Biology subjects

Saini, A.

Publications and source records attributed to Saini, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Simulated microgravity induces aging phenotypes in human organoids

Simulated microgravity provides a controlled perturbation for testing how loss of mechanical load affects human tissues. After 24 hours in rotating-wall vessels, neural, cardiac and tonsil organoids developed tissue-specific transcriptional responses that overlapped published spaceflight datasets, accompanied by higher transcriptomic-age estimates in neural and cardiac models, altered cardiac-organoid contraction dynamics and modified tonsil-organoid antiviral gene-expression signatures. These findings establish a multi-organoid framework for studying acute responses to gravitational unloading.

molecular biology↗

bioRxiv2026

Interpretable machine learning coupled to gene regulatory networks uncovers subcircuits underlying cell fate decisions

Gene regulatory networks (GRNs) model causal linkages that control cell fate decisions and differentiation transitions. Prioritizing regulatory subnetworks underlying cell state differences is of critical importance, but current methods including those reliant on topological metrics introduce circularity as the metrics prioritizing TFs are computed from the same networks whose assumptions they inherit. Separately, interpretable machine learning methods can identify latent factors (LFs) that discriminate cellular states with formal statistical guarantees but do not model regulatory linkages. Here, we present FOCAL (Factor-Outcome Coupling for Assessment of Linkages), a paradigm to prioritize regulatory subnetworks by coupling state-specific and dynamic GRNs with outcome-supervised LFs learned using interpretable machine learning without reference to network topology. This shifts GRN focus from macroscopic TF nodes to state-specific and dynamic TF-gene linkages. In B and T cells, FOCAL identified GIFs (GRNs coupled to Interpretable latent Factors), prioritized regulatory subnetworks underlying established states as well as transient regulatory episodes preceding them. By coupling LFs learnt from perturbation experiments of lineage-defining TFs, FOCAL identified transcriptional predisposition to alternative fates within progenitor cell populations before overt differentiation. This uncovered a novel NFATC2-IRF8 interplay in activated B cells, that was validated by in-vitro and in-vivo genetic perturbations. The two transcription factors act cooperatively to restrain extrafollicular plasmablast differentiation and promote germinal center B cell fate.

systems biology↗

bioRxiv2026

Deep Brain Stimulation Microelectrodes as a Source of Human Subcortical RNA: Validation of a Low-Input Transcriptomic Protocol

