Biology subjects

Chakrabarti, J.

Publications and source records attributed to Chakrabarti, J..

15 recordsLinked to original sources

bioRxiv2026

Patient-Derived Organoids as a Model to Understand Tumor Microenvironment-Driven Nano-Bio Interactions - A Framework Towards Improved Nanomedicine Translation

Nanomedicines that perform well in conventional cell cultures often fail to translate into patients, in part due to the inability to reproduce the protein corona, and hence the biological identity, that nanoparticles acquire within the tumor microenvironment (TME). Here, we establish patient-derived colonic organoids (PDCOs) as a platform for profiling nano-bio interactions under physiologically tumor-relevant conditions using graphene quantum dots (GQD) and Silicon Quantum Dots (SiQD) as material-distinct probes. By combining spectral flow cytometry, confocal imaging, dynamic light scattering, and multi-spectral dimension-reduction analysis of single-cell uptake, we resolve how nanoparticle identity, uptake, and intracellular fate diverge between conventional culture and patient tissue. Conventional two-dimensional culture overestimated internalization by two-fold relative to ex vivo PDCOs, while TME-specific proteins directed nanoparticles to distinct organoid subpopulations. Exploiting this, we engineer the protein corona with tumor-specific proteins to redirect SiQDs to chemoresistant cells, from 3.8% to 70%, an 18-fold increase in targeted delivery to the cells that drive therapy resistance. Internalization was also model-dependent. PDCOs and fibroblasts favored clathrin/dynamin (DNM1)-mediated endocytosis into lysosomes, whereas cancer cells favored caveolae (CAV1)-mediated entry that bypasses lysosomal degradation. This distinction determines whether a nanocarrier is degraded or delivered intact. Collectively, these findings turn the protein corona into a programmable design tool and establish PDCOs as a next-generation platform for precision nano-oncology.

bioengineering↗

bioRxiv2026

pH Induced Changes in Protein Structure and Hydration

The molten globule (MG) state is an intermediate in the unfolding pathway of proteins, typically triggered by denaturing agents such as urea, extreme pH, high pressure, or heat. The microscopic details of such states are far from understood. Here we study the MG states in protein Hen Egg-White Lysozyme (PDB ID: 1AKI) using microscopic constant pH molecular dynamics (CpHMD) simulations and experiments across a wide pH range. We observe that the titratable residues act as key drivers of conformational fluctuations, promoting the emergence of MG states at extreme pH. These states display partial unfolding, and small global structural changes (< 7% deviation). Hydration around the fluctuating acidic residues shows reduced water density and weakened hydrogen bonding at low pH. At high pH, hydration around acidic residues increases relative to pH = 7, whereas hydration around basic residues decreases. The translational and rotational dynamics of hydration water also exhibit pronounced pH dependence: the translational diffusion coefficient (Dtrans) increases linearly with decrease in pH in acidic medium and increases linearly with increasing pH in the basic regime. The rotational diffusion (Drot) shows similar dependencies on pH except a break at pH {approx} 4 corresponding to acidic residue pKa values. Our results may be useful to identify ligand binding of lysozyme in extreme pH conditions.

biophysics↗

bioRxiv2026

Preservation of Human Colonic Stem Cells Requires an ERK Dynamics Checkpoint Mediated by AKT

