Biology subjects

Sinha, S. K.

Publications and source records attributed to Sinha, S. K..

6 recordsLinked to original sources

bioRxiv2026

Early detection of sudden transitions in Notch signalling

Identifying sudden transitions during phenotypic decision-making of complex biological systems can be crucial for our ability to control a cellular state. Yet, prior determination of these sudden transitions or tipping points remains challenging, as biological systems often exhibit only subtle early changes, which are often masked by inherent noise or rapid transition dynamics. Using Notch signaling as a model, we systematically analyze dynamical transitions in Notch-Delta (ND), Notch-Delta-Jagged (NDJ), and Fringe-mediated NDJ systems for both one and two-cell contexts. In the one-cell ND system, critical slowing down (CSD)-based early warning signals (EWSs) reliably capture transitions between sender (S) and receiver (R) states and remain robust to variation in forcing rate. We further find that flickering is a precursor to transitions in one-cell NDJ system. In contrast, flickering does not occur in the two-cell Notch model due to the presence of a supercritical bifurcation. Our analysis also offers insight into how NICD (Notch Intracellular Domain)-driven and Fringe-mediated asymmetries, along with the strength of external signals, control the emergence of flickering. Overall, this study identifies sudden transitions in Notch signaling under demographic noise and can be extended to other noisy biological systems, with potential applications in drug development and targeted therapeutic interventions.

systems biology↗

bioRxiv2025

A Highly Thermostable and Novel GH5 Endoglucanase from Bacillus sp. Strain BS with Enhanced Biomass Saccharification Potential in Seawater

The efficient enzymatic conversion of lignocellulosic biomass to glucose is crucial for biofuel production, with enzyme costs being a significant barrier to commercialization. Multifunctional enzymes that reduce enzyme components can improve efficiency. In this study, we amplified and sequenced a gene from Bacillus sp. strain BS, which showed 89.8% identity with bacterial endoglucanase Q6YK34. This putative novel endoglucanase, BsEG2, is a GH5-like endoglucanase, with optimal activity at pH 6.0 and 55 {square}, and efficiently hydrolyzes amorphous and crystalline cellulose, including pre-treated and untreated sugarcane bagasse. Kinetics on carboxymethyl cellulose (CMC) indicate high substrate binding efficiency (apparent Vmax of 148.9 {micro}M min-1, a Km of 24.9 mg mL-1, and a kcat of 517 s-1). BsEG2 hydrolyzes long cellulosic chains, oligosaccharides, and cellobiose to produce glucose, demonstrating multifunctionality. It exhibits remarkable thermal stability, retaining over 90% activity after 15 days at 55 {degrees}C. BsEG2 also retains activity in high salts and ionic liquids, and is not inhibited by cellobiose, even at 200 mM, unlike other endoglucanases and cellobiohydrolases. Furthermore, BsEG2 functions in seawater, reducing freshwater footprint in biofuel production. BsEG2 is a promising candidate for a cost-effective enzyme formulation for glucose production from lignocellulosic biomass, supporting downstream glucose-fed metabolic pathways.

biochemistry↗

bioRxiv2020

An Inherent Structural Difference Between Serine and Threonine Phosphorylation: Phosphothreonine Prefers an Ordered, Compact, Cyclic Conformation

Phosphorylation and dephosphorylation of proteins by kinases and phosphatases are central to cellular responses and function. The structural effects of serine and threonine phosphorylation were examined in peptides and in proteins, by circular dichroism, NMR spectroscopy, bioinformatics analysis of the PDB, small-molecule X-ray crystallography, and computational investigations. Phosphorylation of both serine and threonine residues induces substantial conformational restriction in their physiologically more important dianionic forms. Threonine exhibits a particularly strong disorder-to-order transition upon phosphorylation, with dianionic phosphothreonine preferentially adopting a cyclic conformation with restricted{phi} ({phi} [~] -60{degrees}) stabilized by three noncovalent interactions: a strong intraresidue phosphate-amide hydrogen bond, an n[->]{pi}* interaction between consecutive carbonyls, and an n[->]{sigma}* interaction between the phosphate O{gamma} lone pair and the antibonding orbital of C-H{beta} that restricts the{chi} 2 side chain conformation. Proline is unique among the canonical amino acids for its covalent cyclization on the backbone. Phosphothreonine can mimic prolines backbone cyclization via noncovalent interactions. The preferred torsions of dianionic phosphothreonine are{phi} ,{psi} = polyproline helix or -helix ({phi} [~] -60{degrees});{chi} 1 = g-;{chi} 2 = eclipsed C-H/O-P bonds. This structural signature is observed in diverse proteins, including the activation loops of protein kinases and protein-protein interactions. In total, these results suggest a structural basis for the differential use and evolution of threonine versus serine phosphorylation sites in proteins, with serine phosphorylation typically inducing smaller, rheostat-like changes, versus threonine phosphorylation promoting larger, step function-like switches, in proteins.

