Biology subjects

Dasgupta, P.

Publications and source records attributed to Dasgupta, P..

At least 19 recordsLinked to original sources

bioRxiv2026

A simplified intermittent fasting regimen robustly extends C. elegans lifespan without FUdR or antibiotic confounds

Fasting-based dietary interventions are conserved regulators of aging that extend lifespan across species, including Caenorhabditis elegans. However, fasting studies in C. elegans are sensitive to experimental variables that can independently influence lifespan and health, including FUdR, antibiotic treatment, germline-less mutants, and the use of UV- or heat-killed bacteria. FUdR can alter lifespan, age-associated pathology, and stress responses, while antibiotics used to prevent bacterial growth during fasting may directly affect worm physiology. To minimize these confounding factors, we developed a simple adult-onset intermittent fasting paradigm that does not require FUdR, antibiotics, or bacterial killing. Wild-type worms were subjected to daily fasting periods of 5 h, 6 h, or 18 h until day 10 of adulthood and compared with continuously fed controls. Daily intermittent fasting robustly extended lifespan by 24-57%, demonstrating that repeated fasting windows during adulthood are sufficient to promote longevity under minimally confounded conditions. These findings establish a straightforward and experimentally tractable intermittent fasting paradigm for C. elegans and underscore the importance of limiting pharmacological and microbial conditions in dietary-intervention experiments.

physiology↗

bioRxiv2026

An Integrated Proteomics and Genomics Approach to Identify Essential Protein Kinases During Human Trophoblast Development

In the developing human placenta, three subtypes of trophoblast cells, cytotrophoblasts (CTBs), extravillous trophoblasts (EVTs), and syncytiotrophoblasts (STBs), mediate critical functions essential for a successful pregnancy. CTBs constitute the stem/progenitor compartment and differentiate into STBs and EVTs within the floating and anchoring villi, respectively. STBs establish the maternal-fetal exchange interface and secrets human chorionic gonadotropin (hCG), a hormone vital for the maintenance of early pregnancy. EVTs anchor the maternal endometrium and invade the uterine tissue to remodel maternal cells, supporting implantation and progression of pregnancy. In this study, we used human trophoblast stem cells (hTSCs) as a model system and performed quantitative, label-free liquid chromatography-tandem Mass Spectrometry (LC-MS/MS) to profile proteome and phosphoproteome in TSC stem state (analogous to undifferentiated CTBs) and following their differentiation to STBs and EVTs. Through a multiomics approach, we integrated our proteomics data with global gene expression profiles to correlate cell-type specific gene and protein expression during human trophoblast development. We also identified global phosphoproteome and analyzed kinases that are specifically active in hTSC stem state, as well as in differentiated STBs and EVTs. We experimentally validated specific kinases, such as BUB1B, PAK6, PKYMT1 and TNIK are essential for maintaining the hTSC stem-state. Additionally, atypical protein kinase C isoforms PKC{zeta} is essential for STB development, while PTK2B, SRC, TRIO and LYN are important for EVT development. Our findings highlight key kinases uniquely required for specific stages of trophoblast development during human placentation and suggest that pharmacological inhibition of these kinases could negatively impact the placentation process during pregnancy.

developmental biology↗

bioRxiv2026

Prioritizing peptides for targeted mass spectrometry experiments using deep learning

One critical step in any targeted mass spectrometry experiment is selecting, from each protein of interest, a small number of peptides that respond well in the mass spectrometer and can serve as reliable proxies for protein quantification. Existing methods select target peptides either by relying on prior empirical measurements, limiting their applicability to previously observed peptides, or using machine learning to predict peptide behavior from sequence alone. However, current machine learning tools suffer from various limitations, including using detectability as an indirect proxy for intensity, relying on small training sets, or ignoring the precursor charge state. In this study, we introduce Bromo, a transformer-based deep learning model that ranks peptide precursors from a given protein by their relative response, taking charge state into account. Trained on millions of annotated peptide pairs derived from large-scale, publicly available data-independent acquisition mass spectrometry data, Bromo consistently outperforms existing sequence-based methods across diverse, independent datasets. Furthermore, we show that fine-tuning Bromo on experiment-specific data can account for differences in sample preparation, sample matrix, and instrument platform, all of which influence which peptides serve as optimal targets. This adaptability makes Bromo a practical tool for selecting target peptides for selected reaction monitoring and parallel reaction monitoring assay development across a wide range of experimental conditions.

