Biology subjects

Rao, B.

Publications and source records attributed to Rao, B..

12 recordsLinked to original sources

bioRxiv2026

Genomic Footprints of Bottlenecks, Isolation, and Inbreeding: A Case Study of Two Vulture Cohorts in India

Genomic erosion as a manifestation of small effective population size (Ne) and consanguinity subverts long-term perpetuation of threatened species by compromising their adaptive potential; however, the integration of genomics remains limited in applied conservation efforts to guide priorities. This study combines non-invasive sampling, double-digest Restriction site-associated DNA sequencing (ddRAD), and population-genomic analyses to assess genetic health in two vulture assemblages-mixed wild enclosure and captive breeding cohorts. Both the geographical locations exhibit signs of populations in distress: low genetic diversity and abundant intermediate-length runs of homozygosity (RoH), consistent with long-term reduced Ne plus recent demographic isolation. Our demographic model runs favoured ancient migration (AM) topology characterised by an ephemeral window of gene flow, taken over by a prolonged population separation period. The mutation quantification results from approximately 59,000 outgroup-polarised SNPs reveal higher additive burden and more homozygous-derived sites in BKN. However, this was later traced to low-impact and non-coding variants rather than a surge in the loss-of-function (LoF) alleles. The data support a genomic profile that carries an elevated risk from polygenic/aggregate deleterious burden in BKN despite a scarcity of high-impact mutations. By highlighting the disconnect between genetic resilience and demographic recovery, our results accentuate the need to incorporate genomics-informed inbreeding and monitoring programs, while also focusing on reducing anthropogenic mortality with genetic augmentation.

genomics↗

bioRxiv2025

Nicotinamide opposes ET-1's adverse effect on uterine decidualization via EDNRB

Endothelin-1 is involved in pathogenesis of preeclampsia. Mice (Edn1H/+) having excess endothelin-1 developed preeclampsia-like phenotypes during pregnancy in a maternal genotype-dependent manner. Here, we investigate whether decidualization is impaired in Edn1H/+ dams, and whether nicotinamide (a potent inhibitor of endothelin-1) exerts beneficial effect. We compared implantation sites between wild type (WT) and Edn1H/+ dams with or without nicotinamide treatment. Implantation sites of Edn1H/+ dams exhibited abnormal ectoplacental cone and sinusoids with reduced vascular density in mesometrial regions of deciduae. There was more VEGF in decidua of Edn1H/+ dams than WT dams. Markers of decidualization were decreased in Edn1H/+ dams. Nicotinamide supplementation corrected this abnormality. During differentiation (decidualization) of cultured human endometrial stomal cells, endothelin-1 impaired the upregulated expression of decidualization markers. The endothelin-1s effect was reversed by nicotinamide. These results show nicotinamide counteracts ET-1s detrimental effects on endometrial decidualization and has potential to improve embryo implantation and subsequent pregnancy outcomes.

developmental biology↗

bioRxiv2024

The biophysical mechanism of mitochondrial pearling

Mitochondrial networks exhibit remarkable dynamics that are driven in part by fission and fusion events. However, there are other reorganizations of the network that do not involve fission and fusion. One such exception is the elusive, "beads-on-a-string" morphological transition of mitochondria. During such transitions, the cylindrical tubes of the mitochondrial membrane transiently undergo shape changes to a string of "pearls" connected along thin tubes. These dynamics have been anecdotally observed in many contexts and given disparate explanations. Here we unify these observations by proposing a common underlying mechanism based on the biophysical properties of tubular fluid membranes for which it is known that, under particular regimes of tension and pressure, membranes reach an instability and undergo a shape transition to a string of connected pearls. First, we use high-speed light-sheet microscopy to show that transient, short-lived pearling events occur spontaneously in the mitochondrial network in every cell type we have examined, including primary fibroblasts, T-cells, neurons, and budding yeast. We present evidence that transient mitochondrial pearling occurs during important biological events, particularly during T cell activation, neuronal firing, and replicative senescence. Using our high-temporal resolution data, we identify two distinct categories of spontaneous pearling, i) internal pressure-driven pearling generated by ionic flux, and ii) external tension-driven pearling generated by the cytoskeleton. By applying live-cell STED and FIB-SEM imaging we document the structural reorganization of inner cristae membranes during mitochondrial pearling and the role of the MICOS complex in regulating the frequency of pearling events. We then establish numerous methods for inducing pearling, including the ability to induce these dynamics with single mitochondrion precision. These methods include ionophores, channel activators, osmotic shock, detergents, laser stimulation, membrane intercalating molecules, chemical fixation, and micro-needle force. These disparate inducers establish three main physical causes of pearling, i) ionic flux producing internal osmotic pressure, ii) membrane packing lowering bending elasticity, and iii) external mechanical force increasing membrane tension. Pearling dynamics thereby reveal a fundamental biophysical facet of mitochondrial biology. We suggest that pearling should take its place beside fission and fusion as a key process of mitochondrial dynamics, with implications for physiology, disease, and aging.

