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Pramanik, D.

Publications and source records attributed to Pramanik, D..

13 recordsLinked to original sources

MifRix: An Integrated microbiome framework for predicting generic and disease-specific risks investigating inter-disease diagnostic cross-talks and intra-disease signature variability

The human gut microbiome is increasingly recognized as a diagnostic indicator across diverse diseases, yet unified frameworks integrating taxonomic and functional features for multi-disease risk assessment, while capturing disease-specific variability in microbiome-alteration signatures, remain limited. Here we present MifRix (Microbiome-inferred Risk-scores with explainability), a two-step ensemble machine-learning framework integrating microbial composition with composition-derived functional signatures to predict generic and disease-specific risk scores across 10 major diseases, coupled with profiling of risk-explainable microbiome features. MifRix was trained using 38,054 gut microbiomes spanning 150 cohorts and 48 nationalities, leveraging taxa abundance and taxa-inferred functional profiles derived from 57,743 functional features mapped across 4,814 species-level taxa. On unseen validation datasets (4,649 microbiomes, 32 cohorts), MifRix outperformed established microbiome health metrics, with disease-specific risk scores achieving strong discrimination (AUC: 0.92-0.99). The explainability module revealed shared microbial signatures across disease pairs, organizing all ten diseases along a continuous gastrointestinal-to-neurological risk gradient, driven not by whole-disease microbiome alteration signatures but by specific, reproducible sub-signatures within each disease. Applied across independent cohorts, MifRix scores further identified population subgroups and individuals at elevated risk for related diseases, flagged precursor/pre-disease states, and tracked therapy-associated response, establishing an interpretable framework for microbiome-based precision diagnostics and disease-risk stratification.

microbiology↗

Molecular rewiring and compensatory mechanisms sustain DNA recognition in mutant ZTA transcription factor: insights from molecular dynamics simulations

Protein-DNA complexes are stabilized by various interactions forming an interaction network between the protein and DNA molecules. Any change in the system - whether through mutations in the protein or DNA, external factors, or protein conformational transitions -- can alter this interaction network, thereby affecting structural and functional aspects. Employing all-atom classical molecular dynamics, we investigated how the interaction network in the ZTA TF-DNA is rewired when key arginine residues in ZTA are mutated to oppositely charged glutamic acids. Using the MMPBSA technique, we calculated per-residue binding energies for all systems and correlated binding affinity with structural features. Our detailed mechanistic study shows that when key arginine residues are mutated, new interactions are formed either around the mutation site and/or in other ZTA monomer. Through load-sharing, the system attempts to counter-balance the interaction load, leading to reorganization of the interaction network. As the number of mutations increases from single-to-double site, the system is able to partially maintain its structural stability. However, with multi-site mutations, even after reorganization of the interaction network, system cannot sustain its structural stability and therefore becomes destabilized. Despite the structural symmetry of the ZTA TF, we observed asymmetric monomer contributions upon mutation. Overall, our rigorous mechanistic studies provide deeper insights into the mechanism of interaction network reorganization in ZTA-DNA system. These comprehensive insights may be useful for tuning binding affinity and structural adaptability under adverse conditions. Since ZTA is a key factor in the Epstein-Barr virus (EBV), this study will be central to understanding DNA recognition and developing drug therapeutics targeting viral transcription factors in EBV.

biochemistry↗

Thermodynamics and unbinding kinetics of A22 at multiple actin binding sites revealed by enhanced sampling simulations

