Biology subjects

Patel, S.

Publications and source records attributed to Patel, S..

At least 199 records · Page 11Linked to original sources

bioRxiv2020

A C. elegans genome-wide RNAi screen for altered levamisole sensitivity identifies genes required for muscle function

At the neuromuscular junction (NMJ), postsynaptic ionotropic acetylcholine receptors (AChRs) transduce a chemical signal released from a cholinergic motor neuron into an electrical signal to induce muscle contraction. To identify regulators of postsynaptic function, we conducted a genome-wide RNAi screen for genes required for proper response to levamisole, a pharmacological agonist of ionotropic L-AChRs at the Caenorhabditis elegans NMJ. A total of 117 gene knockdowns were found to cause levamisole hypersensitivity, while 18 resulted in levamisole resistance. Our screen identified conserved genes important for muscle function including some that are mutated in congenital myasthenic syndrome, congenital muscular dystrophy, congenital myopathy, myotonic dystrophy, and mitochondrial myopathy. Of the genes found in the screen, we further investigated those predicted to play a role in endocytosis of cell surface receptors. Loss of the Epsin homolog epn-1 had opposing effects on the levels of postsynaptic L-AChRs and GABAA receptors, resulting in increased and decreased abundance, respectively. This disrupts the balance of postsynaptic excitatory and inhibitory signaling, causing levamisole hypersensitivity. We also examined other genes that resulted in a levamisole hypersensitive phenotype when knocked down including gas-1, which functions in Complex I of the mitochondrial electron transport chain. Consistent with altered ATP synthesis impacting levamisole response, treatment of wild-type animals with levamisole resulted in L-AChR dependent depletion of ATP levels. These results suggest that the paralytic effects of levamisole ultimately lead to metabolic exhaustion.

genetics↗

bioRxiv2020

Effects of ploidy and salmonid alphavirus infection on the skin and gill microbiome of Atlantic salmon (Salmo salar)

The microbial communities that live in symbiosis with the mucosal surfaces of animals provide the host with defense strategies against pathogens. These microbial communities are largely shaped by the environment and the host genetics. Triploid Atlantic salmon (Salmo salar) are being considered for aquaculture as they are reproductively sterile and thus cannot contaminate the natural gene pool. It has not been previously investigated how the microbiome of triploid salmon compares to that of their diploid counterparts. In this study, we compare the steady-state skin and gill microbiome of both diploid and triploid salmon, and determine the effects of salmonid alphavirus 3 experimental infection on their microbial composition. Our results show limited differences in the skin-associated microbiome between triploid and diploid salmon, irrespective of infection. In the gills, we observed a high incidence of the bacterial pathogen Candidatus Branchiomonas, with higher abundance in diploid compared to triploid control fish. Diploid salmon infected with SAV3 showed greater histopathological signs of epitheliocystis compared to controls, a phenomenon not observed in triploid fish. Our results indicate that ploidy can affect the alpha diversity of the gills but not the skin-associated microbial community. Importantly, during a natural outbreak of Branchiomonas sp. the gill microbiome of diploid Atlantic salmon became significantly more dominated by this pathogen than in triploid animals. Thus, our results suggest that ploidy may play a role on Atlantic salmon gill health and provide insights into co-infection with SAV3 and C. Branchiomonas in Atlantic salmon.

microbiology↗

bioRxiv2020

Integrating long-range regulatory interactions to predict gene expression using graph convolutional neural networks

Long-range spatial interactions among genomic regions are critical for regulating gene expression, and their disruption has been associated with a host of diseases. However, when modeling the effects of regulatory factors, most deep learning models either neglect long-range interactions or fail to capture the inherent 3D structure of the underlying genomic organization. To address these limitations, we present GC-MERGE, a Graph Convolutional Model for Epigenetic Regulation of Gene Expression. Using a graph-based framework, the model incorporates important information about long-range interactions via a natural encoding of spatial interactions into the graph representation. It integrates measurements of both the spatial genomic organization and local regulatory factors, specifically histone modifications, to not only predict the expression of a given gene of interest but also quantify the importance of its regulatory factors. We apply GC-MERGE to datasets for three cell lines - GM12878 (lymphoblastoid), K562 (myelogenous leukemia), and HUVEC (human umbilical vein endothelial) - and demonstrate its state-of-the-art predictive performance. Crucially, we show that our model is interpretable in terms of the observed biological regulatory factors, high-lighting both the histone modifications and the interacting genomic regions contributing to a genes predicted expression. We provide model explanations for multiple exemplar genes and validate them with evidence from the literature. Our model presents a novel setup for predicting gene expression by integrating multimodal datasets in a graph convolutional framework. More importantly, it enables interpretation of the biological mechanisms driving the models predictions. Available at: https://github.com/rsinghlab/GC-MERGE.