biophysics↗

bioRxiv2020

Lipid Sponge Droplets as Programmable Synthetic Organelles

Living cells segregate molecules and reactions in various subcellular compartments known as organelles. Spatial organization is likely essential for expanding the biochemical functions of synthetic reaction systems, including artificial cells. Many studies have attempted to mimic organelle functions using lamellar membrane-bound vesicles. However, vesicles typically suffer from highly limited transport across the membranes and an inability to mimic the dense membrane networks typically found in organelles such as the endoplasmic reticulum. Here we describe programmable synthetic organelles based on highly stable nonlamellar sponge phase droplets that spontaneously assemble from a single-chain galactolipid and non-ionic detergents. Due to their nanoporous structure, lipid sponge droplets readily exchange materials with the surrounding environment. In addition, the sponge phase contains a dense network of lipid bilayers and nanometric aqueous channels, which allows different classes of molecules to partition based on their size, polarity, and specific binding motifs. The sequestration of biologically relevant macromolecules can be programmed by the addition of suitably functionalized amphiphiles to the droplets. We demonstrate that droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled. Our results show that lipid sponge droplets permit the facile integration of membrane-rich environments and self-assembling spatial organization with biochemical reaction systems. Significance statementOrganelles spatially and temporally orchestrate biochemical reactions in a cell to a degree of precision that is still unattainable in synthetic reaction systems. Additionally, organelles such as the endoplasmic reticulum (ER) contain highly interconnected and dense membrane networks that provide large reaction spaces for both transmembrane and soluble enzymes. We present lipid sponge droplets to emulate the functions of organelles such as the ER. We demonstrate that lipid sponge droplets can be programmed to internally concentrate specific proteins, host and accelerate biochemical transformations, and to rapidly and reversibly sequester and release proteins to control enzymatic reactions. The self-assembled and programmable nature of lipid sponge droplets will facilitate the integration of complex functions for bottom up synthetic biology.

synthetic biology↗

bioRxiv2019

Elucidating the regulation of glucose tolerance through the interaction between the reaction product and active site pocket residues of a β-glucosidase from Halothermothrix orenii

{beta}-glucosidase catalyzes the hydrolysis of {beta}-1,4 linkage between two glucose molecules in cello-oligosaccharides and is prone to inhibition by the reaction product glucose. Relieving the glucose inhibition of {beta}-glucosidase is a significant challenge. Towards the goal of understanding how glucose interacts with {beta}-glucosidase, we expressed in Escherichia coli, the Hore_15280 gene encoding a {beta}-glucosidase in Halothermothrix orenii. Our results show that the enzyme is glucose tolerant, and its activity stimulated in the presence of up to 0.5 M glucose. NMR analyses show the unexpected interactions between glucose and the {beta}-glucosidase at lower concentrations of glucose that however does not lead to enzyme inhibition. We identified non-conserved residues at the aglycone-binding and the gatekeeper site and show that increased hydrophobicity at the pocket entrance and a reduction in steric hindrances are critical towards enhanced substrate accessibility and significant improvement in activity. Analysis of structures and in combination with molecular dynamics simulations show that glucose increases the accessibility of the substrate by enhancing the structural flexibility of the active site pocket and may explain the stimulation in specific activity up to 0.5 M glucose. Such novel regulation of {beta}-glucosidase activity by its reaction product may offer novel ways of engineering glucose tolerance.

biochemistry↗

bioRxiv2019

Anticipating critical transitions in epithelial-hybrid-mesenchymal cell-fate determination

In the vicinity of a tipping point, critical transitions occur when small changes in an input condition causes sudden, large and often irreversible changes in the state of a system. Many natural systems ranging from ecosystems to molecular biosystems are known to exhibit critical transitions in their response to stochastic perturbations. In diseases, an early prediction of upcoming critical transitions from a healthy to a disease state by using early warning signals is of prime interest due to potential application in forecasting disease onset. Here, we analyze cell-fate transitions between different phenotypes (epithelial, hybrid epithelial/mesenchymal (E/M) and mesenchymal states) that are implicated in cancer metastasis and chemoresistance. These transitions are mediated by a mutually inhibitory feedback loop microRNA-200/ZEB driven by the levels of transcription factor SNAIL. We find that the proximity to tipping points enabling these transitions among different phenotypes can be captured by critical slowing down based early warning signals, calculated from the trajectory of ZEB mRNA level. Further, the basin stability analysis reveals the unexpectedly large basin of attraction for a hybrid E/M phenotype. Finally, we identified mechanisms that can potentially elude the transition to a hybrid E/M phenotype. Overall, our results unravel the early warning signals that can be used to anticipate upcoming epithelial-hybrid-mesenchymal transitions. With the emerging evidence about the hybrid E/M phenotype being a key driver of metastasis, drug resistance, and tumor relapse; our results suggest ways to potentially evade these transitions, reducing the fitness of cancer cells and restricting tumor aggressiveness.\n\nSignificance StatementEpithelial-hybrid-mesenchymal transitions play critical roles in cancer metastasis, drug resistance, and tumor relapse. Recent studies have proposed that cells in a hybrid epithelial/mesenchymal phenotype may be more aggressive than those on either end of the spectrum. However, no biomarker to predict upcoming transitions has been identified. Here, we show that critical slowing down based early warning signals can detect sudden transitions among epithelial, hybrid E/M, and mesenchymal phenotypes. Importantly, our results highlight how stable a hybrid E/M phenotype can be, and how can a transition to this state be avoided. Thus, our study provides valuable insights into restricting cellular plasticity en route metastasis.

systems biology↗