Biology subjects

Seth, P.

Publications and source records attributed to Seth, P..

13 recordsLinked to original sources

bioRxiv2026

Automated Multimodal Correlative Registration for Organelle-Specific Molecular Imaging

Mapping subcellular drug distribution is essential for understanding trafficking and off-target effects. NanoSIMS enables chemical imaging of labeled therapeutics, but signal interpretation requires ultrastructural correlation with electron microscopy, a manual and laborious process. We present an automated AI-driven pipeline for correlating chemical and ultrastructural images, enabling multiscale, organelle-precise imaging of molecules in cells and tissues. The method integrates bidirectional optical flow, confidence-guided affine transformation, and automated template matching for cross-scale EM alignment. Morphology-rich ion channels (e.g., 32S) estimate transformations that propagate to sparse therapeutic signals (e.g., 79Br, 15N), overcoming low signal-to-noise challenges. We validate this framework across diverse cell and tissue types, tracking oligonucleotide and antibody therapeutics in vitro and in vivo to reveal cell-type- and organelle-specific distribution patterns. This work establishes a generalizable platform for automated multimodal registration and organelle-resolved subcellular pharmacology.

bioinformatics↗

bioRxiv2025

Scale-Specific Viscoelastic Characterization of Hydrogels: Integrated AFM and Finite Element Modeling

Viscoelastic hydrogels mimic the dynamic mechanical properties of native extracellular matrices, making them essential for biomedical applications. However, characterizing their scale-dependent mechanical properties remains challenging, despite their critical influence on cell-material interactions and biomaterial performance. Here, we present an integrated experimental-computational approach to quantify and model the viscoelastic behavior of interpenetrating polymer network hydrogels across micro- and macro-scales. Atomic force microscopy-based stress relaxation tests revealed that microgels exhibit rapid, localized relaxation, while macroscopic bulk gels displayed prolonged relaxation dominated by poroelastic effects. Finite element simulations accurately replicated experimental conditions, enabling the extraction of key parameters: instantaneous elastic modulus, relaxation modulus, and relaxation time constant. We further developed a novel analytical model to predict viscoelastic parameters from experimental data with minimal error (< 5%), significantly streamlining characterization. Our findings highlight the necessity of scale-specific mechanical analysis and provide a robust platform for designing biomaterials with tailored viscoelasticity for tissue engineering and regenerative medicine.

bioengineering↗

bioRxiv2023

The atypical cadherin FAT1 is a novel regulator of STAT1, driving its pro-tumorigenic effect via the STAT1/PDCD4 axis in glioblastoma

FAT1 is an atypical cadherin that has been shown to act both as an oncogene and a tumour suppressor gene (TSG) in different tumor types. We have earlier shown that upregulated FAT1 acts as an oncogene in glial tumors by promoting pro-tumorigenic inflammation and EMT in primary human glioblastoma and in cell lines. One effect was through the suppression of the Tumor Suppressor Gene (TSG), Programmed Cell Death 4 (PDCD4). Here, we have studied how, in glioblastoma, upregulated FAT1 affects downstream events that control PDCD4 expression. In silico analysis of the PDCD4 promoter revealed multiple STAT1 binding sites. We also found a positive correlation in mRNA levels of STAT1 and FAT1 in the Glioblastoma databases, as well as in resected patient derived tumor samples by qPCR. Increased FAT1 as well as STAT1 were associated with poor prognosis in these data bases. In the glioblastoma cell lines LN229 and U87MG, FAT1 knockdown resulted in decreased STAT1 expression. Also, STAT1 knockdown resulted in increased PDCD4 expression, implying that STAT1 may mediate FAT1s role in suppressing PDCD4. Further, ChIP experiments showed that STAT1 protein binds to the PDCD4 promoter and upon FAT1 knockdown, STAT1 binding to the PDCD4 promoter reduces. As for FAT1, STAT1 knockdown also reduces the expression of pro-inflammatory cytokines and EMT markers, also migration and invasion of glioma derived cell lines. This work identifies STAT1 as a novel downstream mediator of FAT1 which mediates its pro-tumorigenic action in suppressing the TSG, PDCD4.