Colonic stem cells reside in a microenvironment enriched in epidermal growth factor, which is essential for their survival and can activate both PI3K-AKT and MAPK-ERK pathways. This predicts co-activation of both pathways within the growth factor-high stem cell compartment at the base of crypts. However, in patient-derived human colonic organoids and normal human tissue, stem cells maintain robust AKT activity while suppressing ERK signaling despite active EGFR engagement. As stem cells differentiate, they activate pulsatile ERK signaling, which is essential for migration, survival, and maintenance of barrier function. We show that AKT-dependent phosphorylation of RAF-1 at serine 259 establishes a post-receptor checkpoint that maintains ERK temporal dynamics in stem cells. Acute activation of ERK in stem cells triggers rapid global differentiation. Disruption of the ERK checkpoint via mutation of serine 259 leads to sustained AKT and ERK co-activation in stem cells. Unlike ERK/AKT coactivation driven by apoptosis, co-activation in the stem cell compartment results in the emergence of a neoplastic, architecturally disorganized cell population dominating the cell fate profile. Incredibly, introducing brief ERK pulses through AKT inhibition or ERK activation triggers re-differentiation of neoplastic cells. Consistent with duration-dependent MAPK encoding principles, these data demonstrate that regardless of baseline signaling amplitude, ERK signaling dynamics are epistatic to total kinase signaling load in human colonic stem cells. SIGNIFICANCEStem cells must balance self-renewal and differentiation while remaining responsive to continuous mitogenic stimulation to preserve tissue homeostasis. When self-renewal is impaired, wound healing and barrier integrity decline, whereas loss of proper differentiation drives tumorigenesis. Our findings demonstrate that this balance in the human colon is achieved through temporal control of kinase signaling rather than modulation of ligand availability. By establishing an AKT-dependent ERK dynamics checkpoint, colonic stem cells suppress differentiation-inducing ERK pulses while maintaining growth factor responsiveness. These results identify kinase dynamics as a fundamental determinant of epithelial homeostasis and suggest that subtle alterations in these dynamics may destabilize tissue organization during regeneration or chronic inflammation. Temporal encoding of kinase activity thus represents a central organizing principle in human stem cell biology.

cell biology↗

bioRxiv2025

Heterogeneous water dynamics in the Hyaluronan-DPPC Interfaces

The synergistic interactions between hyaluronic acid (HA) and other cellular components, particularly lipids and water, play a vital role in maintaining cellular structure and function. Variations in HA concentration and chain length lead to changes in the mechanical and viscoelastic properties of diseased cells, such as those in colon cancer and osteoarthritis. Despite its biological significance, the microscopic dynamics at the HA-water and lipid bilayer interface remain poorly understood. In this study, we investigate the dynamic properties of water molecules at the interface between HA and Dipalmitoylphosphatidyl-choline (DPPC) lipid bilayer using molecular dynamics simulations. The interfacial and hydration water exhibit non-Gaussian self van Hove functions, indicating dynamic hetero-geneity. The diffusivity distributions reveal contrasts in the underlying translational and rotational dynamics between the two regions. In the diffusive interface, while diffusion coefficients decrease with increasing HA concentration, they show only a weak dependence on HA chain length. However, water dynamics in the subdiffusive hydration layer are largely unaffected by the presence of HA chains.

biophysics↗

bioRxiv2025

Structure-Preserving Coarse-Grained Simulationof Proteins in Explicit Solvent

Many biologically relevant processes occur on time and length scales which are far beyond the reach of atomistic simulations. These processes include large protein dynamics and the self-assembly of biological materials. Coarse-grained molecular modeling allows computer simulations on length and time scales 2-3 orders of magnitude larger than atomistic simulations, bridging the gap between the atomistic and mesoscopic scales. However, the structural information involving the dihedral angles is lost in coarse-graining. We develop a simple coarse-grained protein model with structural information in explicit solvent. We represent the center of mass of each residue as a polymer bead and water oxygen as a solvent bead. Each polymer bead has five degrees of freedom: position of the center and two additional variables for the backbone dihedral angles. All interaction parameters for bonded, non-bonded, dihedral coupling and bead-solvent interactions are derived from the equilibrated all-atom molecular dynamics simulation trajectory. We find that our coarse-grained approach reproduces residue-level structural information that closely matches the crystal structures and all-atom simulation results.

