Biology subjects

Mukherjee, D.

Publications and source records attributed to Mukherjee, D..

52 records · Page 3Linked to original sources

bioRxiv2022

Increased CaMKK2 expression is an adaptive response that maintains the fitness of tumor-infiltrating natural killer cells

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a key regulator of energy homeostasis in several cell types. Expression of this enzyme in tumor cells promotes proliferation and migration, and expression in tumor-associated immune cells facilitates M2 macrophage polarization and the development of myeloid-derived suppressor cells. Thus, there has been considerable interest in developing CaMKK2 inhibitors as potential cancer therapeutics. However, the roles of CaMKK2 in other cellular compartments within the tumor immune environment remain to be established, an impediment to the clinical development of these agents. We report that CaMKK2 is expressed at low basal levels in natural killer (NK) cells but is significantly upregulated in tumor-infiltrating NK cells where it suppresses apoptosis and promotes proliferation. It was further demonstrated that NK cell-intrinsic deletion of CaMKK2 increased metastatic progression across several murine models, establishing a critical role for this enzyme in NK cell tumor immunity. Interestingly, ablation of the CaMKK2 protein, but not inhibition of its kinase activity, resulted in decreased NK cell survival. These results indicate an important scaffold function for CaMKK2 in NK cells and suggest that competitive CaMKK2 inhibitors and ligand-directed degraders (LDDs) are likely to have distinct therapeutic utilities. Finally, we determined that intracellular lactic acid is a key driver of CaMKK2 expression, suggesting that upregulated expression of this enzyme is an adaptive mechanism by which tumor-infiltrating NK cells mitigate the deleterious effects of a lactate-rich tumor environment. The findings of this study should inform strategies to manipulate the CaMKK2 signaling axis as a therapeutic approach in cancer.

cancer biology↗

bioRxiv2022

Deep Learning on Multimodal Chemical and Whole Slide Imaging Data for Predicting Prostate Cancer Directly from Tissue Images

Prostate cancer is one of the most common cancers globally and is the second most common cancer in the male population in the US. Here we develop a study based on correlating the H&E-stained biopsy data with MALDI mass-spectrometric imaging of the corresponding tissue to determine the cancerous regions and their unique chemical signatures, and variation of the predicted regions with original pathological annotations. We spatially register features obtained through deep learning from high-resolution optical micrographs of whole slide H&E stained data with MSI data to correlate the chemical signature with the tissue anatomy of the data, and then use the learned correlation to predict prostate cancer from observed H&E images using trained co-registered MSI data. We found that this system is more robust than predicting from a single imaging modality and can predict cancerous regions with[~] 80% accuracy. Two chemical biomarkers were also found to be predicting the ground truth cancerous regions. This will improve on generating patient treatment trajectories by more accurately predicting prostate cancer directly from H&E-stained biopsy images.

cancer biology↗

bioRxiv2022

Outer membrane protein A (OmpA) deficient Salmonella Typhimurium displays enhanced susceptibility towards β-lactam antibiotics: third-generation cephalosporins (ceftazidime) and carbapenems (meropenem)

The invasive non-typhoidal serovar of Salmonella enterica, namely Salmonella Typhimurium ST313, causes bloodstream infection in sub-Saharan Africa. Like other bacterial pathogens, the development of antimicrobial resistance is a severe problem in curing non-typhoidal Salmonella infection. In this work, we have investigated the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD, and OmpF, in resistance against broad-spectrum {beta}-lactam antibiotics-ceftazidime and meropenem. We found that deleting OmpA from Salmonella makes the bacteria susceptible to {beta}-lactam drugs. The MIC for both the antibiotics reduced significantly for STM{Delta} ompA compared to the wild-type and the ompA complemented strains. Despite the presence of antibiotics, the uninterrupted growth of STM{Delta} ompC,{Delta} ompD, and{Delta} ompF endorsed the dispensability of these three porins in antimicrobial resistance. The {beta}-lactam antibiotics caused massive depolarization in the outer membrane of the bacteria in the absence of OmpA. We have proved that none of the extracellular loops but the complete structure of perfectly folded OmpA is required by the bacteria for developing antimicrobial resistance. Our data revealed that STM{Delta} ompA consumed more antibiotics than the wild-type and the complemented strain, resulting in severe damage of the bacterial outer membrane and subsequent killing of the pathogen by antibiotic-mediated oxidative stress. Upon deleting ompA, the steady decrease in the relative proportion of antibiotic-resistant persisters and the clearance of the STM{Delta} ompA from the liver and spleen of C57BL/6 mice upon treatment with ceftazidime proved the role of OmpA in rendering protection against {beta}-lactam antibiotics.

