Biology subjects

Subramanian, K.

Publications and source records attributed to Subramanian, K..

At least 19 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle-bound bacterial toxin pneumolysin triggers membrane engagement and damage beyond canonical pore formation

Pneumolysin (PLY) is a cholesterol-dependent pore-forming toxin and a key virulence factor of Streptococcus pneumoniae, the leading cause of pneumonia worldwide in children aged below 5 years. At sublytic toxin doses, host cells shed PLY-laden extracellular vesicles (EVs) during membrane repair response. However, it remains unclear how these toxin-bearing EVs engage and damage target cell membranes. Here, we combine molecular dynamics simulations, liposome fusion assays, and cell-based experiments to elucidate the membrane interaction potential of vesicle-bound PLY. Simulations indicate that EV-embedded PLY uses an exposed helix to bind target cell membranes, inducing pronounced curvature, bilayer thinning, and water influx. Liposome fusion assays demonstrate that both PLY and membrane cholesterol promote vesicle-membrane interactions. Characterization of vesicle subpopulations released from PLY-challenged monocytes by western blotting and immunogold electron microscopy demonstrated that plasma membrane-derived microvesicles are preferentially enriched in membrane-bound PLY compared to small extracellular vesicles. Consistent with these findings, MVs purified from wild-type PLY-challenged monocytes, but not the toxoid mutant PLYW433F fuse with human immune cells, delivering toxin and causing membrane damage. Together our results reveal a noncanonical, pore-independent mode of toxin dissemination, in which, vesicle-bound pneumolysin fuses and destabilize target cell membranes, representing a new proposal for EV-mediated toxin activity and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2025

Sorafenib, a clinically approved kinase inhibitor attenuates Streptococcus pneumoniae pathogenesis in vivo by targeting serine/threonine kinase StkP

Streptococcus pneumoniae is a respiratory commensal bacterium responsible for over one million annual fatalities globally, particularly among children under five years of age. The rapid emergence of macrolide-resistant strains led the WHO in 2024 to designate S. pneumoniae as a priority pathogen, underscoring the need for alternative strategies such as anti-virulence therapy. Here, we repurposed the FDA-approved cancer drug, sorafenib identified by in silico screening of compounds targeting the bacterial Serine/Threonine kinase protein StkP, an essential regulator of cell division and peptidoglycan synthesis conserved across many bacterial pathogens. Sorafenib interacts with the StkP-kinase domain and exhibited broad-spectrum activity against diverse pneumococcal serotypes including multi-drug-resistant clinical isolates. Ectopic expression of StkP in both wild-type and isogenic mutant strains conferred partial resistance to sorafenib, confirming on-target activity. Scanning electron microscopy revealed aberrant cell-wall morphology, and differential viability staining demonstrated increased membrane permeability. Consistently, sorafenib-treated bacteria showed significantly higher complement C3 deposition and consequent killing in human serum. In human lung epithelial cells, sorafenib reduced bacterial adherence and invasion without detectable host cytotoxicity. Serial passaging at sub-microbicidal concentrations indicated a low propensity for resistance development in vitro. In vivo, sorafenib administration reduced mortality and lung bacterial load in a mouse model of pneumonia. Taken together, our data identify StkP as target of sorafenib in S. pneumoniae and justify its continued preclinical development for therapeutic intervention.

microbiology↗

bioRxiv2025

Could instructor talk drive CURE effectiveness? A comparative study of instructor talk in introductory lab courses

