Biology subjects

Sarkar, P.

Publications and source records attributed to Sarkar, P..

32 records · Page 2Linked to original sources

bioRxiv2023

Scalable, Robust, High-throughput Expression, Purification & Characterization of Nanobodies Enabled by 2-Stage Dynamic Control

Nanobodies are single-domain antibody fragments that have garnered considerable use as diagnostic and therapeutic agents as well as research tools. However, obtaining pure VHHs, like many proteins, can be laborious and inconsistent. High level cytoplasmic expression in E. coli can be challenging due to improper folding and insoluble aggregation caused by reduction of the conserved disulfide bond. We report a systems engineering approach leveraging engineered strains of E. coli, in combination with a two-stage process and simplified downstream purification, enabling improved, robust soluble cytoplasmic nanobody expression, as well as rapid cell autolysis and purification. This approach relies on the dynamic control over the reduction potential of the cytoplasm, in combination with dynamic expression of chaperones and lysis enzymes needed for purification. Collectively, the engineered system results in more robust growth and protein expression, enabling efficient scalable nanobody production, and purification from high throughput microtiter plates, to routine shake flask cultures and larger instrumented bioreactors. We expect this system will expedite VHH development.

synthetic biology↗

bioRxiv2023

Direct ionic stress sensing and mitigation by the transcription factor NFAT5

Homeostatic control of intracellular ionic strength is essential for protein, organelle and genome function, yet mechanisms that sense and enable adaptation to ionic stress remain poorly understood in animals. We find that the transcription factor NFAT5 directly senses solution ionic strength using a C-terminal intrinsically disordered region. Both in intact cells and in a purified system, NFAT5 forms dynamic, reversible biomolecular condensates in response to increasing ionic strength. This self-associative property, conserved from insects to mammals, allows NFAT5 to accumulate in the nucleus and activate genes that restore cellular ion content. Mutations that reduce condensation or those that promote aggregation both reduce NFAT5 activity, highlighting the importance of optimally tuned associative interactions. Remarkably, human NFAT5 alone is sufficient to reconstitute a mammalian transcriptional response to ionic or hypertonic stress in yeast. Thus NFAT5 is both the sensor and effector of a cell-autonomous ionic stress response pathway in animal cells.

cell biology↗

bioRxiv2023

Differential Carbonic Anhydrase Activities Control EBV-Induced B-Cell Transformation and Lytic Cycle Reactivation

Epstein-Barr virus (EBV) contributes to [~]1% of all human cancers including several B-cell neoplasms. A characteristic feature of EBV life cycle is its ability to transform metabolically quiescent B-lymphocytes into hyperproliferating B-cell blasts with the establishment of viral latency, while intermittent lytic cycle induction is necessary for the production of progeny virus. Our RNA-Seq analyses of both latently infected naive B-lymphocytes and transformed B-lymphocytes upon lytic cycle replication indicate a contrasting expression pattern of a membrane-associated carbonic anhydrase isoform CA9, an essential component for maintaining cell acid-base homeostasis. We show that while CA9 expression is transcriptionally activated during latent infection model, lytic cycle replication restrains its expression. Pharmacological inhibition of CA-activity using specific inhibitors retards EBV induced B-cell transformation, inhibits B-cells outgrowth and colony formation ability of transformed B-lymphocytes through lowering the intracellular pH, induction of cell apoptosis and facilitating degradation of CA9 transcripts. Reanalyses of ChIP-Seq data along with utilization of EBNA2 knockout virus and sh-RNA mediated knockdown of CA9 expression we further demonstrate that EBNA2 mediated CA9 transcriptional activation is essential for EBV latently infected B-cell survival. In contrast, during lytic cycle reactivation CA9 expression is transcriptionally suppressed by the key EBV lytic cycle transactivator, BZLF1 through its transactivation domain. Overall, our study highlights the dynamic alterations of CA9 expression and its activity in regulating pH homeostasis act as one of the major drivers for EBV induced B-cell transformation and subsequent B-cell lymphomagenesis.

microbiology↗

bioRxiv2023

The molecular prognostic score, a classifier for risk stratification of high-grade serous ovarian cancer

