Biology subjects

Sridharan, S.

Publications and source records attributed to Sridharan, S..

16 recordsLinked to original sources

bioRxiv2025

ILP4 and InR regulate Paclitaxel-induced hypersensitivity differently in Drosophila larvae

Paclitaxel (PTX), a chemotherapeutic that stabilizes microtubules, induces nociceptive hypersensitivity and sensory neuron damage in humans, mice, and flies. To enhance our basic understanding of PTX-induced effects we undertook a molecular/genetic dissection of PTX-induced nociceptive hypersensitivity. Larvae fed viable doses of PTX exhibited dose-dependent hypersensitivity to sub-noxious thermal stimuli. Hypersensitivity developed rapidly and did not completely resolve at the larval stage. Live imaging of peripheral thermal nociceptors showed that lower doses of PTX (< 10 {micro}M) caused hyper-sprouting of tertiary dendritic branches. At 10 {micro}M and above, dendritic beading was observed. PTX-induced hypersensitivity does not depend on signaling pathways previously implicated in acute injury-induced nociceptive sensitization. However, the insulin-like peptide 4 (ILP4), was required for PTX-induced thermal hypersensitivity at 10 {micro}M PTX. Surprisingly, RNAi targeting the insulin receptor (InR) in nociceptors increased PTX-induced hypersensitivity, suggesting that ILP4 does not activate InR in this context. The salivary gland is likely the primary tissue source of functional ILP4. ILP4 mutant larvae did not exhibit PTX-induced beading (10 {micro}M) but did exhibit hypersprouting at lower PTX concentrations. In summary, our model of PTX-induced hypersensitivity reveals a disconnect between hypersensitivity and neuronal morphology and a genetic separation of ILP4 and InR in PTX-induced hypersensitivity.

genetics↗

bioRxiv2025

Rapid turnover and recurrent structural variation at the 17q21.31 locus in modern and ancient human genomes, and primates

The 17q21.31 locus in humans harbors several complex structural haplotypes including a [~]970kb inversion. Different inversion haplotypes have been associated with susceptibility to microdeletions causing Koolen-de Vries syndrome and variation in fecundity and recombination rates. Here, using 210 haplotype-resolved human genome assemblies and pangenome graph-based approaches we characterize 11 distinct structural haplotypes, several of which have not been previously described. Extending our analyses to a set of haplotype-resolved great-ape genomes, we characterize the structure of an independent inversion in chimpanzees which extends an additional 650kb, encompasses 5 additional genes, and is [~]2 million years younger than the human inversion. We further determine that gorillas exhibit an independent duplication of the KANSL1 gene which may predispose them to Koolen-de Vries syndrome causing microdeletions. Using short read sequencing data we characterize 17q21.31 haplotype diversity worldwide in [~]5174 individuals from 107 populations finding increased frequencies of KANSL1 duplication-containing haplotypes in both European and South Asian populations as well as 8 double recombination events between inverted and non-inverted haplotypes ranging in size from 20-180kb. Finally, using 626 ancient Eurasian human genomes we show the frequency of haplotypes containing KANSL1 duplications has increased [~]6-fold over the past 12 thousand years in Europe. Together, our results highlight the dynamics, complexity, and recurrent, independent evolution of a medically relevant locus across humans and great apes.

genomics↗

bioRxiv2025

Feature-specific inhibitory connectivity augments the accuracy of cortical representations

To interpret complex sensory scenes, animals exploit statistical regularities to infer missing features and suppress redundant or ambiguous information. Cortical microcircuits might contribute to this cognitive goal by either completing or cancelling predictable activity, but it remains unknown whether, and how, a single circuit can implement these antagonistic computations. To address this central question, we used all-optical physiology to simulate sensory-evoked activity patterns in pyramidal cells (PCs) and somatostatin interneurons (SSTs) in the mouses primary visual cortex (V1). In the absence of external visual input, photostimulation of orientation-tuned PC ensembles drove either completion or cancelation of input-matching representations, depending on the number of photostimulated cells. This dual computational capacity arose from the co-existence of like-to-like excitatory interactions between PCs, and a newly discovered like-to-like SST-PC connectivity motif, in which SSTs are preferentially recruited by, and in turn suppress, similarly tuned PCs. Finally, we show that photoactivation of tuned SST ensembles during visual processing improved the discriminability of their preferred visual input by suppressing ambiguous activity. Thus, these complementary feature-specific connectivity motifs allow different strategies of contextual modulation to optimize inference by either completion (through PC-PC interactions) or cancelation (via PC-SST-PC loops) of predictable activity, depending on the structure of the input and the network state.

