Biology subjects

Gupta, K.

Publications and source records attributed to Gupta, K..

103 records · Page 6Linked to original sources

bioRxiv2019

Integrative analysis and machine learning based characterization of single circulating tumor cells

We collated publicly available single-cell expression profiles of circulating tumor cells (CTCs) and showed that CTCs across cancers lie on a near-perfect continuum of epithelial to mesenchymal (EMT) transition. Integrative analysis of CTC transcriptomes also highlighted the inverse gene expression pattern between PD-L1 and MHC, which is implicated in cancer immunotherapy. We used the CTCs expression profiles in tandem with publicly available peripheral blood mononuclear cell (PBMC) transcriptomes to train a classifier that accurately recognizes CTCs of diverse phenotype. Further, we used this classifier to validate circulating breast tumor cells captured using a newly developed microfluidic systems for label-free enrichment of CTCs.

genomics↗

bioRxiv2019

DETECTing Merkel cell Polyomavirus in Merkel Tumours

Merkel cell carcinoma (MCC) is a rare, aggressive skin cancer caused either by Merkel cell polyomavirus (MCV) T antigen expression, post integration ([~]80% cases), or by UV mediated DNA damage. Interestingly, overall survival of patients suffering from MCV positive Merkel cell carcinoma is better, making this differential information of significant diagnostic and prognostic value. Also, MCV as a causative agent also provides a direct target for therapy in virus positive MCC patients. Currently, the methods used for diagnosis of MCV in tumours are often tedious, discordant and unreliable. In this study we used a guided molecular scissors based - DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) technique to develop an in vitro molecular diagnostic tool for MCV positive MCC. DETECTR couples recombinase polymerase based amplification of target MCV DNA with Cas12a mediated detection. CRISPR diagnostics couple specific detection followed by cutting of the pathogenic DNA by the Cas enzyme - gRNA complex, with non-specific cutting of ssDNA that provides a measurable visual cue. To detect MCV DNA in MCC tumours, we designed Cas12a gRNAs targeting the MCV DNA and tested their targeting efficiency, and sensitivity using a fluorophore quencher labeled reporter assay. We show that this sophisticated MCV DETECTR system can detect MCV integrated in Merkel tumour rapidly, specifically and with femto-molar sensitivity. This new MCV DNA detecting system is promising and we hope it can be coupled with histopathological and immunohistochemical studies to diagnose the viral status of MCC in clinics in the near future.

molecular biology↗

bioRxiv2019

Spontaneous oligomerization of BAK/BAX is suppressed by hetero-dimerization with MCL-1

BCL-2 proteins control the intrinsic pathway of programmed cell death. Composed of anti- and pro-apoptotic members, their network of interactions forms a molecular switch that controls mitochondrial outer-membrane permeability. Apoptotic stimulation leads to BAK/BAX oligomerization and pore formation, yet the molecular details of this pivotal step remain poorly understood, and controversy persists regarding the activation mechanism. Here we use native mass spectrometry and kinetics to show that the homo-oligomerization of BAK and BAX is spontaneous in hydrophobic environments. This process is abrogated by hetero-dimerization of both BAK and BAX with the anti-apoptotic BCL-2 protein MCL-1. Pro-apoptotic BH3-only proteins disrupt these hetero-dimers by binding competitively to MCL-1, releasing BAK/BAX for homo-oligomerization. Thus, we infer that their oligomeric states are thermodynamically favored at the membrane. Our approach provides the framework for future quantitative biophysical characterizations of the BCL-2 network, and advances our molecular understanding of apoptosis.

biophysics↗

bioRxiv2019

Loss of function from widely distributed, synonymous mutations at single codons

Mutational tolerance inferred from laboratory-based mutational studies is typically much higher than observed natural sequence variation. Using saturation mutagenesis, we show that the ccdA antitoxin component of the ccdAB toxin-antitoxin system is unusually sensitive to mutation with over 60% of mutations leading to loss of function. Multi-base synonymous mutations at a codon display enhanced propensity to show altered phenotypes, relative to single-base ones. Such mutations modulate RNA structure, leading to altered relative translation efficiencies of the two genes in the operon, and a CcdA:CcdB protein ratio below one. These insights were used to predict and experimentally validate synonymous mutations that lead to loss of function in the unrelated relBE operon as well as the lacZ gene. Thus, synonymous mutations can have significant phenotypic effects, in the absence of overexpression or extraneous reporters. More generally, proteins are likely more sensitive to mutation than inferred from previous saturation mutagenesis studies.

molecular biology↗

bioRxiv2019

Structural characterization of tau in fuzzy tau:tubulin complexes

Tau is a neuronal microtubule (MT) associated protein of significant interest due to its association with several neurodegenerative disorders. Taus intrinsic disorder and the dynamic nature of its interactions with tubulin and MTs make its structural characterization challenging. Here we use an environmentally sensitive fluorophore as a site-specific probe of tau bound to soluble tubulin. Comparison of our results with recently published tau:MT cryo-EM model reveals structural similarities between tubulin- and MT-bound tau. Analysis of residues across the repeat regions reveal a hierarchy in tubulin occupancy, which may be relevant to taus ability to differentiate between tubulin and MTs. As binding to soluble tubulin is a critical first step in MT polymerization, our characterization of the structural features of tau in dynamic, fuzzy tau:tubulin assemblies advances our understanding of how tau functions in the cell and how function may be disrupted in disease.