The clinicopathological parameters such as residual tumor, grade, FIGO score are often used to predict the survival of ovarian cancer patients, but the 5-year survival of high grade serous ovarian cancer (HGSOC) still remains around 30%. In recent years, a gene expression based molecular prognostic score (mPS) was developed that showed improved prognosis in several cancers including ovarian cancer. The feature extraction using LASSO-Cox regression was applied on the training data with 10-fold cross validation to obtain 20 predictor genes along with the coefficients to derive mPS. The mPS based prognosis of HGSOC patients was validated using the log-rank test and receiver operator characteristic curve. The AUC of 20 gene-based mPS in predicting the 5-year overall survival was around 0.7 in both the training (n=491) and test datasets (n=491). It was also validated across HGSOC patients (n=7542), data collected from the Ovarian Tumor Tissue Analysis (OTTA) consortium. The mPS showed significant impact (adjusted HR = 6.1, 95% CI of HR= 3.65-10.3; p <0.001) on prognosis of HGSOC. The performance of mPS for the prognosis of survival of HGSOC was substantially better than conventional parameters: FIGO (adjusted HR=1.1, 95% CI=0.97-1.2, p=0.121), residual disease (adjusted HR=1.3, 95% CI= 1.13-1.4, p<0.001), and age (adjusted HR=1.2, 95% CI= 0.98-1.6, p=0.08). It was found that focal-adhesion, Wnt and Notch signaling pathways were significantly (p<0.001) upregulated, whereas antigen processing and presentation (p<0.001) was downregulated in high risk HGSOC cohorts based on mPS stratification. The molecular prognostic score derived from 20-gene signature is found to be the novel robust prognostic marker of HGSOC. It could potentially be harnessed in clinical settings to determine the overall survival of ovarian cancer. The high risk HGSOC patients based on mPS stratification could be benefited from alternative therapies targeting Wnt/ Notch signaling pathways and also immune evasion. Author summaryThe 20-gene signature based molecular prognostic score (mPS) was found to be associated with risk stratification and hence, predicting the overall survival time of HGSOCs. It was applicable in all training HGSOC datasets and across RNA sequencing platforms that also includes the previous reported studies in HGSOC cohorts (Millstein et al., 2020; Talhouk et al., 2020). The 20-gene signature based mPS for the prognosis of overall survival of HGSOC outperformed conventional parameters: age, residual disease and FIGO score. The high or increased risk group of HGSOC based on our mPS stratification was found to have dysregulated pathways of Wnt, Notch, Akt signaling, and antigen presentation. Thus, treatments targeting these pathways might be beneficial for high risk HGSOC and hence anticipated to improve the over-all survival of HGSOC.

genomics↗

bioRxiv2022

Resting-state aperiodic neural activity as a novel objective marker of daytime somnolence

Study ObjectivesCurrent assessment of excessive daytime somnolence (EDS) requires subjective measurements such as the Epworth Sleepiness Scale (ESS), and/or resource intensive sleep laboratory investigations. Recent work 1,2 has called for more non-performance-based measures of EDS. One promising non-performance-based measure of EDS is the aperiodic component of electroencephalography (EEG). Aperiodic (non-oscillatory) activity reflects excitation/inhibition ratios of neural populations and is altered in various states of consciousness, and thus may be a potential biomarker of hypersomnolence. MethodsWe retrospectively analysed EEG data from patients who underwent a Multiple Sleep Latency Test (MSLT) and determined whether aperiodic neural activity is predictive of EDS. Participants having undergone laboratory polysomnogram and next day MSLT were grouped into MSLT+ (n = 26) and MSLT- (n = 33) groups (mean sleep latency of < 8min and > 10min, respectively) and compared against a non-clinical (Control) group of participants (n = 26). ResultsWhile the MSLT+ and MSLT- groups did not differ in their aperiodic activity, the Control group had a significantly flatter slope and larger offset compared to both MSLT+ and MSLT- groups. Logistic regression machine learning predicted group status (i.e., symptomatic, non-symptomatic) with 90% accuracy based on the aperiodic slope while controlling for age. Slow oscillation-spindle coupling was also significantly stronger in the Control group relative to MSLT+ and MSLT- groups. ConclusionsOur results provide first evidence that aperiodic neural dynamics and sleep-based cross-frequency coupling is predictive of EDS, thereby providing a novel avenue for basic and applied research in the study of sleepiness.

neuroscience↗

bioRxiv2022

Effects of calcium-regulated autophagy on Candidatus Liberibacter solanacearum in carrot psyllid midguts