bioinformatics↗

bioRxiv2026

Enhanced photosynthetic efficiency and ROS modulation promote cold stress tolerance of indica rice

Indica rice, being a tropical crop, is highly sensitive to cold temperature. Cold stress affects vegetative growth, photosynthetic efficiency, along with reproductive features. Genetic resource screening in diverse landraces is an approach for identifying cold-tolerant traits. Here, we have characterised a boro germplasm, CB1, with an efficient germination rate and growth vigour when treated at chilling temperatures. CB1 seedlings show a higher survival rate compared to IR36 when subjected to prolonged chilling stress. Biochemical analyses indicated efficient ROS modulation, higher chlorophyll content, enhanced photosystem II efficiency and unique stomatal traits, leading to higher relative water content in CB1 plants during stress and recovery. Transcriptome analysis supported upregulation of chlorophyll biosynthesis, photosystem, & light harvesting complex and ROS scavenger genes in CB1 seedlings. Interestingly, high D1 protein turnover in CB1 promotes damage-repair of PSII for efficient photosynthesis. Furthermore, key transcription factors for stomatal development and expression of photosynthetic genes were upregulated in CB1 during stress recovery. Notably, higher expression of OsGLK1 and enrichment of GLK1 targets were observed in CB1 plants during chilling stress and recovery. Taken together, our results suggested that CB1 plants exhibit cold tolerance by modulating photosynthesis efficiency and stomatal behavior for better adaptability and survival against chilling temperature. HIGHLIGHTSThe efficient photosynthetic recovery, active ROS scavenging system and maintenance of water content through regulating stomatal traits, enhance the survival of indica germplasm CB1 against chilling stress.

plant biology↗

bioRxiv2026

Probing the role of residues lining the active site in the generation of glucose-tolerant variants of a fungal GH1 enzyme

The hydrolytic breakdown of cellobiose into glucose, catalysed by {beta}-glucosidases, is the last and rate-limiting step in cellulose saccharification for producing fermentable glucose in the bioethanol industry. This limitation arises because {beta}-glucosidase activity is inhibited by factors such as temperature, pH, and glucose accumulation in reactors. Enzyme inactivation leads to the buildup of cello-oligosaccharides, which, in turn, inhibit upstream cellulases. Therefore, glucose-tolerant {beta}-glucosidases are preferred for the formulation of industrial cellulase cocktails. In this study, we have recombinantly expressed, purified, and biochemically characterised a {beta}-glucosidase from the cellulolytic fungus Fusarium odoratissimum (FoBgl-WT). FoBgl-WT exhibits optimal cellobiose hydrolysis over a broad pH range (4.5-7.5), an important and industrially desirable property for its application in bioreactors. However, the glucose tolerance of FoBgl-WT was [~]0.56 M. Structure-based analyses were carried out to map the residues lining the active site of FoBgl, and their roles in stabilising the product glucose (or even the substrate, cellobiose) were elucidated through a series of site-specific mutations, followed by biochemical characterisation of the resulting FoBgl mutants. Among all the mutants generated, FoBgl-K256I-Y325F exhibits >2.5-fold greater glucose tolerance ([~]1.4 M) than FoBgl-WT. Further, we have observed that the FoBgl-K256W and FoBgl-K256I mutants exhibit improved kinetic properties, such as catalytic efficiencies. The structure-based rational engineering efforts improve glucose tolerance and the kinetic properties of FoBgl mutants, making it a useful and promising candidate enzyme for industrial cellulase cocktails.

biochemistry↗

bioRxiv2026

FAF1 and FAF2 enhance human p97-UFD1-NPL4 complex unfoldase activity enabling rational design of p97 activators

VCP/p97 is an AAA+ ATPase that, together with its cofactors UFD1-NPL4 (p97-UN), binds and unfolds ubiquitylated substrates to maintain cellular homeostasis. The human p97-UN complex associates with additional cofactors, but how these cofactors modulate p97-UN activity is not fully understood. Here, we screen for cofactors that enhance p97-UN activity and identify FAF2 as the strongest activator. Using biochemical and structural approaches, we show how FAF2 engages p97-UN and polyubiquitin to promote unfolding. We define a conserved activation motif in FAF2 that contacts both UFD1 and the ubiquitin proximal to the initiator, stabilizing and initiating unfolding in a UFD1-dependent manner. We leverage the features of FAF2 AM to engineer de novo proteins that potently enhance unfolding, providing a rational strategy to boost p97 activity. Our findings reveal how cofactors can provide additional adaptive control, fine-tuning human p97 activity to unfold challenging substrates and those modified with short ubiquitin chains.