microbiology↗

bioRxiv2021

Structural remodeling of SARS-CoV-2 spike protein glycans reveals the regulatory roles in receptor binding affinity

Glycans of the SARS-CoV-2 spike protein are speculated to play functional roles in the infection processes as they extensively cover the protein surface and are highly conserved across the variants. To date, the spike protein has become the principal target for vaccine and therapeutic development while the exact effects of its glycosylation remain elusive. Experimental reports have described the heterogeneity of the spike protein glycosylation profile. Subsequent molecular simulation studies provided a knowledge basis of the glycan functions. However, there are no studies to date on the role of discrete glycoforms on the spike protein pathobiology. Building an understanding of its role in SARS-CoV-2 is important as we continue to develop effective medicines and vaccines to combat the disease. Herein, we used designed combinations of glycoengineering enzymes to simplify and control the glycosylation profile of the spike protein receptor-binding domain (RBD). Measurements of the receptor binding affinity revealed the regulatory effects of the RBD glycans. Remarkably, opposite effects were observed from differently remodeled glycans, which presents a potential strategy for modulating the spike protein behaviors through glycoengineering. Moreover, we found that the reported anti-SARS-CoV-(2) antibody, S309, neutralizes the impact of different RBD glycoforms on the receptor binding affinity. Overall, this work reports the regulatory roles that glycosylation plays in the interaction between the viral spike protein and host receptor, providing new insights into the nature of SARS-CoV-2. Beyond this study, enzymatic remodeling of glycosylation offers the opportunity to understand the fundamental role of specific glycoforms on glycoconjugates across molecular biology. Covert art LegendsThe glycosylation of the SARS-CoV-2 spike protein receptor-binding domain has regulatory effects on the receptor binding affinity. Sialylation or not determines the "stabilizing" or "destabilizing" effect of the glycans. (Protein structure model is adapted from Protein Data Bank: 6moj. The original model does not contain the glycan structure.) SignificanceGlycans extensively cover the surface of SARS-CoV-2 spike (S) protein but the relationships between the glycan structures and the protein pathological behaviors remain elusive. Herein, we simplified and harmonized the glycan structures in the S protein receptor-binding domain and reported their regulatory roles in human receptor interaction. Opposite regulatory effects were observed and were determined by discrete glycan structures, which can be neutralized by the reported S309 antibody binding to the S protein. This report provides new insight into the mechanism of SARS-CoV-2 S protein infection as well as S309 neutralization.

biochemistry↗

bioRxiv2021

Essential Loop Dynamics Modulates Catalytic Activity in α-Chymotrypsin

Conformational dynamics of macromolecules including enzymes are essential for their function. The present work reports the role of essential dynamics in alpha-chymotrypsin (CHT) which correlates with its catalytic activity. Detailed optical spectroscopy and classical molecular dynamics (MD) simulation were used to study thermal stability, catalytic activity and dynamical flexibility of the enzyme. The study of the enzyme kinetics reveals an optimum catalytic efficiency at 308K. Polarization gated fluorescence anisotropy with 8-anilino-1-napthelene sulfonate (ANS) have indicated increasing flexibility of the enzyme with an increase in temperature. Examination of the structure of CHT reveal the presence of five loop regions (LRs) around the catalytic S1 pocket. MD simulations have indicated that flexibility increases concurrently with temperature which decreases beyond optimum temperature. Principal component analysis (PCA) of the eigenvectors manifests essential dynamics and gatekeeping role of the five LRs surrounding the catalytic pocket which controls the enzyme activity.

biophysics↗

bioRxiv2021

A Combined Spectroscopic and Molecular Modeling Study on Structure-Function-Dynamics under Chemical Modification: Alpha-Chymotrypsin with Formalin Preservative

