Biology subjects

Swaminathan, J.

Publications and source records attributed to Swaminathan, J..

9 recordsLinked to original sources

bioRxiv2026

REST-AKT-SOX2 axis controls neuronal lineage state and Midkine-mediated communication in SHH-medulloblastoma.

Stem/progenitor-like cells are known drivers of tumor progression, chemoresistance, and relapse in Sonic-Hedgehog medulloblastoma (SHH-MB), yet the regulatory mechanisms that sustain these resilient cellular states remain incompletely defined. Here, we identify the RE1-silencing transcription factor (REST) as a key transcriptional regulator that preserves the progenitor compartment in SHH-MB through stabilization of the stemness factor SOX2. Mechanistically, REST activates AKT signaling, which in turn enhances SOX2 protein stability, revealing a REST-AKT-SOX2 axis that supports stem/progenitor identity and ongoing tumor maintenance. Beyond maintaining intrinsic stem-like programs, REST also orchestrates the extrinsic communication network of SHH-MB tumors. Single cell transcriptomic profiling and ligand-receptor interaction mapping highlight Midkine (MDK)-mediated signaling as one of the most upregulated intercellular communication routes in MB. We demonstrate that REST drives this signaling cascade through its control of SOX2. Perturbation of REST or SOX2 results in reduced MDK and its receptor, SDC2 expression, and chromatin immunoprecipitation assays show that SOX2 directly binds and regulates MDK/SDC2 expression, establishing and reinforcing a REST/SOX2 centered transcriptional mechanism that coordinates both progenitor maintenance and cell-cell communication in the malignant compartment. Together, these findings position REST as an integrator of intrinsic progenitor cell programs and extrinsic MDK-mediated signaling in SHH-MB. By linking stemness, communication and potential for therapeutic resistance, the REST-AKT-SOX2-MDK signaling axis emerges as a targetable vulnerability to suppress stem/progenitor driven tumor program in REST-driven SHH-MBs.

cancer biology↗

bioRxiv2025

REST elevation-dependent chromatin remodeling and alternative Grk6 transcript synthesis hyperactivates Cxcr4-Sdf1 signaling in cerebellar granule cell progenitors

RE1 Silencing Transcription Factor (REST) is a repressor of transcriptional initiation of genes involved in neurogenesis. Here, we show that conditional REST elevation in cerebellar granule cell progenitors (CGNPs) of RESTTG mice perturbed foliation, increased cell migration, and sustained C-X-C motif receptor 4 (Cxcr4) signaling, a pathway key to postnatal CGNP migration. Mechanistic studies uncovered a novel role for REST in controlling transcript diversity and exon skipping in CGNPs. Alternative transcript expression was detected in known Cxcr4 signaling regulator, G-protein-coupled receptor kinase-6 (Grk6). Further analysis of Grk6s isoform expression revealed an upregulation of a transcript lacking exon 10a (Grk6-207) in RESTTG CGNPs. Grk6-207 expression in wildtype CGNPs hyperactivated Cxcr4 signaling and increased chemotaxis. Structural modeling of Grk6-207 predicted changes in active site conformation and interactions with Cxcr4 and {beta}-Arrestin-1, supporting impairment of Cxcr4 signaling desensitization. Interestingly, REST elevation promoted increased chromatin accessibility at the exon10a-10b junction and exon 10a exclusion. Integrated multiomic analyses identified the enhancer of zeste (Ezh2) as a potential mediator of alternative transcript generation which demonstrated increased occupancy at the exon10a-10b locus in RESTTG CGNPs. Pharmacological inhibition of Ezh2 downregulated Grk6-207, confirming a role for Ezh2 in Grk6 exon10a exclusion and the increased migration in RESTTG CGNPs. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=67 SRC="FIGDIR/small/682654v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@166db32org.highwire.dtl.DTLVardef@1c0c5e0org.highwire.dtl.DTLVardef@ff2d6borg.highwire.dtl.DTLVardef@fed170_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2023

Robust and scalable single-molecule protein sequencing with fluorosequencing

The need to accurately survey proteins and their modifications with ever higher sensitivities, particularly in clinical settings with limited samples, is spurring development of new single molecule proteomics technologies. Fluorosequencing is one such highly parallelized single molecule peptide sequencing platform, based on determining the sequence positions of select amino acid types within peptides to enable their identification and quantification from a reference database. Here, we describe substantial improvements to fluorosequencing, including identifying fluorophores compatible with the sequencing chemistry, mitigating dye-dye interactions through the use of extended polyproline linkers, and developing an end-to-end workflow for sample preparation and sequencing. We demonstrate by fluorosequencing peptides in mixtures and identifying a target neoantigen from a database of decoy MHC peptides, highlighting the potential of the technology for high sensitivity clinical applications.