biophysics↗

bioRxiv2024

Disorganized Inhibitory Dynamics and Functional Connectivity in Hippocampal area CA1 of 22q11.2 Deletion Mutant Mice

Individuals with the 22q11.2 deletion syndrome, one of the strongest genetic risk factors for schizophrenia, demonstrate cognitive impairments, including episodic memory dysfunction. Place cell activity of excitatory pyramidal neurons in the hippocampus supporting episodic memory is impaired in a mouse model for the 22q11.2 deletion (Df(16)A+/-). While excitatory dynamics are under tight inhibitory control by multiple subtypes of GABAergic interneurons, previous studies have predominantly focused on a single subtype of PV-expressing interneurons; there have not yet been studies describing the functional relationships between molecularly identified inhibitory types in Df(16)A+/-mice. Here, we examined interneuron subtype-specific activity dynamics in the dorsal hippocampal area CA1 of Df(16)A+/- mice during random foraging and spatial reward navigation tasks. Capitalizing on 3D acousto-optical deflector two-photon microscopy with post hoc immunohistochemical identification, we found that multiple interneuron types exhibit aberrant responses to reward locations and delayed reward enrichment extinction. Df(16)A+/- inhibitory interneurons also carry markedly reduced spatial information in a subtype-dependent manner. We observed task-dependent changes in the correlation structure and coactivity among multiple GABAergic subtypes, suggesting a broadly disorganized microcircuit functionality in mutant mice. Overall, we identify widespread and heterogeneous subtype-specific alterations in interneuron dynamics during spatial reward navigation, reflecting impaired flexibility and organization in CA1 inhibitory microcircuits. Our study provides critical insights into how schizophrenia-risk mutations affect local-circuit interactions among diverse cell types in the mouse hippocampus during learning and spatial navigation.

neuroscience↗

bioRxiv2023

behaviorMate: An Intranet of Things Approach for Adaptable Control of Behavioral and Navigation-Based Experiments

1Investigators conducting behavioral experiments often need precise control over the timing of the delivery of stimuli to subjects and to collect the precise times of the subsequent behavioral responses. Furthermore, investigators want fine-tuned control over how various multi-modal cues are presented. behaviorMate takes an "Intranet of Things" approach, using a networked system of hardware and software components for achieving these goals. The system outputs a file with integrated timestamp-event pairs that investigators can then format and process using their own analysis pipelines. We present an overview of the electronic components and GUI application that make up behaviorMate as well as mechanical designs for compatible experimental rigs to provide the reader with the ability to set up their own system. A wide variety of paradigms are supported, including goal-oriented learning, random foraging, and context switching. We demonstrate behaviorMates utility and reliability with a range of use cases from several published studies and benchmark tests. Finally, we present experimental validation demonstrating different modalities of hippocampal place field studies. Both treadmill with burlap belt and virtual reality with running wheel paradigms were performed to confirm the efficacy and flexibility of the approach. Previous solutions rely on proprietary systems that may have large upfront costs or present frameworks that require customized software to be developed. behaviorMate uses open-source software and a flexible configuration system to mitigate both concerns. behaviorMate has a proven record for head-fixed imaging experiments and could be easily adopted for task control in a variety of experimental situations.

