Biology subjects

Rakshit, T.

Publications and source records attributed to Rakshit, T..

9 recordsLinked to original sources

bioRxiv2026

A Nanomechanical Fingerprint of Centromeric Methylation Co-Localization in TNBC Chromosomal Instability

Centromeric heterochromatin is maintained by a self-reinforcing loop between DNA methylation (5mC) and histone H3 lysine 9 trimethylation (H3K9me3), yet whether this coupling is preserved at the level of individual chromosomes in cancer has been inaccessible to conventional epigenomic assays, which average signals across cell populations and cannot resolve single-chromosome heterogeneity. Here, we use antibody-functionalized atomic force spectroscopy to simultaneously map 5mC and H3K9me3 at the centromeres of individual metaphase chromosomes from non-tumorigenic, luminal, and triple-negative breast cancer (TNBC) cell models. Co-localized 5mC/H3K9me3 sites are mechanically stiffer than sites carrying either mark alone across the panel, revealing a cooperative biophysical signature of epigenetic crosstalk. Luminal chromosomes acquire the highest mark density, co-localization, and stiffness, whereas TNBC chromosomes show reduced density and diminished, though not absent, co-localization and stiffness, converging toward the low baseline of non-tumorigenic epithelium. TGF-{beta}1-induced epithelial-mesenchymal transition reproduces this attenuated phenotype in luminal cells, linking it directly to a process central to TNBC aggressiveness. These biophysical parameters classify cancer versus non-tumorigenic chromosomes with near-perfect, cross-validated accuracy, and further resolve the two TNBC subtypes using colocalization-derived features alone (macro-F1 = 0.933). These results establish the mechanical consequence of epigenetic mark co-localization as a single-chromosome-resolvable signature of centromeric organization, attenuated in aggressive breast cancer, and demonstrate a force-spectroscopy platform for mechano-epigenetic profiling of cancer chromosomes.

biophysics↗

bioRxiv2026

Hyaluronan Surface Architecture Dictates Colorectal Cancer Progression and Extracellular Vesicle Communication

Hyaluronan (HA) is a principal component of the tumor glycocalyx in colorectal cancer (CRC). However, how the disease progression is linked to HA abundance and its nanoscale organization remains unclear. Single-molecule measurements of surface glycans on cell membranes and extracellular vesicles (EVs) have not yet been correlated. In this work, using single-molecule force spectroscopy, we mapped HA density and chain length on CRC cells and their EVs across Dukes' stages. HA density increased with stage in both compartments, but their organization diverged. Cell-surface HA became progressively fragmented, whereas EVs remained enriched in short HA chains at every stage. EVs, therefore, appear to select HA during formation rather than inherit it from the parent cell. This divergence had mechanical consequences. Both cells and EVs softened with stage, and removing HA reversed this softening. In addition, coarse-grained membrane simulations revealed that both HA chain length and surface density regulate membrane wrapping, with chain length primarily influencing wrapping kinetics and surface density affecting the final wrapping extent. These findings provide a physical basis for the differences we observed in EV uptake. Reprogramming stage D cells with exogenous high-molecular-weight HA reversed this signature, lowering EV surface HA density, stiffening the vesicles, slowing migration, and suppressing EV uptake by recipient cells. These findings establish HA surface architecture as a stage-encoded and experimentally reversible determinant of CRC progression.

biophysics↗

bioRxiv2026

A Covalent Organic Framework-Inspired β-Ketoenamine Crosslinking Strategy for Robust, Injectable Bovine Serum Albumin Hydrogels with pH-Triggered Drug Release

Globular proteins are difficult to convert into robust hydrogels, as their compact, folded structures bury reactive residues, forcing conventional strategies to rely on denaturation or synthetic-polymer reinforcement that compromise the native protein. Inspired by the beta-ketoenamine bond-forming chemistry of covalent organic frameworks (COFs), we report the crosslinking of native bovine serum albumin (BSA) with 1,3,5-triformylphloroglucinol (TFP), a C3-symmetric trialdehyde, into a chemically defined hydrogel. TFP reacts with surface-exposed lysine residues through an irreversible enol-to-keto tautomerization, confirmed by FTIR and NMR spectroscopy, generating stable {beta}-ketoenamine crosslinks under mild aqueous conditions without denaturing the protein, as verified by intrinsic tryptophan fluorescence. The resulting hydrogels are mechanically robust compared to a reversible-imine control, injectable and self-recovering, exhibit reversible shape memory and substantial load-bearing capacity, and remain stable across a broad pH range over extended periods. The network shows consistent swelling behavior at physiological and mildly acidic pH, with modest compaction under strongly basic conditions; scanning electron microscopy reveals a dense, nodular network for the TFP hydrogel versus an open, sheet-like lamellar morphology for the reversible-imine control. The hydrogel efficiently encapsulates doxorubicin and displays pH-triggered, acid-selective release, which comparative kinetic analysis attributes principally to pH-dependent weakening of DOX-BSA binding affinity (linked to the N-to-F conformational transition of BSA near its isoelectric point) rather than to bulk network swelling or degradation. Doxorubicin-loaded hydrogels show enhanced killing of MCF-7 breast cancer cells relative to the free drug while remaining cytocompatible toward normal mammalian cells, and a ciprofloxacin-loaded variant exhibits potent antibacterial activity against both Gram-positive (M. luteus) and Gram-negative (E. coli) bacteria. This work translates reticular {beta}ketoenamine chemistry into a general platform for robust, stimuli-responsive protein biomaterials.