bioinformatics↗

bioRxiv2020

Reconstructing the lineage histories and differentiation trajectories of individual cancer cells in JAK2-mutant myeloproliferative neoplasms

Some cancers originate from a single mutation event in a single cell. For example, blood cancers known as myeloproliferative neoplasms (MPN) are thought to originate through the acquisition of a driver mutation (most commonly JAK2-V617F) in a hematopoietic stem cell (HSC). However, when the mutation first occurs in individual patients and how it impacts the behavior of HSCs in their native context is not known. Here we quantified the impact of the JAK2-V617F mutation on the proliferation dynamics of HSCs and the differentiation trajectories of their progenies in individual MPN patients. We reconstructed the lineage history of individual HSCs obtained from MPN patients using the patterns of spontaneous somatic mutations accrued in their genomes over time. Strikingly, we found that the JAK2-V617F mutation occurred in a single HSC several decades before MPN diagnosis -- at age 9{+/-}2 years in a 34-year-old patient, and at age 19{+/-}3 years in a 63-year-old patient. For each patient, we inferred the number of mutated HSCs over time and computed their fitness. The population of JAK2-mutated HSCs grew exponentially by 63{+/-}15% and 44{+/-}13% every year in the two patients, respectively. To contrast the differentiation trajectories of the JAK2-mutated HSCs with those of healthy HSCs, we simultaneously measured the full transcriptome and somatic mutations in single hematopoietic stem and progenitor cells (HSPCs). We found that the fraction of JAK2-mutant HSPCs varied significantly across different myeloid cell types within the same patient. The erythroid progenitor cells were often entirely JAK2-mutant, even when the peripheral blood JAK2-V617F allele burden was low. The novel biological insights uncovered by this work have implications for the prevention and treatment of MPN, as well as the accurate assessment of disease burden in patients. The technology platforms and computational frameworks developed here are broadly applicable to other types of hematological malignancies and cancers.

cancer biology↗

bioRxiv2020

Mantis shrimp rank the shape of an object over its color during recognition

Mantis shrimp are predatory crustaceans that commonly occupy burrows in shallow, tropical waters worldwide. Most of these animals inhabit structurally complex, benthic environments with an abundance of visual features that are regularly observed, including conspecifics, predators, prey, and landmarks for use in navigation. While these animals are capable of learning and discriminating color and polarization, it is unknown what specific attributes of a visual object are important for its recognition. Here we show that mantis shrimp of the species Neogonodactylus oerstedii can learn the shape of a trained target. Furthermore, when the shape and color of a target which they had been trained to identify were placed in conflict, N. oerstedii significantly chose the target of the trained shape over the target of the trained color. Thus, we conclude that the shape of a target is more important than its color for its recognition by N. oerstedii. Our findings suggest that the shapes of learned structures, such as landmarks or other animals, are important for N. oerstedii during object recognition.

animal behavior and cognition↗

bioRxiv2020

Susceptibility to gingipains and transcriptomic response to P. gingivalis highlights the ribosome, hypothalamus, and cholinergic neurons

Porphyromonas gingivalis, a keystone species in the development of periodontal disease, is a suspected cause of Alzheimers disease. This bacterium is reliant on gingipain proteases, which cleave host proteins after arginine and lysine residues. To characterize gingipain susceptibility, we performed enrichment analyses of arginine and lysine proportion proteome-wide. Proteins in the SRP-dependent cotranslational protein targeting to membrane pathway were enriched for these residues and previously associated with periodontal and Alzheimers disease. These ribosomal genes are up-regulated in prefrontal cortex samples with detected P. gingivalis sequences. Other differentially expressed genes have been previously associated with dementia (ITM2B, MAPI, ZNF267, and DHX37). For an anatomical perspective, we characterized the expression of the P. gingivalis associated genes in the mouse and human brain. This analysis highlighted the hypothalamus, cholinergic neurons, and the basal forebrain. Our results suggest markers of neural P. gingivalis infection and link the gingipain and cholinergic hypotheses of Alzheimers disease.

