Biology subjects

Parmar, S.

Publications and source records attributed to Parmar, S..

14 recordsLinked to original sources

bioRxiv2025

Discovery of small molecule probes targeting the 5' stem-loop in the yeast U4/U6 snRNA

The spliceosome is a large ribonucleoprotein complex that regulates pre-mRNA splicing and has been an intriguing target for drug discovery. Essential to the assembly of the spliceosome are the small nuclear RNAs (snRNAs), which form RNA-RNA and RNA-protein interactions in the intact spliceosome and during its assembly. Here, we study the yeast U4/U6 snRNA assembly and report the rapid discovery of small molecule K-turn ligands via parallel small molecule microarray (SMM) screening of multiple related RNA constructs. For hit validation, biophysical analyses were conducted to confirm the binding and effects on thermodynamic stability and of RNA structure. One analog of the hit molecule (22) exhibited improved affinity towards the yeast U4/U6 snRNA (KD = 3.9 {+/-} 2.2 {micro}M). The specific interaction between 22 and the K-turn region was studied experimentally using deltaSHAPE and in silico with MD simulations. Moreover, this molecule was found to inhibit the binding of the U4 to Snu13 in biochemical assays (IC50 = 3.2 {+/-} 0.4 {micro}M). This work reports new ligands for the U4 snRNA and reveals that a multiplexed, structure-based approach can be used to identify small molecules that bind to specific regions of complex RNAs. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/681657v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@c156f4org.highwire.dtl.DTLVardef@133e90eorg.highwire.dtl.DTLVardef@109b793org.highwire.dtl.DTLVardef@f3301f_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

Whole genome sequencing-based multi-locus association mapping for kernel iron, zinc and protein content in groundnut

Malnutrition is a major global challenge, especially in the developing regions, where improving the nutritional content of staple crops is a significant step towards alleviating hidden hunger. Groundnut, a nutrient rich legume contains several mineral nutrients, high protein, essential amino-acids and vitamins that are required for human health. In this study, multi-season phenotyping data for kernel iron (Fe), zinc (Zn) content and protein content (PC) and whole genome re-sequencing (WGRS) data on mini-core collection, were used to perform genome-wide association study (GWAS) analysis. Phenotypic variability analysis revealed a large variation in the Fe (7.6 - 42.8 ppm), Zn (10.9 - 62.4 ppm) and PC (12.7 - 33.6%). GWAS analysis identified a total of 15 marker-trait associations (MTAs) and 28 candidate genes for pooled season data, as well as 44 MTAs and 62 candidate genes for individual seasons. Key candidate genes like MYB transcription factor (Arahy.QI0PHV, Arahy.1I6ZSS), Zn finger MYM type protein, RING finger MYM type protein (Arahy.7P97F6, Arahy.9R964H, Arahy.I3B88T) and NAC domain protein (Arahy.LV3APC), associated with the Fe and Zn homeostasis pathway, genes related to protein homeostasis, such as protein kinase family protein (Arahy.4D7KBI), and E3 ubiquitin-protein ligase (Arahy.PE3CF6), were also identified within significant MTA regions. These findings provide basis for the detection and characterization of the possible candidate genes related to the nutritional quality traits. Single nucleotide polymorphism (SNP)-based KASP (Kompetitive Allele Specific Polymerase Chain Reaction) markers for 9 SNPs were designed and validated. Of these, three markers (snpAH00636, snpAH00641 and snpAH00644) showed polymorphism which could be deployed in the genomics-assisted breeding for the development of nutrient-rich groundnut varieties.

plant biology↗

bioRxiv2025

Kinase interplay centered on two regulatory elements confines the checkpoint protein Lte1 to the bud cell cortical compartment