neuroscience↗

bioRxiv2025

Integrating Artificial Intelligence-Driven Digital Pathology and Genomics to Establish Patient-Derived Organoids as a Novel Alternative Model for Drug Response in Head and Neck Cancer

Patient-derived organoids (PDOs) are emerging as advanced 3D ex vivo novel alternative method (NAM) preclinical models, offering significant advantages over traditional cell lines and monolayer cultures for therapeutic development. In this study, we established PDOs from surgically resected fresh tissues of human papillomavirus (HPV)-negative head and neck squamous cell carcinoma (HNSCC) across anatomical sites, tumor T-categories, and sample types. These PDOs faithfully recapitulate the tumors pathology, mutational profile, and drug response. To enable rapid classification of PDO identity, we developed a new convolutional neural network (CNN) model, TransferNet-PDO, which accurately distinguished tumor versus normal PDOs in culture using digital histopathology images (AUC[≥]0.88). PDOs maintained stable cultures and were cryopreserved between passages 5 and 12. Immunohistochemistry (IHC) staining (PanCK, p63, Cytokeratin 13, Ki67) confirmed squamous phenotype and histologic aggression of the original tumor. For tumors harboring TP53 mutations by whole-exome sequencing (WES), PDOs retained the corresponding p53 functional status as confirmed by IHC (enhanced or loss of expression). Somatic mutational landscape revealed that PDOs preserved driver somatic mutations, copy number variations (CNVs), and clonal architecture including low-prevalence subclones. Drug sensitivity assessment of PDOs showed that cisplatin reduced cell viability, whereas cetuximab and lenvatinib had minimal effects. Chemoradiation led to greater tumor organoid killing compared to radiation or chemotherapy alone. This study presents an integrated HNSCC PDO platform combining tissue biobanking, organoid establishment, multi-omics characterization, functional drug screening, and AI-driven histopathologic classification, providing a comprehensive and scalable system for translational cancer research.

cancer biology↗

bioRxiv2024

Antagonistic activity of AEA on TAT treated Human Astrocytes identify Inflammaging pathways

Physiologically, the endocannabinoids are known to reduce inflammation by decreasing production of inflammatory factors in immune and glial cells. Astrocytes secrete soluble inflammatory mediators and prolonged activation of astrocytes is associated with accelerated aging in Central Nervous System. Many reports show miRNAs as critical gene regulators in inflammation and astrogliosis in astrocytes. The aim of this study is to investigate the microRNA changes affected by Anandamide on HIV1 TAT (Trans-activator of transcription protein) stimulated normal human astrocytes. We performed global microRNA profile in TAT activated astrocytes and analysed changes on exposure to AEA. To delineate the mechanism of action we assessed with bioinformatic tools miRWalk, KEGG and Cytoscape the global microarray for significantly impacted miRNAs, and their gene targets. TAT activation in astrocytes upregulated 122 miRNAs significantly (p < 0.05). Addition of AEA in activated astrocytes downregulate the expression of 57 miRNAs significantly. Out of 122 upregulated miRNAs on TAT treatment, 37 miRNAs which were common in TAT and TAT+AEA cells showed reversal suggesting clues to critical miRNAs for the AEA-induced mitigation of neuroinflammation. Reversal in expression of selected group of miRNAs identify antagonistic pathways which are promoting anti-inflammatory environment. Pathway analysis of key 37 miRNAs show gene targets that regulate inflammation and senescence.

immunology↗

bioRxiv2023

Artificially inserted G-quadruplex DNA secondary structures induce long-distance chromatin activation