bioengineering↗

bioRxiv2024

Valproic Acid Treatment Enhances Chromosome Flexibility and Electron Transport in MCF7 Breast Cancer Cells

The structural integrity of the chromosomes is essential to every functional process within the eukaryotic nuclei. Chromosomes are DNA-histone complexes essential for the inheritance of genetic information to the offspring and any defect in it is linked to mitotic errors, cancer growth, and cellular aging. Changes in the mechanical properties of a chromosome could lead to its compromised function and stability, leading to chromosome breaks. Here, we studied the changes in chromosome physical properties using metaphase chromosomes isolated from human breast cancer cells (MCF7) exposed to Valproic Acid (VPA), a known epigenetic modifier drug involved in histone hyperacetylation and DNA demethylation. Due to chromosomal structural intricacy, preparative and technical limitations of analytical tools, we employed a label-free atomic force microscopy approach for simultaneously visualizing and mapping single chromosome elasticity. Additionally, we performed electron transport characteristics of metaphase chromosomes to elucidate the effect of VPA. Our multi-parametric strategy of probing physical properties of chromosomes offers a new scope in terms of analytical tools for studying chromosomal structural changes/aberrations and associated structure-function relationships pertinent to cancer.

biophysics↗

bioRxiv2023

E-Protein Protonation Titration-induced Single Particle Chemical Force Spectroscopy for Microscopic Understanding and pI Estimation of Infectious DENV

The ionization state of amino acids on the outer surface of a virus regulates its physicochemical properties toward the sorbent surface. Serologically different strain of dengue virus (DENV) shows different extents of infectivity depending upon their interactions with a receptor on the host cell. To understand the structural dependence of E-protein protonation over its sequence dependence, we have followed E-protein titration kinetics both experimentally and theoretically for two differentially infected dengue serotypes, namely DENV-2 and DENV-4. We have performed an E-protein protonation titration-induced single particle chemical force spectroscopy using an atomic force microscope (AFM) to measure the surface chemistry of DENV in physiological aqueous solutions not only to understand the charge distribution dynamics on virus surface but also to estimate the isoelectric point (pI) accurately for infectious dengue viruses. Cryo-EM structure-based theoretical pI calculations of DENV-2 surface protein were shown to be consistent with the evaluated pI value from force spectroscopy measurements. This is a comprehensive study to understand how the cumulative charge distribution on the outer surface of a specific serotype of DENV regulates a prominent role of infectivity over minute changes at the genetic level.

biophysics↗

bioRxiv2022

DNA-functionalized Gold Nanorods for Targeted Triple-modal Optical Imaging and Photothermal Therapy of Triple-negative Breast Cancer

Targeted imaging and therapy for triple-negative breast cancer (TNBC) in the perioperative period are believed to be imperative for better disease management and improved life expectancy. Still, they are not yet available in clinical settings, and only a few nanoparticle-based theranostic agents potentially offer these capabilities. Herein, we develop an innovative class of biocompatible triple-modality nanoprobes (TMNPs) that offer optical imaging using optoacoustic, fluorescence, and surface-enhanced Raman scattering (SERS), as well as photothermal therapy (PTT) with near-infrared (NIR) light. The TMNPs are fabricated by immobilizing positively charged NIR fluorophores on negatively charged DNA-coated gold nanorods (AuNR), then silica encapsulation. The DNA-based design allows the screening of commercially available positively charged NIR fluorophores for the optimum fluorescence and SERS signals. After the design optimization, we functionalize TMNPs with folate groups to target folate receptor1 (FOLR1)-overexpressing TNBC in vitro and in vivo. Our results reveal that TMNPs preferentially accumulate in the FOLR1 positive tumors in TNBC patient-derived xenograft mouse models and show excellent imaging capabilities with all three imaging modalities. Selective exposure of the tumor with NIR laser further shows efficient thermal tissue ablation without causing systemic toxicity. Collectively, TMNP holds great promise for real-time multiplexed imaging of cancer biomarkers and therapeutic capability.