The cytoskeletal protein plays a major role in various cellular processes. Understanding the interactions of small molecules with cytoskeletal protein therefore might help in the development of therapeutics. Employing combined molecular docking, all-atom molecular dynamics (MD) and infrequent well-tempered metadynamics (iWT-MetaD) we studied bacterial inhibitor A22 and actin protein interaction. Five probable A22 binding sites (S1-S5) were observed in actin and unbiased MD simulations of 100 ns - 500 ns showed structural stability at these sites. Interaction analyses showed A22 to mostly forms transient interactions except at sites S4 and S5 where long-lives interactions were present. Enhanced sampling simulations quantitatively estimated ligand dissociation free energy ({Delta}G) [~] - 2 kcal/mol to - 3.5 kcal/mol for sites S1-S4 with residence times [~] {micro}s to ms. For site S5, we observed the highest binding affinity ({Delta}G [~] - 6.05 {+/-} 0.48 kcal/mol) and longest residence time [~] 0.75 sec. Analyses of the dissociation trajectories predict multiple dissociation pathways for A22 and found key gatekeeper residues at specific-sites facilitating ligand dissociation. Comparison of A22 with other known inhibitors suggest that A22 binds actin with relatively lower affinity, however, exhibits site-specific unbinding kinetics. Thus, our study provides a detailed mechanistic overview of A22-actin interaction, its various binding modes, and unbinding kinetics. It also shows the importance and usage of infrequent metadynamics in exploring rare events like ligand-protein interaction. Deeper insights gained from this study expands our understanding of cytoskeletal ligand dynamics. These knowledges will be paramount in designing drug targeting cytoskeletal protein actin. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/663866v2_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@dea318org.highwire.dtl.DTLVardef@a592a4org.highwire.dtl.DTLVardef@3e8df1org.highwire.dtl.DTLVardef@d8dddb_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

biophysics↗

Influence of DNA sequences on thermodynamic and structural stability of ZTA transcription factor - DNA complex: An all-atom molecular dynamics study

The Epstein-Barr virus (EBV) is one of the cancer-causing gamma type viruses. Although more than 99% people are infected by this virus at some point, it remains in the body in a latent state, typically causing only minor symptoms. Our current understanding is that a known transcription factor (TF), the ZTA protein, binds with dsDNA (double stranded deoxyribonucleic acid) and plays crucial role in mediating the viral latent-to-lytic cycle through binding of specific ZTA responsive elements (ZREs). However, there is no clear understanding of the effect of DNA sequences on the structural stability and quantitative estimation of the binding affinity between the ZTA TF and DNA, along with their mechanistic details. In this study, we employ integrated classical all-atom molecular dynamics (MD) and enhanced sampling simulations to study the ZTA-dsDNA structural properties, thermodynamics, and mechanistic details for the ZTA protein and for two different dsDNA systems: core motif and core motif with flanking end sequences. For each system, we studied three different ZTA responsive elements (ZREs) sequences: ZRE 1, ZRE 2 and ZRE 3. We performed structural analyses, including RMSD and RMSF calculations, to assess conformational stability, along with detailed interaction profiles and hydrogen bond analysis. We conducted residue-level and nucleic acid-level analyses to assess the important protein residues and DNA bases forming interactions between the ZTA and dsDNA systems. We also explored the effect of adding flanking end sequences to the core motif on DNA groove lengths and interstrand hydrogen bonds. Our results indicate that the flanking sequences surrounding the core motif significantly influence the structural stability and binding affinity of the ZTA-dsDNA complex. Among ZRE 1, ZRE 2, and ZRE 3, particularly when paired with their naturally occurring flanking ends, ZRE 3 exhibits higher stability and binding affinity. These findings provide insights into the molecular mechanisms underlying EBV pathogenesis and may indicate potential targets for therapeutic intervention. A detailed of the binding mechanisms will allow for the design of better-targeted therapies against EBV-associated cancers. This study will serve as a holistic benchmark for future studies on these viral protein interactions.

