Biology subjects

Banerjee, S.

Publications and source records attributed to Banerjee, S..

At least 55 records · Page 3Linked to original sources

bioRxiv2026

Epidermal Growth Factor/c-Met receptor signalling crosstalk drives tunneling nanotube formation in A549 lung adenocarcinoma cells

Tunneling nanotubes (TNTs) are actin-based cytoplasmic connections that can mediate intercellular transfer of various cellular cargo and have been implicated in cancer progression and chemoresistance. However, the signalling mechanisms driving their formation remain poorly understood. Given the frequent dysregulation of EGFR and c-Met signalling in non-small cell lung cancer (NSCLC), and prior evidence of TNTs in lung adenocarcinoma patient samples, we investigated the role of EGFR and c-Met receptor signalling crosstalk in TNT induction in A549 lung adenocarcinoma cells. Stimulation with EGF, HGF, or in combination induced a concentration dependent increase in the formation of TNTs. TNTs exhibited typical characteristics, including F-actin expression, non-adherence to the substratum and facilitated intercellular trafficking of lysosomes, mitochondria, and lipid vesicles. EGFR was identified as a novel component of TNTs, but had little co-localisation with the c-Met receptor. Co-stimulation with HGF and EGF did not produce consistent additive or synergistic effects on TNT formation, suggesting shared downstream signalling. Furthermore, although EGFR and c-Met inhibition blocked EGF- and HGF-induced TNTs respectively, inhibition of both receptors was required to suppress TNTs following dual HGF/EGF treatment. Interestingly, blocking the EGF receptor alongside c-Met resulted in a more potent inhibition of HGF-induced TNTs, indicating crosstalk. Furthermore, inhibition of downstream MEK and PI3K pathways reduced HGF- or EGF- induced TNT formation, but dual inhibition was required to completely block TNT formation in HGF+EGF co-stimulated cells. These findings reveal a novel convergence of EGFR and c-Met and their downstream MAPK/PI3K pathways in TNT regulation, which can have important clinical implications in combinatorial receptor and cell signalling pathway targeting in NSCLC.

cancer biology↗

bioRxiv2026

Dual Lineage Tracing Identifies Cellular Mechanisms Underlying Radiation-Associated Changes in Atherosclerotic Lesion Composition

BackgroundPhenotypic plasticity of smooth muscle cells (SMCs) and endothelial cells (ECs) contributes to atherosclerotic plaque composition and stability, yet how shifts in one population influence the contribution and function of the other under conditions of vascular stress, such as irradiation, is poorly understood. A major limitation has been the inability to simultaneously fate-map both cell types within the same lesion, with most studies mapping one lineage while inferring the other using unreliable dynamically changing marker genes, risking false-positive and false-negative assignment. MethodsWe generated dual lineage-tracing Apoe-deficient mice, enabling simultaneous fate mapping of SMCs and ECs. This model was used to extend prior findings from single lineage-tracing models demonstrating irradiation-induced loss of SMC lesion investment and expansion of EC-derived cells. Dual lineage-tracing mice were subjected to irradiation and bone marrow transplantation, followed by Western diet feeding to induce atherosclerosis. Lineage tracing, immunostaining and scRNA-seq analysis were used to define coordinated SMC and EC responses and identify changes relevant to plaque instability. ResultsDual lineage tracing specifically and simultaneously labeled SMC- and EC-derived cells in healthy and atherosclerotic vessels. Irradiation induced divergent responses: SMC-derived cells failed to invest in lesions and upregulated stress-activated inflammatory genes, whereas EC-derived cells expanded and upregulated SMC-associated genes. However, EC-derived cells within lesions failed to induce extracellular matrix genes, and lesions from irradiated mice exhibited reduced collagen content and fewer ACTA2+ cells within the fibrous cap, consistent with reduced plaque stability. ConclusionsDual lineage-tracing of SMCs and ECs demonstrated that irradiation-induced loss of lesional SMC and expansion of EC-derived ACTA2+ cells are not artifacts of false lineage assignment. By resolving SMC and EC fate within the same lesion, we identify irradiation-induced cell dynamics including stress-activated inflammatory reprogramming of SMCs, EC phenotypic modulation, impaired extracellular matrix organization, and reduced ACTA2 fibrous cap cellularity that may contribute to radiotherapy-associated increased atherosclerotic cardiovascular disease risk. Clinical PerspectiveWhat Is New? O_LIWe developed a dual lineage-tracing mouse model that enables simultaneous fate mapping of smooth muscle cells and endothelial cells within the same atherosclerotic lesion. C_LIO_LIThis model reveals coordinated arterial cell wall responses to vascular injury that cannot be resolved using single lineage-tracing approaches. C_LIO_LIExtending prior observations, we show that irradiation-induced inflammatory reprogramming of smooth muscle cells and endothelial-to-mesenchymal transition of endothelial cells towards a smooth muscle cell-like state are associated with reduced total lesion collagen content and decreased overall ACTA2+ fibrous cap cellularity. C_LIO_LIThis dual lineage-tracing mouse establishes a broadly applicable model for investigating arterial wall cell dynamics across diverse vascular disease states. C_LI What Are the Clinical Implications? O_LICancer therapies involving radiotherapy are associated with increased long-term risk of atherosclerotic cardiovascular disease. C_LIO_LIOur findings identify a potential cellular mechanism underlying this risk, in which irradiation-induced smooth muscle cell loss is not functionally compensated by endothelial-to-mesenchymal transition toward a SMC-like state. C_LIO_LIThis dual lineage-tracing model provides a tool to evaluate how cancer therapies and other vascular stressors may alter arterial wall cell fate and indices of plaque stability in atherosclerosis and other vascular diseases. C_LI

