Biology subjects

Das, T.

Publications and source records attributed to Das, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

ALTering cancer by triggering telomere replication stress through the stabilization of promoter G-quadruplex in SMARCAL1

Most of the human cancers are dependent on telomerase to extend the telomeres. But [~]10% of all cancers use a telomerase-independent, homologous recombination mediated pathway called alternative lengthening of telomeres (ALT). Due to poor prognosis, the ALT status is not being considered yet in the diagnosis of cancer. No such specific treatment is available till date for ALT cancers. ALT positive cancers are dependent on replication stress to deploy DNA repair pathways to the telomeres to execute homology recombination mediated telomere extension. SMARCAL1 (SWI/SNF related, matrix-associated, actin-dependent regulator of chromatin, subfamily A-like 1) is associated with the ALT telomeres to resolve replication stress thus providing telomere stability. Thus, the dependency on replication stress regulatory factors like SMARCAL1 made it a suitable therapeutic target for the treatment of ALT positive cancers. In this study, we found a significant downregulation of SMARCAL1 expression by stabilizing the G-quadruplex (G4) motif found in the promoter of SMARCAL1 by potent G4 stabilizers, like TMPyP4 and BRACO-19. SMARCAL1 downregulation led towards the increased localization of PML (promyelocytic leukaemia) bodies in ALT telomeres and triggered the formation of APBs (ALT-associated promyelocytic leukaemia bodies) in ALT positive cell lines, thus increasing telomere replication stress and DNA damage at genomic level. Induction of replication stress and hyper recombinogenic phenotype in ALT cells mediated by G4 stabilizing molecules already highlighted their possible application as a new therapeutic window to target ALT-positive tumors. In accordance with this, our study will also provide a valuable insight towards the development of G4 based ALT-therapeutics targeting SMARCAL1.

biochemistry↗

bioRxiv2023

Amplification of avian influenza viruses along poultry marketing chains in Bangladesh: a controlled field experiment

The prevalence of avian influenza viruses (AIVs) is commonly found to increase dramatically from farms to live bird markets (LBMs). Viral transmission dynamics along marketing chains is, however, poorly understood. To address this gap, we implemented a field experiment altering chicken supply to an LBM in Chattogram, Bangladesh. Chickens traded along altered (intervention) and conventional (control) marketing chains were tested for AIVs. Upon arrival at the LBM, the odds of detecting AIVs did not differ between control and intervention groups. However, 12 hours later, intervention group odds were lower, particularly for broiler chickens, indicating that viral shedding in LBM resulted partly from infections during transport and trade. Curtailing AIV prevalence in LBMs requires mitigating risk in marketing chain nodes preceding chickens delivery at LBMs. Article Summary LineThe high prevalence of avian influenza viruses in marketed chickens cannot be solely attributed to viral transmission within live bird markets but is also influenced by infections occurring prior to the chickens supply to these markets.

ecology↗

bioRxiv2023

SecDATA: Secure Data Access and de novo TranscriptAssembly protocol - To meet the challenge of reliableNGS data analysis

Recent developments in sequencing technologies have created new opportunities to generate high-throughput biological data at an affordable price. Such high-throughput data needs immense computational resources for performing transcript assembly. Further, a high-end storage facility is needed to store the analyzed data and raw data. Here comes the need for centralized repositories to store such mountains of raw and analyzed data. Hence, it is of utmost importance to ensure data privacy for storing the data while performing transcript assembly. In this paper, we have developed a protocol named SecDATA which performs de novo transcript assembly ensuring data security. It consists of two modules. The first module deals with a framework for secured access and storage of data. The novelty of the first module lies in the employment of distributed ledger technology for data storage that ensures the privacy of the data. The second module deals with the development of an optimized graph-based method for de novo transcript assembly. We have compared our results with the state-of-art method de Bruijn graph and the popular pipeline Trinity, for transcript reconstruction, and our protocol outperforms them.

bioinformatics↗

bioRxiv2023

Mechanical imbalance between normal and cancer cells drives epithelial defence against cancer