biophysics↗

Selective promotion of oligonucleotides in the course of evolution

Evolution shapes the structure and content of genomes, yet the contribution of local sequence composition to variant selection remains poorly understood. While traditional models emphasize protein function or cross-species conservation, we propose that intra-genomic patterns of oligonucleotide (k-mer) frequencies also reflect selective forces. To explore this, we developed kGain score, a metric that quantifies the frequency shift of a k-mer upon single-nucleotide substitution, using the surrounding genomic context as a baseline. We hypothesize that variants arising in high-kGain contexts are more likely to persist due to evolutionary favorability. We validated this hypothesis across multiple systems. In E. coli and S. cerevisiae long-term evolution experiments, we found that fixed, essential, and parallel mutations consistently show elevated kGain scores. This trend held in SARS-CoV-2 variants of concern and in an in-house antibiotic adaptation experiment, where a high-kGain fusA Y515N mutation conferred resistance and maintained fitness when overexpressed, demonstrating a causal link between kGain and adaptive potential. To enable cross-species generalization, we trained a transformer-based neural network regressor on LTEE-derived mutations to predict kGain from sequence alone. The model achieved high correlation in held-out in-domain data (Pearson r = 0.81) and accurately predicted kGain trends in out-of-domain data (Pearson r = 0.82), demonstrating that k-mer-based sequence constraints learned from one genome can be effectively transferred to others. Together, our results establish kGain as a biologically meaningful, scalable metric for probing within-genome sequence selection, offering a complementary lens to existing conservation-based frameworks for understanding evolutionary fitness and variant persistence.

evolutionary biology↗

Explainable models using transcription factor binding and epigenome patterns at promoters reveal disease-associated genes and their regulators in the context of cell-types

Understanding genome-wide epigenetic regulation of diseases is important in establishing pathogenic factors and could aid in disease diagnosis, prognosis, and therapeutics. In this study, we have utilized transcription factors (TFs) and co-factor profiles (n=823) as features in machine learning models to link them to various diseases. Further, along with TFs and co-factor profiles, histone modifications ChIP-seq (n = 621), cap analysis gene expression (CAGE) tags (n = 255), and DNase hypersensitivity profiles (n = 255) as features allowed for the modeling of association of coding and non-coding genes to diseases. Such predicted associations could be independently validated using genome-wide association data and survival analysis. However, the unique aspect of our approach is that it highlights the link between TF binding patterns and diseases in the context of cell types. Besides highlighting relevant TF-binding in known cell-types associated with diseases, it also provided their surprising link with TFs expressed in immune cells and other seemingly non-related cells. Further investigation revealed such links to be genuine and potentially useful for prognosis, further revealing the need to deconvolve a set of known genes associated with diseases.

bioinformatics↗

Probing the Molecular Interactions of A22 with Prokaryotic Actin MreB and Eukaryotic Actin: A Computational and Experimental Study

Actin is a major cytoskeletal system that mediates the intricate organization of macromolecules within cells. The bacterial cytoskeletal protein MreB is a prokaryotic actin-like protein governing cell shape and intracellular organization in many rod-shaped bacteria including pathogens. MreB stands as a target for antibiotic development, and compounds like A22 and its analogue, MP265, are identified as potent inhibitors of MreB. The bacterial actin MreB shares structural homology with eukaryotic actin, despite lacking sequence similarity. It is currently not clear whether small molecules that inhibit MreB can act on the eukaryotic actin due to their structural similarity. In this study, we investigate the molecular interactions between A22 and both MreB and eukaryotic actin through molecular dynamics approach. Employing MD simulations and free energy calculations with an all-atom model, we unveil robust A22-MreB interaction and substantial binding affinity with eukaryotic actin. Experimental assays reveal A22s toxicity to eukaryotic cells, including yeast and human glioblastoma cells. Microscopy analysis demonstrates profound effects of A22 on actin organization in human glioblastoma cells. Overall, this integrative computational and experimental study advances our understanding of A22s mode of action and highlights its potential as a versatile tool for probing actin dynamics and as a candidate for therapeutic intervention in pathological conditions like cancer.

biophysics↗

IRI-CCE: A Platform for Evaluating CRISPR-based Genome Editing Tools and Its Components