Although the role of G-quadruplex (G4) DNA structures has been suggested in chromosomal looping this was not tested directly. Here, to test causal function, an array of G4s, or control sequence that does not form G4s, were inserted within chromatin in cells. In vivo G4 formation of the inserted G4 sequence array, and not the control sequence, was confirmed using G4-selective antibody. Compared to the control insert, we observed a remarkable increase in the number of 3D chromatin looping interactions from the inserted G4 array. This was evident within the immediate topologically associated domain (TAD) and throughout the genome. Locally, recruitment of enhancer histone marks and the transcriptional coactivator p300/Acetylated-p300 increased in the G4-array, but not in the control insertion. Resulting promoter-enhancer interactions and gene activation were clear up to 5 Mb away from the insertion site. Together, these show the causal role of G4s in enhancer function and long-range chromatin interactions. Mechanisms of 3D topology are primarily based on DNA-bound architectural proteins that induce/stabilize long-range interactions. Involvement of the underlying intrinsic DNA sequence/structure in 3D looping shown here therefore throws new light on how long-range chromosomal interactions might be induced or maintained.

molecular biology↗

bioRxiv2022

Influenza A virus liquid condensates can undergo pharmacological hardening

Multiple viral infections form biomolecular condensates in the host cell to compartmentalize viral reactions. Accumulating evidence indicates that these viral condensates may be hardened, a strategy with potential for exploitation as novel antiviral therapy, given that viral reactions rely on specific material properties for function. However, there is no molecular understanding on how to specifically and efficiently modify the material properties of viral condensates, a pre-requisite for overcoming off-target effects by rational drug design. In vitro, the material properties of biological condensates are modified by different thermodynamic parameters, including free energy, concentration, and type/strength of interactions. Here, we used influenza A virus liquid cytosolic condensates, A.K.A viral inclusions, to provide a proof of concept that modulating the type/strength of transient interactions among the interactome in IAV inclusions is more efficient at hardening these structures than varying the temperature or concentration, both in in vitro and in in vivo models. This stabilization can be achieved by a known pharmacological sticker that can specifically change the material properties of viral inclusions without affecting host proteome abundance nor solubility. Our work supports the development of antivirals targeting the material properties of biomolecular condensates in viral infections. It also provides a framework for the selection of compounds with this activity for general application and thus provides an advance in disease therapy.

cell biology↗

bioRxiv2022

BMAL1 and MEX3A co-regulate intestinal stem cell succession

Chemotherapeutic agents, such as 5-fluorouracil (5-FU), often induce intestinal mucositis due to toxicity to rapidly dividing intestinal stem cells (ISCs). Drug delivery at different times of day can alter 5-FU toxicity; however, the underlying biology is unclear. We found that homeostasis and succession between two ISC types, fast proliferating Lgr5+ crypt base columnar (CBC) cells and stress-resistant +4 cells (Bmi1+), are controlled by the circadian clock transcription factor BMAL1 and the BMAL1-regulated RNA-binding protein MEX3A via both direct transcriptional regulation and post-transcriptional control of Lgr5 mRNA stability. Bmal1 knockout in Lgr5+ CBCs reduced MEX3A expression and CBC numbers but increased Bmi1 expression in the crypts. Timing 5-FU delivery when crypt cells had low Lgr5 but peak level of Bmi1 protected ISCs from apoptosis. Together, these findings identify a novel role of BMAL1 in ISC homeostasis and provide a biological explanation for chronotherapeutic chemoprotection.

cell biology↗

bioRxiv2021

Cryo-EM structures of the channelrhodopsin ChRmine in lipid nanodiscs

Microbial channelrhodopsins are light-gated ion channels widely used for optogenetic manipulation of neuronal activity. ChRmine is a bacteriorhodopsin-like cation channelrhodopsin (BCCR) more closely related to ion pump rhodopsins than other channelrhodopsins. ChRmine displays unique properties favorable for optogenetics including high light sensitivity, a red-shifted activation spectrum, cation selectivity, and large photocurrents while its slow closing kinetics impede some applications. The structural basis for ChRmine function, or that of any other BCCR, is unknown. Here, we present cryo-EM structures of ChRmine in lipid nanodiscs in apo (opsin) and retinal-bound (rhodopsin) forms. The structures reveal an unprecedented trimeric architecture with a lipid filled central pore. Large electronegative cavities on either side of the membrane facilitate high conductance and selectivity for cations over protons. The retinal binding pocket structure suggests spectral and kinetic properties could be tuned with mutations and we identify ChRmine variants with two-fold increased and ten-fold decreased closing rates. These results provide insight into structural features that generate an ultra-potent microbial opsin and provide a platform for rational engineering of channelrhodopsins with improved properties that could expand the scale, depth, and precision of optogenetic manipulations.

