Biology subjects

Joshi, M.

Publications and source records attributed to Joshi, M..

At least 19 recordsLinked to original sources

bioRxiv2026

Polyglutamine aggregation proceeds by stochastic nucleation in the C. elegans nervous system

Fibrillar protein aggregation is the hallmark of a wide variety of neurodegenerative diseases. It is still poorly understood how soluble proteins transition to fibrillar aggregates, in particular within the complex environment of cells and tissues. Here, we employ the nematode Caenorhabditis elegans to elucidate the molecular mechanisms of polyglutamine aggregation in the nervous system of a multicellular animal. By using fluorescence microscopy in combination with mathematical modelling, we find that polyglutamine aggregation occurs by stochastic nucleation in individual neurons, at a rate that is of the same order of magnitude as previously determined for muscle cells. The J-protein DNAJB6 strongly suppresses polyglutamine aggregation in both tissues, consistent with a mechanism in which aggregation is limited by primary nucleation. We confirm that the same mechanism holds true for a construct including the disease-relevant flanking regions of Huntingtin exon 1. Altogether, our data reveal that polyglutamine aggregation occurs spontaneously and largely independent of the cellular context.

biophysics↗

bioRxiv2026

How variable and stress-sensitive is sleep expression in lizards?

Sleep expression in wild animals is regulated by their ecological context. Yet, how populations of a species differ in their sleep in response to environmental challenges such as urbanization, remains to be fully understood. Robust quantification of sleep in small animals, such as lizards, has so far been methodologically difficult, limiting population-level assessments. Using recently developed miniature loggers, we recorded sleep in 19 wild-caught individuals of the Peninsular rock agama lizard (Psammophilus dorsalis). We compared individuals from urban and rural environments and measured electro-oculogram (EOG)-derived sleep parameters: sleep duration, bout frequency, bout duration, and interval between bouts. We then subjected all individuals to an acute stressor (handling-restraint) and examined changes in sleep expression. We found that this species slept mostly at night, with an average total sleep time of 11h 10min. EOG-derived sleep characteristics did not differ substantially between populations, although daytime sleep bouts were more consolidated (shorter interval between bouts) in urban lizards. Contrary to evidence from mammals, acute restraint stress did not impact sleep, overall or for either population. To complement these measures, we also measured the latency to arousal after exposure to vibration stimuli in a separate set of urban and rural lizards (n = 12 each), and found that urban individuals responded marginally quicker at night compared rural individuals, demonstrating greater vigilance and/or more fragmented sleep. Overall, we show that sleep characteristics of wild animals in a common garden condition can be conserved within a species, with limited context-dependent variation between populations.

animal behavior and cognition↗

bioRxiv2026

Identification of small-molecule enhancers of circadian rhythm amplitude in central and peripheral clocks

The circadian clock regulates daily rhythms in metabolism, behavior, and physiological homeostasis. Damped or disrupted circadian rhythms are linked to physiological malfunctions, including neurodegeneration, metabolic syndrome, and cancers. Enhancing circadian rhythm amplitude can strengthen the processes regulated through the circadian system and offer a potential therapeutic approach. Here, we present eight small-molecule enhancers of circadian amplitude with previously unrecognized roles in circadian regulation. By using a Bmal1 transcription reporter system in NIH3T3 fibroblasts, we screened a drug repurposing library comprising 5,631 diverse compounds. Through which eight molecules were identified to increase circadian amplitude across multiple cycles, in a dose-dependent manner, without significantly perturbing period or phase. We evaluated the physiological potential of these compounds in ex vivo mouse tissues expressing PER2 luminescence reporter. A set of compounds tested in organotypic liver or suprachiasmatic nucleus slices enhanced the circadian amplitude in respective tissues, showing their suitability for peripheral as well as central clocks. Single-cell imaging of suprachiasmatic nucleus slices revealed that amplitude enhancement results from increased expression of clock proteins in individual cells rather than from intercellular synchronization. Together, these compounds and their targets present a new array of circadian amplitude modulators that may present opportunities to pharmacologically enhance clock robustness in physiology and disease. Significance StatementThrough a network of molecular interactions, the circadian clock coordinates rhythms of biochemical and behavioural processes, right from the level of a single cell to organs. Therefore, maintaining a strong circadian clock, through its high-amplitude circadian rhythm, is crucial. However, few pharmacological approaches and targets are known to enhance circadian amplitude. This study presents eight previously uncharacterized amplitude enhancer molecules by conducting a chemical screen on cultured fibroblasts. Identified molecules also induced amplitude enhancement in mouse-derived liver and brain-SCN slices, showing their robust functionality across tissues. In brain-SCN slices, the amplitude enhancement was found to occur on the single-cell level without affecting the synchronization between cells.

