration in the RBD region, further affecting its stability and function. Docking analysis of RBD variants and wild-type strains revealed that these variants have a higher affinity for the ACE2 (angiotensin 2 altered enzymes) receptor. Molecular interaction with CR3022 antibody revealed that binding affinity was less in comparison to wild type, with B.1.617 showing the least binding affinity. Conclusions: The results of the extensive simulations provide novel mechanistic insights into the conformational dynamics and improve our understanding of the enhanced properties of these variants in terms of infectivity, transmissibility, neutralization potential, virulence, and host-viral replication fitness. © 2022 Indian Association of Medical Microbiologists
Impact of exogenous direct electric current on the expression of mRNA related to OPG, in SaOS-2 cells using quantitative reverse transcription polymerase chain reaction: A qualitative and quantitative analysis
(Elsevier B.V., 2025) Aparajita Pandey; Ashish Agrawal; Parimal Das; Ritu Dixit; Prashant Ranjan; Sushit Kumar Sonu
Objective: To assess OPG gene expression in SaOS-2 cells after exposure to pulsed direct current (30 μA/10 s, square wave) using qRT-PCR at different time points (5, 7,12, and 24 h). Materials and methods: The study investigated the effects of direct current (DC) electrical. stimulation on SaOS-2 cells by exposing experimental. groups to DC (30 μA, 10-s pulses) for 5, 7, 12, and 24 h, while control groups received no stimulation. Stainless steel electrodes were utilized, and both groups were cultured under identical. conditions. Qualitative assessments included cell morphology analysis through phase contrast microscopy, while quantitative evaluations involved MTT assays for cell viability and quantitative reverse transcription PCR (qRT-PCR) for osteoprotegerin (OPG) gene expression. RNA was isolated after stimulation, followed by complementary DNA (cDNA) synthesis for gene analysis. Data were analyzed to assess stimulation-induced cellular and genetic responses. Results: Direct current stimulation induced time-dependent cytotoxicity in SaOS-2 cells, with cell death increasing from approximately 10 % at 5 h to about 52 % at 24 h qRT-PCR reveals significant downregulation of OPG expression, nearly eliminated between 12 and 24 h (p < 0.0001), indicating strong inhibitory effects on both cell viability and gene expression. Conclusion: Direct electrical. stimulation downregulated OPG expression in SaOS-2 cells in a time-dependent manner, with a significant decrease observed as early as 5 h. The MTT assay reveals time-dependent cytotoxicity from DC stimulation. Reduced OPG expression suggests potential. enhancement of osteoclastic activity, indicating a possible role of DC stimulation in bone remodelling, which warrants further investigation. The Authors
Implication of rare genetic variants of NODAL and ACVR1B in congenital heart disease patients from Indian population
(Elsevier Inc., 2021) Manohar Lal Yadav; Prashant Ranjan; Parimal Das; Dharmendra Jain; Ashok Kumar; Bhagyalaxmi Mohapatra
NODAL signaling plays an essential role in vertebrate embryonic patterning and heart development. Accumulating evidences suggest that genetic mutations in TGF-β/NODAL signaling pathway can cause congenital heart disease in humans. To investigate the implication of NODAL signaling in isolated cardiovascular malformation, we have screened 300 non-syndromic CHD cases and 200 controls for NODAL and ACVR1B by Sanger sequencing and identified two rare missense (c.152C > T; p.P51L and c.981 T > A; p.D327E) variants in NODAL and a novel missense variant c.1035G > A; p.M345I in ACVR1B. All these variants are absent in 200 controls. Three-dimensional protein-modelling demonstrates that both p.P51L and p.D327E variations of NODAL and p.M345I mutation of ACVR1B, affect the tertiary structure of respective proteins. Variants of NODAL (p.P51L and p.D327E) and ACVR1B (p.M345I), significantly reduce the transactivation of AR3-Luc, (CAGA)12-Luc and (SBE)4-Luc promoters. Moreover, qRT-PCR results have also deciphered a reduction in the expression of cardiac-enriched transcription factors namely Gata4, Nkx2-5, and Tbx5 in both the mutants of NODAL. Decreased expression of, Gata4, Nkx2-5, Tbx5, and lefty is observed in p.M345I mutant of ACVR1B as well. Additionally, reduced phosphorylation of SMAD2/3 in response to these variants, suggests impaired NODAL signaling and possibly responsible for defective cell fate decision and differentiation of cardiomyocytes leading to CHD phenotype. © 2021 Elsevier Inc.
Structural, functional and evolutionary analysis of wheat WRKY45 protein: a combined bioinformatics and MD simulation approach
(Akademiai Kiado ZRt., 2024) Prashant Ranjan; Ashok Yadav; Ananta Keshari Behera; Dhiraj Kumar Singh; Premkant Singh; Ganga Prasad Singh
Bread wheat (Triticum aestivum L.) is the world's as well as India’s second-most important cereal crop. It is an allohexaploid composed of three homeologous sub-genomes (AA, BB, and DD), which is a constraint in determining the complete genome sequence. Several transcription factors have been associated with both abiotic and biotic stress. WRKY transcription factors are among the best characterised in the context of pathogen defence mechanisms. Different members of the WRKY transcription factors have been shown to confer resistance to stress. But very little is known about the wheat WRKY transcription factors. In silico analysis of the TaWRKY45 protein was performed in the present study using several bioinformatics tools like motif scan, CD search, NetPhos, NGlycos, GRAVY, and the SWISS MODEL. The study revealed that TaWRKY45 belongs to the group III family and contains hydrophilic proteins with 19 potential phosphorylation sites. TaWRKY45 protein was found to be orthologous to rice OsWRKY45 by phylogenetic analysis. The catalytic domain was analysed by motif scan which showed that TaWRKY45 has one WRKY domain and a C2-HC zinc finger motif. TaWRKY45's structure was determined to be more stable, more constrained, more compact, and have greater potential to interact with other molecules than OsWRKY45, according to MD simulation analysis. Thus, the in silico analysis of transcription factors in this study highlights the protein function, interaction, and regulatory pathways. © Akadémiai Kiadó Zrt. 2023.