biochemistry↗

bioRxiv2025

Arginine methyltransferase PRMT1 equipoises trophoblast development to prevent early pregnancy loss

Abstract1-2% of all human pregnancies suffer from idiopathic recurrent pregnancy loss (RPL) and underlying molecular causes are poorly understood. Here we show that defective Protein Arginine Methyltransferase 1 (PRMT1) function in trophoblast progenitors is a molecular cause for early pregnancy failure. PRMT1 is conserved in trophoblast progenitors and conditional deletion of PRMT1 in mouse trophoblast progenitors arrests placenta and embryonic development leading to lethality [~]E7.5. Remarkably, a subset of idiopathic RPL is associated with loss of PRMT1 in cytotrophoblast progenitors (CTBs). Experiments with human trophoblast stem cells (hTSCs), derived from these RPL-patients as well as PRMT1-depleted hTSCs revealed that PRMT1 is crucial for trophoblast progenitors self-renewal. Employing RNA-seq and CUT&RUN-sequencing in hTSCs, CTBs and primary mouse trophoblast progenitors we discover that PRMT1 promotes transcription of trophoblast stem-state regulators, like TEAD4 and MYBL2, by directly enriching histone H4 arginine 3 asymmetric di-methylation (H4R3Me2a) at their chromatin loci. PRMT1 is also essential for extravillous trophoblast (EVT) development during human placentation, while loss of PRMT1 in hTSCs spontaneously promotes syncytiotrophoblast (STB) differentiation. Our findings indicate that PRMT1 is an epigenetic governor that orchestrates mammalian trophoblast development and implicate the therapeutic potential of targeting the PRMT1-H4R3Me2a axis to mitigate early pregnancy loss.

developmental biology↗

bioRxiv2024

Hypoxia and loss of GCM1 expression prevents differentiation and contact inhibition in human trophoblast stem cells

The placenta develops alongside the embryo and nurtures fetal development to term. During the first stages of embryonic development, due to low blood circulation, the blood and ambient oxygen supply is very low ([~]1-2% O2) and gradually increases upon placental invasion. While a hypoxic environment is associated with stem cell self-renewal and proliferation, persistent hypoxia may have severe effects on differentiating cells and could be the underlying cause of placental disorders. We find that human trophoblast stem cells (hTSC) thrive in low oxygen, whereas differentiation of hTSC to trophoblast to syncytiotrophoblast (STB) and extravillous trophoblast (EVT) is negatively affected by hypoxic conditions. The pro-differentiation factor GCM1 (human Glial Cell Missing-1) is downregulated in low oxygen, and concordantly there is substantial reduction of GCM1-regulated genes in hypoxic conditions. Knockout of GCM1 in hTSC caused impaired EVT and STB formation and function, reduced expression of differentiation-responsive genes, and resulted in maintenance of self-renewal genes. Treatment with a PI3K inhibitor reported to reduce GCM1 protein levels likewise counteracts spontaneous or directed differentiation. Additionally, chromatin immunoprecipitation of GCM1 showed enrichment of GCM1-specific binding near key transcription factors upregulated upon differentiation including the contact inhibition factor CDKN1C. Loss of GCM1 resulted in downregulation of CDKN1C and corresponding loss of contact inhibition, implicating GCM1 in regulation of this critical process.

cell biology↗

bioRxiv2024

Adaptation to yeast restriction through the evolution of resource acquisition, macronutrient reserve, and ovarian function

Optimization of reproduction under dietary adversity is an important aspect of diet-dependent adaptation. Yet, little is known about the mechanism of such adaptive evolution. Here, we investigated a set of experimentally evolved populations of Drosophila melanogaster where early-life fecundity evolved as an adaptation to chronic protein restriction. We tested the role of resource acquisition and macronutrient storage, and changes in ovarian function that could have allowed such reproductive adaptation. We show that higher early-life fecundity was associated with the increased larval feeding rate, aiding in accumulation of higher protein content at the time of eclosion. Further evidence also suggests increase in reproductively allocated lipid content. Evolved females were found to have larger ovaries that also had a higher number of mature, post-vitellogenic oocytes that seem to readily allow the attainment of higher fecundity. Our results further show the evolution of plasticity in ovariole count (depending on mating status) and total egg-chamber count in females. These results are important in understanding the putative role of genetic variance and covariances that aid or limit the evolution of reproductive optimization, especially under nutritional adversity.