biochemistry↗

bioRxiv2026

Distinct Effects of the Extracellular Matrix on Tumor Organization and Response to Cancer Virotherapy

Extracellular matrix (ECM) regulates tumor architecture and immune accessibility, but its impact on virus-based anticancer therapies is not well understood. In particular, ECM modulation leads to both enhanced and impaired therapeutic outcomes in vivo, reflecting the complexity of matrix-immune-virus interactions. Here, we combine computational modeling with empirical 3D tumor spheroid-immune in vitro co-cultures to examine how ECM composition and abundance shape an Alphavirus replicon-based therapy. We show that ECM-induced changes in tumor spheroid architecture vary by cell-adhesion phenotype but higher matrix abundance consistently reduces cellular density. Notably, increasing abundance of basement membrane extract or Collagen-I reduced tumor spheroid cell density, which generally enhanced viral infection, T cell activation, and IFN{gamma} production. However, reduced immune accessibility in collagen-rich environments suppressed immune responses despite improved infection, highlighting matrix-specific effects. Our findings provide insights into ECMs role in virus-based therapies and propose a bottom-up framework for systematically evaluating its influence on therapeutic efficacy.

cancer biology↗

bioRxiv2025

Structural basis of half-site reactivity in Class Ib ribonucleotide reductases

Ribonucleotide reductases (RNRs) employ radical chemistry to generate deoxyribonucleotides required for DNA synthesis and repair. A notable feature of RNRs is half-site reactivity, where, despite the enzyme being a symmetric 2 dimer, only one active site is catalytically active at a time while the other remains in a "poised" state for substrate binding. This phenomenon is tightly linked to the asymmetric 2{beta}2 interaction required for radical transfer. Here, we determined cryo-EM structures of the -subunit in the apo and holo states, i.e., the complex bound to TTP (effector) and GDP (substrate). The structures reveal asymmetric binding of the effector TTP and the substrate GDP across the dimer, with concomitant stabilization of loops surrounding the ligand-binding site. Interestingly, this asymmetry leads to well-resolved N-terminal density for [~]150 residues in the substrate-bound subunit, but weak density for this region in the effector-bound monomer. N-terminal domains are unresolved in both monomers of the apo structure. Isothermal titration calorimetry supports asymmetric binding of pyrimidine effectors with micromolar affinities. Molecular dynamics simulations and three-dimensional variability analysis reveal synchronous motions of loop 2, which together with the N-terminal domain drive alternate opening and closing of the active sites in the two monomers. These conformational dynamics provide key insights into the mechanistic basis of half-site reactivity. Together, these findings provide new insights into the structural dynamics and thermodynamic principles governing regulation and half-site activity in Class Ib RNRs. Significance statementRibonucleotide reductases (RNRs) are essential enzymes that supply the building blocks required for DNA synthesis and repair, yet the structural basis of their half-site reactivity has remained unclear. Using cryo-electron microscopy, calorimetry, molecular dynamics simulations, and conformational variability analysis, we show that the catalytic -subunit of a Class Ib RNR exhibits asymmetric nucleotide binding and coordinated conformational dynamics between the two monomers. These motions drive alternating opening and closing of the active sites and are linked to differential stabilization of the N-terminal region. Our findings suggest that asymmetric conformational gating and N-terminal sampling regulate productive interaction with the radical-generating {beta}-subunit, providing a mechanistic framework for understanding half-site reactivity and allosteric regulation in RNRs.