biophysics↗

bioRxiv2025

Spatial Biology and Organoid Technologies Reveal a Potential Therapy-Resistant Cancer Stem Cell Population in Pancreatic Ductal Adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies with a 5-year survival rate of less than 10%. Chemotherapy is the current standard-of-care (SOC) for advanced PDAC; however, treatment resistance driven by a complex and immunosuppressive tumor microenvironment (TME) limits its effectiveness. To address mechanisms of resistance, we studied cabozantinib (cabo), a multi-kinase inhibitor approved for several solid tumors. Cabo has shown promise in preclinical PDAC models and clinical trials, particularly when combined with immunotherapy, but its mechanisms of action within the human PDAC TME and its potential to overcome therapy resistance remain unclear. Using CosMx Spatial Molecular Imaging (CosMx SMI) and Orion Multiplex Immunofluorescence (MxIF), we analyzed PDAC tissues collected after first line systemic chemotherapy and the Whipple surgical procedure, as well as from liver or lung metastatic sites from patients with PDAC. To functionally model the TME, we established matched patient-derived organoid (PDO) co-cultures harboring cancer associated fibroblasts (CAFs), and autologous immune cells (IMM) (PDO/CAF/IMM). These models were used to evaluate the effects of cabo and pembrolizumab, an anti-PD1 immune checkpoint inhibitor, with benchmarking of findings to the patients TME. Spatial analysis of post-chemotherapy and metastatic PDAC tissues revealed heterogeneous cellular neighborhoods within the TME, including enrichment of Schwann cells, CAFs, T regulatory cells, and cancer stem cells (CSCs). Distinct niches were observed in metastatic liver tissues, characterized by mesenchymal stem cells, fibroblasts, including myCAFs and iCAFs, and CSCs expressing CD44 and TROP2. In PDO/CAF/IMM co-cultures, treatment with cabo in combination with pembrolizumab enhanced cancer cell death by depleting myeloid-derived suppressor cells (MDSCs) and promoted cytotoxic T lymphocyte proliferation. Across both patient tissues and treated co-cultures, a persistent SOC-resistant cancer stem cell population emerged that expressed CD44 variant 9 (CD44v9). Taken together, these integrative spatial and organoid-based studies demonstrate that cabo can remodel the PDAC TME and potentiate PD-1 immunotherapy in preclinical models, while resistance associates with a CD44v9+ CSC population, revealing a potential therapeutic target.

cancer biology↗

bioRxiv2024

Dynamics of aqueous suspension of short Hyaluronic acid chains near DPPC bilayer

The synergy between Hyaluronic acid (HA) and lipid molecules plays a crucial role in synovial fluids, cell coatings, etc. Diseased cells in cancer and arthritis, show changes in HA concentration and chain size, impacting the viscoelastic and mechanical properties of the cells. Although the solution behavior of HA is known in experiments, a molecular-level understanding of the role of HA on the dynamics at the interface of HA-water and the cellular boundary is lacking. Here we perform atomistic molecular dynamics simulation of short HA chains in explicit water solvent in presence of DPPC bilayer, relevant in pathological cases. We identify a stable interface between HA-water and the bilayer where the water molecules are in contact with the bilayer and the HA chains are located away without any direct contact. Both translation and rotation of the interfacial waters in contact with the lipid bilayer and translation of the HA chains exhibit subdiffusive behavior. The diffusive behavior sets in slightly away from the bilayer, where the diffusion coefficients of water and HA decrease monotonically with increase in HA concentration. On the contrary, the dependence on HA chain size is only marginal due to enhanced chain flexibility as their size increases. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/580078v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@618f6aorg.highwire.dtl.DTLVardef@10e9f53org.highwire.dtl.DTLVardef@fe0efforg.highwire.dtl.DTLVardef@18a8f09_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Conformational stability and order of Hoogsteen base pair induced by protein binding