In budding yeast, Lte1, a component of the spindle position checkpoint, must be confined to the bud for correctly coupling chromosomal segregation across the bud neck with mitotic exit. Bud compartmentalization involves a phospho-dependent program with Ras-GTP acting as the ultimate cortical anchor. Here we have dissected this program by identifying an Lte1 domain participating in Ras binding and two phospho-regulatory motifs, the N- and C-LREs (Lte1 Regulatory Elements). The C-LRE mediates control of Lte1 cortical recruitment by the p21-activated kinase, Cla4. The N-LRE modulates Lte1 binding to Ras more directly and is phosphorylated by the yeast MARK/PAR-1 protein kinases, Kin1/2. Along with previously described CDK phosphorylation sites, the N- and C-LREs are required for Lte1 localization and timely mitotic exit. These studies uncover the coordinated interplay between multiple protein kinases allowing Lte1 to integrate cell polarity and cell cycle cues for strict compartmentalization at the bud cell cortex.

cell biology↗

bioRxiv2024

High-throughput unsupervised quantification of patterns in the natural behavior of marmosets

Recent advances in genetic engineering have accelerated the production of nonhuman primate models for neuropsychiatric disorders. To use these models for preclinical drug testing, behavioral screening methods will be necessary to determine how the model animals deviate from controls, and whether treatments can restore typical patterns of behavior. In this study, we collected a multimodal dataset from a large cohort of marmoset monkeys and described typical patterns in their natural behavior. We found that these behavioral measurements varied substantially across days, and that behavioral state usage was highly correlated to the behavior of cagemates and to the vocalization rate of other animals in the colony. To elicit acute behavioral responses, we presented animals with a panel of stimuli including novel, appetitive, neutral, aversive, and social stimuli. By comparing these behavioral conditions, we demonstrate that outlier detection can be used to identify atypical responses to a range of stimuli. This data will help guide the study of marmosets as models for neuropsychiatric disorders.

animal behavior and cognition↗

bioRxiv2024

Mechanistic Analysis of Riboswitch Ligand Interactions Provides Insights into Pharmacological Control over Gene Expression

Riboswitches are structured RNA elements that regulate gene expression upon binding to small molecule ligands. Understanding the mechanisms by which small molecules impact riboswitch activity is key to developing potent, selective ligands for these and other RNA targets. We report the structure-informed design of chemically diverse synthetic ligands for PreQ1 riboswitches. Multiple X-ray co-crystal structures of synthetic ligands with the Thermoanaerobacter tengcongensis (Tte)-PreQ1 riboswitch confirm a common binding site with the cognate ligand, despite considerable chemical differences among the ligands. Structure probing assays demonstrate that one ligand causes conformational changes similar to PreQ1 in six structurally and mechanistically diverse PreQ1 riboswitch aptamers. Single-molecule force spectroscopy is used to demonstrate differential modes of riboswitch stabilization by the ligands. Binding of the natural ligand brings about the formation of a persistent, folded pseudoknot structure, whereas a synthetic ligand decreases the rate of unfolding through a kinetic mechanism. Single round transcription termination assays show the biochemical activity of the ligands, while a GFP reporter system reveals compound activity in regulating gene expression in live cells without toxicity. Taken together, this study reveals that diverse small molecules can impact gene expression in live cells by altering conformational changes in RNA structures through distinct mechanisms.

biochemistry↗

bioRxiv2024

Deciphering the molecular mechanisms underlying anti-pathogenic potential of a polyherbal formulation Enteropan against multi-drug resistant Pseudomonas aeruginosa

Anti-pathogenic potential of a polyherbal formulation Enteropan(R) was investigated against a multidrug resistant strain of the bacterium Pseudomonas aeruginosa. Enteropan-pre-exposed P. aeruginosa displayed reduced ([~]70%{downarrow}) virulence towards the model host Caenorhabditis elegans. Enteropan affected various traits like biofilm formation, protein synthesis and secretion, quorum-modulated pigment production, antibiotic susceptibility, nitrogen metabolism etc. in this pathogen. P. aeruginosa could not develop complete resistance to the virulence-attenuating activity of Enteropan even after repeated exposure to this polyherbal formulation. Whole transcriptome analysis showed 17% of P. aeruginosa genome to get differentially expressed under influence of Enteropan. Major mechanisms through which Enteropan exerted its anti-virulence activity were found to be generation of nitrosative stress, oxidative stress, envelop stress, quorum modulation, disturbance of protein homeostasis and metal homeostasis. Network analysis of the differently expressed genes resulted in identification of 10 proteins with high network centrality as potential targets from among the down-regulated genes. Differential expression of genes coding for five (rpoA, tig, rpsB, rpsL, and rpsJ) of these targets was validated through RT-PCR too, and they can further be pursued as potential targets by various drug discovery programmes.