Rapid assessment of CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated Cas protein)-based genome editing (GE) tools and their components are critical aspects for successful applications in different organisms. In many bacteria, double-stranded breaks (DSBs) generated by CRISPR/Cas tool generally cause cell death due to the lack of an efficient non-homologous end-joining pathway and restricts its use. CRISPR-based DSB-free base editors (BEs) have been applied for precise nucleotide editing in bacteria, which does not need to make DSBs. However, optimization of newer BE tools in bacteria is challenging owing to the toxic effects of BE reagents expressed using strong promoters. Improved variants of two main BEs capable of converting C-to-T (CBE) and A-to-G (ABE) have been recently developed but yet to be tested in bacteria. Here, we report a platform for in vivo rapid investigation of CRISPR-BE components in Escherichia coli (IRI-CCE) comprising different combinations of promoters/terminators. We demonstrate the use of IRI-CEE to characterize different variants of CBEs (PmCDA1, evoCDA1, APOBEC3A) and ABEs (ABE8e, ABE9e), exhibiting that each independent BE has its specific editing pattern for a given target site and promoter type. Additionally, the IRI-CCE platform offers a rapid way to screen functional gRNAs. In summary, CRISPR-BE components expressed by promoters of different strengths in the IRI-CCE allow an analysis of various BE tools, including cloned biopart modules and gRNAs.

bioengineering↗

ST6GAL1-mediated heterogeneity in sialic acid levels potentiates invasion of breast cancer epithelia

Heterogeneity in phenotypes of malignantly transformed cells and aberrant glycan expression on their surface are two prominent hallmarks of cancers that have hitherto not been linked to each other. In this paper, we identify heterogeneity in a specific glycan linkage: 2,6-linked sialic acids within breast cancer cells in vivo and in culture. Upon sorting out two populations with moderate and relatively higher cell surface 2,6-linked sialic acid levels from the triple negative breast cancer cell line MDA-MB-231, both populations (denoted as medium and high-2,6-Sial cells respectively) stably retained their levels in early passages. Upon continuous culturing, medium 2,6-Sial cells recapitulated the heterogeneity of the unsorted line whereas high 2,6-Sial cells showed no such tendency. Compared with the high 2,6-Sial, the medium 2,6-Sial cells showed greater adhesion to reconstituted extracellular matrices (ECM) as well as invaded faster as single cells. The level of 2,6-linked sialic acids in the two sublines was found to be consistent with the expression of a specific glycosyl transferase, ST6GAL1. Stably knocking down ST6GAL1 in the high 2,6-Sial cells, enhanced their invasiveness. When cultured together, medium 2,6-Sial cells differentially migrated to the edge of growing tumoroid-like cultures, whereas high 2,6-Sial cells formed the central bulk. Simulations in a Cellular Potts model-based computational environment that is calibrated to our experimental findings suggest that the heterogeneity of cell-ECM adhesion, likely regulated by 2,6-linked sialic acids facilitates niches of highly invasive cells to efficiently migrate centrifugally as the invasive front of a malignant tumor. Significance StatementCell-surface sugars are aberrantly expressed in cancer but their contributions to tumor heterogeneity are not known. In this study, we uncover and separate breast cancer populations with distinct 2,6-linked sialic acid levels. The moderately expressing population shows stronger adhesion to extracellular matrix than the high expressing population. It also invades faster through the matrix as single cells. Combining experiments with computational modelling, we show that the heterogeneity in matrix adhesion is vital to accentuating cell invasion. In some conditions, invasion of heterogeneous populations may compare with, or exceed that of, homogeneous moderately expressing populations. Our findings are vital to furthering our understanding of how cancers spread and potentially qualify efforts to manage the disease through glycan-editing or immunotherapeutic approaches.

cancer biology↗

Interactive dynamics of reaction-diffusion and adhesion predict diverse invasion strategies of cancer cells in matrix-like microenvironments