cancer biology↗

bioRxiv2023

DKK1, a negative regulator of Wnt signaling, is a novel inducer of neuroblastoma differentiation

BackgroundThis study aimed to analyze the role of Dickopff1 [DKK1] as a potential differentiating agent for the neuroblastoma cell line SHSY5Y and neurospheres derived from it. Materials and MethodsSHSY5Y neurospheres were formed from undifferentiated adherent cultures. The cellular properties and gene expression were used to study the effect of DKK1 on SHSY5Y-formed neurospheres. Its effect on SHSY5Y neuronal differentiation was also studied. ResultsSHSY5Y adherent undifferentiated cells were grown as neurospheres. Treatment of neurospheres resulted in their fragmentation. It also resulted in reduced mRNA expression of markers of cancer stem cells, pluripotency, and proliferation [p[≤]0.05]. DKK1 treatment also resulted in reduced mRNA expression of {beta}-catenin and TCF genes. There was significantly higher expression of neuronal differentiation genes in SHSY5Y adherent cells grown in complete DMEM media containing DKK1 as compared to cells grown without DKK1. We also found DKK1 synergized with retinoic acid-induced differentiation of neuroblastoma cells. ConclusionDKK1 was able to convincingly abrogate neurosphere formation and promote neuronal differentiation of SHSY5Y cells, including synergy with retinoic acid. This was accompanied by corresponding changes in mRNA markers of cancer stem cells, pluripotency and proliferation. These results may have implications for neuroblastoma stemness and differentiation.

neuroscience↗

bioRxiv2023

Sulfide oxidation promotes hypoxic angiogenesis and neovascularization

Angiogenic programming in the vascular endothelium is a tightly regulated process to maintain tissue homeostasis and is activated in tissue injury and the tumor microenvironment. The metabolic basis of how gas signaling molecules regulate angiogenesis is elusive. Herein, we report that hypoxic upregulation of NO synthesis in endothelial cells reprograms the transsulfuration pathway and increases H2S biogenesis. Furthermore, H2S oxidation by mitochondrial sulfide quinone oxidoreductase (SQOR) rather than downstream persulfides, synergizes with hypoxia to induce a reductive shift, limiting endothelial cell proliferation that is attenuated by dissipation of the mitochondrial NADH pool. Tumor xenografts in whole-body WBCreSQORfl/fl knockout mice exhibit lower mass and reduced angiogenesis compared to SQORfl/fl controls. WBCreSQORfl/fl mice also exhibit reduced muscle angiogenesis following femoral artery ligation, compared to controls. Collectively, our data reveal the molecular intersections between H2S, O2 and NO metabolism and identify SQOR inhibition as a metabolic vulnerability for endothelial cell proliferation and neovascularization. HighlightsO_LIHypoxic induction of *NO in endothelial cells inhibits CBS and switches CTH reaction specificity C_LIO_LIHypoxic interruption of the canonical transsulfuration pathway promotes H2S synthesis C_LIO_LISynergizing with hypoxia, SQOR deficiency induces a reductive shift in the ETC and restricts proliferation C_LIO_LISQOR KO mice exhibit lower neovascularization in tumor xenograft and hind limb ischemia models C_LI

biochemistry↗

bioRxiv2023

Zika virus E protein alters blood-brain barrier by modulating brain microvascular endothelial cell and astrocyte functions.

Neurotropic viruses can cross the otherwise dynamically regulated blood-brain barrier (BBB) and affect the brain cells. Zika virus (ZIKV) is an enveloped neurotropic Flavivirus known to cause severe neurological complications, such as encephalitis and foetal microcephaly. In the present study, we used human brain microvascular endothelial cells (hBMECs) and human progenitor derived astrocytes to form a physiologically relevant BBB model. We used this model to investigate the effects of ZIKV envelope (E) protein on properties of cells comprising the BBB. E protein is the principal viral protein involved in interaction with host cell surface receptors, facilitating the viral entry. Our findings show that ZIKV E protein results in activation of both hBMECs and astrocytes. hBMECs showed reduced expression of endothelial junction proteins - ZO-1, Occludin and VE-Cadherin, which are crucial in establishing and maintaining the BBB. As a result, ZIKV E protein triggered alteration in BBB integrity and permeability. We also found upregulation of genes involved in leukocyte recruitment along with increased proinflammatory chemokines and cytokines upon exposure to E protein. Furthermore, E protein resulted in astrogliosis as seen by increased expression of GFAP and Vimentin. Both BBB cell types exhibited inflammatory response following exposure to E protein which may influence viral access into the central nervous system (CNS), resulting in infection of other CNS cells. Overall, our study provides valuable insights into the transient changes that occur at the site of BBB upon ZIKV infection.