BackgroundUnderstanding the molecular basis of Parkinsons disease (PD) phenotypic heterogeneity maybe improved by in vivo access to deep brain tissue. Deep brain stimulation (DBS) surgery offers a unique opportunity: microelectrodes traversing the subthalamic nucleus (STN) carry adherent brain tissue upon withdrawal, providing a source of RNA from subcortical regions in living patients without additional invasive procedures. AimTo develop and validate a low-input RNA extraction and transcriptomic profiling protocol using DBS microelectrodes in post-mortem human brain, to confirm that recovered RNA is of brain rather than blood origin, and to characterise its sub-regional and cellular identity. MethodologyDBS microelectrodes were inserted without image guidance or guide tube into three unfixed post-mortem human brains targeting the STN trajectory. In clinical practice, a guide tube shields the electrode from cortical tissue; its absence here means tissue from the full insertion trajectory may contribute to recovered RNA. Thirty-eight microelectrodes were evaluated across single and pooled strategies. RNA was extracted using a modified RNeasy Micro low-input protocol; libraries prepared using NEBNext Single Cell/Low Input RNA Library Kit and sequenced on Illumina NovaSeq 6000 (paired-end, 2x150 bp). Tissue identity was validated against GTEx v10 (54 tissues) and Allen Human Brain Atlas (ABA, 19 subcortical regions including STN) using Pearson correlation with permutation testing (1,000 permutations) and BH-FDR correction. Transcriptional overlap between subcortical and cortical reference regions was quantified and subcortical-enriched gene filtering performed. ResultsTwenty-five RNA isolates were obtained from 38 microelectrodes; 54.5% of Bioanalyzer-assessed samples achieved RIN [≥]5 (median 7.1; range: 5.9-7.8). Fifteen libraries passed sequencing QC (mean depth 43.0 {+/-} 14.1 million read pairs; mean Q30 83.2 {+/-} 5.6 %). PCA of rlog transformed expression data resolved samples by donor identity. All samples confirmed brain tissue origin by GTEx {tau}-index tissue specificity correlation (n=996 brain and blood specific markers). Brain correlations were highest for frontal cortex (mean r= 0.683 {+/-} 0.072), anterior cingulate cortex (mean r= 0.658 {+/-} 0.069), amygdala (mean r= 0.615 {+/-} 0.068), and basal ganglia including caudate (mean r= 0.553 {+/-} 0.059), putamen (mean r= 0.546 {+/-} 0.059), and nucleus accumbens (mean r= 0.544 {+/-} 0.057); all BH-FDR < 0.05. Cerebellum showed the lowest brain correlations (cerebellar hemisphere: mean r = 0.327 {+/-} 0.039; cerebellum: mean r = 0.314 {+/-} 0.037). Whole blood correlation was strongly negative (mean r= -0.250 {+/-} 0.079), confirming non-haematological origin. ABA genome-wide analysis confirmed positive STN correlation (r=0.54-0.62; permutation p<0.001). MuSiC cell-type deconvolution against the Allen Brain Atlas HMBA-BG snRNA-seq reference identified oligodendrocytes (33.5 {+/-} 17.2%), frontal cortical neurons (9.1 {+/-} 7.0%), STR D1 MSNs (6.0 {+/-} 4.9%), and dopaminergic neurons (1.1{+/-} 2.7%) as the principal cell types, independently corroborating bulk transcriptomic findings at single-cell resolution. ConclusionThis study validates a low-input RNA extraction protocol for DBS microelectrodes, confirming brain-specific transcriptomic profiles consistent with STN-adjacent subcortical sampling. The 96% transcriptional overlap between cortical and subcortical regions, combined with absence of a guide tube in this post-mortem model, limits sub-regional specificity; clinical application with a guide tube would enrich the subcortical signal. These findings provide the methodological framework for in vivo molecular profiling of the human basal ganglia during DBS surgery in PD patients.

genetics↗

bioRxiv2026

Multiple molecular and cellular properties jointly affect protein and site-specific evolutionary rates

Protein evolutionary rates vary widely across proteins and among sites within proteins, reflecting multiple molecular, cellular, and functional constraints. While protein-level properties, such as expression and essentiality, and site-level structural and functional constraints, are known to influence evolutionary rates, how these constraints combine across scales to determine site-specific evolutionary rates remains unclear. Moreover, because many protein features are strongly correlated, it is difficult to disentangle their individual contributions to evolutionary rate variance, and unified predictive models that integrate these properties are still lacking. Here, we use neural networks to predict protein evolutionary rates across multiple scales based on multiple molecular and cellular features. At the protein level, integrating molecular and cellular descriptors explains substantial variance in evolutionary rates across proteins in multiple eukaryotic species, including nearly 50% of the variance in humans and substantial fractions of the variance in other eukaryotic species. The model also allows us to identify proteins whose evolutionary rates deviate from expectations based on their molecular and cellular properties. At the site level, we found that structural and functional features explain a comparable fraction of the variance in relative evolutionary rates. By integrating protein-level and site-level predictors, the model explains up to 37% of the variance in site-specific evolutionary rates across proteins. Our analysis demonstrates that constraints at these two scales combine largely additively, with protein-level properties setting the overall evolutionary context and site-level properties shaping variation within proteins. Together, these results provide a quantitative framework for understanding protein evolution across biological scales.

evolutionary biology↗

bioRxiv2026

Introduction to Single-cell Physiologically-Based Pharmacokinetic (scPBPK) Models

The current investigation introduces single-cell physiologically-based pharmacokinetic (scPBPK) models to gain insight into drug disposition at the cellular scale. The transition from standard PBPK (sPBPK) models to scPBPK models required depiction of expression-dependent (ED) processes, such as drug metabolism or membrane transport. ED processes utilize weighting functions - a defined or data-driven distribution -that yield heterogeneity in individual cell kinetics. Two scPBPK model examples are provided, one involving a drug (AZD1775) subject to 3 ED blood-brain barrier transport processes, and another drug (midazolam) with a single ED process of metabolism by hepatocytes. For both examples, the weighting function for each ED process was defined by a negative binomial distribution that is often used in scRNAseq analytics. The AZD1775 model simulations indicated a large degree of single cell drug concentration heterogeneity, whereas those for midazolam did not, due to high membrane transport relative to metabolism. scPBPK models offer a means to probe cellular pharmacokinetics compatible with modern omic technologies and may be extended to pharmacodynamic models. TeaserThe modeling framework to predict drug concentrations in single cells is presented.