Candidatus Liberibacter solanacearum (CLso) transmitted by the carrot psyllid, Bactericera trigonica causes carrot yellows in Israel, and has recently gained much importance due to the excessive economical loss. Understanding the interactions between CLso and the psyllid at the cellular level is fundamental for the disease management. Here, we demonstrate the role of calcium ATPase, cytosolic calcium and most importantly Beclin1 in regulating autophagy and its association with Liberibacter. Presence of CLso generates reactive oxygen species and induces the expression of the detoxification enzymes in the psyllid midguts. CLso also induces the expression of both sarco/endoplasmic reticulum Ca2+ pump (SERCA) and 1,4,5-trisphosphate receptors (ITPR) in the midguts, followed by high levels of calcium in the cytosol. Silencing these proteins individually disrupted the calcium levels in the cytosol leading to direct effects on autophagy and thus on Liberibacter. On the other hand, inhibiting Beclin1-phosphorylation through different calcium induced kinases altered the expression of autophagy and CLso abundance. This study establishes a direct correlation between cytosolic calcium levels, autophagy and CLso in the carrot psyllid midgut.

molecular biology↗

bioRxiv2022

Multi-modal Imaging of Disease Progression in TH-MYCN Mouse Models of Neuroblastoma

MYCN is a major driver for neuroblastoma (NB) and the tyrosine hydroxylase (TH)-MYCN transgenic mouse model is extensively used for preclinical NB studies. However, spatio-temporal NB progression in the TH-MYCN model has not been studied, and questions remain about the value of implanted models as a surrogate for transgenic mice. In this work, we used magnetic resonance imaging (MRI) to study tumor progression and nanoparticle contrast-enhanced computed tomography (n-CECT) to assess tumor vascular architecture in TH-MYCN transgenic mice (2-7 weeks of age) and TH-MYCN+/+-derived orthotopic allograft and syngeneic mice (2-5 weeks post-tumor implantation). Tumors in TH-MYCN transgenic mice became evident in the abdominal paraspinal region at week 5. A delayed thoracic paraspinal mass became evident at week 6 and most mice succumbed by week 7. In allograft and syngeneic mice, single mass tumor growth was restricted to the peritoneal cavity. N-CECT revealed a predominantly microvascular network in TH-MYCN tumors while implanted tumors exhibited heterogeneous and tortuous vessels. N-CECT quantitative analysis demonstrated high vascularity (tumor fractional blood volume ~ 0.12) in all models. Multi-modal imaging of TH-MYCN transgenic and implanted models revealed differences in growth patterns and vascular architecture that should be considered in designing preclinical studies.

cancer biology↗

bioRxiv2021

Liberibacter solanacearum interacts with host psyllid vitellogenin with its membrane proteins

Candidatus Liberibacter solanacearum (CLso) haplotype D, transmitted by the carrot psyllid Bactericera trigonica is a major constraint in carrot production in Israel. Understanding the molecular interactions between the psyllid vector and CLso can facilitate non-chemical approaches for controlling CLso caused-diseases. In this study, we used CLso outer membrane protein (OmpA) and flagellin as baits to screen for psyllid interacting proteins in a yeast-two hybrid assay. We identified psyllid vitellogenin (Vg) protein to interact with both OmpA and flagellin of CLso. As Vg is often involved in innate immunity with its expression tightly linked to autophagy, a major component of the immune response in the cell, we also analyzed the expression of autophagy-related genes to further elucidate this interaction. We used the juvenile hormone (JH-III) to induce the expression of Vg, thapsigargin for suppressing autophagy, and rapamycin for inducing autophagy. The results revealed that Vg negatively regulates autophagy and vice versa. JH-III induced Vg expression significantly suppressed autophagy and, the levels of CLso significantly increased resulting in a significant mortality of the insect. Although the specific role of Vg remains obscure, the findings presented here identify Vg as an important component in the insect immune responses against CLso and may help in understanding the initial molecular response in the vector against Liberibacter.