Course-based undergraduate research experiences (CUREs) are thought to enhance students motivation to continue in college, in science, and in research. Yet, how CUREs enhance student motivation is largely undefined. Theories of instructor immediacy, self-efficacy, and task values suggest that CURE instructors may talk in ways that influence students motivational beliefs. We characterized the non-content related talk of instructors teaching 48 introductory biology lab courses, half CUREs and half non-CUREs. We identified 14 types of instructor talk that fit these theoretical perspectives: fostering students closeness with their instructor (i.e., immediacy talk), building students confidence in their scientific abilities (i.e., self-efficacy talk), and promoting students sense of worth in their work (i.e., task value talk). Course type had a medium effect on talk type, with CURE instructors utilizing more immediacy, self-efficacy, and task values talk than non-CURE instructors but also showing more variation in these types of talk. Our results suggest that motivation-related instructor talk is more prevalent in CUREs than non-CUREs, but wide variation in CURE instructor talk indicates additional investigation is needed before non-content talk can be considered a mechanism for the motivational influences of CUREs. HIGHLIGHTThis study compares non-content instructor talk in CURE and non-CURE lab courses using immediacy, self-efficacy, and task value theories. CURE instructors use more talk than non-CURE instructors, but variation in CURE instructor talk leaves open the question of whether talk is a causal factor in the motivational influence of CURE instruction.

scientific communication and education↗

bioRxiv2023

Bacterial pore-forming toxin pneumolysin drives pathogenicity through shed toxin-loaded host extracellular vesicles

Streptococcus pneumoniae is a global priority respiratory pathogen that kills over a million people annually and produces the pore-forming cytotoxin, pneumolysin (PLY). Host cells expel membrane assembled toxin by shedding microvesicles, but the composition and pathophysiological sequelae of the toxin-induced host extracellular vesicles (EVs) are unknown. Here, we found that EVs shed from PLY-challenged monocytes (PLY-EVs) harbor membrane-bound toxin that induced cytotoxicity upon fusion with recipient cells. EVs from human monocytes challenged with recombinant PLY as well as PLY-expressing pneumococcal strains, but not the isogenic PLY mutant, primed dendritic cells and evoked higher pro-inflammatory cytokines upon infection. Proteomic analysis revealed that PLY-EVs are enriched for key antimicrobial and inflammatory host proteins such as IFI16, NLRC4, PTX3 and MMP9. In vivo, zebrafish and mice administered with PLY-EVs showed mortality, pericardial edema, tissue damage and inflammation. Our findings show that host EVs bearing the cytotoxin PLY constitute a previously unexplored mechanism of pneumococcal pathogenesis.

microbiology↗

bioRxiv2022

PERK-ATAD3A interaction protects mitochondrial proteins synthesis during ER stress

Widespread repression of protein synthesis rates is a key feature of Endoplasmic Reticulum (ER) stress, mediated by the ER sensor kinase PERK. While select transcripts escape this repression, global translational down-regulation impacts crucial protein levels in all cellular compartments, beyond the ER. How the cell manages this paradox is unclear. PERK has a unique cytoplasmic loop within its kinase domain that binds PERKs target, eIF2. We identified the mitochondrial protein, ATAD3A, as a new interactor of the loop, binding to a highly conserved region within it. During ER stress, increased interaction between ATAD3A and PERK attenuates PERK signalling to eIF2, removing the translational block on several mitochondrial proteins. This occurs at novel context-dependent, mitochondria-ER contact sites. The interaction provides a previously unknown mechanism for fine-tuning translational repression at a local level, mitigating the impact of ER stress on mitochondria. Further, it represents a new target for selective modulation of PERK-eIF2 signalling in diseases from cancer to neurodegeneration. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/501280v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@f74252org.highwire.dtl.DTLVardef@1234633org.highwire.dtl.DTLVardef@d5dc9aorg.highwire.dtl.DTLVardef@8606fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2022

An oblique plane microscope for mesoscopic imaging of freely moving organisms with cellular resolution

Several important questions in biology require non-invasive and three-dimensional imaging techniques with appropriate spatiotemporal resolution that permit live organisms to move in an unconstrained fashion over an extended field-of-view. While selective-plane illumination microscopy (SPIM) has emerged as a powerful method to observe live biological specimens at high spatio-temporal resolution, typical implementations often necessitate constraining sample mounting or lack the required volumetric speed. Here, we report on an open-top, dual-objective oblique plane microscope (OPM) capable of observing millimeter sized, freely moving animals at cellular resolution. We demonstrate the capabilities of our mesoscopic OPM (MesOPM) by imaging the behavioural dynamics of the sea anemone Nematostella vectensis over 1.56 x 1.56 x 0.25 mm at 1.5 x 2.8 x 5.3{micro}m resolution and 0.5Hz volume rate.