evolutionary biology↗

bioRxiv2024

A Single Trophoblast Layer Acts as the Gatekeeper at the Endothelial-Hematopoietic Crossroad in the Placenta

During embryonic development the placental vasculature acts as a major hematopoietic niche, where endothelial to hematopoietic transition ensures emergence of hematopoietic stem cells (HSCs). However, the molecular mechanisms that regulate the placental hematoendothelial niche are poorly understood. Using a parietal trophoblast giant cell (TGC)-specific knockout mouse model and single-cell RNA-sequencing, we show that the paracrine factors secreted by the TGCs are critical in the development of this niche. Disruptions in the TGC-specific paracrine signaling leads to the loss of HSC population and the concomitant expansion of a KDR+/DLL4+/PROM1+ hematoendothelial cell-population in the placenta. Combining single- cell transcriptomics and receptor-ligand pair analyses, we also define the parietal TGC- dependent paracrine signaling network and identify Integrin signaling as a fundamental regulator of this process. Our study elucidates novel mechanisms by which non-autonomous signaling from the primary parietal TGCs maintain the delicate placental hematopoietic- angiogenic balance and ensures embryonic and extraembryonic development.

developmental biology↗

bioRxiv2024

METTL3 shapes m6A epitranscriptomic landscape for successful human placentation

Methyltransferase-like 3 (METTL3), the catalytic enzyme of methyltransferase complex for m6A methylation of RNA, is essential for mammalian development. However, the importance of METTL3 in human placentation remains largely unexplored. Here, we show that a fine balance of METTL3 function in trophoblast cells is essential for successful human placentation. Both loss-of and gain-in METTL3 functions are associated with adverse human pregnancies. A subset of recurrent pregnancy losses and preterm pregnancies are often associated with loss of METTL3 expression in trophoblast progenitors. In contrast, METTL3 is induced in pregnancies associated with fetal growth restriction (FGR). Our loss of function analyses showed that METTL3 is essential for the maintenance of human TSC self-renewal and their differentiation to extravillous trophoblast cells (EVTs). In contrast, loss of METTL3 in human TSCs promotes syncytiotrophoblast (STB) development. Global analyses of RNA m6A modification and METTL3-RNA interaction in human TSCs showed that METTL3 regulates m6A modifications on the mRNA molecules of critical trophoblast regulators, including GATA2, GATA3, TEAD1, TEAD4, WWTR1, YAP1, TFAP2C and ASCL2, and loss of METTL3 leads to depletion of mRNA molecules of these critical regulators. Importantly, conditional deletion of Mettl3 in trophoblast progenitors of an early post-implantation mouse embryo also leads to arrested self-renewal. Hence, our findings indicate that METLL3 is a conserved epitranscriptomic governor in trophoblast progenitors and ensures successful placentation by regulating their self-renewal and dictating their differentiation fate.

developmental biology↗

bioRxiv2023

GATA-regulated transcriptional program dictate cell fate equilibrium to establish the maternal-fetal exchange interface and fetal development

The placenta establishes a maternal-fetal exchange interface to transport nutrients and gases between the mother and the fetus. Establishment of this exchange interface relies on the development of multinucleated syncytiotrophoblasts (SynT) from trophoblast progenitors and defect in SynT development often leads to pregnancy failure and impaired embryonic development. Here, we show that mouse embryos with conditional deletion of GATA2 and GATA3 in labyrinth trophoblast progenitors have underdeveloped placenta and die [~] embryonic day 9.5 (E9.5). Single cell RNA Seq (scRNA-Seq) analysis revealed excessive accumulation of multipotent labyrinth trophoblast progenitors upon conditional deletion of GATA factors. The GATA factor-deleted multipotent progenitors were unable to differentiate to matured SynTs. We also show that the GATA factor-mediated priming of trophoblast progenitors for SynT differentiation is a conserved event during human placentation. Loss of either GATA2 or GATA3 in cytotrophoblast (CTB)-derived human trophoblast stem cells (human TSCs) drastically inhibits SynT differentiation potential. Identification of GATA2 and GATA3 target genes along with comparative bioinformatics analyses revealed that GATA factors directly regulate hundreds of common genes in human TSCs, including genes that are essential for SynT development and implicated in preeclampsia and fetal growth retardation. Thus, our study uncovers a conserved molecular mechanism, in which coordinated function of GATA2 and GATA3 promote trophoblast progenitor-to-SynT commitment ensuring establishment of the maternal-fetal exchange interface.