molecular biology↗

bioRxiv2023

Short-term intermittent hypoxia therapy promotes gliogenesis in a rat model of middle cerebral artery occlusion (MCAO) stroke

AimAfter focal cerebral ischemia, intermittent hypoxia therapy (IHT) could be used as a non-invasive method to stabilize and stimulate neurogenesis in the innate stem cell niche in the brain, and disrupt the glial scar around the infarct to increase neuroblast migration in the striatal infarct area. MethodsWe induced focal cerebral ischemia in Wistar albino rats using the MCAo model. A week later, animals were subjected to intermittent hypoxia (12%O2, 4hr/day) for a period of 14 days. Post-treatment analysis of functional recovery and cellular regeneration was done using immunofluorescence analysis of multiple neuronal cell markers including Doublecortin (DCX), Nestin, and Vimentin among others. ResultsObservations of GFAP-positive cells revealed that IH treatment facilitates gliogenesis in the infarct striatal region of a rat model of MCAo stroke. The percentage of DCX and GFAP double-positive cells was increased in the IH-treated group. Also, there was a significant difference in the morphology of vimentin-positive cells and microglia cells between the stroke groups. ConclusionThese outcomes suggest that exposure of MCAo stroke-affected rats to intermittent hypoxia results in an increase in migrated neuroblasts resulting in a subsequent altered glial scar integrity in the infarct region, thus suggesting an alternative non-invasive method against the common stem cell transplant techniques, to increase endogenous neuroblasts in the infarct area after stroke.

neuroscience↗

bioRxiv2023

Design and Evaluation of an Affordable Hypoxic Chamber with Comprehensive Environmental Control for Research Applications

PurposeIn-vivo Hypoxia chamber is sought after for research on sleep apnea, among others. Recently hypoxia chambers are utilized to create intermittent hypoxia, thereby utilizing it as a treatment strategy. The commonly used Invivo hypoxic chambers only monitor oxygen levels and fail to read up on vital factors like CO2 buildup, and humidity among others. In addition, these devices are expensive and make them unaffordable for many labs. MethodsHere we report a lab-designed and assembled Arduino microcontroller-based hypoxic chamber with automatic maintenance of O2 level, CO2 level, and Humidity. The entire 650$ setup is inclusive of an Arduino module with sensors for oxygen, CO2, humidity, pressure, and temperature. A detailed software program was also written to help efficiently carry out the parameters set out for use. ResultsThe equipment is capable of automatically regulating the inner environment based on the set parameters. The combined cluster of regulators, efficiently controlled the oxygen levels, CO2 levels, and humidity levels within the experimental parameters. ConclusionThis equipment thus acts as one of the easily reproducible simple assembly units with the integration of complex parameters, thus monitoring and controlling the inner environment with high precision.

bioengineering↗

bioRxiv2022

Dissociation protocols used for sarcoma tissues bias the transcriptome observed in single-cell and single-nucleus RNA sequencing

BackgroundSingle-cell RNA-seq has emerged as an innovative technology used to study complex tissues and characterize cell types, states, and lineages at a single-cell level. Classification of bulk tumors by their individual cellular constituents has also created new opportunities to generate single-cell atlases for many organs, cancers, and developmental models. Despite the tremendous promise of this technology, recent evidence studying epithelial tissues and diverse carcinomas suggests the methods used for tissue processing, cell disaggregation, and preservation can significantly bias gene expression and alter the observed cell types. To determine whether sarcomas - tumors of mesenchymal origin - are subject to the same technical artifacts, we profiled patient-derived tumor explants (PDXs) propagated from three aggressive subtypes: osteosarcoma, Ewing sarcoma (ES), desmoplastic small round cell tumor (DSRCT). Given the rarity of these sarcoma subtypes, we explored whether single-nuclei RNA-seq from more widely available archival frozen specimens could accurately be identified by gene expression signatures linked to tissue phenotype or pathognomonic fusion proteins. ResultsWe systematically assessed dissociation methods across different sarcoma subtypes. We compared gene expression from single-cell and single-nucleus RNA-sequencing of 125,831 whole-cells and nuclei from ES, DSRCT, and osteosarcoma PDXs. We detected warm dissociation artifacts in single-cell samples and gene length bias in single-nucleus samples. Classic sarcoma gene signatures were observed regardless of dissociation method. In addition, we showed that dissociation method biases can be computationally corrected. ConclusionsWe highlighted transcriptional biases, including warm dissociation and gene-length biases, introduced by the dissociation method for various sarcoma subtypes. This work is the first to characterize how the dissociation methods used for sc/snRNA-seq may affect the interpretation of the molecular features in sarcoma PDXs.