Enzyme conformations can be altered via modification of its amino acid residues, side chains and large-scale domain modifications, which are closely linked to its function. Herein, we have addressed the role of residue modification in catalytic activity and molecular recognition of an enzyme alpha-chymotrypsin (CHT) in presence of covalent cross-linker formalin. Optical spectroscopy studies exhibit reduced catalytic activity of the enzyme with increased formalin concentration. Polarization gated anisotropy studies of a fluorophore 8-anilino-1- napthelenesulfonic acid (ANS) in CHT show a dip rise pattern in presence of formalin which is consistent with the generation of multiple ANS binding sites in the enzyme owing to modifications of its local amino acid residues. Molecular docking study on minimal local residue modifications in CHT reveals formation of a stable enzyme-substrate complex even with the serine-histidine cross-linked enzyme which prohibits product formation giving rise to reduced catalytic activity.

biophysics↗

bioRxiv2021

Targeting "Immunogenic Hotspots" in Dengue and Zika Virus: A Novel Approach to a Common Vaccine

Diseases caused by Dengue (DENV) and Zika (ZIKV) viruses cause significant mortality and illness globally. Due to the high sequence similarity of the viral proteins and the purported cross-reactive immune responses against the viruses, we envisioned a common multi-epitope vaccine (MEV) against both viruses by adopting a novel approach of identifying "immunogenic hotspots". These stretches of the structural and non-structural proteins are enriched with MHC class I and class II supertype-restricted T cell epitopes, and B cell epitopes, in addition to being highly conserved between different DENV serotypes and ZIKV. Such an approach ensures inclusion of multiple overlapping T and B cell epitopes common to both viruses, and also warrants high population coverage. Importantly, epitopes known to cause antibody-dependent-enhancement of infection have been excluded. These immunogenic hotspots have then been stitched together with linkers in-silico along with an adjuvant, CTxB to develop the MEV candidate. Four structural models of the MEV were selected on the basis of conformational preservation of CTxB, and their biophysical parameters, which also conserved the immunogenicity of the multiple epitopes. Importantly, each of the MEV candidates were found to interact with TLR4-MD2 complex by molecular docking studies, indicative of their ability to induce TLR-mediated immune responses.

immunology↗

bioRxiv2021

Microstructure Aware Modeling Of Biochemical Transport In Arterial Blood Clots

Flow-mediated transport of biochemical species is central to thrombotic phenomena. Comprehensive three-dimensional modeling of flow-mediated transport around realistic macroscale thrombi poses challenges owing to their arbitrary heterogeneous microstructure. Here, we develop a microstructure aware model for species transport within and around a macroscale thrombus by devising a custom preconditioned fictitious domain formulation for thrombus-hemodynamics interactions, and coupling it with a fictitious domain advection-diffusion formulation for transport. Microstructural heterogeneities are accounted through a hybrid discrete particle-continuum approach for the thrombus interior. We present systematic numerical investigations on unsteady arterial flow within and around a three-dimensional macroscale thrombus; demonstrate the formation of coherent flow structures around the thrombus which organize advective transport; illustrate the role of the permeation processes at the thrombus boundary and subsequent intra-thrombus transport; and characterize species transport from bulk flow to the thrombus boundary and vice versa.

physiology↗

bioRxiv2020

Egr2 induction in Drd1+ ensembles of the ventrolateral striatum supports the development of cocaine reward

Drug addiction develops due to brain-wide plasticity within neuronal ensembles, mediated by dynamic gene expression. Though the most common approach to identify such ensembles relies on immediate early gene expression, little is known of how the activity of these genes is linked to modified behavior observed following repeated drug exposure. To address this gap, we present a broad-to-specific approach, beginning with a comprehensive investigation of brain-wide cocaine-driven gene expression, through the description of dynamic spatial patterns of gene induction in subregions of the striatum, and finally address functionality of region-specific gene induction in the development of cocaine preference. Our findings reveal differential cell-type specific dynamic transcriptional recruitment patterns within two subdomains of the dorsal striatum following repeated cocaine exposure. Furthermore, we demonstrate that induction of the IEG Egr2 in the ventrolateral striatum, as well as the cells within which it is expressed, are required for the development of cocaine seeking. Impact statementVLS ensembles are dynamically recruited by cocaine experiences to mediate cocaine reward.

neuroscience↗

bioRxiv2020

Computational Investigation Of Blood Flow And Flow-mediated Transport In Arterial Thrombus Neighborhood