Several experimental studies have shown that in the presence of proteins, the Hoogsteen (HG) base pair (bp) becomes stabilized. The molecular mechanism underlying this stabilization is not well known. This leads us to use all-atom molecular dynamics simulation to examine the stability of the HG bp in duplex DNA in both the absence and presence of proteins. We use conformational thermodynamics to investigate the stability of a HG bp in duplex DNA at the molecular level. We also compute the changes in the conformational free energy and entropy of DNA when DNA adopts a HG bp in its bp sequence rather than a Watson-Crick (WC) bp in both naked DNA and protein-bound DNA complex. We observe that the HG bp and the entire DNA duplex conformation are stabilized and ordered in the presence of proteins. Sugar-phosphate, sugar-base, and sugar-pucker torsion angles play key roles in stabilizing and ordering the HG bp in the protein-bound DNA complex.

biophysics↗

bioRxiv2023

Fluctuation dominated ligand binding in molten globule protein

A molten globule (MG) state is an intermediate state of protein observed during the unfolding of the native structure. In MG states, milk protein -Lactalbumin (aLA) binds to oleic acid (OLA). This MG-aLA-OLA complex, popularly known as XAM-LET performs cytotoxic activities against cancer cell lines. However, the microscopic understanding of ligand recognition ability in MG state of protein is not yet explored. Motivated by this, we explore binding of bovine aLA with OLA (BAMLET) using all atom molecular dynamics(MD) simulations. We find the binding mode between MG-aLA and OLA using the conformational thermodynamics method. We also estimate the binding free energy using the umbrella sampling (US) method for both MG state and neutral state. We find that the binding free energy obtained from US is comparable with earlier experimental results. We characterize the dihedral fluctuations as the ligand is liberated from the active site of the protein using steered molecular dynamics. The long-live fluctuations occur near the ligand binding site, which eventually transfers towards Ca2+ binding site as the ligand is taken away from the protein. TOC Graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/538683v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1e374f7org.highwire.dtl.DTLVardef@142d1aorg.highwire.dtl.DTLVardef@9d3636org.highwire.dtl.DTLVardef@152542b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Unraveling the Interactions between Human DPP4 Receptor, SARS-CoV-2 Variants, and MERS-CoV, converged for Pulmonary Disorders Integrating through Immunoinformatics and Molecular Dynamics

Human coronaviruses like MERS CoV are known to utilize dipeptidyl peptidase 4 (DPP4), apart from angiotensin-converting enzyme 2(ACE2) as potential co-receptor for viral cell entry. DPP4, ubiquitous membrane-bound aminopeptidase is closely associated with elevation of disease severity in comorbidities. In SARS-CoV-2, there is inadequate evidence for combination of spike protein variants with DPP4, and underlying adversity in COVID19. To elucidate this mechanistic basis, we have investigated interaction of spike protein variants with DPP4 through molecular docking and simulation studies. The possible binding interactions between receptor binding domain (RBD) of different spike variants of SARS-CoV-2 and DPP4 have been compared with interactions observed in experimentally determined structure of complex of MERS-CoV with DPP4. Comparative binding affinity confers that Delta-CoV-2:DPP4 shows close proximity with MERS-CoV:DPP4, as depicted from accessible surface area, radius of gyration, number of hydrogen bonding and energy of interactions. Mutation in delta variant, L452R and T478K, directly participate in DPP4 interaction enhancing DPP4 binding. E484K in alpha and gamma variant of spike protein is also found to interact with DPP4. Hence, DPP4 interaction with spike protein gets more suitable due to mutation especially due to L452R, T478K and E484K. Furthermore, perturbation in the nearby residues Y495, Q474 and Y489 is evident due to L452R, T478K and E484K respectively. Virulent strains of spike protein are more susceptible to DPP4 interaction and are prone to be victimized in patients due to comorbidities. Our results will aid the rational optimization of DPP4 as a potential therapeutic target to manage COVID-19 disease severity.

bioinformatics↗

bioRxiv2022

Genetically Engineered Human Pituitary Corticotroph Tumor Organoids Exhibit Divergent Responses To Glucocorticoid Receptor Modulators