Cell competition in epithelial tissue eliminates transformed cells expressing activated oncoproteins to maintain epithelial homeostasis. Although the process is now understood to be of mechanochemical origin, direct mechanical characterization and associated biochemical underpinnings are lacking. Here, we employ tissue-scale stress and compressibility measurements and theoretical modeling to unveil a mechanical imbalance between normal and transformed cells, which drives cell competition. In the mouse intestinal epithelium and epithelial monolayer, transformed cells get compacted during competition. Stress microscopy reveals an emergent compressive stress at the transformed loci leading to this compaction. A cell-based self-propelled Voronoi model predicts that this compressive stress originates from a difference in the collective compressibility of the competing populations. A new collective compressibility measurement technique named gel compression microscopy then elucidates a two-fold higher compressibility of the transformed population than the normal population. Mechanistically, weakened cell-cell adhesions due to reduced junctional abundance of E-cadherin in the transformed cells render them collectively more compressible than normal cells. Taken together, our findings unveil a mechanical basis for epithelial homeostasis against oncogenic transformations with implications in epithelial defense against cancer.

biophysics↗

bioRxiv2023

Multivalent GU-rich oligonucleotides sequester TDP-43 in the nucleus by inducing high molecular weight RNP complexes

TDP-43 nuclear clearance and cytoplasmic aggregation are hallmarks of TDP-43 proteinopathies. We recently demonstrated that binding to endogenous nuclear GU-rich RNAs sequesters TDP-43 in the nucleus by restricting its passive nuclear export. Here, we tested the feasibility of synthetic RNA oligonucleotide-mediated augmentation of TDP-43 nuclear localization. Using biochemical assays, we compared the ability of GU-rich oligonucleotides to engage in multivalent, RRM-dependent binding with TDP-43. When transfected into cells, (GU)16 attenuated TDP-43 mislocalization induced by transcriptional blockade or RanGAP1 ablation. Clip34nt and (GU)16 accelerated TDP-43 nuclear re-import after cytoplasmic mislocalization. RNA pulldowns confirmed that multivalent GU-oligonucleotides induced high molecular weight RNP complexes, incorporating TDP-43 and possibly other GU-binding proteins. Transfected GU-repeat oligos disrupted TDP-43 cryptic exon repression, likely by diverting TDP-43 from endogenous RNAs, except for Clip34nt which contains interspersed A and C. Thus, exogenous multivalent GU-RNAs can promote TDP-43 nuclear localization, though pure GU- repeat motifs impair TDP-43 function.

cell biology↗

bioRxiv2023

Matrix stiffening promotes perinuclear clustering of mitochondria

Mechanical cues from the tissue microenvironment, such as the stiffness of the extracellular matrix, modulate cellular forms and functions. As numerous studies have shown, this modulation depends on the stiffness-dependent remodeling of cytoskeletal elements. In contrast, very little is known about how the intracellular organelles such as mitochondria respond to matrix stiffness and whether their form, function, and localization change accordingly. Here, we performed an extensive quantitative characterization of mitochondrial morphology, subcellular localization, dynamics and membrane tension on soft and stiff matrices. This characterization revealed that while matrix stiffness affected all these aspects, matrix stiffening most distinctively led to an increased perinuclear clustering of mitochondria. Subsequently, we could identify the matrix stiffness-sensitive perinuclear localization of filamin as the key factor dictating this perinuclear clustering. Photo-conversion labeling and fluorescent recovery after photobleaching experiments revealed that perinuclear and peripheral mitochondrial populations differed in their motility on the soft matrix but surprisingly they did not show any difference on the stiff matrix. Finally, perinuclear mitochondrial clustering appeared to be crucial for priming human mesenchymal stem cells towards osteogenesis on the stiff matrix. Taken together, we elucidate a dependence of mitochondrial localization on matrix stiffness, which possibly enables a cell to adapt to its microenvironment.

cell biology↗

bioRxiv2023

Collective heterogeneity of mitochondrial potential in contact inhibition of proliferation