developmental biology↗

bioRxiv2023

Evaluation of physiological responses in selected tissues of a freshwater fish (Cyprinus carpio var koi) maintained in hard waters: A report on glucose, oxidative stress and antioxidant biomarkers

Fishes endemic to freshwater habitat are strongly influenced by water hardness with physiological consequences. The present study aimed to understand the effects of four-fold sequential increase from soft to hard waters, on selected tissues of Koi carp, a popular ornamental freshwater fish. Secondary stress markers - Glucose, Oxidative stress (Malondialdehyde - Lipid Peroxidation damage) and Antioxidants (Catalase, Glutathione-S-Transferase and Glutathione) were quantified in gills and white muscle after 14 days of exposure to hardness of 75 (Soft - TS), 150 (Moderate - TM), 225 (Hard - TH) to 300 (Very Hard - TV) mg CaCO3/L. Both the examined tissues were distinctly affected by soft and moderate waters. Glucose in gills (p < 0.05) was proportional to the rise in hardness concentration. Soft, moderate and very hard waters (75, 150 and 300 mg CaCO3/L) affected gills and muscle due to elevated MDA concentrations (p < 0.05). CAT and GST provided antioxidative protection to the tissues. The study results showed tissue-specific differential responses and more importantly, concentrations below 225 mg CaCO3/L elicited strong oxidative impairment in both gill and muscle.

zoology↗

bioRxiv2022

A bead-based GPCR phosphorylation immunoassay for high-throughput ligand profiling and GRK inhibitor screening

Analysis of agonist-driven phosphorylation of G protein-coupled receptors (GPCRs) can provide valuable insights into the receptor activation state and ligand pharmacology. However, to date, assessment of GPCR phosphorylation using high-throughput applications has been challenging. We have developed and validated a bead-based immunoassay for the quantitative assessment of agonist-induced GPCR phosphorylation that can be performed entirely in multiwell cell culture plates. The assay involves immunoprecipitation of affinity-tagged receptors using magnetic beads followed by protein detection using phosphorylation-state-specific and phosphorylation state-independent anti-GPCR antibodies. As proof of concept, five prototypical GPCRs (MOP, C5a1, D1, SST2, CB2) were treated with different agonists and antagonists, and concentration-response curves were generated. We then extended our approach to establish selective cellular GPCR kinase (GRK) inhibitor assays, which led to the rapid identification of a selective GRK5/6 inhibitor (LDC8988) and a highly potent pan-GRK inhibitor (LDC9728). In conclusion, this versatile GPCR phosphorylation assay can be used extensively for ligand profiling and inhibitor screening.

pharmacology and toxicology↗

bioRxiv2022

Germline protein, Cup, non-cell autonomously limits migratory cell fate in Drosophila oogenesis.

Attaining migratory fate from a stationary cell population is complex and indispensable both for the multicellular organism development as well for the pathological condition like tumor metastasis. Though widely prevalent in the metazoans, the molecular understanding of this phenomenon remains elusive. Specification of migratory border cells from the follicular epithelium during Drosophila oogenesis has emerged as one of the excellent model systems to study how motile cell are specified. JAK-STAT activation in 6-10 anterior most follicle cells of the Drosophila egg chamber transforms them to a migratory cluster called the border cells. We show that a nurse cell protein, Cup, non-cell autonomously restricts the domain of JAK-STAT activation in the anterior follicle cells. Further examination suggests that Cup functions through Rab11GTPase to regulate Delta trafficking in the nurse cells potentiating Notch activation in the anterior follicle cells. Since Notch activity in the follicle cells modulates the JAK-STAT, any perturbation in Notch activation affects the border cell fate. Altogether, we propose that germline Cup affects the border cell fate through appropriate activation of Notch and JAK-STAT signaling in the follicle cells.

developmental biology↗

bioRxiv2021

Evolution of reduced mate-harming tendency of males in Drosophila melanogaster populations selected for faster life history