A pathologically formed blood clot or thrombus is central to major cardiovascular diseases like heart attack and stroke. Detailed quantitative evaluation of flow and flow-mediated transport processes in the thrombus neighborhood within large artery hemodynamics is crucial for understanding disease progression and assessing treatment efficacy. This, however, remains a challenging task owing to the complexity of pulsatile viscous flow interactions with arbitrary shape and heterogeneous microstructure of realistic thrombi. Here, we address this challenge by conducting a systematic parametric simulation based study on characterizing unsteady hemodynamics and flow-mediated transport in the neighborhood of an arterial thrombus. We use a hybrid particle-continuum based finite element approach to handle arbitrary thrombus shape and microstructural variations. Results from a cohort of 50 different unsteady flow scenarios are presented, including unsteady vortical structures, pressure-gradient across the thrombus boundary, finite time Lyapunov exponents, and dynamic coherent structures that organize advective transport. We clearly illustrate the combined influence of three key parameters - thrombus shape, microstructure, and extent of wall disease - in terms of: (a) determining hemodynamic features in the thrombus neighborhood; and (b) governing the balance between advection, permeation, and diffusion to regulate transport processes in the thrombus neighborhood.

biophysics↗

bioRxiv2020

Development of a novel allograft model of prostate cancer: a new tool to inform clinical translation

BackgroundAllograft models enable characterisation of genomic drivers and treatment responses by modelling immune and micro-environmental changes more accurately than xenografts. Despite this, few models are available to the prostate cancer (PCa) community. This study presents a novel allograft model of high-risk, localised PCa. In characterising this model we have focused on its response to radiotherapy (RT). MethodsThe DVL3 cell line was derived from a transgenic trp53-/-/Pten-/- mouse model and characterised both in vitro and in vivo. The DVL3 cells were allografted and response to RT was investigated and compared to the TRAMP-C1 model. Extensive tumour profiling in the DVL3 model was performed using flow cytometry, immunohistochemistry and RNA-seq. ResultsIn vitro the DVL3 cells expressed basal and luminal markers. DVL3 cells formed tumours with distinct glandular morphology which expressed androgen receptor, similar to human localised PC. DVL3 tumour growth was delayed following administration of fractionated RT, with infiltration of myeloid derived suppressor cells (MDSC). ConclusionsThe DVL3 allograft model represents substantial progress in PCa modelling, displaying; luminal differentiation, strong AR expression, and an immunosuppressive microenvironment, similar to observations in high-risk PCa patients. This model is ideally suited for development and validation of novel therapeutics, in particular immune-modulatory agents in combination with RT.

cancer biology↗

bioRxiv2018

Quantitative modelling explains distinct STAT1 and STAT3 activation dynamics in response to both IFNγ and IL-10 stimuli and predicts emergence of reciprocal signalling at the level of single cells

IFN{gamma} and IL10 pathways elicit functionally opposing cell fates, however, these two stimuli activate common transcription factors like STAT1 and STAT3 (S/1/3). How the two stimuli regulate the dynamics of S/1/3 activation remains less understood. Here, we experimentally measured the signaling dynamics of S/1/3 in response to IFN{gamma} and IL10 and found that STAT3, in particular, exhibits a bell-shaped response to both stimuli with maximal activation in an intermediate dose. We built a mathematical model, which quantitatively captured the S/1/3 dynamics and by model analysis, we identified the primary regulators controlling the bell-shaped STAT3 responses in both pathways. As the STATs are activated in response to both stimuli, in a scenario when cells are subjected to co-stimulation of IFN{gamma} and IL10, an interpathway competition to activate the common substrate is plausible. Additionally, a strong transcriptional induction of SOCS1 (a negative regulator of IFN{gamma} pathway) was observed upon IL10 stimulation, suggesting that IL10 pathway can potentially inhibit the IFN{gamma} pathway via SOCS1 induction. To quantitatively understand the S/1/3 responses to co-stimulation we next simulated the same which predicted STAT3 activation and SOCS1 induction dynamics would robustly remain IL10 driven. Subsequent experiments validated the model predictions. Further, the model analysis identified the primary regulators controlling the robustness of IL10-STAT3 axis in co-stimulation. Together, our data-driven model quantitatively captured the dynamics of the STATs in response to both pro (IFN{gamma}) and anti (IL10)-inflammatory stimuli and identifies distinct regulatory elements controlling STAT3 signaling in individual and co-stimulation conditions.

