Biology subjects

Ghosh, B.

Publications and source records attributed to Ghosh, B..

5 recordsLinked to original sources

bioRxiv2018

A pipeline for rapidly generating genetically engineered mouse models of pancreatic cancer using in vivo CRISPR-Cas9 mediated somatic recombination

Genetically engineered mouse models (GEMMs) that recapitulate the major genetic drivers in pancreatic ductal adenocarcinoma (PDAC) have provided unprecedented insights into the pathogenesis of this lethal neoplasm. Nonetheless, generating an autochthonous model is an expensive, time consuming and labor intensive process, particularly when tissue specific expression or deletion of compound alleles are involved. In addition, many of the current PDAC GEMMs cause embryonic, pancreas-wide activation or loss of driver alleles, neither of which reflects the cognate human disease scenario. The advent of CRISPR/Cas9 based gene editing can potentially circumvent many of the aforementioned shortcomings of conventional breeding schema, but ensuring the efficiency of gene editing in vivo remains a challenge. Here we have developed a pipeline for generating PDAC GEMMs of complex genotypes with high efficiency using a single \"workhorse\" mouse strain expressing Cas9 in the adult pancreas under a p48 promoter. Using adeno-associated virus (AAV) mediated delivery of multiplexed guide RNAs (sgRNAs) to the adult murine pancreas of p48-Cre; LSL-Cas9 mice, we confirm our ability to express an oncogenic Kras G12D allele through homology-directed repair (HDR), in conjunction with CRISPR-induced disruption of cooperating alleles (Trp53, Lkb1 and Arid1A). The resulting GEMMs demonstrate a spectrum of precursor lesions (pancreatic intraepithelial neoplasia [PanIN] or Intraductal papillary mucinous neoplasm [IPMN] with eventual progression to PDAC. Next generation sequencing of the resulting murine PDAC confirms HDR of oncogenic KrasG12D allele at the endogenous locus, and insertion deletion (\"indel\") and frameshift mutations of targeted tumor suppressor alleles. By using a single \"workhorse\" mouse strain and optimal AAV serotype for in vivo gene editing with combination of driver alleles, we have created a facile autochthonous platform for interrogation of the PDAC genome.

cancer biology↗

bioRxiv2018

Simvastatin mediates inhibition of exosome synthesis, localization, and secretion via multicomponent interventions.

Discovery of exosomes as modulator of cellular communication has added a new dimension to our understanding of biological processes. Exosomes influence the biological systems by mediating trans-communication across tissues and cells, which has important implication for health and disease. Identification of strategies for exosome modulation may pave the way towards better understanding of exosome biology and development of novel therapeutics. In absence of well-characterized modulators of exosome biogenesis, an alternative option is to target pathways generating important exosomal components. Cholesterol represents one such essential component required for exosomal biogenesis. We initiated this study to test the hypothesis that owing to its cholesterol lowering effect, simvastatin, a HMG CoA inhibitor, might be able to alter exosome formation and secretion. Using previously established protocols for detecting secreted exosomes in biological fluids, simvastatin was tested for its effect on exosome secretion under various in-vitro and in-vivo settings. Murine model of AAI was used for further validation of our findings. Utilizing aforementioned systems, we demonstrate exosome-lowering potential of simvastatin in various in-vivo and in-vitro models, of AAI and atherosclerosis. We believe that the knowledge acquired in this study holds potential for extension to other exosome dominated pathologies and model systems.

cell biology↗

bioRxiv2018

GnasR201C Induces Murine Pancreatic Cystic Neoplasms through Suppression of YAP1 Signaling and Transcriptional Reprogramming