pharmacology and toxicology↗

bioRxiv2026

Nuclear blebs are composed of variable chromatin states but consistently enrich transcription initiation relative to elongation

Nuclear blebs are herniations of the nucleus that occur in many human conditions including aging, heart disease, muscular dystrophy, and many cancers. Nuclear blebbing causes nuclear rupture and cellular dysfunction. However, understanding the formation, stability, and identification of nuclear blebs remains an ongoing challenge. Our previous studies reveal that nuclear blebs are best hallmarked by decreased DNA density. To determine if chromatin decompaction underlies decreased DNA density in nuclear blebs, we investigated the histone composition of nuclear blebs across multiple cell lines. Time lapse and immunofluorescence imaging revealed that global histone H2B and H3 levels are decreased in the nuclear bleb relative to the nuclear body. Next, we imaged histone modification states of euchromatin and heterochromatin, which respectively track decompact and compact states of chromatin. Overall, we find that nuclear blebs display variable histone modification state across cell lines, as euchromatin does not consistently enrich nor is heterochromatin consistently depleted. Nuclear blebs did consistently show active RNA Pol II initiation is enriched relative to elongation. Thus, we find that the local histone modification state is not an essential component of nuclear blebs while transcription initiation enrichment over elongation is reproducible across cell lines and conditions. Summary statementWe measured histones and their modification states in nuclear blebs. We find that chromatin state is variable while transcription initiation is consistently enriched relative to elongation in nuclear blebs.

cell biology↗

bioRxiv2026

Simulated Microgravity Recapitulates Aspects of Biological Aging in Humans

Spaceflight and microgravity profoundly affect human physiology and have been proposed to recapitulate key features of biological aging, yet the underlying mechanisms remain incompletely understood. Here, we performed whole-genome transcriptomic profiling to define immune cell alterations associated with both natural aging and simulated microgravity. Leveraging the longitudinal nature of the Stanford 1,000 immunomes Project, we compared peripheral blood mononuclear cells (PBMCs) exposed to rotating wall vessel bioreactor with matched samples collected up to 9 years later from the same individuals. We quantified changes across aging hallmarks, molecular pathways, gene modules, cellular energetics, disease risk and vaccine-response signatures. Microgravity-induced transcriptional closely tracked subject-level aging trajectories spanning across disease risk domains including those affecting the metabolic, musculoskeletal and circulatory systems, and multiple aging hallmarks involving nutrient sensing, intrinsic capacity, chronic inflammation, proteostasis, cellular senescence and metabolic regulation. Independent validation using Single-Cell Energetic Metabolism by Profiling Translation Inhibition (SCENITH) profiling confirmed these observed metabolic adaptations and revealed reduced mitochondrial dependence with minimal compensatory glucose dependence across immune cell subsets, features that strongly parallel aging biology. Consistent with previous findings, longitudinal changes indicated that close of 1/3 of participants do not follow population trajectories but these can be partly predicted with simulated microgravity exposure. Together, this within-donor framework establishes simulated microgravity as a scalable and experimentally tractable platform to model aspects of biological aging in humans and accelerating the prioritization of candidate countermeasures for spaceflight and aging on Earth.

immunology↗

bioRxiv2025

Non-human primate LIBRA-Seq accelerates neutralizing antibody discovery in RM vaccinated against HIV-1