neuroscience↗

bioRxiv2022

Targeted high throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

The spliceosome is a staggeringly complex machine comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and interrogated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A-complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B binding site but also to the G-patch (ATPase activator) domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

molecular biology↗

bioRxiv2021

Structural basis of ATP-dependent high-fidelity epigenome maintenance

Epigenetic evolution occurs over million-year timescales in Cryptococcus neoformans and is mediated by DNMT5, the first maintenance-type cytosine methyltransferase identified in the fungal or protist kingdoms. DNMT5 requires ATP and displays exquisite hemimethyl-DNA specificity. To understand these novel properties, we solved cryo-EM structures of CnDNMT5 in three states. These studies reveal an elaborate allosteric cascade in which hemimethylated DNA first activates the SNF2 ATPase domain by a large rigid body rotation while the target cytosine partially flips out the DNA duplex. ATP binding then triggers a striking structural reconfiguration of the methyltransferase catalytic pocket that enables cofactor binding, completion of base-flipping, and catalysis. Unmethylated DNA binding fails to open cofactor pocket and subsequent ATP binding triggers its ejection to ensure fidelity. This chaperone-like, enzyme-remodeling role of the SNF2 domain illuminates how energy can be used to enable faithful epigenetic memory. HighlightsO_LIStructures of DNMT5 reveal mechanism of ATP-dependent DNA methylation C_LIO_LIHemimethylated CpG recognition triggers partial base flipping of the target cytosine C_LIO_LIHemimethylated DNA induces rigid body rotation to activate the SNF2 ATPase domain C_LIO_LIMTase catalytic pocket is remodeled by the SNF2 ATPase to achieve specificity C_LI

molecular biology↗

bioRxiv2021

Coupling of spliceosome complexity to intron diversity

We determined that over 40 spliceosomal proteins are conserved between many fungal species and humans but were lost during the evolution of S. cerevisiae, an intron-poor yeast with unusually rigid splicing signals. We analyzed null mutations in a subset of these factors, most of which had not been investigated previously, in the intron-rich yeast Cryptococcus neoformans. We found they govern splicing efficiency of introns with divergent spacing between intron elements. Importantly, most of these factors also suppress usage of weak nearby cryptic/alternative splice sites. Among these, orthologs of GPATCH1 and the helicase DHX35 display correlated functional signatures and copurify with each other as well as components of catalytically active spliceosomes, identifying a conserved G-patch/helicase pair that promotes splicing fidelity. We propose that a significant fraction of spliceosomal proteins in humans and most eukaryotes are involved in limiting splicing errors, potentially through kinetic proofreading mechanisms, thereby enabling greater intron diversity.

molecular biology↗

bioRxiv2020

Functional Connectome Fingerprinting Using Shallow Feedforward Neural Networks

Although individual subjects can be identified with high accuracy using correlation matrices computed from resting-state functional magnetic resonance imaging (rsfMRI) data, the performance significantly degrades as the scan duration is decreased. Recurrent neural networks can achieve high accuracy with short duration (72s) data segments but are designed to use temporal features not present in the correlation matrices. Here we show that shallow feedforward neural networks that rely solely on the information in rsfMRI correlation matrices can achieve state-of-the-art identification accuracies ([≥] 99.5%) with data segments as short as 20s and across a range of input data-size combinations when the total number of data points (# regions x # time points) is on the order of 10,000.

neuroscience↗

bioRxiv2020

Coordinate genomic association of transcription factors controlled by an imported quorum sensing peptide in Cryptococcus neoformans

