Biology subjects

Ravisankar, P.

Publications and source records attributed to Ravisankar, P..

9 recordsLinked to original sources

bioRxiv2026

Prime editing enables high-throughput identification and characterization of selective MutSβ inhibitors

DNA mismatch repair (MMR) maintains genome stability but paradoxically drives expansion of pathogenic repeats responsible for trinucleotide repeat disorders. Attenuating repeat instability through targeted inhibition of MMR is therefore a therapeutic strategy of interest; however, progress is constrained by a lack of robust methods for defining and isolating MMR subpathways. We exploited features of prime editing to profile the effects of core MMR genes on thousands of programmed DNA mispairs in cells and resolved distinct MMR processes, including a noncanonical, loop-directed mechanism. Guided by these results, we built a set of scalable reporters that detect loss of separable MMR processes and screened ~72,000 small molecules for activity against MutS{beta}-directed MMR. This effort identified a novel series of MutS{beta} inhibitors that reduced (CAG)n expansion in a cell model. Altogether, this study establishes several methods for systematically interrogating MMR, with demonstrated capacity to identify inhibitors of therapeutic value.

bioengineering↗

bioRxiv2026

Deconvolution of the mechanisms of T cell drug response in multiple myeloma induction therapy

Lenalidomide (Revlimid), bortezomib (Velcade), and dexamethasone, are used alone or in combination (RVd therapy) as first-line therapies for hematologic malignancies including the plasma cell dyscrasia, Multiple Myeloma (MM). The effects of RVd treatment on tumor cells have been documented, but little is known about their impacts on "healthy" immune cells. New therapeutics, including chimeric antigen receptor T cell (CAR-T) and bispecific T-cell engagers (BiTEs) depend on robust T cell function but these are often harvested for cellular therapies after or in tandem with RVd therapy. Understanding the molecular effects of these drugs on healthy T cells is therefore of particular importance. Since the molecular effects of each drug includes regulation of key transcription factors, we used a tri-modal, single-cell assay (TEA-seq), simultaneously profiling mRNA transcripts, cell surface proteins, and chromatin accessibility in primary human peripheral blood T cells treated for 4, 24, or 72 hours in vitro. Synergies and conflicts potentially arising from combinatorial therapy were identified and validated by flow cytometry. Doublet and triplet drug combinations were used to assess effects on cell-surface marker expression and on T cell activation. Our results suggest that T cell function can be optimized by the administration of these drugs individually and that sequential administration may offer an opportunity to enhance T cell function by affecting localization and altering T cell states. This work provides those interested in immunology, oncology, and cell therapies an opportunity to explore molecular pathways that could be manipulated by these commonly used drugs to modify T cell function.

immunology↗

bioRxiv2026

Multi-omic profiling of human antibody-secreting cells reveals diverse subsets sustain durable humoral immunity

Antibody-secreting cells (ASCs) provide humoral immunity that can mediate lifelong protection against pathogens. Current classifications cannot delineate the heterogenous functionalities, tissue residencies, and lifespans of human ASC subsets, impeding clinical translation. We applied multi-omic sequencing, spatial proteomics, and functional assays to discover and characterize human bone marrow (BM) ASC subsets. We identified two peripheral subsets (ASCp) also present in blood and three BM-resident subsets (ASCr), comprising a maturation continuum associated with increased mitochondrial networking, diminished antibody secretion, differential transcription factor motif accessibility, and preferential co-localization in homotypic niches. CD19+9+ASCr and CD19-ASCr exhibited poor recovery years after BM transplantation, indicating a strong dependence on supportive niches. Childhood vaccine antigens were recognized by long-lived ASCr subsets in adults and by immature HLA-DR+ASCp, implying ASCs can differentiate without recent antigen exposure. Our results provide new insights into ASC identity, maturation, and longevity and a generalizable framework for study and manipulation of human ASCs.

immunology↗

bioRxiv2024

Systemic inflammation and lymphocyte activation precede rheumatoid arthritis

Some autoimmune diseases, including rheumatoid arthritis (RA), are preceded by a critical subclinical phase of disease activity. Proactive clinical management is hampered by a lack of biological understanding of this subclinical at-risk state and the changes underlying disease development. In a cross-sectional and longitudinal multi-omics study of peripheral immunity in the autoantibody-positive at-risk for RA period, we identified systemic inflammation, proinflammatory-skewed B cells, expanded Tfh17-like cells, epigenetic bias in naive T cells, TNF+IL1B+ monocytes resembling a synovial macrophage population, and CD4 T cell transcriptional features resembling those suppressed by abatacept (CTLA4-Ig) in RA patients. Our findings characterize pathogenesis prior to clinical diagnosis and suggest the at-risk state exhibits substantial immune alterations that could potentially be targeted for early intervention to delay or prevent autoimmunity. We provide a suite of tools at https://apps.allenimmunology.org/aifi/insights/ra-progression/ to facilitate exploration and enhance accessibility of this extensive dataset. One Sentence SummaryACPA+ at-risk individuals show RA-like inflammation and multi-compartment immune dysregulation during transition to clinically active RA