genomics↗

bioRxiv2020

Nanotrap(R) particles improve detection of SARS-CoV-2 for pooled sample methods, extraction-free saliva methods, and extraction-free medium methods

Here we present a rapid and versatile method for capturing and concentrating SARS-CoV-2 from transport medium and saliva using affinity-capture magnetic hydrogel particles. We demonstrate that the method concentrates virus prior to RNA extraction, thus significantly improving detection of the virus using a real-time RT-PCR assay across a range of viral titers, from 100 to 1,000,000 viral copies/mL; in particular, detection of virus in low viral load samples is enhanced when using the method coupled with the IDT 2019-nCoV CDC EUA Kit. This method is compatible with commercially available nucleic acid extraction kits, as well with a simple heat and detergent method. Using transport medium diagnostic remnant samples that previously had been tested for SARS-CoV-2 using either the Abbott RealTime SARS-CoV-2 EUA Test (n=14) or the Cepheid Xpert Xpress SARS-CoV-2 EUA Test (n=35), we demonstrate that our method not only correctly identifies all positive samples (n = 17) but also significantly improves detection of the virus in low viral load samples. The average improvement in cycle threshold (Ct) value as measured with the IDT 2019-nCoV CDC EUA Kit was 3.1; n = 10. Finally, to demonstrate that the method could potentially be used to enable pooled testing, we spiked infectious virus or a confirmed positive diagnostic remnant sample into 5 mL and 10 mL of negative transport medium and observed significant improvement in the detection of the virus from those larger sample volumes.

microbiology↗

bioRxiv2020

Whole genome doubling confers unique genetic vulnerabilities on tumor cells

Whole genome doubling (WGD) occurs early in tumorigenesis and generates genetically unstable tetraploid cells that fuel tumor development. Cells that undergo WGD (WGD+) must adapt to accommodate their abnormal tetraploid state; however, the nature of these adaptations, and whether they confer vulnerabilities that can subsequently be exploited therapeutically, is unclear. Using sequencing data from [~]10,000 primary human cancer samples and essentiality data from [~]600 cancer cell lines, we show that WGD gives rise to common genetic traits that are accompanied by unique vulnerabilities. We reveal that WGD+ cells are more dependent on spindle assembly checkpoint signaling, DNA replication factors, and proteasome function than WGD- cells. We also identify KIF18A, which encodes for a mitotic kinesin, as being specifically required for the viability of WGD+ cells. While loss of KIF18A is largely dispensable for accurate chromosome segregation during mitosis in WGD- cells, its loss induces dramatic mitotic errors in WGD+ cells, ultimately impairing cell viability. Collectively, our results reveal new strategies to specifically target WGD+ cancer cells while sparing the normal, non-transformed WGD- cells that comprise human tissue.

cancer biology↗

bioRxiv2020

Preferential Pharmacological Inhibition of Nav1.6, but not Nav1.1, Abolishes Epileptiform Activity Induced by 4-AP in Mouse Cortical Slices

Brain isoforms of voltage-gated sodium channels (VGSCs) have distinct cellular and subcellular expression patterns as well as functional roles that are critical for normal physiology as aberrations in their expression or activity lead to pathophysiological conditions. In this study, we asked how inhibition of select isoforms of VGSCs alters epileptiform activity to further parse out the roles of VGSCs in the brain. We first determined the relative selectivity of recently discovered small molecule, aryl sulfonamide, inhibitors (ICA-121431 and Compound 801) against Nav1.1, Nav1.2, and Nav1.6 activity using whole-cell patch clamp recordings obtained from HEK293 cells. To test the effects of these inhibitors on epileptiform activity, we obtained multielectrode array (MEA) recordings from mouse cortical slices in the presence of 4-aminopyridine (4-AP) to induce epileptiform activity. We found that the ICA-121431 and Compound 801 compounds are relatively selective for Nav1.1 and Nav1.6, respectively. From the MEA recordings, we found that inhibition of Nav1.6 and Nav1.2 with 500nM of the Compound 801 compound completely abolishes ictal local field potentials induced by 4-AP, whereas inhibition of Nav1.1 with 500nM of the ICA-121431 compound had minimal effect on epileptiform activity induced by 4-AP. Due to the prominent expression of Nav1.1 in inhibitory neurons, we asked whether inhibition of Nav1.1 alone alters activity. We found that, indeed, inhibition of Nav1.1 with the ICA-121431 compound increased basal activity in the absence of 4-AP. These findings expand our current understanding of the roles of VGSC isoforms in the brain and suggest that selective targeting of Nav1.6 may be a more efficacious treatment strategy for epileptic syndromes.