biophysics↗

bioRxiv2019

Post-ictal neuronal network remodeling and Wnt pathway dysregulation in the intra-hippocampal kainate mouse model of temporal lobe epilepsy

Mouse models of mesial temporal lobe epilepsy recapitulate aspects of human epilepsy, which is characterized by neuronal network remodeling in the hippocampal dentate gyrus. Observational studies suggest that this remodeling is associated with altered Wnt pathway signaling, although this has not been experimentally examined. We used the well-characterized mouse intrahippocampal kainate model of temporal lobe epilepsy to examine associations between post-seizure hippocampal neurogenesis and altered Wnt signaling. Tissue was analyzed using immunohistochemistry and confocal microscopy, and transcriptome analysis was performed on RNA extracted from anatomically micro-dissected dentate gyri. Seizures increased neurogenesis and dendritic arborization of newborn hippocampal dentate granule cells in peri-ictal regions, and decreased neurogenesis in the ictal zone, 2-weeks after kainate injection. Interestingly, administration of the novel canonical Wnt pathway inhibitor XAV939 daily for 2-weeks after kainate injection further increased dendritic arborization in peri-ictal regions after seizure, without an effect on baseline neurogenesis in control animals. Transcriptome analysis of dentate gyri demonstrated significant canonical Wnt gene dysregulation in kainate-injected mice across all regions for Wnt3, 5a and 9a. Intriguingly, certain Wnt genes demonstrated differential patterns of dysregulation between the ictal and peri-ictal zones, most notably Wnt5B, 7B and DKK-1. Together, these results demonstrate regional variation in Wnt pathway dysregulation in the early post-ictal period, and surprisingly, suggest that some Wnt-mediated effects might actually temper aberrant neurogenesis after seizures. The Wnt pathway may therefore provide suitable targets for novel therapies that prevent network remodeling and the development of epileptic foci in high-risk patients.

neuroscience↗

bioRxiv2019

A smart polymer for sequence-selective binding, pulldown and release of DNA targets

Selective isolation of DNA is crucial for applications in biology, bionanotechnology, clinical diagnostics and forensics. We herein report a smart methanol-responsive polymer (MeRPy) that can be programmed to bind and separate single- as well as double-stranded DNA targets. Captured targets are quickly isolated and released back into solution by denaturation (sequence-agnostic) or toehold-mediated strand displacement (sequence-selective). The latter mode allows 99.8% efficient removal of unwanted sequences and 79% recovery of highly pure target sequences. We applied MeRPy for the depletion of insulin, glucagon, and transthyretin cDNA from clinical next-generation sequencing (NGS) libraries. This step improved data quality for low-abundance transcripts in expression profiles of pancreatic tissues. Its low cost, scalability, high stability and ease of use make MeRPy suitable for diverse applications in research and clinical laboratories, including enhancement of NGS libraries, extraction of DNA from biological samples, preparative-scale DNA isolations, and sorting of DNA-labeled non-nucleic acid targets.

molecular biology↗

bioRxiv2019

The Evolution of Placental Invasion and Cancer Metastasis are Causally Linked

Among mammals, the extent of placental invasion is correlated with vulnerability to malignancy. Animals with more invasive placentation (e.g. humans) are more vulnerable to malignancy, whereas animals with a non-invasive placenta (e.g. ruminants) are less likely to develop malignant cancer. To explain this correlation, we propose the hypothesis of Evolved Levels of Invasibility (ELI) positing that the permissiveness of stromal tissue to invasion is a unitary character affecting both placental and cancer invasion. We provide evidence for this hypothesis by contrasting invasion of human and bovine cancer and placental cells into a lawn of stromal cells from different species. We find that both bovine endometrial and skin fibroblasts are more resistant to invasion of placental and cancer cells than their human counterparts. Gene expression profiling identified genes with high expression in human but not bovine fibroblasts. Knocking down of a subset of them in human fibroblasts leads to significantly stronger resistance to cancer cell invasion. Comparative analysis of gene expression among mammals suggests that humans evolved higher vulnerability to malignancy than the eutherian ancestor, possibly as a correlate of more invasive placentation, and boroeutherians evolved to decrease stromal invasibility. Identifying the evolutionary determinants of stromal invasibility can provide significant insights to develop rational anti-metastatic therapeutics.

cancer biology↗

bioRxiv2018

Non-Communicable Diseases and their determinants: A Cross-sectional State-Wide STEPS Survey, Haryana, North India