Background & AimsSomatic \"hotspot\" mutations of GNAS, which encodes for the alpha subunit of stimulatory G-protein, are present in ~60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. There are currently no cognate animal models that recapitulate the biology of mutant Gnas-induced IPMNs, and the underlying mechanisms that lead to the cystic pathway of neoplasia in the pancreas remain unknown.\n\nMethodsWe generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras; Gnas mice) where pancreas-specific GnasR201C expression was induced by doxycycline administration. In this model, mutant Kras is constitutively expressed, and control mice were produced through absence of doxycycline. Separate cohorts of mice were utilized for timed necropsies and for Kaplan-Meier survival analysis. Isogenic cell lines (with doxycycline inducible mutant Gnas expression) were propagated from the resulting pancreatic ductal adenocarcinoma (PDAC).\n\nResultsCo-expression of KrasG12D and GnasR201C resulted in the development of pancreatic cystic lesions resembling human IPMNs in 100% of mice, with higher grades of epithelial dysplasia observed over time. Approximately one-third of Kras; Gnas mice developed PDAC at a median of 38 weeks post doxycycline induction. GnasR201C did not accelerate oncogenic transformation with KrasG12D, but rather, reprogrammed Ras-induced neoplasms towards a well-differentiated phenotype. GnasR201C induction led to activation of the inhibitory Hippo kinase cascade and cytoplasmic sequestration of phosphorylated YAP1 protein, a phenomenon that was also observed in human IPMN with GNAS mutations.\n\nConclusionsGNASR201C functions not as a traditional oncogene, but rather as an \"oncomodulator\" of KRAS-mediated pancreatic neoplasia, through suppression of YAP1 and transcriptional reprogramming towards a differentiated (large ductal) phenotype.

cancer biology↗

bioRxiv2017

Optical Coherence Tomography Reveals Mechanobiologically Stable Self-Organizing Di-Fork Architecture of Mice Cutaneous Scars

Scientific studies report crucial impacts of biomechanical effectors to modulate wound healing either by scarring or regeneration. Further, the biological decision to predominantly favor the former is still cryptic. Real-time visualization of biomechanical manifestations in situ in scarring is hence necessary. Endorsed by nanostructural testing, synthetic phantom analysis, and computational simulations, we found strong mechanobiological correlates for Swept Source Optical Coherence Tomography (SS-OCT) speckles in mice cutaneous repair (full-thickness) up to 10 months. The theoretical basis of the optomechanics to provide insights into scar form-factor and evolution is proposed. Optomechanical changes have been considered as the resultant of intrinsic (e.g. fiber elastic modulus) and gross tissue mechanics (extracellular matrix (ECM)) in maturing scars. Non-invasive optomechanics supported with microscopic findings reveal scars cross-sectional self-organizing di-fork architecture. Dual-compartment heterogeneity of di-fork exhibits stress-evading features with a dichotomy in inhabitant cellular stress-fiber distributions. This differential interactivity of scar with adjoining tissues reflects its architectural intelligence to compensate tissue loss (hypodermis/muscle) by assembling into a di-fork. Gradual establishment of baseline shifted lasting mechanobiological steady-state, later in scarring, expose scar as an alternate stable state within the skin. Significance StatementWound repair in mammals, predominantly culminates into function compromising scar that is occasionally fatal in vital organs. How the biological system often adopts scarring over a restorative regeneration is yet a conundrum. Wound and ambient mechanics play a pivotal role in deciding the healing fate. SS-OCT is hence demonstrated here as a non-invasive window to such mechanical manifestations during skin wound healing. This exposed gradual emergence of temporally maintained and stress-resilient di-fork architecture of the scar with differential neighborhood interfaces. Accommodation of such an alternate self-organizing steady-state of scar sheds light on its sustenance and paradoxical selection.

biophysics↗

bioRxiv2017

Comparative Analysis of Non-linear Behaviour with Power Spectral Intensity Response Between Normal and Epileptic EEG Signals

Epilepsy is a neurological condition which affects the nervous system. It is a general term used for a group of disorders in which nerve cells of the brain discharge anomalous electrical impulses from time to time, causing a temporary malfunction of the other nerve cells of the brain. EEG signal provides an important cue for diagnosis and interpretation related to prognosis of epilepsy. In this work we envisage to provide novel tool which can be used to detect the prognosis of epileptic disorder by comparing linear and nonlinear modalities of EEG analysis conventionally used Power spectral analysis and a robust non linear method, Detrended Fluctuation Analysis (DFA). Publicly available dataset is used for this work consisting of 100 normal patients EEG data as control group and 100 epileptic patients EEG data for comparison. Response for different frequency bands (alpha, theta, beta) of the EEG spectrum have been analyzed using Detrended Fluctuation Analysis (DFA) and Power Spectral Intensity (PSI). The comparison of the DFA scaling exponent with the spectral power data is calculated for all the 3 different frequency bands of EEG signal provide new and interesting results which have been discussed in detail.

neuroscience↗