bioengineering↗

bioRxiv2022

The Codon Statistics Database: a Database of Codon Usage Bias

MotivationMost amino acids can be encoded by a set of synonymous codons. Often, for any given amino acid, certain codons are significantly more used than others, a phenomenon known as codon usage bias. The genomes of different species differ in the frequencies at which they use each codon (e.g., a codon that is highly used in one species may be lowly used in another species). In addition, within any given genome, genes differ in their degree of codon bias, with highly expressed genes being more likely to use preferred codons. Knowing the codons that are preferred by a certain genome, and the amount of codon bias exhibited by each gene, has multiple applications (e.g., in heterologous expression, gene prediction, or phylogenetic inference). ResultsWe have developed the Codon Statistics Database, an online database that contains codon usage statistics for all the species with reference or representative genomes in RefSeq. The user can search for any species and access two sets of tables. One set lists, for each codon, the frequency, the Relative Synonymous Codon Usage (RSCU), and whether the codon is preferred. Another set of tables lists, for each gene, its GC content, Effective Number of Codons (ENC), Codon Adaptation Index (CAI), and frequency of optimal codons (Fop). Equivalent tables can be accessed for 1) all nuclear genes, 2) nuclear genes encoding ribosomal proteins, 3) mitochondrial genes and 4) chloroplastic genes (if available in the relevant assembly). The user can also search for any taxonomic group (e.g., "primates") and obtain a table comparing all the species in the group. AvailabilityThe database is free to access without registration at http://codonstatsdb.unr.edu.

bioinformatics↗

bioRxiv2021

Multiplexed and reproducible high content screening of live and fixed cells using the Dye Drop method

High-throughput measurement of cells perturbed using libraries of small molecules, gene knockouts, or different microenvironmental factors is a key step in functional genomics and pre-clinical drug discovery. However, it remains difficult to perform accurate single-cell assays in 384-well plates, limiting many studies to well-average measurements (e.g. CellTiter-Glo(R)). Here we describe a public domain "Dye Drop" method that uses sequential density displacement and microscopy to perform multi-step assays on living cells. We use Dye Drop cell viability and DNA replication assays followed by immunofluorescence imaging to collect single-cell dose-response data for 67 investigational and clinical-grade small molecules in 58 breast cancer cell lines. By separating the cytostatic and cytotoxic effects of drugs computationally, we uncover unexpected relationships between the two. Dye Drop is rapid, reproducible, customizable, and compatible with manual or automated laboratory equipment. Dye Drop improves the tradeoff between data content and cost, enabling the collection of information-rich perturbagen-response datasets.

systems biology↗

bioRxiv2021

Ethology of morphogenesis reveals the design principles of cnidarian size and shape development

During development, organisms interact with their natural habitats while undergoing morphological changes, yet it remains unclear whether the interplay between developing systems and their environments impacts animal morphogenesis. Here, we use the cnidarian Nematostella vectensis as a developmental model to uncover a mechanistic link between organism size, shape and behavior. Using quantitative live imaging, including extensive behavioral profiling, combined with molecular and biophysical experiments, we demonstrate that the muscular hydraulic machinery that controls body movement directly drives larva-polyp morphogenesis. Unexpectedly, size and shape development are differentially controlled by antagonistic muscles. A simple theoretical model shows how a combination of slow-priming and fast-pumping pressures generated by muscular hydraulics acts as a global mechanical regulator that coordinates tissue remodeling. Altogether, our findings illuminate how dynamic behavioral modes in the environment can be harnessed to drive morphogenetic trajectories, establishing ethology as a critical component of organismal morphogenesis - termed ethology of morphogenesis.