neuroscience↗

bioRxiv2021

Condensate-mediated reactivation of mumps virus infection under stress

Many viruses can establish long-term persistent infections in the human host and cause chronic diseases. Here we combine cell biology, whole-cell proteomics and cryo-electron tomography to uncover how cellular stress disrupts the host-virus equilibrium in persistent infection and induces viral replication, using a model of negative-stranded RNA viruses, the mumps virus. We show that phosphorylation of the largely disordered viral phosphoprotein coincides with increased partitioning of viral polymerase into pre-formed liquid-like condensates and the formation of a stable replication machinery. By obtaining the first atomic models for the authentic mumps virus nucleocapsid, we elucidate a concomitant conformational change that exposes the viral genome to its replication machinery. These events that occur within viral condensates upon stress, together with concerted down-regulation of the host antiviral defense, provide an environment that supports up-regulation of viral replication and constitute a stress-mediated switch that disrupts the host-virus equilibrium in persistent infection. In BriefA multi-scale approach uncovers molecular and structural basis of how cellular stress provokes activation of persistent viral infection mediated by biomolecular condensates.

cell biology↗

bioRxiv2021

High performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks

Patterned optogenetic activation of defined neuronal populations in the intact brain can reveal fundamental aspects of the neural codes of perception and behavior. The biophysical properties of existing optogenetic tools, however, constrain the scale, speed, and fidelity of precise optical control. Here we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity in vivo and in vitro. We benchmark these new opsins against existing optogenetics tools with whole-cell electrophysiology and all-optical physiology and provide a detailed biophysical characterization of a diverse family of microbial opsins under two-photon illumination. This establishes a toolkit and a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for cell-specific holographic photo-stimulation, we demonstrate the simultaneous co-activation of several hundred spatially defined neurons with a single hologram, and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.

neuroscience↗

bioRxiv2020

Cryo-EM structure of the SARS-CoV-2 3a ion channel in lipid nanodiscs

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes three putative ion channels: E, 8a, and 3a1,2. 3a is expressed in SARS patient tissue and anti-3a antibodies are observed in patient plasma3-6. 3a has been implicated in viral release7, inhibition of autophagy8, inflammasome activation9, and cell death10,11 and its deletion reduces viral titer and morbidity in mice1, raising the possibility that 3a could be an effective vaccine or therapeutic target3,12. Here, we present the first cryo-EM structures of SARS-CoV-2 3a to 2.1 [A] resolution and demonstrate 3a forms an ion channel in reconstituted liposomes. The structures in lipid nanodiscs reveal 3a dimers and tetramers adopt a novel fold with a large polar cavity that spans halfway across the membrane and is accessible to the cytosol and the surrounding bilayer through separate water- and lipid-filled openings. Electrophysiology and fluorescent ion imaging experiments show 3a forms Ca2+-permeable non-selective cation channels. We identify point mutations that alter ion permeability and discover polycationic inhibitors of 3a channel activity. We find 3a-like proteins in multiple Alphacoronavirus and Betacoronavirus lineages that infect bats and humans. These data show 3a forms a functional ion channel that may promote COVID-19 pathogenesis and suggest targeting 3a could broadly treat coronavirus diseases.

biophysics↗

bioRxiv2020

Computational analysis of ligand dose range thermal proteome profiles

Detecting ligand-protein interactions in living cells is a fundamental challenge in molecular biology and drug research. Proteome-wide profiling of thermal stability as a function of ligand concentration promises to tackle this challenge. We present a statistical analysis method with reliable control of the false discovery rate and apply it to several datasets probing epigenetic drugs. We detect off-target drug engagement in unrelated protein families.

bioinformatics↗

bioRxiv2018

Proteome-wide solubility and thermal stability profiling reveals distinct regulatory roles for ATP