Broadly neutralizing antibodies (bNAbs) exhibit protective efficacy against HIV-1 infection making them an ideal archetype for HIV-1 vaccine design. Presently, no vaccine candidate has induced antibody responses capable of meaningful protection against the swathe of circulating, difficult to neutralize tier 2 HIV-1 viruses. However, the development of stabilized, native-like envelope (Env) trimers such as BG505.SOSIP.664.T332N (BG505 SOSIP) has marked a significant advancement in vaccine design, due to their ability to elicit NAbs that neutralize tier 2 viruses in rhesus macaques (RM). NAb development following envelope trimer immunization in RM remains poorly understood, with hypothesized contributions from genetic variation at the IG loci, naive B cell repertoire, and differential gene expression in B cell lineages. To address these knowledge gaps, we have developed a set of BG505 SOSIP probes capable of recovering paired clonotype identity, antigen specificity, and gene expression of B cells in a high throughput fashion. These probes were constructed by conjugating biotinylated BG505 SOSIP to streptavidin covalently linked to both sc-RNA-Seq compatible DNA oligonucleotides and flow cytometry compatible fluorophores. Using these reagents, we isolated and sequenced BG505 SOSIP specific memory B cells from the PBMCs of an RM that developed high titers of neutralizing antibodies. To benchmark the accuracy of our technology, we compared our recovered heavy and light chain sequences to those identified from the same animal using conventional methodology and recovered 100% of previously identified NAbs. We then applied this technology to recover BG505 SOSIP specific memory B cells from five additional vaccinated RMs, cloned 34 antibodies for functional characterization, and identified ten antibodies with autologous neutralizing activity. Author SummaryUnderstanding how effective antibodies arise after HIV vaccination is essential for developing a protective vaccine, yet studying these responses in non-human primates has been limited by low- throughput methods. In this study, we adapted a high-throughput single-cell sequencing approach to identify HIV envelope-specific antibodies from vaccinated rhesus macaques. This method allowed us to recover paired antibody sequences together with their antigen specificity from thousands of individual B cells. We successfully identified known neutralizing antibodies and discovered additional antibodies capable of neutralizing HIV across multiple animals. Our analysis revealed that vaccine-elicited antibody responses were dominated by a small number of expanded lineages, with shared genetic features among animals with stronger neutralization. These findings demonstrate that this approach can efficiently define the antibody repertoires generated by HIV vaccines and provide a powerful tool for evaluating and improving immunogens in preclinical vaccine studies.

immunology↗

bioRxiv2025

CRISPRi Screening Identifies SON and MAP4K1 as Regulators of Type III Cytokine Expression in Innate Lymphoid Cells

The cytokines interleukin (IL)-22 and IL-17 are secreted by innate and adaptive immune cells to drive "type III" responses that protect against extracellular pathogens, promote mucosal barrier integrity, and foster microbiota homeostasis. However, dysregulation of IL-22 and/or IL-17 contributes to autoimmunity, chronic inflammation, and malignancy. Thus, a deeper understanding of mechanisms regulating type III cytokine production could provide new therapeutic targets for a spectrum of immune-mediated diseases. Toward this goal, we performed a genome-wide CRISPR inhibition (CRISPRi) screen to identify factors that regulate IL-22/IL-17 expression in a murine type III innate lymphoid cell (ILC3) model, MNK3, following stimulation with IL-23 and IL-1b. In addition to previously known regulators of type III cytokines, including IL-23 receptor components IL23R and IL12RB1, the screen identified a large set of new factors that either potentiate or attenuate expression of IL-22 and/or IL-17. A subset of these novel factors was chosen for validation, from which two were selected for further study. The nuclear protein, SON, which binds both DNA and RNA, impaired expression of IL12RB1 at the levels of de novo transcription and RNA processing. The second, MAP4K1 (HPK1), is a serine/threonine kinase that is required for IL-22 but not IL-17 expression. Depletion of MAP4K1 in MNK3 also enhanced expression of the type I cytokine, IFNg, which was co-expressed with IL-17, a phenotype reminiscent of pathogenic Th17 cells. Together, results from the CRISPRi screen broaden our understanding of the factors involved in type III immune responses and offer new targets for modulating IL-22/17 expression.

immunology↗

bioRxiv2025

Investigation of CD36 interactome provides insights into multimolecular complexes necessary for anti-angiogenic signalling