physiology↗

bioRxiv2026

Prime editing of an evolutionarily informed PEPC residue enhances photosynthesis and grain nutrition in C₃ rice

C4 photosynthesis evolved through adaptive changes in key metabolic enzymes that enhance carbon fixation efficiency, yet transferring such traits into C3 crops has met limited success. Using precision prime editing, we introduced an evolutionarily conserved single amino acid substitution (Arg[->]Gly) into phosphoenolpyruvate carboxylase (PEPC) in rice. Edited plants exhibited enhanced PEPC activity in the presence of the feedback inhibitor malate, increased chlorophyll content, elevated photosynthetic rates under ambient CO2, and increased seed size and weight. Remarkably, seeds from the edited lines showed simultaneous enrichment of zinc (Zn), iron (Fe), and protein, hereafter referred to as ZiP rice. This work demonstrates that a single, evolutionarily guided residue change can reprogram core carbon metabolism in a C3 crop, enabling concurrent improvements in photosynthesis and grain nutritional quality.

plant biology↗

bioRxiv2026

Protocol for using an ELISA assay to detect total α-synuclein levels in Drosophila melanogaster lines expressing human α-synuclein point mutations

In Parkinsons disease (PD), aggregation of alpha-synuclein (-syn) contributes to neuronal dysfunction and death, particularly in dopaminergic neurons, driving disease progression. Several pathogenic point mutations have been identified in human -syn such as A30P, E46K, H50Q, G51D, A53T and A53E that have been associated with PD. In this study, a sandwich ELISA assay was developed to quantify total -syn levels in various Drosophila melanogaster genotypes expressing A30P, E46K, G51D and A53T. Using this approach, we compared -syn levels across wild-type (WT) and mutant forms of the protein. The E46K and A53T mutations exhibited higher total -syn concentrations compared to WT and the G51D mutation. In addition to characterizing mutationdependent differences in -syn levels, this assay was applied to evaluate smallmolecule modulators that inhibit -syn aggregation. These findings demonstrate that the ELISA-based approach provides a useful platform for quantifying -syn and assessing potential therapeutic compounds targeting syn pathology.