Nucleotide triphosphates (NTPs) regulate numerous biochemical processes in cells as (co-)substrates, allosteric modulators, biosynthetic precursors, and signaling molecules1-3. Apart from its roles as energy source fueling cellular biochemistry, adenosine triphosphate (ATP), the most abundant NTP in cells, has been reported to affect macromolecular assemblies, such as protein complexes4,5 and membrane-less organelles6-8. Moreover, both ATP and guanosine triphosphate (GTP) have recently been shown to dissolve protein aggregates9. However, system-wide studies to characterize NTP-interactions under conditions approximating the native cellular environment are lacking, which limits our perspective of the diverse physiological roles of NTPs. Here, we have mapped and quantified proteome-wide NTP-interactions by assessing thermal stability and solubility of proteins using mechanically disrupted cells. Our results reveal diverse biological roles of ATP depending on its concentration. We found that ATP specifically interacts with proteins that utilize it as substrate or allosteric modulator at doses lower than 500 M, while it affects protein-protein interactions of protein complexes at mildly higher concentrations (between 1-2 mM). At high concentrations (> 2 mM), ATP modulates the solubility state of a quarter of the insoluble proteome, consisting of positively charged, intrinsically disordered, nucleic acid binding proteins, which are part of membrane-less organelles. The extent of solubilization depends on the localization of proteins to different membrane-less organelles. Furthermore, we uncover that ATP regulates protein-DNA interactions of the Barrier to autointegration factor (BANF1). Our data provides the first quantitative proteome-wide map of ATP affecting protein structure and protein complex stability and solubility, providing unique clues on its role in protein phase transitions.

systems biology↗

bioRxiv2018

Complementary subnetworks of cortical somatostatin interneurons

The neocortex is organized into discrete layers of excitatory neurons: layer 4 receives the densest bottom up projection carrying external sensory data, while layers 2/3 and 5 receive top down inputs from higher cortical areas that may convey sensory expectations and behavioral goals. A subset of cortical somatostatin (SST) neurons gate top down input and control sensory computation by inhibiting the apical dendrites of pyramidal cells in layers 2/3 and 5. However, it is unknown whether an analogous inhibitory mechanism separately and specifically controls activity in layer 4. We hypothesized that distinct SST circuits might exist to inhibit specific cortical layers. By enforcing layer-specific inhibition, distinct SST subnetworks could mediate pathway-specific gain control, such as regulating the balance between bottom up and top down input. Employing a combination of high precision circuit mapping, in vivo optogenetic perturbations, and single cell transcriptional profiling, we reveal distinct and complementary SST circuits that specifically and reciprocally interconnect with excitatory cells in either layer 4 or layers 2/3 and 5. Our data further define a transcriptionally distinct SST neuronal sub-class that powerfully gates bottom up sensory activity during active sensation by regulating layer 4 activity. This integrated paradigm further represents a potentially generalizable approach to identify and characterize neuronal cell types and reveal their in vivo function.

neuroscience↗

bioRxiv2017

Simultaneous cell traction and growth measurements using light

Understanding cell mechanotransduction is important for discerning matrix structure-cell function relationships underlying health and disease. Despite the crucial role of mechanochemical signaling in phenomena such as cell migration, proliferation, and differentiation, measuring the cell-generated forces at the interface with the extracellular matrix during these biological processes remains challenging. An ideal method would provide continuous, non-destructive images of the force field applied by cells, over broad spatial and temporal scales, while simultaneously revealing the cell biological process under investigation. Toward this goal, we present the integration of a new real-time traction stress imaging modality, Hilbert phase dynamometry (HPD), with the technique of spatial light interference microscopy (SLIM) for label free monitoring of cell growth. HPD relies on extracting the displacement field in a deformable substrate, which is chemically patterned with a fluorescent grid. The displacements introduced by the cell are captured by the phase of the periodic signal associated with the grid, borrowing concepts from holography. The displacement field is uniquely converted into forces by solving an elasticity inverse problem. Because the measurement of displacement only uses the epi-fluorescence channel of an inverted microscope, we can simultaneously achieve measurements in transmission. We performed SLIM and extracted cell mass on the same field of view in addition to the measured displacement field. We used this technique to study mesenchymal stem cells and found that cells undergoing osteogenesis and adipogenesis exerted larger and more dynamic stresses than their precursor. Our results indicate that the MSCs develop the smallest forces and growth rates. We anticipate that simultaneous cell growth and traction measurements will improve our understanding of mechanotransduction, particularly during dynamic processes where the matrix properties provide context to guide cells towards a physiological or pathological outcome, e.g., tissue morphogenesis, or cancer metastasis.

cell biology↗