In the epithelium, cell density and cell proliferation are closely connected to each other through contact inhibition of proliferation (CIP). Depending on cell density, CIP proceeds through three distinct stages, namely the free-growing stage at low density, the pre-epithelial transition stage at medium density, and the post-epithelial transition stage at high density. Previous studies have elucidated how cell morphology, motion, and mechanics vary in these stages. However, it remains unknown whether cellular metabolism also has a density-dependent behavior. By measuring the mitochondrial membrane potential at different cell densities, here we reveal a heterogeneous landscape of metabolism in the epithelium, which appears qualitatively distinct in three stages of CIP. Moreover, it did not follow the trend of other CIP-associated parameters, which increase or decrease monotonically with increasing cell density. Importantly, epithelial cells established a collective heterogeneity in mitochondrial potential exclusively in the pre-epithelial transition stage, where the multicellular clusters of high and low-potential cells emerged. However, in the post-epithelial transition stage, the potential field became relatively homogeneous. The collective metabolic heterogeneity in the pre-epithelial transition stage was independent of the mitochondrial content and spatially correlated with the local cell density. Next, to study the underlying dynamics, we constructed a system-biological model, which predicted the role of cell proliferation in metabolic potential towards establishing collective heterogeneity. Further experiments revealed that the metabolic pattern indeed spatially correlated with the proliferative capacity of cells, as measured by the nuclear localization of a pro-proliferation protein, YAP. Finally, experiments perturbing the actomyosin contractility revealed that while metabolic heterogeneity was maintained in absence of actomyosin contractility, its ab initio emergence depended on the latter. Taken together, our results revealed a density-dependent collective heterogeneity in the metabolic field of a pre-epitheli al transition stage epithelial monolayer, which may have significant implications for epithelial form and function. STATEMENT OF SIGNIFICANCEEpithelial contact inhibition of proliferation (CIP) plays a key role in tissue homeostasis, morphogenesis, and development. The biochemical changes in cells during different stages of CIP are not as well-documented as the biophysical changes. We unveil a heterogeneous landscape of metabolism which appears distinct in different stages of CIP. Importantly, in the pre-epithelial transition stage, the epithelial cells establish a collective metabolic heterogeneity wherein multicellular clusters of high and low-potential cells emerge, despite the uniform genetic and nutrient conditions for the cells. The collective heterogeneity is correlated to the local fluctuations in geometrical parameters and the proliferative capacity of cells. Finally, we demonstrate the role of cell mechanics in the establishment of collective heterogeneity.

biophysics↗

bioRxiv2022

Species-specific LUBAC-mediated M1 ubiquitination counteracts necroptosis by segregating the cellular distribution and fate of activated MLKL

Plasma membrane accumulation of phosphorylated mixed lineage kinase domain-like (MLKL) is a hallmark of necroptosis, leading to membrane rupture and inflammatory cell death. Pro-death functions of MLKL are tightly controlled by several checkpoints, including phosphorylation. Endocytosis and exocytosis limit MLKL membrane accumulation and counteract necroptosis, but the exact mechanisms remain poorly understood. Here, we identify linear ubiquitin chain assembly complex (LUBAC)-mediated M1 poly-ubiquitination (poly-Ub) as novel checkpoint for necroptosis regulation downstream of activated MLKL in human cells. Loss of LUBAC activity inhibits necroptosis, without affecting necroptotic signaling, but by preventing membrane accumulation of activated MLKL. Flotillin-1/2 act as putative necroptotic M1 poly-Ub targets that inhibit necroptosis suppression induced by LUBAC inhibition. Finally, we confirm LUBAC-dependent activation of necroptosis in primary human pancreatic organoids. Our findings identify LUBAC as species-specific regulator of necroptosis which promotes MLKL membrane accumulation and pioneer primary human organoids to model necroptosis in near-physiological settings.

cell biology↗

bioRxiv2022

Prediction of Golgi Polarity in Collectively Migrating Epithelial Cells Using Graph Neural Network

In the stationary epithelium, the Golgi apparatus assumes an apical position, above the cell nucleus. However, during wound healing and morphogenesis, as the epithelial cells starts migrating, it relocalizes closer to the basal plane. On this plane, the position of Golgi with respect to the cell nucleus defines the organizational polarity of a migrating epithelial cell, which is crucial for an efficient collective migration. Yet, factors influencing the Golgi polarity remain elusive. Here we constructed a graph neural network-based deep learning model to systematically analyze the dependency of Golgi polarity on multiple geometric and physical factors. In spite of the complexity of a migrating epithelial monolayer, our simple model was able to predict the Golgi polarity with 75% accuracy. Moreover, the model predicted that Golgi polarity predominantly correlates with the orientation of maximum principal stress. Finally, we found that this correlation operates locally since progressive coarsening of the stress field over multiple cell-lengths reduced the stress polarity-Golgi polarity correlation as well as the predictive accuracy of the neural network model. Taken together, our results demonstrated that graph neural networks could be a powerful tool towards understanding how different physical factors influence collective cell migration. They also highlighted a previously unknown role of physical cues in defining the intracellular organization.