bioengineering↗

bioRxiv2022

Mobility of nucleosomes is regulated by their binding partners

Chromatin accessibility is modulated in a variety of ways to create open and closed chromatin states, both of which are critical for eukaryotic gene regulation. At the single molecule level, how accessibility is regulated in the chromatin fiber composed of canonical or variant nucleosomes is a fundamental question in the field. Here, we developed a single-molecule tracking method where we could analyze thousands of canonical H3 and centromeric variant nucleosomes imaged by high-speed atomic force microscopy. This approach allowed us to investigate how changes in nucleosome dynamics in vitro inform us about chromatin accessibility in vivo. By high-speed atomic force microscopy, we tracked chromatin dynamics in real time and determined the MSD and diffusion constant for the variant centromeric CENP-A nucleosome. Furthermore, an essential kinetochore protein CENP-C reduces the diffusion constant and mobility of centromeric nucleosomes along the chromatin fiber. We subsequently interrogated how CENP-C modulates CENP-A chromatin dynamics in vivo. Overexpressing CENP-C resulted in reduced centromeric transcription and impaired loading of new CENP-A molecules. Thus, changes which alter chromatin accessibility in vitro, also correspondingly alter transcription in vivo. These data suggest a model in which variant nucleosomes encode their own diffusion kinetics and mobility, and where binding partners can suppress or enhance mobility.

biophysics↗

bioRxiv2019

CENP-C stabilizes the conformation of CENP-A nucleosomes within the inner kinetochore at human centromere

The centromere is the chromosomal locus that seeds the kinetochore, allowing for a physical connection between the chromosome and the mitotic spindle. At the heart of the centromere is the centromere-specific histone H3 variant CENP-A/CENH3. Throughout the cell cycle the constitutive centromere associated network is bound to CENP-A chromatin, but how this protein network modifies CENP-A nucleosome dynamics in vivo is unknown. Here, we purify kinetochore associated native centromeric chromatin and analyze its biochemical features using a combinatorial approach. We report that kinetochore bound chromatin has strongly reduced DNA accessibility and a distinct stabilized nucleosomal configuration. Disrupting the balance between CENP-A and CENP-C result in reduced centromeric occupancy of RNA polymerase 2 and impaired de novo CENP-A loading on the centromeric chromatin fiber, correlating with significant mitotic defects. CENP-A mutants that restore the ratio rescue the mitotic defects. These data support a model in which CENP-C bound centromeric nucleosomes behave as a barrier to the transcriptional machinery and suggest that maintaining the correct ratio between CENP-A and CENP-C levels is critical for centromere homeostasis.

biochemistry↗

bioRxiv2018

Elastic and Rigidified CENP-A Nucleosomes govern Centromeric Chromatin Plasticity

Histone variants fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. Whether and how nucleosome variants encode unique mechanical properties to their cognate chromatin structures remains elusive. Here, using novel in silico and in vitro nanoindentation methods, extending to in vivo dissections, we report that histone variant nucleosomes are intrinsically more elastic than their canonical counterparts. Furthermore, binding proteins which discriminate between histone variant nucleosomes suppress this innate elasticity and also compact chromatin. Interestingly, when we overexpress the binding proteins in vivo, we also observe increased compaction of chromatin enriched for histone variant nucleosomes, correlating with diminished access. Together, these data suggest a plausible link between innate mechanical properties possessed by histone variant nucleosomes, the adaptability of chromatin states in vivo, and the epigenetic plasticity of the underlying locus.\n\nSignificanceNucleosomes are the base unit which organize eukaryotic genomes. Besides the canonical histone, histone variants create unique local chromatin domains that fine-tune transcription, replication, DNA damage repair, and faithful chromosome segregation. We developed computational and single-molecule nanoindentation tools to determine mechanical properties of histone variant nucleosomes. We found that the CENP-A nucleosome variant is more elastic than the canonical H3 nucleosome but becomes stiffer when bound to its partner CENP-C. In addition, CENP-C induces cross-array clustering, creating a chromatin state that less accessible. These data suggest that innate material properties of nucleosomes can influence the ultimate chromatin state, thereby influence biological outcomes.

biochemistry↗