Cushings disease (CD) is a serious endocrine disorder attributed to an ACTH-secreting pituitary neuroendocrine tumor (PitNET) that subsequently causes chronic hypercortisolemia. PitNET regression has been reported following treatment with the investigational selective glucocorticoid receptor (GR) modulator relacorilant, but the mechanisms behind that effect remain unknown. Human PitNET organoid models were generated from induced human pluripotent stem cells (iPSCs) or fresh tissue obtained from CD patient PitNETs (hPITOs). Genetically engineered iPSC derived organoids were used to model the development of corticotroph PitNETs expressing USP48 (iPSCUSP48) or USP8 (iPSCUSP8) somatic mutations. Organoids were treated with the GR antagonist mifepristone or the GR modulator relacorilant with or without somatostatin receptor (SSTR) agonists pasireotide or octreotide. In iPSCUSP48 and iPSCUSP8 cultures, mifepristone induced the predominant expression of SSTR2 with a concomitant increase in ACTH secretion and tumor cell proliferation. Relacorilant predominantly induced SSTR5 expression and tumor cell apoptosis with minimal ACTH induction. Hedgehog signaling mediated the induction of SSTR2 and SSTR5 in response to mifepristone and relacorilant. Relacorilant sensitized PitNET organoid responsiveness to pasireotide. Therefore, our study identified the potential therapeutic use of relacorilant in combination with somatostatin analogs and demonstrated the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD patients. HIGHLIGHTSO_LICushing disease (CD) is a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) that leads to chronic hypercortisolemia C_LIO_LIMifepristone (Korlym(R)), a non-selective glucocorticoid receptor (GR) antagonist, is an approved treatment for patients with Cushing disease, and competes with the binding of cortisol to the GR as well as the binding of progesterone to the progesterone receptor. C_LIO_LIRelacorilant is an investigational selective GR modulator in development for the treatment of Cushing syndrome that, unlike mifepristone, does not bind to the other hormone receptors. C_LIO_LIUnlike mifepristone, relacorilant does not significantly raise systemic cortisol levels, and cases of PitNET regression with relacorilant have been reported. However, the mechanisms behind these clinical differences remained unknown. C_LIO_LIPitNET organoids were generated from: 1) CRISPR-Cas9 gene editing of patient iPSCs, and 2) CD patient corticotroph PitNETs (hPITOs) and used to compare the diverse effects of mifepristone and relacorilant in a human-relevant model that recapitulates the PitNET microenvironment in vitro. C_LIO_LIMifepristone and relacorilant have different effects on the induction of somatostatin receptor (SSTR) SSTR2 and SSTR5 expression, ACTH secretion and PitNET organoid proliferation and apoptosis. C_LI BRIEF COMMENTARYO_ST_ABSBackgroundC_ST_ABSCushings disease (CD), a serious endocrine disorder caused by an adrenocorticotropic hormone (ACTH)-secreting pituitary neuroendocrine tumor (PitNET) leads to chronic hypercortisolemia. Approved for the treatment for CD, Mifepristone (Korlym(R)) is a non-selective glucocorticoid receptor (GR) antagonist with additional competitive binding with progesterone for the progesterone receptor. Relacorilant, an investigational selective GR modulator in development for the treatment of CD, does not bind to the other hormone receptors. Translational SignificancePatient-derived PitNET organoids recapitulate the tumor microenvironment in vitro. PitNET organoids revealed the advantages of relacorilant over mifepristone, supporting its further development for use in the treatment of CD.