cell biology↗

bioRxiv2022

Mechanosensitive dynamics of lysosomes regulates the emergence of leader cells during collective cell migration

Collective cell migration during embryonic development, wound healing, and cancer metastasis entails the emergence of leader cells at the migration front. These cells with conspicuous lamellipodial structures provide directional guidance to the collective. Despite their physiological relevance, the mechanisms underlying the emergence of leader cells remain elusive. Here we report that in diverse model systems for wound healing, including cultured epithelial monolayer, Drosophila embryo, and mouse embryonic skin, leader cells display a peripheral accumulation of lysosomes. This accumulation appears essential for leader cell emergence, involves lysosomal movement along microtubules, and depends on the actomyosin contractility-generated cellular forces. Peripheral lysosomes associate with inactive Rac1 molecules to remove them from the leading periphery, which increases local Rac1-activity, triggering actin polymerization and promoting lamellipodium formation. Taken together, we demonstrate that beyond their catabolic role, lysosomes act as the intracellular platform that links mechanical and biochemical signals to control the emergence of leader cells.

cell biology↗

bioRxiv2021

Spatial-transcriptomics reveals unique defining molecular features of fluorescence-sorted 5-aminolevulinic acid+ infiltrative tumor cells associated with glioblastoma recurrence and poor survival

Spatiotemporal-heterogeneity of glioblastoma (GBM) originating from the genomic and transcriptional variation in spatially distinct intra-tumor sites, may contribute to subtype switching in GBM prior to and upon recurrence. Fluorescence-guided neurosurgical resection utilizing 5-aminolevulinic acid (5ALA) has enabled the isolation of infiltrative margin tumor cells (5ALA+ cells) from a background of non-neoplastic cells. We have explored the spatial-transcriptomic (ST) landscape to interrogate molecular signatures unique to infiltrating 5ALA+ cells in comparison to GBM core, rim, and invasive margin non-neoplastic cells. ST analysis reveals that GBM molecular subtype plasticity is not restricted to recurrence, but manifests regionally in a cell-type-specific manner. Whilst GBM core and rim are highly enriched with Classical and Proneural subtypes, the unique enrichment of the Mesenchymal subtype (MES) in 5ALA+ cells supports the hypothesis that MES 5ALA+ cells may drive GBM recurrence. Upregulation of the wound response pathway in 5ALA+ cells signifies the possibility of hijacking the wound healing pathway of neural cells to promote tumor growth. Exon-intron split analysis revealed an upregulation of exonic counts for MES and wound-response genes in 5ALA+ cells, implying that these genes are under active post-transcriptional control. Network analysis suggests that wound response genes, including chemokine CCL2 that recruits regulatory T-cells and monocytic myeloid-derived suppressor cells, are controlled by an IRF8-mediated transcriptional program in 5ALA+ cells. A higher stemness signature both in 5ALA+ cells and non-neoplastic cells of the invasive margin emphasizes the role of this microenvironment in stemness acquisition and defines 5ALA+ cells as a rare sub-population of GBM stem cells. Finally, we establish a link between the unique molecular signatures of 5ALA+ cells and poor survival and GBM recurrence. Characterization of the 5ALA+ infiltrative sub-population offers an opportunity to develop more effective GBM treatments and urges focus away from the GBM proliferative core, upon which failed targeted therapies have been predicated.