biophysics↗

bioRxiv2025

Mutations in antimicrobial peptides differently affect sleep and plasticity

Molecules implicated in host defense can, independent of their roles as immune mediators, regulate sleep. In mammals, cytokines such as Tumor Necrosis Factor (TNF ) and Interleukin 1{beta} (IL1{beta}) promote sleep. In Drosophila, infection stimulates the systemic release of a battery of anti-microbial peptides (AMPs). These fly immune effectors have been understudied as sleep modulators. We obtained mutants in different AMPs and characterized their functions in sleep and sleep dependent outputs. AMP mutants sleep less with a longer sleep latency, and lowered arousal thresholds. Group C mutant flies, doubly mutant for the anti-fungal peptides Metchinikowin (Mtk) and Drosomycin (Drs), showed the greatest impairments. These Group C mutants also showed an exaggerated sleep rebound. Sleep rebound was unaltered in other mutants. Different sets of AMP mutants exhibited specific disruptions in socialization and rocking induced sleep. These data are a detailed characterization of sleep regulation in AMP mutants. We also evaluated sleep functions. Group C mutants uniquely exhibited normal learning and memory, and lower synapse abundance, despite sleeping the least. Group C mutants are thus able to carry out some sleep functions without sleeping very much. Glial knockdown of Metchnikowin and Drosomycin mimicked the sleep phenotypes of the null mutants, these genes thus act from glia. Sleep and memory defects in AMP mutants was reversible - enhancing sleep of Bomanin mutants pharmacologically or behaviorally improved learning and memory. Together, these data suggest that AMPs are potent sleep modulators and that different classes of AMPs differently affect sleep and sleep dependent outcomes.

neuroscience↗

bioRxiv2025

Human tau compromises neuronal structural integrity in C. elegans promoted by age, stress and phosphorylation

Tauopathies are a group of progressive neurodegenerative diseases amongst which Alzheimers disease is the most prevalent. They typically have a late age of onset and lead to cognitive decline. Tau is a highly soluble, intrinsically disordered microtubule binding protein that aggregates in these diseases. The mechanisms by which age-associated changes alter tau functionality and compromise neuronal structural integrity remain poorly understood. Here, we show with the use of single-copy gene insertion Caenorhabditis elegans models that human tau preferentially localises to neuronal processes in the nematode, consistent with its function of binding to microtubules. The expression of tau in the nervous system leads to age-associated changes in neuronal structural integrity. The observed "buckling" phenotype was exacerbated by exposure of the animals to different stressors which are known to enhance tau phosphorylation. Expressing phosphomimic tau or co-expressing human kinases both severely worsened the buckling phenotype. Kinase expression additionally induced tau inclusions in a small proportion of animals. These results point to an interplay between ageing, stress and phosphorylation leading to structural changes in neuronal processes prior to widespread tau aggregation.

neuroscience↗

bioRxiv2025

Transcriptomic profiling of desert tree Prosopis cineraria under heat stress reveals potential role of multiple gene families in its high thermotolerance

The static nature of plants restrains their potential to evade heat stress and requires them to withstand stress through inherent defence abilities. Prosopis cineraria is a leguminous phreatophyte distributed across arid and semi-arid regions of India and can tolerate very high temperatures due to its adaptive physiological and biochemical mechanisms. Therefore, P. cineraria represents a repository of genes for abiotic stress tolerance. Two-months-old P. cineraria plants were subjected to heat stress at two different temperature regimes and transcriptome sequencing was performed to identify differentially expressed genes (DEGs). A total of 1151 and 1562 DEGs were observed in response to 45 and 55 heat stress compared to control, respectively, indicating that 55 treatment has a pronounced effect on P. cineraria. The transcriptomic data highlighted the potential role of multiple gene families and their interactions for high thermotolerance of P. cineraria. The expression of a few representative heat stress-responsive genes was validated with real-time qPCR. The in-depth bioinformatic analysis provided the detailed transcriptome profiling, supported by its validation, and new insights for important abiotic stress-related genes from thermotolerant P. cineraria, which can be used for crop improvement.

plant biology↗

bioRxiv2024

Comparative and population genomics analyses of the regulatory Transcription Factor (TF)-DNA interaction

Natural selection heavily influences the evolutionary trajectories of species by impacting their genotype to phenotype transitions. On the molecular level, these genotype to phenotype transitions are shaped by the regulatory sequences (RSs). In this study, we employed a combination of population and comparative genomics to investigate how natural selection affects specific RS classes involved in the regulatory transcription factor (TF)-DNA interactions. These interactions consist of two motifs, namely: TF-binding domains (TF-BDs) and TF-binding sites (TF-BSs). Using publicly available annotation data, we constructed the species-specific lists of the TF-BD and TF-BS regions. We also constructed a non-synonymous equivalent class for the TF-BS regions for making comparative analyses across the coding and non-coding regions. On applying some of the commonly used summary statistics, we found a consistent signal of purifying selection acting on TF-BDs, consistent with their functional importance. Interestingly, we also observed that non-coding TF-BS regions showed similar levels of constraint to that of coding regions for populations with large Ne. Concerning positive selection, we showed that the regulatory motifs of large Ne species are, overall, subject to an increased intensity of positive selection. We found an overall positive correlation between the intensity of purifying and positive selection and the species-specific Ne, which is expected under drift-selection equilibrium. Finally, we contrasted the signal of positive selection in H. sapiens and D. melanogaster (outcrossing species) with A. thaliana (selfing species) and noted that selfing plays a key role in influencing the action of positive selection.