Signal transduction is a fundamental process that enables cells to adapt to external cues and organize adequate responses including survival, death, growth, and homeostasis. A key mechanism modulating signal transduction relies on the formation of multimolecular complexes optimized for specificity, modularity and signal amplification. The scavenger receptor CD36, which binds diverse ligands in different cellular contexts, illustrates this principle. To uncover the nature of CD36 multimolecular complexes, we employed a proximity biotinylation labeling approach on human endothelial cells, where CD36 binds to thrombospondin-1 (TSP-1) to initiate a signaling cascade promoting programmed cell death. Using biotin capture and mass spectrometry protein identification, we uncovered a list of proteins in the vicinity of CD36. This list of candidates was refined by proximity ligation assays. The relationship between key CD36 interacting molecules, in particular active integrin beta-1 (ITGB1) and CD9, was further decoded by conditional colocalization analysis, providing support for their association within a tri-molecular complex. The implication of selected candidates in the signaling function of CD36 was further evaluated using shRNA knockdown, revealing that active ITGB1 is essential for Fyn activation downstream of CD36, with the tetraspanin playing a connecting role between CD36 and active ITGB1. Our approach to investigating CD36 complexes emphasizes the complexity and fundamental role of protein-protein interactions and coordination in the context of transmembrane signal transduction.

cell biology↗

bioRxiv2025

Faithful Modeling of Terminal CD8 T Cell Dysfunction and Epigenetic Stabilization In Vitro

Epigenetic scarring of terminally dysfunctional CD8 T cells hinders long-term protection and response to immune checkpoint blockade during chronic infections and cancer. We developed a faithful in vitro model for CD8 T cell terminal dysfunction as a platform to advance T cell immunotherapy. Using TCR-transgenic CD8 T cells, we found that 1-week peptide stimulation, mimicking conditions in previous models, failed to induce a stable exhaustion program in CD8 T cells. In contrast, prolonged stimulation for 2-3 weeks induced T cell dysfunction but triggered activation-induced cell death, precluding long-term investigation of exhaustion programs. To better mimic in vivo exhaustion, we provided post-effector, chronic TGF{beta}1 signals, enabling survival of chronically stimulated CD8 T cells for over 3 weeks. These conditions induced a stable state of terminal dysfunction (TDysf), marked by a stable loss of effector, cytotoxicity, and memory programs, along with mitochondrial stress and impaired protein translation. Importantly, transcriptomic and epigenetic analyses confirmed the development of terminal exhaustion-specific signatures in TDysf cells. Adoptive transfer of TDysf cells revealed their inability to recall effector functions or proliferate after acute LCMV rechallenge. This novel tractable model system enables investigation of molecular pathways driving T cell terminal dysfunction and discovery of new therapeutic targets for cancer or chronic infections.

immunology↗

bioRxiv2025

Twinfilin is a potent uncapper of actin capping protein and modulates actomyosin contractility in the C. elegans spermatheca

The actin cytoskeleton is dynamically remodelled by conserved regulators to control cellular and tissue mechanics. While the functions of these proteins are well studied, how they drive tissue-specific contractility remains unclear. Twinfilin, an actin uncapper and depolymerase, has not previously been linked to tissue contractility. Here, we show that the sole twinfilin ortholog in C. elegans, TWF-2, regulates actomyosin contractility in the spermatheca. TWF-2 localizes to the spermathecal cortex via interactions with -spectrin (SPC-1) and {beta}-spectrin (UNC-70). In vitro, TWF-2 promotes barbed-end depolymerization and rapidly removes CAP-1 from actin filaments. In vivo, embryonic lethality caused by CAP-1 depletion is partially rescued by simultaneous loss of TWF-2. Similarly, loss of the contractility regulator SPV-1 leads to elevated F-actin and phosphorylated myosin, causing hypercontractility. Notably, removing TWF-2 suppresses this hypercontractility by reducing F-actin levels-- without affecting myosin or its phosphorylation--highlighting a specific role in F-actin regulation. Together, these findings show that TWF-2 modulates actin dynamics in a tissue-specific manner. This work provides the first in vivo evidence that twinfilin regulates contractility, and reveals how its interactions with capping protein and spectrins help maintain balanced actomyosin levels in the spermatheca.

cell biology↗

bioRxiv2025

Hb-EGF directs systemic muscle repair

Regenerative capacity varies between tissues, species, and stages of the life cycle. What is less appreciated is that regenerative capacity also varies with the magnitude of the injury, even within a single tissue. Vertebrate skeletal muscle efficiently regenerates following minor injuries; however, extensive injuries may result in incomplete repair, which can be debilitating. To understand if small- and large-scale muscle injuries activate distinct regenerative programs, we developed a systemic muscle injury model in zebrafish. Transcriptomic analysis of muscle and non-muscle tissues revealed that systemic and local muscle injuries elicit distinct molecular responses, both quantitatively and qualitatively. Systemic muscle injury activated the expression of Heparin binding epidermal-like growth factor (Hb-EGF) in the epidermis, and Hb-EGF is necessary for systemic muscle repair. Conversely, local muscle injury did not induce Hb-EGF expression and Hb-EGF was not required for local muscle repair. These studies suggest that large- and small-scale muscle injuries activate different regenerative programs, resulting in either systemic or local repair.