microbiology↗

bioRxiv2021

Interrogating RNA-small molecule interactions with structure probing and AI augmented-molecular simulations

While there is increasing interest in the study of RNA as a therapeutic target, efforts to understand RNA-ligand recognition at the molecular level lag far behind our understanding of protein-ligand recognition. This problem is complicated due to the more than ten orders of magnitude in timescales involved in RNA dynamics and ligand binding events, making it not straightforward to design experiments or simulations. Here we make use of artificial intelligence (AI)-augmented molecular dynamics simulations to directly observe ligand dissociation for cognate and synthetic ligands from a riboswitch system. The site-specific flexibility profiles from our simulations are compared with in vitro measurements of flexibility using Selective 2 Hydroxyl Acylation analyzed by Primer Extension and Mutational Profiling (SHAPE-MaP). Our simulations reproduce known relative binding affinities for the cognate and synthetic ligands, and pinpoint how both ligands make use of different aspects of riboswitch flexibility. On the basis of our dissociation trajectories, we also make and validate predictions of pairs of mutations for both the ligand systems that would show differing binding affinities. These mutations are distal to the binding site and could not have been predicted solely on the basis of structure. The methodology demonstrated here shows how molecular dynamics simulations with all-atom force-fields have now come of age in making predictions that complement existing experimental techniques and illuminate aspects of systems otherwise not trivial to understand.

biophysics↗

bioRxiv2021

Immune microenvironment subtypes and association with tumor cell mutations and antigen expression in follicular lymphoma.

Follicular lymphoma (FL) is a B-cell lymphoma with a complex tumor microenvironment that is rich in non-malignant immune cells. We applied single-cell RNA-sequencing to characterize the diverse tumor and immune cell populations of FL and identified major phenotypic subsets of FL T-cells including a novel cytotoxic CD4 T-cell population. Their relative proportions of T-cells defined four major FL subtypes, characterized by differential representation or relative depletion of distinct T-cell subsets. By integrating exome sequencing, we observed that somatic mutations are associated with, but not definitive for, reduced antigen presentation on FL cells. In turn, expression of MHC class II genes by FL cells was associated with significant differences in the proportions and targetable immunophenotypic characteristics. This provides a classification framework of the FL microenvironment, their association with FL genotypes and antigen presentation, and informs different potential immunotherapeutic strategies based upon tumor cell MHC class II expression. Statement of significanceWe have characterized the FL-infiltrating T-cells, identified cytotoxic CD4 T-cells as an important component, showed that the abundance of these T-cell populations is associated with tumor-cell-intrinsic characteristics, and identified sets of targetable immune checkpoints on T-cells that differed between FLs with normal versus low antigen presentation.

cancer biology↗

bioRxiv2020

Patterns of within-host genetic diversity in SARS-CoV-2

Monitoring the spread of SARS-CoV-2 and reconstructing transmission chains has become a major public health focus for many governments around the world. The modest mutation rate and rapid transmission of SARS-CoV-2 prevents the reconstruction of transmission chains from consensus genome sequences, but within-host genetic diversity could theoretically help identify close contacts. Here we describe the patterns of within-host diversity in 1,181 SARS-CoV-2 samples sequenced to high depth in duplicate. 95% of samples show within-host mutations at detectable allele frequencies. Analyses of the mutational spectra revealed strong strand asymmetries suggestive of damage or RNA editing of the plus strand, rather than replication errors, dominating the accumulation of mutations during the SARS-CoV-2 pandemic. Within and between host diversity show strong purifying selection, particularly against nonsense mutations. Recurrent within-host mutations, many of which coincide with known phylogenetic homoplasies, display a spectrum and patterns of purifying selection more suggestive of mutational hotspots than recombination or convergent evolution. While allele frequencies suggest that most samples result from infection by a single lineage, we identify multiple putative examples of co-infection. Integrating these results into an epidemiological inference framework, we find that while sharing of within-host variants between samples could help the reconstruction of transmission chains, mutational hotspots and rare cases of superinfection can confound these analyses.