neuroscience↗

bioRxiv2021

Hypoxia induces early neurogenesis in human fetal neural stem cells by activating the WNT pathway.

Fetal neural stem cells (FNSCs) are present in the brain of human fetuses that differentiate into cells of neuronal and glial lineages. Difference in oxygen concentration between maternal and fetal circulation indicates that the developing fetus may be exposed to lower oxygen concentrations compared to adults. This physiological hypoxia may influence the growth and differentiation of the FNSCs. This study aimed to evaluate the effect of hypoxia on the differentiation potential of human FNSCs isolated from the sub-ventricular zone of aborted fetal brains (n=5). FNSCs were isolated, expanded, and characterized by Nestin and Sox2 expression, using immunocytochemistry and flowcytometry respectively. These FNSCs were exposed to 20% oxygen (normoxia) and 0.2% oxygen (hypoxia) concentrations for 48 hours, and hypoxia exposure (n=5) was evaluated by a panel of markers (CA9, PGK1 and VEGF). Whole human genome transcriptomic analyses (Genespring GX13) of FNSCs exposed to hypoxia (Agilent 4x44K human array slides), highlighted that genes associated with neurogenesis were getting enriched. The pathway analysis of these enriched genes (using Metacore) showed that WNT signaling played a role in determining the cell fate of FNSCs exposed to hypoxic environment. Microarray analyses was validated using neuronal and glial lineage commitment markers such as Ngn1, Ngn2, ASCL1, DCX, GFAP, Olig2 and Nkx2.2 using qPCR (n=9). This demonstrated upregulation of the neuronal commitment markers on hypoxia exposure, while no change was observed in astrocytic and oligodendrocyte lineage commitment markers. Increased expression of downstream targets of the WNT signaling pathway, TCF4 and ID2, by qPCR (n=9), indicated its involvement in mediating neuronal differentiation on exposure to hypoxia.

neuroscience↗

bioRxiv2021

Human fetal neural stem cell-derived astrocytes maintain glutamate transport after hypoxic injury in vitro

Astrocytes are the most abundant glial cells that play many critical roles in the central nervous system physiology including the uptake of excess glutamate from the synapse by Excitatory Amino Acid Transporters (EAATs). Among the EAATs, EAAT2 are predominantly functional, astrocyte-specific glutamate transporters in the forebrain. Hypoxic brain injury is a pathological phenomenon seen in various clinical conditions including stroke and neonatal hypoxic ischemic encephalopathy. Glutamate excitotoxicity is an important cause of neuronal cell death in disorders involving hypoxic brain injury. As findings from rodent models cannot always be reliably extrapolated to humans, we aimed to develop a homogenous population of primary human astrocytes to study the effect of hypoxic injury on astrocyte function, especially glutamate uptake. We successfully isolated, established and characterized cultures of human fetal neural stem cells (FNSCs) from aborted fetal brains. FNSCs were differentiated into astrocytes, and characterized by increased expression of the astrocyte markers, glial fibrillary acidic protein (GFAP), EAAT1 and EAAT2. A concomitant decrease in neural stem cell marker, Nestin, was observed. Differentiated astrocytes were exposed to various oxygen concentrations mimicking normoxia (20% and 6%), moderate and severe hypoxia (2% and 0.2% respectively). Interestingly, no change was observed in the expression of glutamate transporter, EAAT2 and glutamate uptake by astrocytes, even after exposure to hypoxia. Our novel model of human FNSC derived astrocytes exposed to hypoxic injury, establishes that astrocytes are able to maintain glutamate uptake even after exposure to severe hypoxia for 48 hours, and thus provides evidence for the neuroprotective role of astrocytes in hypoxic injury.