neuroscience↗

bioRxiv2020

Epithelial-mesenchymal transition sensitizes breast cancer cells to cell death via the fungus-derived sesterterpenoid ophiobolin A

The epithelial-mesenchymal transition (EMT) imparts properties of cancer stem-like cells, including resistance to frequently used chemotherapy, necessitating the identification of molecules that induce cell death specifically in stem-like cells with EMT properties. Herein, we demonstrate that breast cancer cells enriched for EMT features are more sensitive to cytotoxicity induced by ophiobolin A (OpA), a sesterterpenoid natural product. Using a model of experimentally induced EMT in human mammary epithelial (HMLE) cells, we show that EMT is both necessary and sufficient for OpA sensitivity. Moreover, prolonged, sub-cytotoxic exposure to OpA is sufficient to reduce migration, sphere formation, and resistance to doxorubicin. OpA is well-tolerated in mice and treatment with OpA alone reduces tumor burden. These data identify a driver of EMT-driven cytotoxicity with significant potential for use either in combination with standard chemotherapy or for tumors enriched for EMT features.

cancer biology↗

bioRxiv2020

Disseminating cells in human tumours acquire an EMT stem cell state that is predictive of metastasis

Cancer stem cells (CSCs) undergo epithelial-mesenchymal transition (EMT) to drive metastatic dissemination in experimental cancer models. However, tumour cells undergoing EMT have not been observed disseminating into the tissue surrounding human tumour specimens, leaving the relevance to human cancer uncertain. We have previously identified both EpCAM and CD24 as markers of EMT CSCs with enhanced plasticity. This afforded the opportunity to investigate whether retention of EpCAM and CD24 alongside upregulation of the EMT marker Vimentin can identify disseminating EMT CSCs in human tumours. Examining disseminating tumour cells in over 12,000 imaging fields from 84 human oral cancer specimens, we see a significant enrichment of single EpCAM, CD24 and Vimentin co-stained cells disseminating beyond the tumour body in metastatic specimens. Through training an artificial neural network, these predict metastasis with high accuracy (cross-validated accuracy of 87-89%). In this study, we have observed single disseminating EMT CSCs in human oral cancer specimens, and these are highly predictive of metastatic disease.

cancer biology↗

bioRxiv2020

CReSCENT: CanceR Single Cell ExpressioN Toolkit

CReSCENTCanceR Single Cell ExpressioN Toolkit (https://crescent.cloud), is an intuitive and scalable web portal incorporating a containerized pipeline execution engine for standardized analysis of single-cell RNA sequencing (scRNA-seq) data. While scRNA-seq data for tumour specimens are readily generated, subsequent analysis requires high-performance computing infrastructure and user expertise to build analysis pipelines and tailor interpretation for cancer biology. CReSCENT uses public data sets and preconfigured pipelines that are accessible to computational biology non-experts and are user-editable to allow optimization, comparison, and reanalysis for specific experiments. Users can also upload their own scRNA-seq data for analysis and results can be kept private or shared with other users.

bioinformatics↗

bioRxiv2020

An Inherent Structural Difference Between Serine and Threonine Phosphorylation: Phosphothreonine Prefers an Ordered, Compact, Cyclic Conformation