neuroscience↗

bioRxiv2026

Selective estradiol sensitivity in 12Z human endometriotic epithelial cell line

Estradiol (E2) is a potent estrogen molecule that plays a crucial role in regulating numerous healthy and pathophysiological processes. To model estrogen-dependent cellular activities in vitro in a physiologically meaningful way, cells chosen for experimentation should exhibit sensitivity to E2. The 12Z human endometriotic epithelial cell line is gaining attention as a model for estrogen-dependent conditions, but its functional responsiveness to E2 has not been well characterized. In the following study, we found that E2 does not influence 12Z proliferation, collective migration, or single-cell migration. However, 12Zs were selectively responsive to E2 in 3D migration models where the cells were physically confined. Upregulating ER, the primary mediator of estrogenic action, in the 12Zs did not enhance their functional E2 sensitivity. To delineate the underpinning of these behaviors, RNA sequencing was performed and revealed a differential expression of pseudogenes and non-coding RNAs in E2-treated 12Zs compared to vehicle control-treated 12Zs. Several signal transduction genes were significantly downregulated in ESR1-overexpressing 12Zs compared to normal 12Zs, which may play a role in their persistent lack of E2 sensitivity. These results prompt us to question whether the 12Z cell line, which mostly lacks functional E2 responsiveness, should be used in vitro as a model of estrogen-dependent processes and conditions.

cell biology↗

bioRxiv2026

Fibrotic substrate stiffness enhances endometriotic epithelial cell motility

Fibrosis is a common pathological feature of inflammatory conditions across various organ systems, leading to a marked increase in matrix stiffness. Although substrate stiffness is known to increase cell migration in cancer and stromal cell populations, it is not well understood how it affects benign epithelial cell motility, particularly within the pelvic cavity. We used an endometriotic epithelial cell line and polyacrylamide hydrogels with tunable stiffness--with glass as an extreme stiffness reference--to model mechanically driven single-cell and multicellular migration. We found that stiff substrates promoted cell speed, actin stress fiber formation, focal adhesion presentation, and spheroid expansion compared to the soft substrate. Increasing cellular contractility on the soft substrate and decreasing contractility on the stiff substrate led to an increase and decrease in cell speed, respectively. These findings provide mechanistic insight on how fibrosis as a biomechanical state regulates epithelial cell migration, with additional relevance to the pathogenesis of benign yet invasive conditions.

cell biology↗

bioRxiv2025

Seizures, increased interhemispheric synchrony, altered brain transcriptomics and a leaky blood-brain barrier result from loss of ap3b2 in a CRISPR tadpole model of DEE48

Loss-of-function variants in AP3B2, a neuronal adaptor protein required for synaptic vesicle formation, cause a severe early-onset neurodevelopmental epilepsy known as Developmental and Epileptic Encephalopathy 48 (DEE48). To investigate how AP3B2 loss alters brain development, leading to increased seizure susceptibility, we generated a Xenopus laevis model by targeting the orthologous gene using CRISPR/Cas9. Ap3b2.S-/- (mosaic) F0 tadpoles displayed increased locomotor activity with frequent seizure-like episodes when compared to sibling controls. Visualisation of forebrain and midbrain activity using the genetically encoded Ca2+ sensor GCaMP6s detected spontaneous, large amplitude, prolonged and widespread neural activity, alongside increased interhemispheric synchrony of both regions. Comparison of whole-brain transcriptomes from ap3b2 CRISPants and unedited sibling controls detected mainly downregulation of brain expressed genes, with significant over-representation of pathways involved in ion transport, axon formation and guidance, inhibitory (GABA) neurotransmission, and transport across the blood-brain barrier (BBB). In a simple assay for BBB integrity, CRISPant tadpoles were confirmed to have faster leakage of sodium fluoresceinAcute exposure to the angiotensin receptor blocker losartan significantly reduced locomotor hyperactivity, and CRISPant cohorts treated with losartan tended to have lower neural activity, indicating incomplete rescue of the ap3b2.S CRISPant phenotype. These findings demonstrate how AP3B2 loss of function alters brain development and the establishment of the BBB, with the resulting alterations in neurotransmitter pathways predisposing the brain to spontaneous seizures. Our results suggest that traditional anti-seizure medications designed to alter ion transport and GABA metabolism could be augmented with drugs targeting neuroinflammation, as adjunct seizure control options in infants with DEE48.

developmental biology↗

bioRxiv2025

Mitochondrial ROS-induced metabolic alterations differentially regulate ferroptosis sensitivity