neuroscience↗

bioRxiv2021

Copper dependent ERK1/2 phosphorylation is essential for the viability of neurons and not glia

Intracellular Copper [Cu(I)] has been hypothesized to play role in the differentiation of the neurons. This necessitates understanding the role of Cu(I) not only in the neurons but also in the glia considering their anatomical proximity, contribution towards ion homeostasis, neuronal physiology, and neurodegeneration. In this study we did a systematic investigation of the changes in the cellular copper homeostasis during neuronal and glial differentiation and the pathways triggered by them. Our study demonstrates increased mRNA for the plasma membrane copper transporter CTR1 leading to an increased Cu(I) during neuronal (PC-12) differentiation. ATP7A is retained in the Trans Golgi Network (TGN) despite high Cu(I) demonstrating its utilization in triggering the pathways towards the neuronal differentiation. One of these pathways is ERK1/2 phosphorylation accompanying the differentiation of both PC-12 and human fetal brain derived neuronal progenitor cells. The study demonstrates that the ERK1/2 phosphorylation is essential for the viability of the neurons. In contrast, differentiated C-6 (glia) cells contain low intracellular copper and significant downregulation of the ERK1/2 phosphorylation. Interestingly ATP7A shows vesicular localization despite the low copper in the glia. In addition to the TGN in the perinuclear region, ATP7A localizes into RAB11 positive recycling endosomes in the glial neurites, not observed in the neurons. Our study demonstrates role of the copper dependent ERK1/2 phosphorylation in the neuronal differentiation. Whereas glial differentiation largely involves sequestration of Cu(I) into the endosomes potentially (i) for ready release to the neurons (ii) rendering cytosolic copper unavailable for pathways like the ERK1/2 activation.

cell biology↗

bioRxiv2021

iRGD-liposomes enhance tumor delivery and therapeutic efficacy of antisense oligonucleotide drugs against primary prostate cancer and bone metastasis

Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.

cancer biology↗

bioRxiv2020

Vortex chip incorporating an orthogonal turn for size-based isolation of circulating cells

Label-free separation of rare cells (e.g. circulating tumor cells (CTCs)) based on their size is attractive due to its wider applicability, simpler sample preparation, faster turnaround, better efficiency and higher purity. Amongst cognate protocols for the same, vortex-trapping based techniques offer high throughput but operate at high flow velocities where the resulting hydrodynamic shear stress is likely to damage cells and compromise their viability for subsequent assays. We present here an orthogonal vortex chip which can carry out size-differentiated trapping at significantly lower (38% of previously reported) flow velocities. Fluid flowing through the chip is constrained to exit the trapping chamber at right angles to that of its entry. Such a flow configuration leads to the formation of vortex in the chamber. Above a critical flow velocity, larger particles are trapped in the vortex whereas smaller particles get ejected with the flow: we call this phenomenon the turn-effect. We have characterized the critical velocities for trapping of cells and particles of different sizes on chips with distinct entry-exit configurations. Optimal architectures for stable vortex trapping at low flow velocities are identified. We explain how shear-gradient lift, centrifugal and Dean flow drag forces contribute to the turn-effect by acting on cells which pushes them into specific vortices in a size- and velocity-dependent fashion. Finally, we demonstrate selective trapping of human breast cancer cells mixed with whole blood at low-concentration. Our findings suggest that the device shows promise for the gentle isolation of rare cells from blood.

bioengineering↗

bioRxiv2019

Origin of a novel CYP20A1 lncRNA through 23 Alu exaptations in the human lineage creates a potential multi-miRNA sponge