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted{phi} ({phi} [~] -60{degrees}) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n[->]{pi}* interaction between consecutive carbonyls, and an n[->]{sigma}* interaction between the phosphate O{gamma} lone pair and the antibonding orbital of C-H{beta} that restricts the{chi} 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic prolines backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are{phi} ,{psi} = polyproline helix or -helix ({phi} [~] -60{degrees});{chi} 1 = g-;{chi} 2 = eclipsed C-H/O-P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

biophysics↗

bioRxiv2020

Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

Occupational noise-induced hearing loss (NIHL) affects millions of people worldwide and presents a large social and personal burden. Some genetic variants in the mitochondrial oxidative stress response correlate strongly with susceptibility to NIHL in both humans and mice. Here we test the hypothesis that SIRT3, a regulator of the mitochondrial oxidative stress response, is required in mice for endogenous recovery of auditory thresholds after a sub-traumatic noise exposure. We expose homozygous Sirt3-KO mice and their wild-type littermates to a noise dose that confers a temporary threshold shift, but is not sufficient to permanently reduce cochlear function, compromise cell survival, or damage synaptic structures. We find no difference in hearing function after recovery from noise exposure between the two genotypes, when measured by either auditory brainstem response (ABR) or distortion product otoacoustic emissions (DPOAE). We show that SIRT3-specific immunoreactivity is present in outer hair cells, around the post-synaptic regions of inner hair cells, and faintly within inner hair cells. Nonetheless, outer hair cells and auditory synapses show no increase in loss after noise exposure in the homozygous Sirt3-KO mouse. These data show that SIRT3-dependent processes are not necessary for endogenous hearing recovery after a single, sub-traumatic noise exposure. They demonstrate the existence of cellular mechanisms of cochlear homeostasis in addition to the mitochondrial oxidative stress response. We also present a novel statistical analysis for identifying differences between peak 1 amplitude progressions in ABR waveforms.

neuroscience↗

bioRxiv2020

A novel cross talk of AtRAV1, an ethylene responsive transcription factor with MAP kinases imparts broad spectrum disease resistance in plants

Plant diseases pose a serious threat to sustainable agriculture as controlling them in eco-friendly manner remains a challenge. In this study, we establish RAV1 as a master transcriptional regulator of defense genes in model plant Arabidopsis. The overexpression of AtRAV1 provided disease resistance against necrotrophic fungal pathogen (Rhizoctonia solani) infection in A. thaliana. The transgenic lines exhibited enhanced expression of several defense genes including mitogen associated protein kinases (MAPKs) and the amplitude of their expression was further enhanced upon pathogen infection. Conversely, the atrav1 mutant plants were unable to induce the expression of these defense genes and were highly susceptible to infection. Our data suggests that upon pathogen attack, AtRAV1 transcriptionally upregulate the expression of MAPKs (AtMPK3, AtMPK4 and AtMPK6) and AtMPK3 and AtMPK6 are essential for AtRAV1 mediated disease resistance. Further, we demonstrate that AtRAV1 is a phosphorylation target of AtMPK3 (but not AtMPK6) and the phospho-defective variants of AtRAV1 are unable to induce disease resistance in A. thaliana. Considering the presence of AtRAV1 orthologs in diverse plant species, we propose that they can be gainfully deployed to control economically important diseases. In deed we observe that overexpression of tomato ortholog of AtRAV1 (SlRAV1) provides broad spectrum disease resistance against bacterial (Ralstonia solanacearum), fungal (R. solani) and viral (Tomato leaf curl virus) infections in tomato.

plant biology↗

bioRxiv2019

Transcriptomic analysis of brain hypometabolism links the cholinergic and gingipain hypotheses of Alzheimer's disease pathogenesis

Alzheimers disease (AD) starts decades before clinical symptoms appear. Low glucose utilization in regions of the cerebral cortex marks early AD and is clinically useful. To identify these regions, we conducted a voxel-wise meta-analysis of positron emission tomography studies that compared AD patients with healthy controls. This meta-analysis included 27 studies that assayed glucose utilization in 915 AD patients and 715 healthy controls. The resulting map marks hypometabolism in the posterior cingulate, middle frontal, angular gyrus, middle and inferior temporal regions. Using the Allen Human Brain Atlas, we identified genes with expression patterns associated with this hypometabolism pattern in the cerebral cortex. Of the six brains in the Atlas, one demonstrated a strong spatial association with the hypometabolism pattern. Previous neuropathological assessment of this brain from a 39-year-old male noted a neurofibrillary tangle in the entorhinal cortex. Using the transcriptomic data, we estimate lower proportions of neurons and more microglia in the hypometabolic regions when compared with the other five brains. Within this single brain, signal recognition particle (SRP)-dependent cotranslational protein targeting genes, which primarily encode cytosolic ribosome proteins, are highly expressed in the hypometabolic regions. Analyses of human and mouse data show that expression of these genes progressively increases across AD-associated states of microglial activation. In addition, genes involved in cell killing, chronic inflammation, ubiquitination, tRNA aminoacylation, and vacuole sorting are associated with the hypometabolism map. These genes suggest disruption of the protein life cycle and neuroimmune activation. Taken together, our molecular characterization of cortical hypometabolism reveals a molecular link to AD associated hypometabolism that may be relevant to preclinical stages.