Ferroptosis is an iron-catalyzed lipid peroxidation (LP)-dependent cell death. Induction of mitochondrial ROS (mtROS) is crucial in the execution of ferroptosis, but the underlying mechanism remains unclear. Through utilizing the hepatocyte model and RNA-seq analysis, we determined mtROS-dependent metabolic changes that modulate ferroptosis sensitivity. Elevated mtROS production and LP suppressed glycolysis, fatty acid oxidation, and citric acid cycle activity, representing adaptive responses that protect cells from ferroptosis. On the other hand, mtROS-driven signaling impaired glutathione biosynthesis and downregulated genes involved in coenzyme Q10 (CoQ) biosynthesis, including those in the mevalonate pathway and CoQ8A, a key stabilizer of the CoQ biosynthetic complex. Importantly, silencing CoQ8A expression enhanced, whereas overexpression of CoQ8A reduced, ferroptosis susceptibility of hepatocytes and various cancer cell types. The mtROS-mediated downregulation of CoQ8A was dependent on farnesoid X receptor (FXR) and retinoid X receptors (RXRs). Collectively, our findings highlight that mtROS promotes ferroptosis, at least in part, by suppressing glutathione and CoQ biosynthesis.

cell biology↗

bioRxiv2025

Learning Epithelial Elasticity via Local Tension Remodeling

Biological materials, like epithelial tissues, exhibit remarkable adaptability to mechanical stresses, dynamically remodeling their structure in response to external and internal forces. A key challenge is understanding how these tissues store a memory of past mechanical stimuli. Here, we investigate this memory using an active Vertex Model of epithelial sheets incorporating a local, mechanosensitive tension-remodeling rule where junctional tension updates depend on strain, acting as a slow, history-dependent variable. We demonstrate three hallmark mechanical consequences of this memory mechanism. First, a localized, short contractile cue permanently reprograms the global shear modulus, with the direction of change (stiffening or softening) controlled by the tension remodeling rate. Second, the tissue stores a long-range mechanical memory: a prior stimulus at one site modulates the tissues response to a subsequent, distant stimulus, mediated by coupling across the entire junctional network. Finally, we show that simple cyclic bulk deformation acts as a training protocol that autonomously tunes the tissues constitutive properties, including programming the Poisson ratio to auxetic (negative) values. These findings position epithelial mechanics within the framework of unsupervised physical learning, identifying the mechanosensitive remodeling rates as powerful control parameters for designing programmable tissue-scale rheology.

biophysics↗

bioRxiv2025

Potential probiotic application of a novel commensal E. coli with antagonistic activity against different enteric pathogens

Diarrheal diseases remain a leading cause of global morbidity and mortality, and treatment options are increasingly compromised by the global rise in multidrug-resistant (MDR) pathogens. Furthermore, the absence of effective vaccines against many causative agents renders large populations vulnerable. This critical gap necessitates the development of novel alternatives that can function both prophylactically to prevent infection and therapeutically to treat established diseases. Here, we investigated the commensal Escherichia coli strain HK220822 (HK5), which was isolated from a healthy human, as a potential live biotherapeutic. Comprehensive genomic and phenotypic characterization established a robust safety profile, confirming the absence of key virulence genes while demonstrating essential probiotic traits, including high tolerance to gastrointestinal stressors, such as acid and bile. The functional efficacy of Escherichia coli HK5 has been validated in murine models of infection. Prophylactic administration before pathogen exposure significantly inhibited intestinal colonization by Salmonella Typhimurium, Shigella flexneri 2a, and Vibrio cholerae (El Tor O1 strain). Moreover, HK5 demonstrated therapeutic potential by significantly reducing pathogen shedding when administered to previously infected animals. These findings establish commensal E. coli (HK5) as a potent candidate with dual preventative and therapeutic efficacy, offering a promising non-antibiotic strategy to combat diarrheal diseases.

microbiology↗

bioRxiv2025

Reinforcement learning for adaptive control of phenotypically heterogeneous bacterial populations

Bacterial populations display extraordinary resilience to antibiotic stress, driven by diverse physiological states that allow some cells to persist and later repopulate. This phenotypic heterogeneity, amplified by environmental fluctuations, undermines the effectiveness of conventional fixed-dose treatment regimens. To address this challenge, we introduce a reinforcement learning (RL) framework that discovers adaptive treatment strategies using only experimentally accessible, population-level measurements. The RL agent learns to infer the hidden physiological state of the population and leverages this knowledge to maintain control even under conditions not encountered during training. Moreover, when granted control over nutrient availability, an important driver of physiological change often overlooked in antibiotic treatment protocols, the agent consistently drives population extinction, surpassing adaptive protocols based solely on drug dynamics. This computational framework offers a powerful, data-driven approach for designing adaptive treatment strategies to counter the growing threat of antimicrobial resistance.