developmental biology↗

bioRxiv2021

Position-dependent Codon Usage Bias in the Human Transcriptome

All life depends on the reliable translation of RNA to protein according to complex interactions between translation machinery and RNA sequence features. While ribosomal occupancy and codon frequencies vary across coding regions, well-established metrics for computing coding potential of RNA do not capture such positional dependence. Here, we investigate positiondependent codon usage bias (PDCUB), which dynamically accounts for the position of proteincoding signals embedded within coding regions. We demonstrate the existence of PDCUB in the human transcriptome, and show that it can be used to predict translation-initiating codons with greater accuracy than other models. We further show that observed PDCUB is not accounted for by other common metrics, including position-dependent GC content, consensus sequences, and the presence of signal peptides in the translation product. More importantly, PDCUB defines a spectrum of translational efficiency supported by ribosomal occupancy and tRNA adaptation index (tAI). High PDCUB scores correspond to a tAI-defined translational ramp and low ribosomal occupancy, while low PDCUB scores exhibit a translational valley and the highest ribosomal occupancy. Finally, we examine the relationship between PDCUB intensity and functional enrichment. We find that transcripts with start codons showing the highest PDCUB are enriched for functions relating to neuropeptide signaling and nucleosome assembly, as well as development of musculoskeletal, cardiovascular, neurological, gastrointestinal, sensory, and other body systems. Furthermore, transcripts with high PDCUB are depleted for functions related to immune response and detection of chemical stimulus. These findings lay important groundwork for advances in our understanding of the regulation of translation, the calculation of coding potential, and the classification of RNA transcripts.

bioinformatics↗

bioRxiv2021

A LINCS microenvironment perturbation resource for integrative assessment of ligand-mediated molecular and phenotypic responses

The phenotype of a cell and its underlying molecular state is strongly influenced by extracellular signals, including growth factors, hormones, and extracellular matrix. While these signals are normally tightly controlled, their dysregulation leads to phenotypic and molecular states associated with diverse diseases. To develop a detailed understanding of the linkage between molecular and phenotypic changes, we generated a comprehensive dataset that catalogs the transcriptional, proteomic, epigenomic and phenotypic responses of MCF10A mammary epithelial cells after exposure to the ligands EGF, HGF, OSM, IFNG, TGFB and BMP2. Systematic assessment of the molecular and cellular phenotypes induced by these ligands comprise the LINCS Microenvironment (ME) perturbation dataset, which has been curated and made publicly available for community-wide analysis and development of novel computational methods (synapse.org/LINCS_MCF10A). In illustrative analyses, we demonstrate how this dataset can be used to discover functionally related molecular features linked to specific cellular phenotypes.

systems biology↗

bioRxiv2021

Metallothionein 3-zinc axis suppresses caspase-11 inflammasome activation and impairs antibacterial immunity

Non-canonical inflammasome activation by mouse caspase-11 (or human CASPASE- 4/5) is crucial for the clearance of certain gram-negative bacterial infections, but can lead to severe inflammatory damage. Factors that promote non-canonical inflammasome activation are well recognized, but less is known about the mechanisms underlying its negative regulation. Herein, we identify that the caspase-11 inflammasome in mouse and human macrophages (M{phi}) is negatively controlled by the zinc (Zn2+) regulating protein, metallothionein 3 (MT3). Upon challenge with intracellular lipopolysaccharide (iLPS), M{phi} increased MT3 expression that curtailed the activation of caspase-11 and its downstream targets caspase-1 and interleukin (IL)-1{beta}. Mechanistically, MT3 increased intramacrophage Zn2+ to downmodulate the TRIF-IRF3-STAT1 axis that is prerequisite for caspase-11 effector function. MT3 suppressed activation of the caspase-11 inflammasome, while caspase-11 and MT3 synergized in impairing antibacterial immunity. The present study identifies an important yin-yang relationship between the non-canonical inflammasome and MT3 in controlling inflammation and immunity to gram- negative bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=149 HEIGHT=200 SRC="FIGDIR/small/454033v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@15070f1org.highwire.dtl.DTLVardef@27174forg.highwire.dtl.DTLVardef@6b48beorg.highwire.dtl.DTLVardef@174b850_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2021