Qsp1 is a secreted quorum sensing peptide required for virulence of the fungal meningitis pathogen Cryptococcus neoformans. Qsp1 functions to control cell wall integrity in vegetatively growing cells and also functions in mating. Rather than acting on a cell surface receptor, Qsp1 is imported to act intracellularly via the predicted oligopeptide transporter Opt1. Here, we identify a transcription factor network as a target of Qsp1. Using whole-genome chromatin immunoprecipitation, we find Qsp1 controls the genomic associations of three transcription factors to genes whose outputs are regulated by Qsp1. One of these transcription factors, Cqs2, is also required for the action of Qsp1 during mating, indicating that it might be a shared proximal target of Qsp1. Consistent with this hypothesis, deletion of CQS2 impacts the binding of other network transcription factors specifically to Qsp1-regulated genes. These genetic and genomic studies illuminate mechanisms by which an imported peptide acts to modulate eukaryotic gene expression. AUTHOR SUMMARYFor many fungal pathogens, the ability to adapt to changing and diverse environments forms the basis for their ability to infect and survive inside macrophages and other niches in the human body, and these changes are accomplished by transcription factors. Many pathogenic microbes coordinate their gene expression as a function of cell density in a process known as quorum sensing. Here, in the human fungal meningitis pathogen Cryptococcus neoformans, we find that an imported eukaryotic quorum sensing peptide that is important for virulence, Qsp1, controls the binding of three different transcription factors to promoters, thereby modulating the expression of Qsp1-regulated genes. This discovery reveals the mechanism for how an imported peptide affects gene expression.

microbiology↗

bioRxiv2019

A non-Dicer RNase III and four other novel factors required for RNAi-mediated transposon suppression in the human pathogenic yeast C. neoformans

The human pathogenic yeast Cryptococcus neoformans silences transposable elements using endo-siRNAs and an Argonaute, Ago1. Endo-siRNAs production requires the RNA-dependent RNA polymerase, Rdp1, and two partially redundant Dicer enzymes, Dcr1 and Dcr2, but is independent of histone H3 lysine 9 methylation. We describe here an insertional mutagenesis screen for factors required to suppress the mobilization of the C. neoformans HARBINGER family DNA transposon HAR1. Validation experiments uncovered five novel genes (RDE1-5) required for HAR1 suppression and global production of suppressive endo-siRNAs. Loss of the RDE genes does not impact transcript levels, suggesting the endo-siRNAs do not act by impacting target transcript synthesis or turnover. RDE3 encodes a non-Dicer RNase III related to S. cerevisiae Rnt1, RDE4 encodes a predicted terminal nucleotidyltransferase, while RDE5 has no strongly predicted encoded domains. Affinity purification-mass spectrometry studies reveal that Rde3 and Rde5 are physically associated. RDE1 encodes a G-patch protein homologous to the S. cerevisiae Sqs1/Pfa1, a nucleolar protein that directly activates the essential helicase Prp43 during rRNA biogenesis. Rde1 copurifies Rde2, another novel protein obtained in the screen, as well as Ago1, a homolog of Prp43, and numerous predicted nucleolar proteins. We also describe the isolation of conditional alleles of PRP43, which are defective in RNAi. This work reveals unanticipated requirements for a non-Dicer RNase III and presumptive nucleolar factors for endo-siRNA biogenesis and transposon mobilization suppression in C. neoformans.

molecular biology↗

bioRxiv2017

Spliceosome profiling visualizes the operations of a dynamic RNP in vivo at nucleotide resolution

Tools to understand how the spliceosome functions in vivo have lagged behind advances in its structural biology. We describe methods to globally profile spliceosome-bound precursor, intermediates and products at nucleotide resolution. We apply these tools to three divergent yeast species that span 600 million years of evolution. The sensitivity of the approach enables detection of novel cases of non-canonical catalysis including interrupted, recursive and nested splicing. Employing statistical modeling to understand the quantitative relationships between RNA features and the data, we uncover independent roles for intron size, position and number in substrate progression through the two catalytic stages. These include species-specific inputs suggestive of spliceosome-transcriptome coevolution. Further investigations reveal ATP-dependent discard of numerous endogenous substrates at both the precursor and lariat-intermediate stages and connect discard to intron retention, a form of splicing regulation. Spliceosome profiling is a quantitative, generalizable global technology to investigate an RNP central to eukaryotic gene expression.\n\nHighlightsO_LIMeasurement of spliceosome-bound precursor and intermediate in three species\nC_LIO_LINon-canonical splicing events revealed\nC_LIO_LIStatistical modeling uncovers substrate features that predict catalytic efficiency\nC_LIO_LIDiscard of suboptimal substrates occurs in vivo and predicts intron-retained mRNAs\nC_LI

molecular biology↗