genomics↗

bioRxiv2020

An Integrated Clinical, Omic, and Image Atlas of an Evolving Metastatic Breast Cancer

Mechanisms of therapeutic resistance manifest in metastatic cancers as tumor cell intrinsic alterations and extrinsic microenvironmental influences that can change during treatment. To support the development of methods for the identification of these mechanisms in individual patients, we present here an Omic and Multidimensional Spatial (OMS) Atlas generated from four serial biopsies of a metastatic breast cancer patient during 3.5 years of therapy. This resource links detailed, longitudinal clinical metadata including treatment times and doses, anatomic imaging, and blood-based response measurements to exploratory analytics including comprehensive DNA, RNA, and protein profiles, images of multiplexed immunostaining, and 2- and 3-dimensional scanning electron micrographs. These data reveal aspects of therapy-associated heterogeneity and evolution of the cancers genome, signaling pathways, immune microenvironment, cellular composition and organization, and ultrastructure. We present illustrative examples showing how integrative analyses of these data provide insights into potential mechanisms of response and resistance, and suggest novel therapeutic vulnerabilities.

cancer biology↗

bioRxiv2020

A blueprint for the implementation of a validated approach for the detection of SARS-Cov2 in clinical samples in academic facilities

The COVID-19 pandemic is expanding at an unprecedented rate. As a result, diagnostic services are stretched to their limit, and there is a clear need for the provision of additional diagnostic capacity. Academic laboratories, many of which are closed due to governmental lockdowns, may be in a position to support local screening capacity by adapting their current laboratory practices. Here, we describe the process of developing a SARS-Cov2 diagnostic workflow in a conventional academic Containment Level 2 (CL2) laboratory. Our outline includes simple SARS-Cov2 deactivation upon contact, the methods for a quantitative real-time reverse transcriptase PCR (qRT-PCR) detecting SARS-Cov2, a description of process establishment and validation, and some considerations for establishing a similar workflow elsewhere. This was achieved under challenging circumstances through the collaborative efforts of scientists, clinical staff, and diagnostic staff to mitigate to the ongoing crisis. Within 14 days, we created a validated COVID-19 diagnostics service for healthcare workers in our local hospital. The described methods are not exhaustive, but we hope may offer support to other academic groups aiming to set up something comparable in a short time frame.

molecular biology↗

bioRxiv2019

Close range vocal interaction in the common marmoset (Callithrix Jacchus)

Vocal communication in animals often involves taking turns vocalizing. In humans, turn taking is a fundamental rule in conversation. Among non-human primates, the common marmoset is known to engage in antiphonal calling using phee calls and trill calls. Calls of the trill type are the most common, yet difficult to study, because they are not very loud and uttered in conditions when animals are in close proximity to one another. Here we recorded trill calls in captive pair-housed marmosets using wearable microphones, while the animals were together with their partner or separated, but within trill call range. Trills were exchanged mainly with the partner and not with other animals in the room. Animals placed outside the home cage increased their trill call rate and uttered more trills in response more to their partner. The fundamental frequency, F0, of trills increased when animals were placed outside the cage. Our results indicate that trill calls can be monitored using wearable audio equipment. Relatively minor changes in social context affect trill call interactions and spectral properties of trill calls, indicating that marmosets can communicate subtle information to their partner vocally.

animal behavior and cognition↗

bioRxiv2019

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends in cells

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a [~]6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the conversion of growing microtubule ends from a tapered into a blunt configuration resulted in reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

cell biology↗

bioRxiv2018

Deep Convolutional Network for Animal Sound Classification and Source Attribution using Dual Audio Recordings

We introduce an end-to-end feedforward convolutional neural network that is able to reliably classify the source and type of animal calls in a noisy environment using two streams of audio data after being trained on a dataset of modest size and imperfect labels. The data consists of audio recordings from captive marmoset monkeys housed in pairs, with several other cages nearby. Our network can classify both the call type and which animal made it with a single pass through a single network using raw spectrogram images as input. The network vastly increases data analysis capacity for researchers interested in studying marmoset vocalizations, and allows data collection in the home cage, in group housed animals.

animal behavior and cognition↗