The influence of new SARS-CoV-2 variant Omicron (B.1.1.529) on vaccine efficacy, its correlation to Delta variants: A computational approach
(Academic Press, 2022) Prashant Ranjan; Neha; Chandra Devi; Kaaviyapriya Arulmozhi Devar; Parimal Das
The newly discovered COVID variant B.1.1.529 in Botswana has more than 30 mutations in spike and many other in non-spike proteins, far more than any other SARS-CoV-2 variant accepted as a variant of concern by the WHO and officially named Omicron, and has sparked concern among scientists and the general public. Our findings provide insights into structural modification caused by the mutations in the Omicrons receptor-binding domain and look into the effects on interaction with the hosts neutralizing antibodies CR3022, B38, CB6, P2B–2F6, and REGN, as well as ACE2R using an in silico approach. Computational analysis revealed that the Omicron variant has a higher binding affinity for the human ACE2 receptor than the wild and Delta (AY.1 and AY.2 strains), but lower than the Delta AY.3 strain. MD simulation and docking analysis suggest that the omicron and Delta AY.3 were found to have relatively unstable RBD structures and hampered interactions with antibodies more than wild and Delta (AY.1 and AY.2), which may lead to relatively more pathogenicity and antibody escape. In addition, we observed lower binding affinity of Omicron for human monoclonal antibodies (CR3022, B38, CB6, and P2B2F6) when compared to wild and Delta (AY.1 & AY.2). However, the binding affinity of Omicron RBD variants for CR3022, B38, and P2B2F6 antibodies is lower as compared to Delta AY.3, which might promote immune evasion and reinfection and needs further experimental investigation. © 2022 Elsevier Ltd
Understanding the impact of missense mutations on the structure and function of the EDA gene in X-linked hypohidrotic ectodermal dysplasia: A bioinformatics approach
(John Wiley and Sons Inc, 2022) Prashant Ranjan; Parimal Das
X-linked hypohidrotic dysplasia (XLHED), caused by mutations in the EDA gene, is a rare genetic disease that affects the development and function of the teeth, hair, nails, and sweat glands. The structural and functional consequences of caused by an ectodysplasin-A (EDA) mutations on protein phenotype, stability, and posttranslational modifications (PTMs) have not been well investigated. The present investigation involves five missense mutations that cause XLHED (L56P, R155C, P220L, V251M, and V322A) in different domains of EDA (TM, furin, collagen, and tumor necrosis factor [TNF]) from previously published papers. The deleterious nature of EDA mutant variants was identified using several computational algorithm tools. The point mutations induce major drifts in the structural flexibility of EDA mutant variants and have a negative impact on their stability, according to the 3D protein modeling tool assay. Using the molecular docking technique, EDA/EDA variants were docked to 10 EDA interacting partners, retrieved from the STRING database. We found a novel biomarker CD68 by molecular docking analysis, suggesting all five EDA variants had lower affinity for EDAR, EDA2R, and CD68, implying that they would affect embryonic signaling between the ectodermal and mesodermal cell layers. In silico research such as gene ontology, subcellular localization, protein–protein interaction, and PTMs investigations indicates major functional alterations would occur in EDA variants. According to molecular simulations, EDA variants influence the structural conformation, compactness, stiffness, and function of the EDA protein. Further studies on cell line and animal models might be useful in determining their specific roles in functional annotations. © 2021 Wiley Periodicals LLC.
Urinary extracellular vesicles miRNA—A new era of prostate cancer biomarkers
(Frontiers Media S.A., 2023) Garima Jain; Parimal Das; Prashant Ranjan; Neha; Ferran Valderrama; Clara Cieza-Borrella
Prostate cancer is the second most common male cancer worldwide showing the highest rates of incidence in Western Europe. Although the measurement of serum prostate-specific antigen levels is the current gold standard in PCa diagnosis, PSA-based screening is not considered a reliable diagnosis and prognosis tool due to its lower sensitivity and poor predictive score which lead to a 22%–43% overdiagnosis, unnecessary biopsies, and over-treatment. These major limitations along with the heterogeneous nature of the disease have made PCa a very unappreciative subject for diagnostics, resulting in poor patient management; thus, it urges to identify and validate new reliable PCa biomarkers that can provide accurate information in regard to disease diagnosis and prognosis. Researchers have explored the analysis of microRNAs (miRNAs), messenger RNAs (mRNAs), small proteins, genomic rearrangements, and gene expression in body fluids and non-solid tissues in search of lesser invasive yet efficient PCa biomarkers. Although the presence of miRNAs in body fluids like blood, urine, and saliva initially sparked great interest among the scientific community; their potential use as liquid biopsy biomarkers in PCa is still at a very nascent stage with respect to other well-established diagnostics and prognosis tools. Up to date, numerous studies have been conducted in search of PCa miRNA-based biomarkers in whole blood or blood serum; however, only a few studies have investigated their presence in urine samples of which less than two tens involve the detection of miRNAs in extracellular vesicles isolated from urine. In addition, there exists some discrepancy around the identification of miRNAs in PCa urine samples due to the diversity of the urine fractions that can be targeted for analysis such as urine circulating cells, cell-free fractions, and exosomes. In this review, we aim to discuss research output from the most recent studies involving the analysis of urinary EVs for the identification of miRNA-based PCa-specific biomarkers. Copyright © 2023 Jain, Das, Ranjan, Neha, Valderrama and Cieza-Borrella.