developmental biology↗

bioRxiv2025

Cell type-specific enhancers regulate IL-22 expression in innate and adaptive lymphoid cells

IL-22, a signature cytokine of type 3 lymphoid cells, mediates epithelial homeostasis and protective pathogen responses in barrier tissues, while its deregulated expression drives chronic inflammation associated with colitis and psoriasis. Despite its therapeutic value, little is known about regulatory elements for IL-22 expression. We identify two conserved enhancers, E22-1 and E22-2, which differentially regulate Il22 in type 3 lymphoid subsets. These enhancers are required for steady-state expression of gut antimicrobial peptides, protection from C. rodentium infection, and development of IL-22-mediated psoriasis. E22-1 resembles many known enhancers, functioning in both Th-ILC counterparts. However, E22-2 is only required for IL-22 expression in ILC3s. Its ILC3 restriction relies on multiple Runx3 sites, combined with the lack of a functional ROR{gamma}t motif, which is present in E22-1. Thus, although responding to similar stimuli, type 3 lymphoid cells use distinct cis-elements for IL-22 expression, with E22-2 likely serving as a homeostatic enhancer in barrier tissues.

immunology↗

bioRxiv2025

Integrated model of the protein molecular clock across mammalian species

One of the foundational concepts in molecular evolution is the protein molecular clock [1-3], encapsulating the observation that proteins tend to accumulate amino acid substitutions at approximately constant and protein-specific rates over long evolutionary timescales. According to the neutral theory of molecular evolution, the majority of protein substitutions have neutral or nearly neutral effects on species fitness. The neutral theory suggests the existence of a strong generation time effect, positing that species with shorter generation times should accumulate protein substitutions faster per unit time compared to species with longer generation times. However, earlier studies failed to detect a significant generation time effect for amino acid substitutions in vertebrates, raising questions about core tenets of the neutral theory. In this study, we take advantage of the recently accumulated evolutionary and genomics data to investigate the scaling of substitution rates with generation time among many dozens of mammalian species and thousands of proteins. Our findings reveal a strong generation time effect both for synonymous and non-synonymous substitutions. We demonstrate that the empirically observed generation time effect is fully consistent with several mechanisms, such as functional and mutation rate selection. Building on these results, we develop an integrated model of protein evolution that incorporates changes in substitution rates due to variation in species generation times throughout their evolutionary history. Overall, our analysis shows that protein evolution is consistent with neutral or nearly neutral theories and explains time-like behavior of the protein molecular clock across deep phylogenetic lineages.

evolutionary biology↗

bioRxiv2023

Microevolution of clade II isolates of Candida auris highlights multifaceted intra-clade heterogeneity in acquiring resistance towards amphotericin B

Candida auris exhibits high-level resistance to amphotericin B (AmB). Mechanisms such as ergosterol biosynthesis malfunction, oxidative damage mismanagement, and increased drug efflux contribute to AmB resistance in C. auris. In this study, we experimentally evolved two East Asian drug-susceptible clade II isolates of C. auris (P2428 and CBS10913T) isolated from different geographical locations to develop resistance against AmB. We analysed alterations in karyotype, genome sequence, and gene expression profiles to uncover the mechanisms driving AmB resistance. The independently evolved clade II adaptors displayed up to 4-16-fold higher MIC50, as compared to the parental cells. AOX2 (alternative oxidase) and the cell wall integrity pathway have been identified as critical in the development of AmB resistance. However, we noted certain intra-clade heterogeneity in the associated mechanisms. While in P2428 adaptors (P-lines), the ergosterol and sphingolipid pathways appear to play a crucial role, this was not the case for CBS10913T adaptors (A-lines), which acquired resistance independent of lipid-associated changes. The transcriptomic, WGS, and phenotypic analyses also confirm that the evolved AmB-resistant isolates follow distinct trajectories for adaptation, Furthermore, unlike the fluconazole-resistant isolates, as reported previously, changes in ploidy do not seem to contribute to the differential mechanisms of AmB resistance. Overall, this study not only provides insights into the mechanisms and pathways involved in AmB resistance but also highlights intra-clade-heterogeneity that exists within clade II of C. auris. ImportanceCandida auris demonstrates significant resistance to amphotericin B (AmB) that stems from factors like alteration of ergosterol biosynthesis, perturbation of the oxidative damage response, etc. A comprehensive understanding of underlying mechanisms can be studied in a holistic manner by subjecting resistant as well as susceptible clinical isolates to a comparative genome-level analysis. An alternate and more dynamic approach is to expose susceptible isolates to a certain concentration of drug which is not lethal but can trigger the resistance mechanisms. In the present study, we evolved C. auris towards AmB and observed novel and differential mechanisms of resistance development, in two different isolates despite belonging to the same clade. This study provides insights into the intra-clade heterogeneous behavior of C. auris towards AmB.