physiology↗

bioRxiv2022

Conformational fluctuations in molten globule state of α-lactalbumin

Molten globule (MG) state is an intermediate state of protein observed during folding into native structure. MG state of protein is induced by various denaturing agent (like Urea), extreme pH, pressure and heat. Experiments suggest that MG state of some protein is functionally relevant even if there is no well-defined tertiary structure. Earlier experimental and theoretical studies suggest that MG state of the protein is dynamic in nature, where conformational states are interconverted in nanosecond time scales. These observations lead us to study and compare conformational fluctuations of MG state to those of intrinsic disordered protein (IDP). We consider -Lactalbumin(aLA) protein, which shows MG state at low pH upon removal of calcium (Ca2+) ion. We use constant pH molecular dynamics simulation (CpHMD) to maintain low pH during simulation. We use the dihedral principal component analysis, the density based clustering method and the machine learning technique to identify the conformational fluctuations. We observe metastable states in the MG state. The residues containing the essential coordinates responsible for metastability belong to stable helix in crystal structure, but most of them prefer unstructured or bend conformation in MG state. These residues control the exposure of the putative binding residues for fatty acids. Thus, the MG state of protein behaves as intrinsic disorder protein, although the disorder here is induced by external conditions.

biophysics↗

bioRxiv2022

Effect on the conformations of spike protein of SARS-CoV-2 due to mutation

The spike protein of SARS CoV-2 mediates receptor binding and cell entry and is the key immunogenic target for virus neutralization and the present attention of many vaccine layouts. It exhibits significant conformational flexibility. We study the structural fluctuations of spike protein among the most common mutations appeared in variant of concerns (VOC). We report the thermodynamics of conformational changes in mutant spike protein with respect to the wildtype from the distributions of the dihedral angles obtained from the equilibrium configurations generated via all-atom molecular dynamics simulations. We find that the mutation causes the increase in distance between N-terminal domain and receptor binding domain leading to an obtuse angle cosine{theta} distribution in the trimeric structure in spike protein. Thus, increase in open-state is conferred to the more infectious variants of SARS-CoV-2. The thermodynamically destabilized and disordered residues of receptor binding motif among the mutant variants of spike protein are proposed to serve as better binding sites for host factor. We identify a short stretch of region connecting the N-terminal domain and receptor binding domain forming linker loop where many residues undergo stabilization in the open state compared to the closed one.

biophysics↗

bioRxiv2022

Live-Cell Imaging in Human Colonic Monolayers Reveals Erk Waves Limit the Stem Cell Compartment to Maintain Epithelial Homeostasis

The establishment and maintenance of different cellular compartments in tissues is a universal requirement across all metazoans. Maintaining the correct ratio of cell types in time and space allows tissues to form patterned compartments and perform complex functions. Patterning is especially evident in the human colon, where tissue homeostasis is maintained by stem cells in crypt structures that balance proliferation and differentiation. Here we developed a 2D patient derived organoid (PDO) screening platform to study tissue patterning and kinase pathway dynamics in single cells across hundreds of conditions. Using this system, we discovered that waves of Erk signaling induced by apoptotic cells play a critical role in maintaining tissue patterning and homeostasis. If Erk is activated acutely across all cells instead of in wavelike patterns, then tissue patterning, and stem cell maintenance are lost. Conversely, if Erk activity is inhibited, then stem cells become unrestricted and expand dramatically. This work demonstrates that the colonic epithelium requires coordinated Erk signaling dynamics to maintain patterning and tissue homeostasis. Our work reveals how Erk can antagonize stem cells yet support cell replacement and the function of the gut.

cell biology↗

bioRxiv2021

Immune escape facilitation by mutations of epitope residues in RdRp of SARS-CoV-2

SARS-CoV-2 has considerably higher mutation rate. SARS-CoV-2 possesses a RNA dependent RNA polymerase (RdRp) which helps to replicate its genome. The mutation P323L in RdRp is associated with the loss of a particular epitope (321-327) from this protein which may influence the pathogenesis of the concern SARS-CoV-2 through the development of antibody escape variants. We consider the effect of mutations in some of the epitope regions including the naturally occurring mutation P323L on the structure of the epitope and their interface with paratope using all-atom molecular dynamics (MD) simulation studies. P323L mutations cause conformational changes in the epitope region by opening up the region associated with increase in the radius of gyration and intramolecular hydrogen bonds, making the region less accessible. Moreover, the fluctuations in the dihedral angles in the epitope:paratope (IgG) interface increase which destabilize the interface. Such mutations may help in escaping antibody mediated immunity of the host.

bioinformatics↗