bioinformatics↗

bioRxiv2025

Design and fabrication of demountable 3D microphysiological systems for modeling barrier function and underlying tissue interactions

Several recent advances in microphysiological systems (MPSs) or organ-on-chip technology have demonstrated its potential for replacing traditional in vitro and animal models in the coming years. Despite the physiological relevance and cost-effectiveness of organ chips, there are several hurdles that must be overcome for widespread adoption for biological studies. Many shortcomings of manufacturing and scalability have been overcome by a transition from PDMS to thermoplastics. However, challenges have arisen in these sealed, brittle systems related to end-point tissue analyses, harvest, and high-resolution imaging, which is particularly difficult for multi-layer organ chips. Here, we present low-cost organ chips that are fluidically sealed but demountable, fabricated using a cut-and-assemble method without the need for cleanroom technologies. We have validated the capabilities of this method by demonstrating the culture of human aortic smooth muscle cells and induced pluripotent stem cell-derived neural cells, encapsulated in gelatin methacryloyl (GelMA) hydrogel on chip, for up to 27 days. The 3D culture layer of the organ chip was removed, and high-resolution images were obtained via immunostaining. Furthermore, these organ chips facilitate rapid redesign and manufacture for alternative tissue and/or interface systems. To our knowledge, this is the first innervated organ chip with multiple removable cell culture layers, as well as the first humanized nerve-artery model that includes a three-dimensional hydrogel culture. In future work, these unique features of our platform can be utilized for investigating the crosstalk mechanisms between different cell types in co-culture. Impact StatementWe present here a new method for fabricating low-cost demountable organ-on-a-chip platforms. This method leverages our recent cut & assemble method for layered 3D organ chips comprised of gas impermeable thermoplastics.

bioengineering↗

bioRxiv2025

The Liver is an Inflammatory Mediator of Pulmonary Arterial Hypertension

The livers contribution to pulmonary arterial hypertension (PAH) pathogenesis remains unclear. We hypothesized that the liver promotes inflammatory injury to the pulmonary endothelium. PAH patients without liver disease with pulmonary artery endothelial cell (PAEC) biopsies were included. Liver serologies and imaging were analyzed by unsupervised classification and regression tree (CART) to identify subclinical liver dysfunction clusters. Two machine-learning models predicted cluster assignment and informed differential expression. PAEC transcriptomes were compared to liver and lung data from monocrotaline and Sugen-Hypoxia rats. Liver fibrosis was assessed in rat and human PAH livers. Among 25 PAH patients (76% female, median age 61 [30 - 84] years), CART identified clusters distinguished by Model for End-Stage Liver Disease Sodium (MELD-Na) [≥]12, predicting higher pulmonary vascular resistance ({beta}=0.5 Wood units per point increase in MELD-Na, 95% CI 0.2-0.8, p=0.005) after adjustment for right atrial pressure. Subjects with MELD-Na [≥]12 had decreased 6-minute walk distance (353 [120 - 576] m vs. 411[300 - 600] m, p=0.03), with upregulation of apelin, beta-catenin, and immune signaling. Rat lung ECs demonstrated survival and hepatic growth-factor signaling, while rat livers showed immune activation. Rat (20.8 vs 16.6 % area stained, p=0.09) and human PAH livers revealed fibrosis despite absent right ventricular failure, supporting a pathogenic lung-liver axis in PAH.

cell biology↗

bioRxiv2025

Candidatus Phytoplasma-induced Retrogressive Morphogenesis in Sesame (Sesamum indicum L.): Tissue-Specific Metabolic and Transcriptomic Reprogramming