bioinformatics↗

bioRxiv2019

Regulation of nerve growth and patterning by cell surface protein disulphide isomerase

Contact repulsion of growing axons is an essential mechanism for spinal nerve patterning. In birds and mammals the embryonic somites generate a linear series of impenetrable barriers, forcing axon growth cones to traverse one half of each somite as they extend towards their body targets. This study shows that protein disulphide isomerase provides a key component of these barriers, mediating contact repulsion at the cell surface in half-somites. Repulsion is reduced both in vivo and in vitro by a range of methods that inhibit enzyme activity. The activity is critical in initiating a nitric oxide/S-nitrosylation-dependent signal transduction pathway that regulates the growth cone cytoskeleton. Rat forebrain grey matter extracts contain a similar activity, and the enzyme is expressed at the surface of cultured human astrocytic cells and rat cortical astrocytes. We suggest this system is co-opted in the brain to counteract and regulate aberrant nerve terminal growth.

developmental biology↗

bioRxiv2019

A novel method to detect intracellular metabolite alterations in MCF-7 cells by doxorubicin induced cell death

Metabolic reprogramming within cancer cells is suggested as a potential barrier to chemotherapy. Additionally, metabolic tumor heterogeneity is one of factor behind discernible hallmarks such as drug resistance, relapse of tumor and the formation of secondary tumors. In this paper, cell based assays including PI/annexin V staining and immunoblot assay were performed to show the apoptotic cell death in MCF-7 cells treated with DOX. Further, MCF-7 cells were lysed in hypotonic buffer and whole cell lysate was purified by a novel and specifically designed metabolite (100 to 1000 Da) fractionation system as vertical tube gel electrophoresis (VTGE). Further, purified intracellular metabolites were subjected to identification by LC-HRMS technique. The authors show the presence of cleaved PARP 1 in MCF-7 cells treated with DOX. Concomitantly, data show the absence of active caspase 3 in MCF-7 cells. Novel findings are to identify key intracellular metabolites assisted by VTGE system that include lipid (CDP-DG, phytosphingosine, dodecanamide), non-lipid (N-acetyl-D-glucosamine, N1-acetylspermidine and gamma-L-glutamyl-L-cysteine) and tripeptide metabolites in MCF-7 cells treated by DOX. Interestingly, the authors report a first evidence of doxorubicinone, an aglycone form of DOX in MCF-7 cells that is potentially linked to the mechanism of cell death in MCF-7 cells. This paper reports on novel methods and processes that involve VTGE system based purification of hypotonically lysed novel intracellular metabolites of MCF-7 cells treated by DOX. Here, these identified intracellular metabolites corroborate to caspase 3 independent and mitochondria induced apoptotic cell death in MCF-7 cells.\n\nSIGNIFICANCE STATEMENTMetabolic reprogramming in cancer cells is implicated in various tumor hallmarks. Interestingly, thousands of research have addressed the molecular basis of drug treatment and resistance in chemotherapy. But, there is a significant gap in the precise methodologies and approaches in addressing intracellular metabolite alterations. This paper reports on a novel approach that helped reveal new findings on intracellular metabolite changes in case of doxorubicin (DOX) induced cell death in MCF-7 cells. This paper highlights the additional insights on debatable findings available in literature in the contexts of DOX induced cell death mechanisms. In this paper, novel and specifically designed vertical tube gel electrophoresis (VTGE) system is claimed to purify intracellular metabolites and this method is compatible with other biological system.

cancer biology↗