BackgroundRecent studies have documented high variation in epidemiologic transition levels among Indian states with noncommunicable disease epidemic rising swiftly. However, the estimates suffer from non-availability of reliable data for NCDs from sub populations. In order to fill the knowledge gap, the distribution and determinants of NCD risk factors were studied along with awareness, treatment and control of NCDs among the adult population in Haryana, India.\n\nMethodsNCD risk factors survey was conducted among 5078 residents, aged 18-69 years during 2016-17. Behavioural risk factors were assessed using STEPS instrument, administered through an android software (mSTEPS). This was followed by physical measurements using standard protocols. Finally, biological risk factors were determined through the analysis of serum and urine samples.\n\nResultsMales were found to be consuming tobacco and alcohol at higher rates of 38.9% (95% CI: 35.3-42.4) and 18.8% (95% CI: 15.8-21.8). One-tenth (11%) (95% CI: 8.6-13.4) of the respondents did not meet the specified WHO recommendations for physical activity for health. Around 35.2% (95%CI: 32.6-37.7) were overweight or obese. Hypertension and diabetes were prevalent at 26.2% (95% CI: 24.6-27.8) and 15.5% (95% CI: 11.0-20.0). 91.3% (95% CI: 89.3-93.3) of the population had higher salt intake than recommended 5gms per day.\n\nConclusionThe documentation of strikingly high and uniform distribution of different NCDs and their risk factors in state warrants urgent need for evidence based interventions and advocacy of policy measures.

epidemiology↗

bioRxiv2018

Sequential digestion with Trypsin and Elastase in cross-linking/mass spectrometry

Cross-linking/mass spectrometry has become an important approach for studying protein structures and protein-protein interactions. The amino acid composition of some protein regions impedes the detection of cross-linked residues, although it would yield invaluable information for protein modelling. Here, we report on a sequential digestion strategy with trypsin and elastase to penetrate regions with a low density of trypsin cleavage sites. We exploited intrinsic substrate recognition properties of elastase to specifically target larger tryptic peptides. Our application of this protocol to the TAF4-12 complex allowed us to identify cross-links in previously inaccessible regions.

biochemistry↗

bioRxiv2018

Bile canaliculi contract autonomously by releasing calcium into hepatocytes via mechanosensitive calcium channel

Bile canaliculi (BC) are the smallest vessels of the biliary tree. They are formed from the apical surfaces of adjoining hepatocytes, resulting in lumenal conduits for bile flow. Bile is propelled along the BC by hepatocyte contractions that arise from cyclic waves of apico-basal Ca2+, but the source and regulation of Ca2+ has been unclear. We report that BC contraction correlates with cyclic transfer of Ca2+ from BC lumen to apico-basal Ca2+ waves in adjacent hepatocytes, and does not correlate with endoplasmic reticulum Ca2+. BC contractility was triggered by ionophore A23187 and unaffected by Thapsigargin. The cycles of Ca2+ transfer could be blocked by the mechanosensitive calcium channel inhibitor GsMTx-4, resulting in cholestatic generation of BC-derived vesicles. The mechanosensitive calcium channel Piezo-1 is preferentially localized at BC membranes, and its hyper-activation by Yoda1 causes increased Ca2+ transfer and increased BC contractility. We propose that canaliculi achieve biomechanical homeostasis through the following feedback system: the pressure of accumulated bile is sensed by mechanosensitive channel, which transmit biliary calcium into adjacent hepatocytes for contraction of the BC lumen and propulsion of the bile.

cell biology↗

bioRxiv2018

Physics of lumen growth

We model the dynamics of formation of intercellular secretory lumens. Using conservation laws, we quantitatively study the balance between paracellular leaks and the build-up of osmotic pressure in the lumen. Our model predicts a critical pumping threshold to expand stable lumens. Consistently with experimental observations in bile canaliculi, the model also describes a transition between a monotonous and oscillatory regime during luminogenesis as a function of ion and water transport parameters. We finally discuss the possible importance of regulation of paracellular leaks in intercellular tubulogenesis.

biophysics↗

bioRxiv2017

Architecture of TAF11/TAF13/TBP complex suggests novel regulatory state in General Transcription Factor TFIID function

General transcription factor TFIID is a key component of RNA polymerase II transcription initiation. Human TFIID is a megadalton-sized complex comprising TATA-binding protein (TBP) and 13 TBP-associated factors (TAFs). TBP binds to core promoter DNA, recognizing the TATA-box. We identified a ternary complex formed by TBP and the histone fold (HF) domain-containing TFIID subunits TAF11 and TAF13. We demonstrate that TAF11/TAF13 competes for TBP binding with TATA-box DNA, and also with the N-terminal domain of TAF1 previously implicated in TATA-box mimicry. In an integrative approach combining crystal coordinates, biochemical analyses and data from cross-linking mass-spectrometry (CLMS), we determine the architecture of the TAF11/TAF13/TBP complex, revealing TAF11/TAF13 interaction with the DNA binding surface of TBP. We identify a highly conserved C-terminal TBP-binding domain (CTID) in TAF13 which is essential for supporting cell growth. Our results thus have implications for cellular TFIID assembly and suggest a novel regulatory state for TFIID function.

biochemistry↗