BackgroundCandidatus Phytoplasma infection is among the most destructive plant diseases, characterized by phyllody, witches broom, virescence etc. Sesame (Sesamum indicum L.), one of the oldest cultivated oilseed crops, valued for its nutritional and medicinal properties, is highly susceptible to phytoplasma infection, causing substantial annual yield losses. The present study aimed to investigate the metabolomic and molecular alterations in sesame plants as a consequence of phytoplasma infection in two distinct tissue types, leaves and flowers, using a multi-omics approach. ResultsThe study revealed that phytoplasma infection significantly alters the expression of floral homeobox and meristem identity genes in an antagonistic manner, resulting in the development of leaf-like traits in floral tissues. Integrated analyses of transcriptomic and metabolomic datasets showed that control leaves, control flowers, infected leaves, and infected flowers each displayed distinct metabolomic and transcriptomic profiles. Metabolomic profiling demonstrated major changes in pathways such as porphyrin metabolism, brassinosteroid metabolism, and phenylpropanoid biosynthesis. Complementary KEGG pathway enrichment analysis of transcriptome data confirmed strong enrichment of secondary metabolite biosynthesis in both tissue types upon infection. Tissue-specific responses were evident. Floral tissues accumulated green pigments due to increased porphyrin biosynthesis and reduced degradation, while leaves showed simultaneous upregulation of both biosynthesis and breakdown pathways of porphyrins. Floral tissues exhibited stronger stress-associated responses, including upregulation of genes related to stress enzymes, phenylpropanoids, and lignification-related metabolites. In contrast, certain compounds such as lignans were specifically accumulated in leaves upon infection. These observations were further supported by biochemical, histological, and qRT-PCR assays. ConclusionThis study provides the first clear evidence of tissue-specific metabolic reprogramming in sesame under Ca. Phytoplasma infection through integrated transcriptomic and metabolomic analyses. These findings improve our understanding of host-pathogen interactions and offer a basis for strategies to reduce phytoplasma-induced yield losses.

plant biology↗

bioRxiv2025

Deep Neurite Analysis Tool (DeNAT): A machine-learning framework for precise automated neurite outgrowth measurement

Accurate quantification of neurite sprouting after injury is a critical step in axon regeneration research. Yet it remains a major bottleneck, as the current gold standard relies on manual counting by multiple blinded observers. This process is slow, labor-intensive, and prone to variability. While some software can measure total neurite length, they arent made to specifically measure new growth in complicated images from real-life injury models, like the thoracic crush and pyramidotomy model. Existing software can measure total neurite length in culture, but it is not designed to capture new growth in complex images from injury models, such as thoracic crush or pyramidotomy. Crucially, these tools lack the ability to selectively analyze growth within user-defined regions, a key requirement for injury paradigms. To address this need, we developed the Deep Neurite Analysis Tool (DeNAT), an accessible deep-learning-based platform that automatically measures neurite outgrowth after injury. DeNAT allows users to define regions of interest to specifically quantify sprouting in images from common spinal cord injury paradigms. We benchmarked its performance against manual scoring and conventional automated approaches. DeNAT achieved 87 percent accuracy in detecting neurite sprouts relative to manual counts, while reducing variability and labor. By combining user-guided region selection with automated deep learning analysis, DeNAT offers an accurate, reproducible, and efficient solution for measuring neurite outgrowth in injury models.

bioinformatics↗

bioRxiv2025

Local translation of circular RNAs is required for synaptic activity and memory

Circular RNAs (circRNAs) are enriched in synapses and implicated in cognitive processes, and recent studies have shown that circRNAs can encode micropeptides, which suggests that there may be novel synaptic proteoforms in the brain that await discovery. Here we report widespread learning-induced local circRNA translation in the prefrontal cortex of male C57BL/6 mice. More than 1500 synapse-enriched circRNAs contain active IRES elements, with 842 interacting with the ribosome and 241 exhibiting direct evidence of activity-induced translation. We discovered a synapse-enriched micropeptide (P1) that is derived from a single exon circRNA, the mRNA host of which encodes an enzyme associated with protein repair. Although P1 is only a third of the size of the full-length protein, it is locally expressed, enzymatically active, and interacts with plasticity-related proteins, including CaMKII. In addition to direct effects on synaptic activity, targeted P1 knockdown impairs whereas its overexpression enhances fear extinction memory. These findings shed new light on the dark proteome in the brain and reveal local circRNA translation as a novel mechanism of plasticity and memory.

neuroscience↗

bioRxiv2025

dCas9 targeted proteome profiling reveals p300-mediated reciprocal regulation of SMAD and SP1 as a driver of GM2 synthase transcription in renal cell carcinoma