systems biology↗

bioRxiv2018

Its not you, its me: Corollary discharge in precerebellar nuclei of sleeping infant rats

In week-old rats, somatosensory input arises predominantly from stimuli in the external environment or from sensory feedback associated with myoclonic twitches during active (REM) sleep. A previous study of neural activity in cerebellar cortex raised the possibility that the brainstem motor structures that produce twitches also send copies of motor commands (or corollary discharge, CD) to the cerebellum. Here, by recording from two precerebellar nuclei--the inferior olive and lateral reticular nucleus--we demonstrate that CD does indeed accompany the production of twitches. Within both structures, the CD signal comprises a surprisingly sharp activity peak within 10 ms of twitch onset. In the inferior olive, this sharp peak is attributable to the opening of slow potassium channels. We conclude that a diversity of neural activity is conveyed to the developing cerebellum preferentially during sleep-related twitching, enabling cerebellar processing of convergent input from CD and reafferent signals.

neuroscience↗

bioRxiv2017

Salient Experiences are Represented by Unique Transcriptional Signatures in the Brain

Inducible transcription is essential for consolidation of salient experiences into long-term memory. However, the question of whether inducible transcription relays information representing the identity of the experience being encoded, has not been explored. To this end, we have analyzed transcription across multiple brain regions, induced by a variety of rewarding and aversive experiences. Our results define robust transcriptional signatures uniquely characterizing individual salient experiences. A subset of these induced transcriptional markers suffice for near-perfect decoding of the identity of recent experiences at the level of individual mice. Furthermore, experiences with shared attributes display commonalities in their transcriptional representation, exemplified in the representation of valence, habituation and reinforcement. Taken together, our results demonstrate the existence of a neural transcriptional code that represents the encoding of experiences in the mouse brain. This code is comprised of distinct transcriptional signatures that correlate to the affective attributes of the experiences that are being encoded.

neuroscience↗

bioRxiv2017

The Role Of Circle Of Willis Anatomy Variations In Cardio-embolic Stroke - A Patient-specific Simulation Based Study.

We describe a patient-specific simulation based investigation on the role of Circle of Willis anatomy in cardioembolic stroke. Our simulation framework consists of medical image-driven modeling of patient anatomy including the Circle, 3D blood flow simulation through patient vasculature, embolus transport modeling using a discrete particle dynamics technique, and a sampling based approach to incorporate parametric variations. A total of 24 (four patients and six Circle anatomies including the complete Circle) models were considered, with cardiogenic emboli of varying sizes and compositions released virtually and tracked to compute distribution to the brain. The results establish that Circle anatomical variations significantly influence embolus distribution to the six major cerebral arteries. Embolus distribution to MCA territory is found to be least sensitive to the influence of anatomical variations. For varying Circle topologies, differences in flow through cervical vasculature are observed. This incoming flow is recruited differently across the communicating arteries of the Circle for varying anastomoses. Emboli interact with the routed flow, and can undergo significant traversal across the Circle arterial segments, depending upon their inertia and density ratio with respect to blood. This interaction drives the underlying biomechanics of embolus transport across the Circle, explaining how Circle anatomy influences embolism risk.

bioengineering↗

bioRxiv2017

Bacterial Colonization Stimulates A Complex Physiological Response In The Immature Human Intestinal Epithelium

The human gastrointestinal tract is immature at birth, yet must adapt to dramatic changes such as oral nutrition and microbial colonization. The confluence of these factors can lead to severe inflammatory disease in premature infants; however, investigating complex environment-host interactions is diZcult due to limited access to immature human tissue. Here, we demonstrate that the epithelium of human pluripotent stem cell-derived human intestinal organoids is globally similar to the immature human epithelium and we utilize HIOs to investigate complex host-microbe interactions in this naive epithelium. Our findings demonstrate that the immature epithelium is intrinsically capable of establishing a stable host-microbe symbiosis. Microbial colonization leads to complex contact and hypoxia driven responses resulting in increased antimicrobial peptide production, maturation of the mucus layer, and improved barrier function. These studies lay the groundwork for an improved mechanistic understanding of how colonization influences development of the immature human intestine.

developmental biology↗