evolutionary biology↗

bioRxiv2024

Phenotypic landscape of a fungal meningitis pathogen reveals its unique biology

Cryptococcus neoformans is the most common cause of fungal meningitis and the top-ranked W.H.O. priority fungal pathogen. Only distantly related to model fungi, C. neoformans is also a powerful experimental system for exploring conserved eukaryotic mechanisms lost from specialist model yeast lineages. To decipher its biology globally, we constructed 4328 gene deletions and measured--with exceptional precision--the fitness of each mutant under 141 diverse growth-limiting in vitro conditions and during murine infection. We defined functional modules by clustering genes based on their phenotypic signatures. In-depth studies leveraged these data in two ways. First, we defined and investigated new components of key signaling pathways, which revealed animal-like pathways/components not predicted from studies of model yeasts. Second, we identified environmental adaptation mechanisms repurposed to promote mammalian virulence by C. neoformans, which lacks a known animal reservoir. Our work provides an unprecedented resource for deciphering a deadly human pathogen.

microbiology↗

bioRxiv2024

Spatial distribution of poultry farms using point pattern modelling: a method to address livestock environmental impacts and disease transmission risks

The distribution of farm locations and sizes is paramount to characterize patterns of disease spread. With some regions undergoing rapid intensification of livestock production, resulting in increased clustering of farms in peri-urban areas, measuring changes in the spatial distribution of farms is crucial to design effective interventions. However, those data are not available in many countries, their generation being resource-intensive. Here, we develop a farm distribution model (FDM), which allows the prediction of locations and sizes of poultry farms in countries with scarce data. The model combines (i) a Log-Gaussian Cox process model to simulate the farm distribution as a spatial Poisson point process, and (ii) a random forest model to simulate farm sizes (i.e. the number of animals per farm). Spatial predictors were used to calibrate the FDM on intensive broiler and layer farm distributions in Bangladesh, Gujarat (Indian state) and Thailand. The FDM yielded realistic farm distributions in terms of spatial clustering, farm locations and sizes, while providing insights on the factors influencing these distributions. Finally, we illustrate the relevance of modelling realistic farm distributions in the context of epidemic spread by simulating pathogen transmission on an array of spatial distributions of farms. We found that farm distributions generated from the FDM yielded spreading patterns consistent with simulations using observed data, while random point patterns underestimated the probability of large outbreaks. Indeed, spatial clustering increases vulnerability to epidemics, highlighting the need to account for it in epidemiological modelling studies. As the FDM maintains a realistic distribution of farm location and sizes, its use to inform mathematical models of disease transmission is particularly relevant for regions where these data are not available.

bioinformatics↗

bioRxiv2023

Paternally-induced environmental programming of placenta development, offspring birth weight and health phenotypes in a mouse model

Mounting evidence suggests that environmentally induced epigenetic inheritance occurs in mammals and that traits in the progeny can be shaped by parental environmental experiences. Epidemiological studies link parental exposure to environmental toxicants, such as the pesticide DDT, to health phenotypes in the progeny, including low birth and increased risk of chronic diseases later in life. Here, we show that the progeny of male mice exposed to DDT in the pre-conception period are born smaller and exhibit sexual dimorphism in metabolic function, with male offspring developing both glucose intolerance and insulin resistance compared to controls. These phenotypes in DDT offspring were linked to reduced fetal growth and placenta size as well as placenta-specific reduction of glycogen levels and the nutrient sensor and epigenetic regulator OGT, with more pronounced phenotypes observed in male placentas. However, placenta-specific genetic reduction of OGT only partially replicates the metabolic phenotype observed in offspring of DDT-exposed males. Our findings reveal a role for paternal pre-conception environmental experiences in shaping placenta development and in fetal growth restriction. While many questions remain, our data raise the tantalizing possibility that placenta programming could be a mediator of environmentally induced intergenerational epigenetic inheritance of phenotypes and needs to be further evaluated.