molecular biology↗

bioRxiv2020

Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

ER+ breast cancer exhibits a strong bone-tropism in metastasis. How the bone microenvironment impacts the ER signaling and endocrine therapies remains poorly understood. Here, we discover that the osteogenic niche transiently reduces ER expression and activities specifically in bone micrometastases (BMMs), leading to endocrine resistance. This is mediated by gap junctions and paracrine FGF/PDGF signaling, which together generate a stable "memory": cancer cells extracted from bone remain resistant to endocrine therapies for several generations. Using single cell-derived populations (SCPs), we demonstrated that this process is independent of clonal selection, and represents an EZH2-mediated epigenomic reprogramming. EZH2 drives ER+ BMMs toward a basal and stem-like state. EZH2 inhibition reverses endocrine resistance. Our data demonstrates how epigenomic adaptation to the bone microenvironment drives phenotypic plasticity of metastatic seeds and alters their therapeutic responses together with clonal selection, and provides insights into the clinical enigma of ER+ metastatic recurrences despite endocrine therapies.

cancer biology↗

bioRxiv2020

New insights into TNFα/PTP1B and PPARγ pathway through RNF213- a link between inflammation, obesity, insulin resistance and Moyamoya disease

Diabetic patients are always at a higher risk of ischemic diseases like coronary artery diseases. One such ischemic carotid artery disease is Moyamoya. Moyamoya disease (MMD) has been associated with diabetes Type-I and II and the causality was unclear. RNF213 is the major susceptible gene for MMD. To understand the association between diabetes mellitus and MMD we chose the major players from both the anomalies, insulin and RNF213. But before establishing a role of RNF213 in insulin regulating pathway we had to understand the involvement of RNF213 within different biological systems. For this we have adopted a preliminary computational approach to understand the prominent interactions of RNF213. Our first objective was to construct an interactome for RNF213. We have analyzed several curated databases and adapted a list of RNF213 interacting partners to develop its interactome. Then to understand the involvement of this interactome in biological functions we have analyzed major biological pathways, biological processes and prominent clusters related to this interactome through computational approach. Then to develop a pathway that might give clue for RNF213 involvement in insulin regulatory pathway we have validated the intercluster and intracluster predictions and identified a regulatory pathway for RNF213. RNF213 interactome was observed to be involved in adaptive immunity with 4 major clusters; one of the cluster involved TNFα. Immune system involves several pathways, and therefore at this point we have chosen an event-based strategy to obtain an explicit target. Immunity is mediated by many pro-inflammatory cytokines like TNFα. TNFα-mediated inflammation, obesity and insulin resistance are associated. Therefore we chose to explore the role of RNF213 in TNFα-mediated inflammation in macrophages and inflammation-mediated insulin-resistance in adipocytes. We have observed an enhancement of RNF213 gene expression by LPS mediated pro-inflammatory stimuli and suppression by PPARγ-mediated anti-inflammatory, insulin sensitizing stimuli in macrophages. A more significant response was observed in adipocytes as well. Administration of the pro-inflammatory cytokine TNFα was able to impede the reduction in RNF213 expression during adipogenesis and this effect was observed to be mediated by PTP1B. Inactivation of PTP1B abolished RNF213 expression which in turn enhanced the adipogenesis process through enhanced PPARγ. Constitutive expression of RNF213 suppressed the adipocyte differentiation by the inhibition of PPARγ. We could show the expression of RNF213 has been regulated by TNFα/PTP1B pathway and PPARγ. The constitutive expression of RNF213 during adipogenesis appears to be an adipostatic measure that obese patients acquire to inhibit further adipogenesis. This is verified in silico by analyzing the gene expression data obtained from Gene Expression Omnibus database, which showed a higher expression of RNF213 in adipose tissue samples of obese people. Overall this study gives new insights in the TNFα-mediated pathway in adipogenesis and suggests a role of RNF213 in adipogenesis via this pathway.View Full Text

bioinformatics↗

bioRxiv2020

Differential levels of dermatan sulfate generate distinct Collagen I gel architectures

Collagen I is the most abundant extracellular matrix (ECM) protein in vertebrate tissues. As an endogenously synthesized fibrillar biopolymer or as a synthetic hydrogel, it provides mechanical durability to tissue microenvironments and regulates cell function. Predominant regulators of its fibrillogenesis are dermatan sulfate proteoglycans (DSPGs), proteins conjugated with iduronic acid containing DS glycosaminoglycans (GAGs). Although DS is known to regulate tissue function through its modulation of Collagen I architecture, a precise quantifiable understanding of the latter remains elusive. We investigate this problem by visualizing the pattern of structural elements within fixed Collagen I gels polymerized in the presence of varying concentrations of DS (50-, 200- and 400- g/mL) using second harmonic generation microscopy (SHG). Measuring four independent imaging parameters: fibril density, mean SHG signal (which estimates the ordering of the fibrils), surface occupancy (which estimates the space occupied by fibrils), and the fibril width allows us to construct an informative model of the effects of DS on Collagen I element architecture. Supported by confocal microscopy, our observations indicate that the effect on collagen fibril pattern of DS is contextual upon its concentrations. Lower levels of DS result in more numerous disorganized fibrils; higher levels restore organization, but at lower fibril densities. Such Collagen I gel pattern-tuning of DS is likely of relevance for understanding its functions in disease progression and biomaterial applications.