cancer biology↗

bioRxiv2021

Fluid intelligence and naturalistic task impairments after focal brain lesions

Classical executive tasks, such as Wisconsin card-sorting and verbal fluency, are widely used as tests of frontal lobe control functions. Since the pioneering work of Shallice and Burgess (1991), it has been known that complex, naturalistic tasks can capture deficits that are missed in these classical tests. Matching this finding, deficits in several classical tasks are predicted by loss of fluid intelligence, linked to damage in a specific cortical "multiple-demand" (MD) network, while deficits in a more naturalistic task are not. To expand on these previous results, we examined the effect of focal brain lesions on three new tests - a modification of the previously-used Hotel task, a new test of task switching after extended delays, and a test of decision-making in imagined real-life scenarios. As potential predictors of impairment we measured volume of damage to a priori MD and default mode (DMN) networks, as well as cortical damage outside these networks. Deficits in the three new tasks were substantial, but were not explained by loss of fluid intelligence, or by volume of damage to either MD or DMN networks. Instead, deficits were associated with diverse lesions, and not strongly correlated with one another. The results confirm that naturalistic tasks capture cognitive deficits beyond those measured by fluid intelligence. We suggest, however, that these deficits may not arise from specific control operations required by complex behaviour. Instead, like everyday activities, complex tasks combine a rich variety of interacting cognitive components, bringing many opportunities for processing to be disturbed.

neuroscience↗

bioRxiv2021

Can active hydrodynamic fluctuations affect barrier crossing during enzymatic catalysis?

The cellular milieu is teeming with biochemical nano-machines whose activity is a strong source of correlated non-thermal fluctuations termed active noise. Essential elements of this circuitry are enzymes, catalysts that speed up the rate of metabolic reactions by orders of magnitude, thereby making life possible. Here, we examine the possibility that active noise in the cell, or in vitro, affects enzymatic catalytic rate by accelerating or decelerating the crossing rate of energy barriers during the reaction. Considering hydrodynamic perturbations induced by biochemical activity as a source of active noise, we evaluate their impact on the enzymatic cycle using a combination of analytic and numerical methods. Our estimates show that the fast component of the active noise spectrum enhances the rate of enzymes, while reactions remain practically unaffected by the slow noise spectrum. Revisiting the physics of barrier crossing under the influence of active hydrodynamic fluctuations suggests that the biochemical activity of macromolecules such as enzymes is coupled to active noise. Thus, we propose that enzymatic catalysis is a collective, many-body process in which enzymes may affect each others activity via long-range hydrodynamic interaction, with potential impact on biochemical networks in living and artificial systems alike.

biophysics↗

bioRxiv2021

Label-free screening of brain tissue myelin content using phase imaging with computational specificity (PICS)

Inadequate myelination in the central nervous system is associated with neurodevelopmental complications. Thus, quantitative, high spatial resolution measurements of myelin levels are highly desirable. We used spatial light interference microcopy (SLIM), a highly sensitive quantitative phase imaging (QPI) technique, to correlate the dry mass content of myelin in piglet brain tissue with dietary changes and gestational size. We combined SLIM micrographs with an AI classifying model that allows us to discern subtle disparities in myelin distributions with high accuracy. This concept of combining QPI label-free data with AI for the purpose of extracting molecular specificity has recently been introduced by our laboratory as phase imaging with computational specificity (PICS). Training on nine thousand SLIM images of piglet brain tissue with the 71-layer transfer learning model Xception, we created a two-parameter classification to differentiate gestational size and diet type with an accuracy of 82% and 80%, respectively. To our knowledge, this type of evaluation is impossible to perform by an expert pathologist or other techniques.

bioinformatics↗

bioRxiv2020

Site-specific lipidation enhances IFITM3 membrane interactions and antiviral activity

Interferon-induced transmembrane proteins (IFITMs) are S-palmitoylated proteins in vertebrates that restrict a diverse range of viruses. S-palmitoylated IFITM3 in particular directly engages incoming virus particles, prevents their cytoplasmic entry, and accelerates their lysosomal clearance by host cells. However, the precise molecular mechanisms of action for IFITM-mediated viral restriction are still unclear. To investigate how site-specific S-palmitoylation controls IFITM3 antiviral activity, here we employed computational, chemical, and biophysical approaches to demonstrate that site-specific lipidation of IFITM3 at highly conserved cysteine 72 modulates its conformation and interaction with lipid membranes leading to enhanced antiviral activity of IFITM3 in mammalian cells. Collectively, our results demonstrate that site-specific S-palmitoylation of IFITM3 directly alters its biophysical properties and activity in cells to prevent virus infection.

biochemistry↗

bioRxiv2020

Hypoxia induced sex-difference in zebrafish brain proteome profile reveals the crucial role of H3K9me3 in recovery from acute hypoxia