cancer biology↗

bioRxiv2021

Photoredox-catalyzed decarboxylative C-terminal differentiation for bulk and single molecule proteomics

Methods for the selective labeling of biogenic functional groups on peptides are being developed and used in the workflow of both current and emerging proteomics technologies, such as single-molecule fluorosequencing. To achieve successful labeling with any one method requires that the peptide fragments contain the functional group for which the labeling chemistry is designed. In practice, only two functional groups are present on every peptide fragment regardless of the protein cleavage site, namely, an N-terminal amine and a C-terminal carboxylic acid. Developing a global-labeling technology, therefore, requires one to specifically target the N- and/or C-terminus of peptides. In this work, we showcase the first successful application of photocatalyzed C-terminal decarboxylative-alkylation for peptide mass-spectrometry and single molecule protein sequencing, that can be broadly applied to any proteome. We demonstrate that peptides in complex mixtures generated from enzymatic digests from bovine serum albumin, as well as protein mixtures from yeast and human cell extracts, can be site-specifically labeled at their C-terminal residue with a Michael acceptor. Using two distinct analytical approaches, we characterize C-terminal labeling efficiencies of greater than 50% across complete proteomes and document the proclivity of various C-terminal amino acid residues for decarboxylative-labeling, showing histidine and tryptophan to be the most disfavored. Finally, we combine C-terminal decarboxylative labeling with an orthogonal carboxylic acid labeling technology in tandem, to establish a new platform for fluorosequencing.

biochemistry↗

bioRxiv2020

Solid-Phase Peptide Capture and Release for Bulk and Single-Molecule Proteomics

The field of proteomics has expanded recently with more sensitive techniques for the bulk measurement of peptides as well as single-molecule techniques. One limiting factor for some of these methods is the need for multiple chemical derivatizations and highly pure proteins free of contaminants. We demonstrate a solid-phase capture strategy suitable for the proteolysis, purification, and subsequent chemical modification of peptides. We use this resin on an HEK293T cell lysate and perform one-pot proteolysis, capture, and derivatization to generate a cellular proteome that identified over 40,000 bead-bound peptides. We also show that this capture can be reversed in a traceless manner, such that it is amenable for single-molecule proteomics techniques. With this technique, we perform a fluorescent labeling and C-terminal derivatization on a peptide and subject it to fluorosequencing, demonstrating that washing the resin is sufficient to remove excess dyes and other reagents prior to single-molecule protein sequencing.

biochemistry↗

bioRxiv2019

EpiSAFARI: Sensitive detection of valleys in epigenetic signals for enhancing annotations of functional elements

The genomewide signal profiles from functional genomics experiments are dense information sources for annotating the regulatory elements. These profiles measure epigenetic activity at the nucleotide resolution and they exhibit distinct patterns along the genome. Most notable of these patterns are the valley patterns that are prevalently observed in many epigenetic assays such as ChIP-Seq and bisulfite sequencing. Valleys mark locations of cis-regulatory elements such as enhancers. Systematic identification of the valleys provides novel information for delineating the annotation of regulatory elements using epigenetic data. Nevertheless, the valleys are generally not reported by analysis pipelines. Here, we describe EpiSAFARI, a computational method for sensitive detection of valleys from diverse types of epigenetic profiles. EpiSAFARI employs a novel smoothing method for decreasing noise in signal profiles and accounts for technical factors such as sparse signals, mappability, and nucleotide content. In performance comparisons, EpiSAFARI performs favorably in terms of accuracy. The histone modification and DNA methylation valleys detected by EpiSAFARI exhibit high conservation, transcription factor binding, and they are enriched in nascent transcription. In addition, the large clusters of histone valleys are found to be enriched at the promoters of the developmentally associated genes.

bioinformatics↗