physiology↗

bioRxiv2019

Asynchronous mixing of kidney progenitor cells potentiates nephrogenesis in organoids

Recent years have seen rapid advances in directed differentiation of human pluripotent stem cells (PSCs) to kidney cells. However, a fundamental difficulty in emulating kidney tissue formation is that kidney development is iterative. Recent studies argue that the human nephron forms through gradual contribution of nephron progenitor cells whose differentiation fates depend on the time at which they are recruited. We show that the majority of PSC-derived nephron progenitor cells differentiated in a short wave in organoid formation and to improve fidelity of PSC-derived organoids, we emulated the asynchronous mix found in the fetal kidney by combining cells differentiated at different times in the same organoid. Asynchronous mixing promoted nephrogenesis, and lineage marking data showed that proximal and distal nephron components preferentially derive from cell populations differentiated at distinct times. When engrafted under the kidney capsule these heterochronic organoids were vascularized and displayed essential features of kidney tissue. Micro-CT and injection of a circulating vascular marker demonstrated that engrafted kidney tissue was connected to the systemic circulation by 2 weeks after engraftment. Proximal tubule glucose uptake was confirmed using intravenous injection of fluorescent dextran. Despite these promising measures of graft function, overgrowth of stromal cells prevented long-term study, and we propose that this is a technical feature of the engraftment procedure rather than a specific shortcoming of the directed differentiation because kidney organoids derived from primary cells and whole embryonic kidneys develop the same stromal overgrowth when engrafted under the kidney capsule.

developmental biology↗

bioRxiv2018

Cryo-Electron tomography 3D structure and nanoscale model of Arabidopsis thaliana cell wall

Using cryo-electron tomography of vitrified sections of one month-old Arabidopsis thaliana inflorescence stem tissue, we visualized primary and secondary cell walls of xylem tissue. Extensive quantitative and statistical analysis of segmented 3D tomographic data allowed geometrically idealized 3D-CAD model building of prototypic microfibrils, cross-links, and their supramolecular microfibril 3D organization. We propose a prototypic microfibril model where a cellulose core is heavily decorated by a thin sheath of hemicellulose with infrequent but sturdy hemicellulose-based cross-links. Such prototypic microfibrils then adopt a rather unexpected 3D supramolecular organization of high order and complexity. We discuss a possible new role for lignin in plant cell walls at low concentrations with lignin not acting as a matrix but rather as a reinforcement of microfibrils and cross-links. Extensive computational simulations of mechanical properties further revealed that this 3D organization of the cell wall is not optimized for load bearing but instead for flexibility and ductility.\n\nOne Sentence SummaryCryo-electron tomography and mechanical simulations revealed cell wall 3D architecture, optimized for flexibility/ductility.

plant biology↗

bioRxiv2017

Phenotypic consequences of RNA polymerase dysregulation in Escherichia coli

Many bacterial adaptive responses to changes in growth conditions due to biotic and abiotic factors involve reprogramming of gene expression at the transcription level. The bacterial RNA polymerase (RNAP), which catalyzes transcription, can thus be considered as the major mediator of cellular adaptive strategies. But how do bacteria respond if a stress factor directly compromises the activity of the RNAP? We used a phage-derived small protein to specifically perturb bacterial RNAP activity in exponentially growing Escherichia coli. Using cytological profiling, tracking RNAP behavior at single-molecule level and transcriptome analysis, we reveal that adaptation to conditions that directly perturb bacterial RNAP performance can result in a biphasic growth behavior and thereby confer the adapted bacterial cells an enhanced ability to tolerate diverse antibacterial stresses. The results imply that while synthetic transcriptional rewiring may confer bacteria with the intended desirable properties, such approaches may also collaterally allow them to acquire undesirable traits.

microbiology↗