HSP90 inhibitors reduce cholesterol storage in Niemann-Pick type C1 mutant fibroblasts

Niemann Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder caused by mutations of the NPC1 gene. More than 300 disease-associated mutations are reported in patients, resulting in abnormal accumulation of unesterified cholesterol, glycosphingolipids and other lipids in late endosomes and lysosomes (LE/Ly) of many cell types. Previously, we showed that treatment of many different NPC1 mutant fibroblasts with histone deacetylase inhibitors resulted in reduction of cholesterol storage, and we found that this was associated with enhanced exit of the NPC1 protein from the endoplasmic reticulum and delivery to LE/Ly. This suggested that histone deacetylase inhibitors may work through changes in protein chaperones to enhance the folding of NPC1 mutants, allowing them to be delivered to LE/Ly. In this study we evaluated the effect of several HSP90 inhibitors on NPC1I1061T skin fibroblasts. We found that HSP90 inhibition resulted in clearance of cholesterol from LE/Ly, and this was associated with enhanced delivery of the mutant NPC1I1061T protein to LE/Ly. We also observed that inhibition of HSP90 increased the expression of HSP70, and overexpression of HSP70 also reduced cholesterol storage in NPC1I1061T fibroblasts. However, we did not see correction of cholesterol storage by arimoclomol, a drug that is reported to increase HSP70 expression, at doses up to 0.5 mM. These results indicate that manipulation of molecular chaperones may lead to effective treatments for NPC1 disease, but further investigation of mechanisms will be required.

cell biology↗

bioRxiv2020

Proteomic profiling of breast cancer cell lines and models

We performed quantitative proteomics on 61 human-derived breast cancer cell lines to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics.

cancer biology↗

bioRxiv2020

DNA barcoding analysis of more than 1000 marine yeast isolates reveals previously unrecorded species

For the yeast population and diversity, marine habitats are the least explored niches and the lack of validated database is considered to be a drawback for yeast research. The aim of the present study is to create a comprehensive DNA barcode library for marine derived yeast species isolated from organic burial hotspots such as coastal sediment in mangrove and continental shelf ecosystems. As we enriched, isolated and ITS gene sequenced 1017 marine derived yeast isolates belonging to 157 marine species in 55 genera, 28 families, 14 orders, 8 classes of 2 Phyla (viz., Ascomycota and Basidiomycota) of which 13 yeast species were first time barcoded. We witnessed yeast species of both terrestrial and marine endemic origin in the barcode datasets. Due to the large volume of sequencing trace files, the variable length of extracted sequences, and the lack of reference sequences in public databases, difficulties were faced in taxonomic sequence validation. The length of the majority of the sequences (99.42%) were more than or equal to 600 base pairs. BLAST analysis revealed that 13 yeast species were barcoded for the first time. The genus, Candida was the speciose genera isolated in this study. K2P intra-species distance analysis performed for selective groups yielded an average of 0.33%, well below the previously proposed yeast barcode gap. ITS gene NJ-tree based identification conducted for selective species in Ascomycota and Basidomycota, precisely clustered the same species into one group, indicating the efficacy of ITS gene in yeast species delineation. Besides isolating some of the common marine yeast species such as Candida oceani, Bandonia marina and Yarrowia lipolytica, we found approximately 60% of the yeast species isolates were previously unrecorded from the marine environment (example; Cystobasidiopsis lactophilus, Slooffia cresolica, Udeniozyma ferulica, Colacogloea falcatus and Pichia guilliermondii), of which 16.5% were recognised as potential human pathogens (example; Candida orthopsilosis, C. rugosa, Debaryomyces fabryi and Yamadazyma triangularis). Apart from releasing the barcode data in GenBank, provisions were made to access the entire dataset along with meta-data in the Barcode of life database (http://dx.doi.org/10.5883/DS-MYIC). This research constitutes the largest dataset to date for collecting marine yeast isolates and their barcodes. As meta- and environmental barcoding analysis were expanding its scope including environmental assessment and monitoring, the datasets such as ours will be more useful.