developmental biology↗

bioRxiv2023

Unravelling Genomic Origin of Lumpy Skin Disease Virus in Recent Outbreaks

Lumpy skin disease virus (LSDV) belongs to the genus Capripox virus and family Poxviridae. LSDV was endemic in most of Africa, Middle east and Turkey, but since 2015, several outbreaks have been reported in Asian countries. In this study we used Whole Genome Sequence (WGS) approach to investigate the origin of the outbreak and understand the genomic landscape of the virus. Our study showed that the LSDV strain of 2022 outbreak exhibited many genetic variations, compared to the Reference Neethling strain sequence and the previous field strains from India. A total of 1819 variations were found in 22 genome sequences, which includes 399 extragenic mutations, 153 insertion frameshift mutations, 234 deletion frameshift mutations, 271 Single nucleotide polymorphisms (SNPs) and 762 silent SNPs. 38 genes have more than 2 variations per gene and these genes belong to viral-core protein, viral binding proteins, replication and RNA polymerase proteins. We highlight the importance of several SNPs in various genes which may play an essential role in pathogenesis of LSDV. Phylogenetic analysis performed on all whole genome sequences of LSDV showed two types of variants in India. One group of the variant with fewer mutations was found to lie closer to the LSDV 2019 strain from Ranchi while the other group clustered with previous Russian outbreaks from 2015. Our study highlights the importance of genomic characterization of viral outbreaks to not only monitor the frequency of mutations but also address its role in pathogenesis of LSDV as the outbreak continues.

microbiology↗

bioRxiv2023

Penetration of Topically Applied Polymeric Nanoparticles across the Epidermis of Thick skin

The natural barrier function of the epidermal skin layer poses a significant challenge to nanoparticle-mediated topical delivery. A key factor in this barrier function is the thickness of the stratum corneum (SC) layer within the epidermis, which varies across different anatomical sites. The epidermis from the palms and soles, for instance, have thicker SC compared to those from other areas. Previous studies have attempted to bypass the SC layer for nanoparticle penetration by using physical disruption; however, these studies have mostly focused on non-thick skin. In this study, we investigate the role of mechano-physical strategies on SC of thick skin for transdermal nanoparticle penetration. We characterize and compare two mechano-physical strategies, namely tape-stripping and microneedle abrasion, for epidermal disruption in both thick and thin skin. Furthermore, we examine the impact of SC disruption in thick and thin skin on the penetration of topically applied 100 nm sized polystyrene nanoparticles using an ex-vivo model. Our findings show that tape-stripping reduced the overall thickness of SC in thick skin by 87%, from 67.4 {+/-} 17.3 {micro}m to 8.2 {+/-} 8.5 {micro}m, whereas it reduced thin skin SC by only 38%, from 9.9 {+/-} 0.6 {micro}m to 6.2 {+/-} 3.2 {micro}m. Compared to non-thick skin, SC disruption in thick skin resulted in higher nanoparticle diffusion. Tape-stripping effectively reduces SC thickness of thick skin and can be potentially utilized for enhanced penetration of topically applied nanoparticles in skin conditions that affect thick skin.

bioengineering↗

bioRxiv2023

Patterns of speciation in a parapatric pair of Saturnia moths as revealed by Target Capture

The focus of this study is to understand the evolutionary relationships and taxonomy of widely distributed parapatric species pair of wild silk moths, Saturnia pavonia and Saturnia pavoniella (Lepidoptera: Saturniidae) in Europe. To address species delimitation challenges associated with many parapatric taxa, target enrichment and mtDNA sequencing was employed alongside phylogenetic, species delimitation, admixture and introgression analyses. The dataset included individuals from both species, two hybrids generated in the lab, as well as individuals from outside the contact zone. Nuclear markers strongly supported both S. pavonia and S. pavoniella as two distinct species, with the hybrids grouping together as intermediate and separate from both species. However, the maximum likelihood (ML) tree generated from mtDNA sequencing data presented a different picture, showing both taxa to be phylogenetically intermixed. This inconsistency may be attributed to mitonuclear discordance, which can arise from biological factors (e.g., introgressive hybridization or incomplete lineage sorting) or alternatively operational factors (e.g., incorrect species delimitation). We further provide the evidence of past introgression to have taken place, but no evidence of current admixture between the two species. Finally, we discuss our results from evolutionary point of view taking into consideration the past climatic oscillations that has likely shaped the present dynamics between the species. Overall, this study demonstrated the effectiveness of the target enrichment approach in resolving the phylogenetic relationships between closely related parapatric species and providing insights into their taxonomic delimitation.