immunology↗

bioRxiv2024

A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Leveraging several recent advances, we assembled a prime editing platform capable of high-efficiency substitution editing across a set of engineered prime editing guide RNAs (epegRNAs) and corresponding target sequences (80% median intended editing). Then, using a custom library of 240,000 epegRNAs targeting >17,000 codons with 175 different substitution types, we benchmarked our platform for functional interrogation of small substitution variants (1-3 nucleotides) targeted to essential genes. Resulting data identified negative growth phenotypes for nonsense mutations targeted to [~]8,000 codons, and comparing those phenotypes to results from controls demonstrated high specificity. We also observed phenotypes for synonymous mutations that disrupted splice site motifs at 3' exon boundaries. Altogether, we establish and benchmark a high-throughput prime editing approach for functional characterization of genetic variants with simple readouts from multiplexed experiments.

genomics↗

bioRxiv2022

Nr2f1a maintains atrial nkx2.5 expression to repress pacemaker identity within venous atrial cardiomyocytes

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor (TF) Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of isolated atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5, a critical repressor of PC differentiation. At the arterial pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous pole, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC identity in nr2f1a mutant atria and analysis of chromatin accessibility identified a Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias.

developmental biology↗

bioRxiv2021

Elevated Hoxb5b expands vagal neural crest pool and blocks enteric neuronal development in zebrafish

Neural crest cells (NCCs) are a migratory, transient, and multipotent stem cell population essential to vertebrate embryonic development, contributing to numerous cell lineages in the adult organism. While great strides have been made in elucidating molecular and cellular events that drive NCC specification, comprehensive knowledge of the genetic factors that orchestrate NCC developmental programs is still far from complete. We discovered that elevated Hoxb5b levels promoted an expansion of zebrafish NCCs, which persisted throughout multiple stages of development. Correspondingly, elevated Hoxb5b also specifically expanded expression domains of the vagal NCC markers foxd3 and phox2bb. Increases in NCCs were most apparent after pulsed ectopic Hoxb5b expression at early developmental stages, rather than later during differentiation stages, as determined using a novel transgenic zebrafish line. The increase in vagal NCCs early in development led to supernumerary Phox2b+ enteric neural progenitors, while leaving many other NCC-derived tissues without an overt phenotype. Surprisingly, these NCC-derived enteric progenitors failed to expand properly into sufficient quantities of enterically fated neurons and stalled in the gut tissue. These results suggest that while Hoxb5b participates in vagal NCC development as a driver of progenitor expansion, the supernumerary, ectopically localized NCC fail to initiate expansion programs in timely fashion in the gut. All together, these data point to a model in which Hoxb5b regulates NCCs both in a tissue specific and temporally restricted manner.

developmental biology↗

bioRxiv2021

Mapping the Genetic Landscape of DNA Double-strand Break Repair

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways that are critical for maintaining genomic integrity. Here we present Repair-seq, a high-throughput screening approach that measures the effects of thousands of genetic perturbations on the distribution of mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair outcomes induced by two programmable nucleases (Cas9 and Cas12a) after knockdown of 476 genes involved in DSB repair or associated processes in the presence or absence of oligonucleotides for homology-directed repair (HDR). The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. Systematic interrogation of these dependencies then uncovered unexpected relationships among DSB repair genes and isolated incompletely characterized repair mechanisms. This work provides a foundation for understanding the complex pathways of DSB repair and for optimizing genome editing across modalities.

genomics↗

bioRxiv2020

Cilia-independent requirements for Ccdc103 promoting proliferation and migration of myeloid cells

The pathology of primary ciliary dyskinesia (PCD) is predominantly attributed to impairment of motile cilia. However, PCD patients also have perplexing functional defects in myeloid cells, which lack motile cilia. Here, we show Coiled-coiled domain containing protein 103 (CCDC103), mutations in which underlie PCD, is required for the proliferation and directed migration of myeloid cells. CCDC103 co-localizes with cytoplasmic microtubules in human myeloid cells. Zebrafish ccdc103/schmalhans (smh) mutants have reduced macrophage and neutrophil proliferation, rounded cell morphology, and an inability to migrate efficiently to the site of sterile wounds. Furthermore, we demonstrate that direct interactions between CCDC103 and Sperm associated antigen 6 (SPAG6), which also promotes microtubule stability, are abrogated by CCDC103 mutations from PCD patients, and that spag6 zebrafish mutants recapitulate the myeloid defects of smh mutants. In summary, we have illuminated a mechanism, independent of motile cilia, to explain functional defects in myeloid cells from PCD patients. Summary StatementWe show Ccdc103 regulates myeloid migration and proliferation independent of cilia in zebrafish and that mutations in CCDC103 that cause primary ciliary dyskinesia abrogate interactions with the microtubule-stabilizing protein SPAG6.

developmental biology↗