Glycolipids constitute an important component of the plasma membrane based on both abundance as well as function. Gangliosides, being a class of structurally diverse and functionally varied glycolipids, can act both as a receptor as well as a ligand and therefore is established as a crucial player in several normal cellular processes. In certain diseases, and in particular cancer, select gangliosides are over-expressed often leading to disease manifestation. GM2-synthase, the enzyme responsible for the formation of a pro-tumorigenic ganglioside, GM2 is well reported to be over-expressed across various cancer tissues and cell lines. This over-expression of GM2-synthase has been linked with increased migration, invasion and epithelial to mesenchymal transition (1) as well as induction of a local and systemic host immune suppression in cancer. Despite only a handful of studies demonstrating an epigenetic regulation underlying the transcriptional regulation of GM2-synthase (B4GalNT1) gene, the detailed mechanism still remains unclear. Here we identified the total proteome associated with the GM2-synthase promoter through a two-step CRISPR-dCas9 based proteome profiling approach by categorizing all the identified proteins leading to a detailed elucidation of the molecular drivers behind GM2-synthase transcription. While the previous study identified an acetylation-dependent de-repression of the transcription factor SP1 causing GM2-synthase activation, the underlying molecular mechanism driving its activation wasnt clear. This study demonstrated that the histone acetyl transferase (2), p300 acts as a pivotal factor which on one hand cause acetylation-mediated degradation of SP1, and on the other hand activates SMAD2/4 to have a direct positive impact on GM2-synthase gene transcription. We identified p300 to have an activator role in GM2-synthase gene transcription through knock out, knock down and over-expression experiments. Furthermore, SP1 degradation, SMAD activation and their DNA binding patterns show the reciprocal role of p300 on SP1 and SMAD complexes. Altogether we have identified SMAD 2/4 as an activator complex, p300 as a positive regulator and uncovered a critical p300-SMAD-SP1 regulatory axis in GM2-synthase transcriptional regulation.

molecular biology↗

bioRxiv2025

Long non-coding RNA Cerox1 targets components of the mitochondrial electron transport chain to regulate the memory impairment caused by sleep deprivation

AbstractSleep deprivation (SD) impairs long-term memory, but the molecular mechanisms underlying the impact of sleep loss on memory are poorly understood. Molecular changes driven by SD have thus far focused on transcription and translation. Long non-coding RNAs (lncRNAs), a class of regulatory RNAs, have recently been recognized as an important player in memory research. However, it remains unclear how sleep deprivation modulates the expression of lncRNAs or their targets to lead to memory impairment. In this study, we explored the role of lncRNAs in the disruption of spatial memory caused by SD. We examined a set of synapse-associated lncRNAs that were identified through a transcriptome analysis after SD. Among them, we discovered that the lncRNA Cerox1 is downregulated in dorsal hippocampus following SD, and its levels recover after 2.5 hours of rebound sleep. Sleep is critical for the regulation of metabolism and sleep loss impairs mitochondrial function. Both sleep deprivation and Cerox1 knockdown were found to reduce complex I activity of the mitochondrial electron transport chain. This reduction of complex I activity is linked to the decrease in expression of a subset of complex I subunits including Ndufs1, Ndufs3, Ndufa3 and Ndufs6. Overexpression of Cerox1 has the opposite effect, leading to increased complex I activity. Sleep deprivation reduced ATP levels in the dorsal hippocampus, while Cerox1 overexpression restored it. SD disrupted memory consolidation, and this impairment was rescued when Cerox1 was overexpressed. Cerox1 transcript contains multiple miRNA binding sites that regulate the activity of the lncRNA. Notably, overexpression of Cerox1 transcript lacking miRNA binding sites did not rescue the memory deficit caused by SD. Our findings demonstrated that the impairment of memory consolidation after SD is linked to lncRNA-mediated control of mitochondrial electron transport chain activity essential for sustaining energy requirements. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/676326v1_ufig1.gif" ALT="Figure 1000"> View larger version (48K): org.highwire.dtl.DTLVardef@6d72d5org.highwire.dtl.DTLVardef@18e3590org.highwire.dtl.DTLVardef@15e83c9org.highwire.dtl.DTLVardef@1e1f92_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