Detrimental effect of males on female, often termed mate harm, is a hallmark of sexual conflict. Allowed to evolve unchecked, mate harming traits are predicted to bring down average fitness of a population, unless mitigated by the evolution of resistance in females. In addition, life history may also modulate sexual conflict, but the mechanism is not clearly understood. Here we investigated the evolution of mate harm in a set of experimentally evolved laboratory populations of Drosophila melanogaster wherein a faster aging has evolved in response to >1000 generations of selection for faster development and early reproduction. We quantified mortality and fecundity of Oregon R females held with evolved (ACO) and ancestral males (CO) to show that the evolved males are significantly less detrimental to their mates. We compared our results from the ACO males with that from a phenocopied version of the ancestral regime (CCO) to show that only part of the observed difference in mate harm can be attributed to the evolved difference in body size. We further show that the reduction in mate harming ability evolved despite an increase in courtship activity, especially early in life. We discuss the causative role of an evolved reproductive schedule and altered breeding ecology. Significance statementSexually antagonistic male effects can significantly bring down female fitness. Along with female counter evolution of resistance traits, life history has been conjectured to impose constrains on the evolution of such harming ability in males. Here, we report the evolution of mate harming ability in males of a set of five replicate Drosophila melanogaster populations that evolved smaller size and faster aging as a result of >1000 generations of experimental evolution for faster development and early reproduction. We show that in spite of ample scope of sexual selection, the faster aging males have evolved reduced mate harming ability despite being more active in courting their mates. To the best of our knowledge, this is one of the first clear evidences demonstrating the causal relationship between evolution of life history and reduction in sexual antagonism in a population.

evolutionary biology↗

bioRxiv2021

Evolution of increased early-life fecundity but faster reproductive senescence in Drosophila melanogaster females through adaptation to chronic live-yeast deprivation

Reproductive output is often constrained by availability of macronutrients, especially protein. Long term protein restriction, therefore, is expected to select for traits that maximize reproductive output in spite of such nutritional challenge. We subjected four replicate populations of Drosophila melanogaster to a complete deprivation of live-yeast supplement, thereby mimicking a protein restricted ecology. As yeast supplement is a key source of protein, such an ecology is expected to strongly limit reproductive output, especially in females. Following 24 generations of experimental evolution, compared to their matched controls, females from experimentally evolved populations showed increase in reproductive output early in life, both in presence and absence of yeast supplement. The observed increase in reproductive output was not associated with any accommodating alteration in egg size; and development time, pre-adult survivorship, and body mass at eclosion of the progeny. There was no evidence for evolution of lifespan and lifelong cumulative reproductive output in females. However, females from experiment regime were found to have a significantly faster rate of reproductive senescence, as indicated by a faster rate of age related decline in reproductive output following the attainment of the reproductive peak. Therefore, adaptation to yeast deprivation ecology in our study involved a novel reproductive strategy whereby females attained higher reproductive output early in life followed by faster reproductive aging. To the best of our knowledge, this set of results is one of the most clear demonstration of optimization of fitness by fine tuning of reproductive schedule during adaptation to a prolonged nutritional deprivation. Supplementary informationA separate file that includes three figures and six tables.

evolutionary biology↗

bioRxiv2020

Medium-chain acyl-CoA dehydrogenase, a gatekeeper of mitochondrial function in glioblastoma multiforme

Glioblastoma (GBM) is among the deadliest of human cancers. Despite extensive efforts, it has proven to be highly resistant to chemo- and immune-based therapeutic strategies, and little headway has been made with targeted inhibitors. Like many cancers, metabolism is dysregulated in GBM. Thus, to identify new vulnerabilities and drug targets in GBM, we conducted genetic screens using pooled RNAi libraries targeting metabolic enzymes. We screened multiple glioma stem cell-derived (GSC) xenograft models, which revealed that several enzymes involved in the mitochondrial metabolism of fatty acids were required for tumor cell proliferation. From among these, we focused on medium-chain acyl-CoA dehydrogenase (MCAD), which oxidizes medium-chain fatty acids, due to its consistently high score across all of our screens, as well as its high expression level in multiple GSC models and its upregulation in GBM compared to normal brain. In this manuscript, we describe the dependence of GBM on sustained fatty acid metabolism to actively catabolize lipid species that would otherwise damage the mitochondrial structure. The uptake of mediumchain fatty acids lacks negative feedback regulation; therefore, in the absence of MCAD, medium-chain fatty acids accumulate to toxic levels, inducing reactive oxygen species (ROS), mitochondrial damage and failure, and apoptosis. Taken together, our findings uncover a previously unappreciated protective role exerted by MCAD in GBM cells, making it a unique and therapeutically exploitable vulnerability.

cancer biology↗