BackgroundPrimate-specific Alus contribute to transcriptional novelties in conserved gene regulatory networks. Alu RNAs are present at elevated levels in stress conditions and consequently leads to transcript isoform specific functional role modulating the physiological outcome. One of the possible mechanisms could be Alu nucleated mRNA-miRNA interplay. ResultUsing combination of bioinformatics and experiments, we report a transcript isoform of an orphan gene, CYP20A1 (CYP20A1_Alu-LT) through exaptation of 23 Alus in its 9kb 3UTR. CYP20A1_Alu-LT, confirmed by 3RACE, is an outlier in length and expressed in multiple cell lines. We demonstrate its presence in single nucleus RNA-seq of [~]16000 human cortical neurons (including rosehip neurons). Its expression is restricted to the higher primates. Most strikingly, miRanda predicts [~]4700 miRNA recognition elements (MREs; with threshold< -25kcal/mol) for [~]1000 miRNAs, which have majorly originated within the 3UTR-Alus post exaptation. We hypothesized that differential expression of this transcript could modulate mRNA-miRNA networks and tested it in primary human neurons where CYP20A1_Alu-LT is downregulated during heat shock response and upregulated upon HIV1-Tat treatment. CYP20A1_Alu-LT could possibly function as a miRNA sponge as it exhibits features of a sponge RNA such as cytosolic localization and [≥]10 MREs for 140 miRNAs. Small RNA-seq revealed expression of nine miRNAs that can potentially be sponged by CYP20A1_Alu-LT in neurons. Additionally, CYP20A1_Alu-LT expression was positively correlated (low in heat shock and high in Tat) with 380 differentially expressed genes that contain cognate MREs for these nine miRNAs. This set is enriched in genes involved in neuronal development and hemostasis pathways. ConclusionWe demonstrate a potential role for CYP20A1_Alu-LT as miRNA sponge through preferential presence of MREs within Alus in a transcript isoform specific manner. This highlights a novel component of Alu-miRNA mediated transcriptional modulation leading to physiological homeostasis.

evolutionary biology↗

bioRxiv2019

Identification and classification of hubs in miRNA target gene networks in human neural stem/progenitor cells following Japanese encephalitis virus infection

Micro RNA dysregulation is observed in many viral diseases. RNA viruses modulate host miRNA machinery for their own benefit. JEV, a neurotropic RNA virus has been reported to manipulate several miRNAs in neuron or microglia. However, no report indicates a complete sketch of the miRNA profile of NSPCs contributing to viral persistence; hence being focused in our current study. We performed a miRNA array of 84 miRNAs in human neuronal progenitor cell line and primary neural precursor cells isolated from aborted foetus. Several fold down-regulation of hsa-miR-9-5p, hsa-miR-22-3p, hsa-miR-124-3p and hsa-miR-132-3p were found in both of the cells. Subsequently, we screened for the target genes of these miRNAs and looked for the important biological pathways the genes significantly regulate. Then we sorted out the target genes which are involved in two or more than two pathways. We constructed a protein-protein interaction (PPI) network of the miRNA target genes based on their interaction patterns. A binary adjacency matrix for each gene network was prepared. Different modules or communities were identified in those networks using community detection algorithms. Mathematically, we identified the hub genes by analyzing their degree centrality and participation co-efficient in the network. The hub genes were classified either as provincial (P<0.4) or connector hubs (P>0.4). We validated the expression of hub genes in both cell line and primary cells through qRT-PCR post JEV infection and respective miR-mimic transfection. Taken together, our findings highlight the importance of specific target gene networks of miRNAs affected by JEV infection in NSPCs.\n\nImportanceJEV damages the neural stem/progenitor cell population of mammalian brain. However, JEV induced alteration in miRNA expression pattern of the cell population remains an open question, hence warrants our present study. In this study, we specifically address the down-regulation of four miRNAs and we prepared a protein-protein interaction network of miRNA target genes. We identified two types of hub genes in the PPI network namely connector hubs and provincial hubs. These two types of miRNA target hub genes critically influence the participation strength in the networks and thereby significantly influence up and down regulation in several key biological pathways. Computational analysis of the PPI networks identifies key protein interactions and hubs in those modules which opens up the possibility of precise identification and classification of host factors for viral infection in NSPCs and how RNA viruses modulate host miRNA machinery for their own benefit post JEV infection to the cells.

microbiology↗