evolutionary biology↗

bioRxiv2023

Species Delimitation Under Allopatry: Genomic Divergences Within and Across Continents in Lepidoptera

Delimitation of allopatric populations into species remains subjective and largely arbitrary. Many cold-adapted species from the subarctic and Central and Southern European Mountain systems display frequent allopatry with disjunct distributions of their populations. The same concerns Holarctic species which are many and which almost regularly show various degrees of differentiation between the continents. In this study, we analyze high- throughput target enrichment data for ten groups of arctic-alpine and Holarctic lepidopteran species sampled from four main regions across the Holarctic realm - Fennoscandia, North America, Alps and Altai. We first aimed to assess whether the genetic differences in the nuclear genome reflect observed DNA barcode divergences and second, whether the gap between population and species-level differences can be dissected using genomic data. We compared the phylogenetic trees and uncorrected pairwise genetic distances obtained from target enrichment and the mitochondrial cytochrome oxidase I (COI) barcodes for each of the study species. Additionally, we also performed a suite of population genetic and species delimitation analyses to further shed light on patterns of intraspecific variation using a large number of nuclear markers. We observed that in about one half of the cases, DNA barcodes tended to show phylogenetic relationships similar to the target enrichment markers. We report varying levels of nuclear genetic differentiation among the populations analyzed, starting from low differentiation of geographically separated populations to the deeper separation of some Nearctic population and further arctic-alpine disjunction. Given that no single consistent pattern emerged across different case studies, we demonstrate that the delimitation of allopatric populations into species could be done much more efficiently and in a consistent manner if based on a large set of universal genetic loci, which would help in reaching standards for taxonomic delimitation of allopatric populations.

evolutionary biology↗

bioRxiv2023

Development of a multifunctional toolkit of intrabody-based biosensors recognizing the V5 peptide tag: highlighting applications with G protein-coupled receptors

ABSTRACT/SUMMARYProtein-protein interactions (PPIs) form the underpinnings of any cellular signaling network. PPIs are highly dynamic processes and often, cell-based assays can be essential for their study as they closely mimic the biological intricacies of cellular environments. Since no sole platform can perform all needed experiments to gain a thoroughly comprehensive understanding into these processes, developing a versatile toolkit is much needed to address this longstanding gap. The use of small peptide tags, such as the V5-tag, has been extensively used in biological and biomedical research, including labeling the C-termini of one of the largest human genome-wide open-reading frame collections. However, these small peptide tags have been primarily used in vitro and lack the in vivo traceability and functionality of larger specialized tags. In this study, we combined structural studies and computer-aided maturation to generate an intracellular nanobody, interacting with the V5-tag. Suitable for assays commonly used to study protein-protein interactions, our nanobody has been applied herein to interrogate G protein-coupled receptor signalling. This novel serviceable intrabody is the cornerstone of a multipurpose intracellular nanobody-based biosensors toolkit, named iBodyV5, which will be available for the scientific community at large.

molecular biology↗

bioRxiv2023

DNA metabarcoding using new rbcL and ITS2 metabarcodes collectively enhance detection efficiency of medicinal plants in single and polyherbal formulations

With the widespread adoption of barcoding and next-generation sequencing, metabarcoding is emerging as a potential tool for detecting labelled and unlabelled plant species in herbal products. In this study, newly designed rbcL and ITS2 metabarcode primers were validated for metabarcoding using in-house mock controls of medicinal plant gDNA pools and biomass pools. The applicability of the multi-barcode sequencing approach was evaluated on 17 single drugs and 15 polyherbal formulations procured from the Indian market. The rbcL metabarcode demonstrated detection efficiencies of 86.7% and 71.7% in gDNA plant pools and biomass mock controls, respectively, while the ITS2 metabarcode demonstrated 82.2% and 69.4%. In the gDNA plant pool and biomass pool mock controls, the cumulative detection efficiency increased by 100% and 90%, respectively. A total of 79% cumulative detection efficiency of both metabarcodes was observed in single drugs, while 76.3% was observed in polyherbal formulations. An average fidelity of 83.6% was observed for targeted plant species present within mock controls as well as in herbal formulations. Our results demonstrated the applicability of multilocus strategies in metabarcoding as a potential tool for detecting labelled and unlabelled plant species in herbal formulations.

molecular biology↗