Biology subjects

Sasmal, D. K.

Publications and source records attributed to Sasmal, D. K..

5 recordsLinked to original sources

bioRxiv2025

Curcumin Conjugated Carbon Quantum Dots: A Theranostic Probe to Study BSA Interaction, Cellular Imaging, and RICS-Based Intracellular Transport

Plant-based products such as jamun leaves and herbaceous perennials like turmeric are replete with carbon-rich elements, and our recent focus has been directed to synthesizing Carbon Quantum Dots (CQDs) in a low-cost, straightforward, and sustainable method, with their biological and therapeutic applications. A valuable insight we have gained from our results is the environmentally friendly production of CQDs with their distinct physicochemical characteristics, in addition to excellent water solubility and remarkable stability. The as-synthesized CQDs manifest good quantum yields with many potential applications. Moreover, for the stability and enhancement of the solubilization of the hydrophobic drug curcumin, to improve its uptake and bio accessibility, CQDs are a unique choice. After the conjugation of CQDs with curcumin (C-CQDs), the findings we have obtained with partially unfolded BSA demonstrate a modest interaction involving dynamic quenching, as exemplified by steady-state and time-resolved fluorescence quenching driven by Forster Resonance Energy Transfer (FRET). In addition to that, our research also delved into the application of in vivo bioimaging, and by using that fluorescence imaging technique, we introduced Raster Image Correlation Spectroscopy (RICS) techniques to find the diffusion of both CQDs and C-CQDs in the live cell. The study also reaffirms the inherent biocompatibility and antibacterial ability confirmed by the MTT-based cytotoxicity assay and MIC study.

biophysics↗

bioRxiv2025

Economical Method to Construct a Prism-based TIRF Setup on an Existing Confocal Microscope to Perform smFRET Experiments

Total internal reflection fluorescence (TIRF) microscopy enables observation of complex bio-assemblies and macromolecular dynamics in high spatial-temporal resolution at single-molecule level in real-time. With TIRF illumination, fluorophores near a sample substrate are excited by an evanescent field, thereby circumventing the axial diffraction limit of light. Prism-based TIRF (p-TIRF) microscopes are comparatively easy to use. They may be readily adjusted to meet the requirements of a broad range of experimental applications, such as to examine of macromolecular complexes, to study of the behaviour of vesicles and small organelles, the study of protein-DNA complexes at the single-molecule level. These experiments can give unique insights into the mechanisms driving the molecular interactions that underline many fundamental activities within the cell by providing information on fluctuation distributions and unusual events. Here, we report a detailed method to build a p-TIRF setup inexpensively using an existing confocal microscope where the same light source can be used for both systems. Furthermore, we provide a brief overview that aims to give the readers a stepwise tutorial protocol for building, assembling, aligning, and preparing the specimen to conduct single-molecule fluorescence resonance energy transfer (smFRET) experiments using a custom-built p-TIRF setup. We believe that this article will be of assistance to labs that already have a confocal microscope and want to perform TIRF experiments for potential future applications. Research HighlightWe report here a simple and cost-effective method to build a prism-based TIRF setup on a laser scanning confocal microscope. The setup is affordable, and users can use both confocal and TIRF modes in the same setup with the same light source and optical components. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/639412v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@e476e5org.highwire.dtl.DTLVardef@139d7b8org.highwire.dtl.DTLVardef@81ac21org.highwire.dtl.DTLVardef@14bd8f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Enzyme-Regulated Non-Thermal Fluctuations Enhance Ligand Diffusion and Receptor-Mediated Endocytosis

Active enzymes during catalyzing chemical reactions, have been found to generate significant mechanical fluctuations, which can influence the dynamics of their surroundings. These phenomena open new avenues for controlling mass transport in complex and dynamically inhomogeneous environments through localized chemical reactions. To explore this potential, we studied the uptake of transferrin molecules in retinal pigment epithelium (RPE) cells via clathrin-mediated endocytosis. In the presence of enzyme catalysis in the extracellular matrix, we observed a significant enhancement in the transport of fluorophore-tagged transferrin inside the cells. Fluorescence correlation spectroscopy measurements showed substantial increase in transferrin diffusion in the presence of active fluctuations. This study sheds light on the possibility that enzyme-substrate reactions within the extracellular matrix may induce long-range mechanical influences, facilitating targeted material delivery within intracellular milieu more efficiently than passive diffusion. These insights are expected to contribute to the development of better therapeutic strategies by overcoming limitations imposed by slow molecular diffusion under complex environments.

biophysics↗

bioRxiv2018

TCR-pMHC bond length controls TCR ligand discrimination

T-cell receptors (TCRs) detect specifically and sensitively a small number of agonist peptide-major histocompatibility complexes (pMHCs) from an ocean of structurally similar self-pMHCs to trigger antigen-specific adaptive immune responses1-4. Despite intense efforts, the mechanism underlying TCR ligand discrimination remains a major unanswered question in immunology. Here we show that a TCR discriminates between closely related peptides by forming TCR-pMHC bonds with different lengths, which precisely control the accessibility of CD3{zeta} immunoreceptor tyrosine-based activation motifs (ITAMs) for phosphorylation. Using in situ fluorescence resonance energy transfer (FRET)3,5, we measured the intermolecular length of single TCR-pMHC bonds and the intramolecular distance of individual TCR-CD3{zeta} complexes at the membrane of live primary T cells. We found that an agonist forms a short TCR-pMHC bond to pull the otherwise sequestered CD3{zeta} off the inner leaflet of the plasma membrane, leading to full exposure of its ITAMs for strong phosphorylation. By contrast, a structurally similar weaker peptide forms a longer bond with the TCR, resulting in partial dissociation of CD3{zeta} from the membrane and weak phosphorylation. Furthermore, we found that TCR-pMHC bond length determines 2D TCR binding kinetics and affinity, T-cell calcium signaling and T-cell proliferation, governing the entire process of signal reception, transduction and regulation. Thus, our data reveal the fundamental mechanism by which a TCR deciphers the structural differences between foreign antigens and self-peptides via TCR-pMHC bond length to initiate different TCR signaling for ligand discrimination.

immunology↗

bioRxiv2016

T cell co-stimulatory receptor CD28 is a primary target for PD-1-mediated inhibition

Programmed death-1 (PD-1) is a co-inhibitory receptor that suppresses T cell activation and is an important cancer immunotherapy target. Upon activation by its ligand PD-L1, PD-1 is thought to suppress signaling through the T cell receptor (TCR). Here, by titrating the strength of PD-1 signaling in both biochemical reconstitution systems and in T cells, we demonstrate that the coreceptor CD28 is strongly preferred over the TCR as a target for dephosphorylation by PD-1- recruited Shp2 phosphatase. We also show that PD-1 colocalizes with the costimulatory receptor CD28 in plasma membrane microclusters but partially segregates from the TCR. These results reveal that PD-1 suppresses T cell function primarily by inactivating CD28 signaling, suggesting that costimulatory pathways may play unexpected roles in regulating effector T cell function and therapeutic responses to anti-PD-L1/PD-1.

biochemistry↗