microbiology↗

bioRxiv2020

Nucleus accumbens melanin-concentrating hormone signaling promotes feeding in a sex-specific manner

Melanin-concentrating hormone (MCH) is an orexigenic neuropeptide produced in the lateral hypothalamus and zona incerta that increases food intake. The neuronal pathways and behavioral mechanisms mediating the orexigenic effects of MCH are poorly understood, as is the extent to which MCH-mediated feeding outcomes are sex-dependent. Here we investigate the hypothesis that MCH-producing neurons act in the nucleus accumbens shell (ACBsh) to promote feeding behavior and motivation for palatable food in a sex-dependent manner. We utilized ACBsh MCH receptor (MCH1R)-directed pharmacology as well as a dual virus chemogenetic approach to selectively activate MCH neurons that project to the ACBsh. Results reveal that both ACBsh MCH1R activation and activating ACBsh-projecting MCH neurons increase consumption of standard chow and palatable sucrose in male rats without affecting motivated operant responding for sucrose, general activity levels, or anxiety-like behavior. In contrast, food intake was not affected in female rats by either ACBsh MCH1R activation or ACBsh-projecting MCH neuron activation. To determine a mechanism for this sexual dimorphism, we investigated whether the orexigenic effect of ACBsh MCH1R activation is reduced by endogenous estradiol signaling. In ovariectomized female rats on a cyclic regimen of either estradiol (EB) or oil vehicle, ACBsh MCH1R activation increased feeding only in oil-treated rats, suggesting that EB attenuates the ability of ACBsh MCH signaling to promote food intake. Collective results show that that MCH ACBsh signaling promotes feeding in an estrogen- and sex-dependent manner, thus identifying novel neurobiological mechanisms through which MCH and female sex hormones interact to influence food intake.

neuroscience↗

bioRxiv2020

Recapitulating evolutionary divergence in a single regulatory element causes expression changes of the lens gene Tdrd7

Mutations in cis-regulatory elements play important roles for phenotypic changes during evolution. Eye degeneration in the blind mole rat (BMR) and other subterranean mammals is significantly associated with widespread divergence of eye regulatory elements, but the effect of these regulatory mutations on eye development and function has not been explored. Here, we investigate the effect of mutations observed in the BMR sequence of a conserved non-coding element upstream of Tdrd7, a pleiotropic gene required for lens development and spermatogenesis. We first show that this conserved element is a transcriptional repressor in lens cells and that the BMR sequence partially lost repressor activity. Next, we recapitulated the evolutionary changes by precisely replacing the endogenous regulatory element in a mouse line by the orthologous BMR sequence with CRISPR-Cas9. Strikingly, this repressor element has a large effect, causing a more than two-fold up-regulation of Tdrd7 in developing lens. Interestingly, the increased mRNA level does not result in a corresponding increase in TDRD7 protein nor an obvious lens phenotype, likely explained by buffering at the posttranscriptional level. Our results are consistent with eye degeneration in subterranean mammals having a polygenic basis where many small-effect mutations in different eye-regulatory elements collectively contribute to phenotypic differences.

evolutionary biology↗

bioRxiv2019

Rod nuclear architecture determines contrast transmission of the retina and behavioral sensitivity in mice

Rod photoreceptors of nocturnal mammals display a striking inversion of nuclear architecture, which has been proposed as an evolutionary adaptation to dark environments. However, the nature of visual benefits and underlying mechanisms remains unclear. It is widely assumed that improvements in nocturnal vision would depend on maximization of photon capture, at the expense of image detail. Here we show that retinal optical quality improves 2-fold during terminal development, which, confirmed by a mouse model, happens due to nuclear inversion. We further reveal that improved retinal contrast-transmission, rather than photon-budget or resolution, leads to enhanced contrast sensitivity under low light condition. Our findings therefore add functional significance to a prominent exception of nuclear organization and establish retinal contrast-transmission as a decisive determinant of mammalian visual perception. One Sentence SummaryOur study reveals that chromatin compaction in rod cells augments contrast sensitivity in mice.

biophysics↗