Understanding the molecular basis of sex differences in neural response to acute hypoxic insult has profound implications for the effective prevention and treatment of ischemic stroke. Global hypoxic-ischemic induced neural damage has been studied recently under the well-controlled, non-invasive, reproducible conditions using zebrafish model. Our earlier report on sex difference in global acute hypoxia induced neural damage and recovery in zebrafish prompted us for comprehensive study on the mechanisms underlying the recovery. An omics approach for studying quantitative changes in brain proteome upon hypoxia insult following recovery was undertaken using iTRAQ-based LC-MS/MS approach. The results shed light on altered expression of many regulatory proteins in zebrafish brain upon acute hypoxia following recovery. The sex difference in differentially expressed proteins along with the proteins expressed in uniform direction in both the sexes was studied. Core expression analysis by Ingenuity Pathway analysis (IPA) showed a distinct sex difference in the disease function heatmap. Most of the upstream regulators obtained through IPA were validated at the transcriptional level. Translational upregulation of H3K9me3 in male led us to elucidate the mechanism of recovery by confirming transcriptional targets through ChIP-qPCR. The upregulation of H3K9me3 level in male at 4 hr post-hypoxia appears to affect the early neurogenic markers nestin, klf4 and sox2, which might explain the late recovery in male, compared to female. Acute hypoxia-induced sex-specific comparison of brain proteome led us to reveal many differentially expressed proteins, which can be further studied for the development of novel targets for better therapeutic strategy.

neuroscience↗

bioRxiv2020

Molecular Basis of Sex Difference in Neuroprotection induced by Hypoxia preconditioning in zebrafish

Hypoxia, the major cause of ischemia, leads to debilitating disease in infants via birth asphyxia and cerebral palsy, whereas in adults via heart attack and stroke. A widespread, natural protective phenomenon termed Hypoxic Preconditioning occurs when prior exposures to hypoxia eventually results in robust hypoxia resistance. Accordingly, we have developed a novel model of sex-specific hypoxic preconditioning in adult zebrafish to mimic the tolerance of mini stroke(s) in human, which appears to protect against the severe damage inflicted by a major stroke event. Remarkable difference in the progression pattern of neuroprotection between preconditioning hypoxia followed by acute hypoxia (PH) group, and acute hypoxia (AH) group were observed with noticeable sex difference. Since gender difference has been reported in stroke, it was pertinent to investigate whether any such sex difference also exists in PHs protective mechanism against acute ischemic stroke. In order to elucidate the neural molecular mechanisms behind sex difference in neuroprotection induced by PH, a high throughput proteomics approach utilizing iTRAQ was performed, followed by protein enrichment analysis using Ingenuity Pathway Analysis. Out of thousands of altered proteins in zebrafish brain, the ones having critical role either in neuroglial proliferation/differentiation or neurotrophic functions, were validated by analyzing their expression levels in PH, AH and normoxia groups. Results indicate that female zebrafish brains are more protected against the severity of AH. The study also sheds light on the involvement of many signaling pathways underlying sex difference in pre-conditioning induced neuroprotective mechanism, which can be further validated for the therapeutic approach.

neuroscience↗

bioRxiv2020

Quantifying myelin content in brain tissue using color spatial light interference microscopy (cSLIM)

Deficient myelination of the brain is associated with neurodevelopmental delays, particularly in high-risk infants, such as those born small in relation to their gestational age (SGA). New methods are needed to further study this condition. Here, we employ Color Spatial Light Interference Microscopy (cSLIM), which uses a brightfield objective and RGB camera to generate pathlength-maps with nanoscale sensitivity in conjunction with a regular brightfield image. Using tissue sections stained with Luxol Fast Blue, the myelin structures were segmented from a brightfield image. Using a binary mask, those portions were quantitatively analyzed in the corresponding phase maps. We first used the CLARITY method to remove tissue lipids and validate the sensitivity of cSLIM to lipid content. We then applied cSLIM to brain histology slices. These specimens are from a previous MRI study, which demonstrated that appropriate for gestational age (AGA) piglets have increased internal capsule myelination (ICM) compared to small for gestational age (SGA) piglets and that a hydrolyzed fat diet improved ICM in both. The identity of samples was blinded until after statistical analyses.

neuroscience↗