microbiology↗

bioRxiv2022

Genomic landscape of the DHA1 family in the newly emerged pathogen Candida auris and mapping substrate repertoire of the prominent member CauMdr1

The last decade has witnessed the rise of extremely threatening healthcare-associated multidrug resistant non-albicans Candida (NAC) species, Candida auris. Thus, understanding the molecular basis of antifungal resistance has emerged as the single most important goal amongst the research community. Since besides target alterations, efflux mechanisms contribute maximally to antifungal resistance, it is imperative to investigate their contributions in this pathogen. Of note, within the Major Facilitator Superfamily (MFS) of efflux pumps, Drug/H+ antiporter family 1 (DHA1) has been established as a predominant contributor towards xenobiotic efflux. Our study provides a complete landscape of DHA1 transporters encoded in the genome of C. auris. This study identifies 14 DHA1 transporters encoded in the genome of the pathogen. We also construct deletion and heterologous overexpression strains for the most important DHA1 drug transporter, viz., CauMdr1 to map the spectrum of its substrates. While the knockout strain did not show any significant changes in the resistance patterns against the majority of the tested substrates, the ortholog when overexpressed in a minimal background S. cerevisiae strain, AD1-8u-, showed significant enhancement in the Minimum inhibitory concentrations (MICs) against a large panel of antifungal molecules. Altogether, the present study provides a comprehensive template for investigating the role of DHA1 members of C. auris in antifungal resistance mechanisms.

microbiology↗

bioRxiv2022

Genome-wide analysis of bromodomain gene family in Arabidopsis and Rice

The bromodomain containing proteins (Brd-proteins) belongs to family of epigenetic mark readers, integral to epigenetic regulation. The Brd-members contain a conserved bromodomain (BRD/BRD-fold: interacts with acetylated-lysine in histones), and several additional domains, making them structurally/functionally diverse. Like animals, plants also contain multiple Brd-homologs, however the extent of their diversity and impact of molecular events (genomic duplications, alternative splicing, AS) therein, is relatively less explored. The present genome-wide analysis of Brd-families of Arabidopsis thaliana and Oryza sativa showed extensive diversity in structure of genes/proteins, regulatory elements, expression pattern, domains/motifs, and the bromodomain (length/sequence, location) among Brd-members. Orthology analysis identified 13 ortholog groups, three paralog groups and four singleton members. While more than 40% Brd-genes were affected by genomic duplication events in both plants, AS-events affected 60% A. thaliana and 40% O. sativa genes. These molecular events affected various regions (promoters, untranslated regions, exons) of different Brd-members with potential impact on expression/structure-function characteristics. RNA-Seq data analysis of Brd-members, and RT-qPCR of duplicate Brd-genes showed differences in tissue-specificity and response to salinity. Phylogenetic analysis based on conserved BRD-region placed the A. thaliana and O. sativa homologs into clusters/sub-clusters, consistent with ortholog/paralog groups. The bromodomain-region displayed conserved signatures in key BRD-fold elements (-helices, loops), and variations (1-20 sites) including indels among the Brd-duplicates. Homology modeling and superposition identified structural variations in BRD-folds of divergent and duplicate Brd-members, which might affect their interaction with chromatin and associated functions. The study also showed contribution of various duplication events in Brd-family expansion among diverse plants, including monocots and dicots.

plant biology↗