The metastasis of malignant epithelial tumors begins with the egress of transformed cells from the confines of their basement membrane to their surrounding collagenous stroma. Invasion can be morphologically diverse, ranging from dispersed mesenchymal cells to multicellular collectives. When breast cancer cells are cultured within basement membrane-like matrix (BM), or Type 1 collagen, or a combination of both, they exhibit collective-, dispersed mesenchymal-, and hybrid collective-dispersed (multiscale) invasion, respectively. In this paper, we asked how distinct these invasive modes are with respect to the cellular and microenvironmental cues that drive them. A rigorous computational exploration of invasion was performed within an experimentally motivated Cellular Potts-based modeling environment. The model comprises of adhesive interactions between cancer cells, BM- and collagen-like extracellular matrix (ECM), and reaction-diffusion-based remodeling of ECM. The model outputs were parameters cognate to dispersed- and collective- invasion. Input sweeps gave rise to a spatial output distribution that consisted of dispersed-, collective- and multiscale- invasion. K-means clustering of the output distribution followed by silhouette analysis revealed three optimal clusters: one signifying indolent invasion and two representing multiscale invasions, which we call collective-multiscale (CMI), and dispersed multiscale invasion (DMI), respectively. Constructing input-output mapped phenotypic spaces suggested that adhesion to BM- and collagen- matrix specify CMI and DMI respectively. Parameter perturbations confirmed these associations and revealed how the cellular phenotype may transition between the three states. Our systems-level analysis provides quantitative insights into how the diversity in matrix microenvironments may steer invasion into distinct phenotypic modes during metastasis.

cancer biology↗

An interplay between reaction-diffusion and cell-matrix adhesion regulates multiscale invasion in early breast carcinomatosis

The progression of cancer in the breast involves multiple reciprocal interactions between malignantly transformed epithelia, surrounding untransformed but affected stromal cells, and the extracellular matrix (ECM) that is remodelled during the process. A quantitative understanding of the relative contribution of such interactions to phenotypes associated with cancer cells can be arrived at through the construction of increasingly complex experimental and computational models. Herein, we introduce a multiscale 3D organo-and patho-typic model that approximates, to an unprecedented extent, the histopathological complexity of a tumor disseminating into its surrounding stromal milieu via both bulk and solitary motility dynamics. End-point and time-lapse microscopic observations of this model allow us to study the earliest steps of cancer invasion as well as the dynamical interactions between the epithelial and stromal compartments. We then construct an agent-based Cellular Potts model that incorporates constituents of the experimental model, as well as places them in similar spatial arrangements. The computational model, which comprises adhesion between cancer cells and the matrices, cell proliferation and apoptosis, and matrix remodeling through reaction-diffusion-based morphogen dynamics, is first trained to phenocopy controls run with the experimental model, wherein one or the other matrices have been removed. The trained computational model successfully predicts phenotypes of the experimental counterparts that are subjected to pharmacological treatments (inhibition of N-linked glycosylation and matrix metalloproteinase activity) and scaffold modulation (alteration of collagen density). Our results suggest that specific permissive regimes of cell-cell and cell-matrix adhesions operating in the context of a reaction-diffusion-regulated ECM dynamics, promote multiscale invasion of breast cancer cells and determine the extent to which they migrate through their surrounding stroma.

cancer biology↗

Can one trust kinetic and thermodynamic observables from biased metadynamics simulations: detailed quantitative benchmarks on millimolar drug fragment dissociation

Obtaining atomistic resolution of ligand dissociation from a protein is a much sought after experimental and computational challenge. Structural details of the dissociation process are in general hard to capture in experiments, while the relevant timescales are far beyond molecular dynamics (MD) simulations even with the most powerful super-computers. As such many different specialized enhanced sampling methods have been proposed that make it possible to efficiently calculate the dissociation mechanisms in protein-ligand systems. However, accurate benchmarks against long unbiased MD simulations are either not reported yet or simply not feasible due to the extremely long timescales. In this manuscript, we consider one such recent method "infrequent metadynamics", and benchmark in detail the various thermodynamics and kinetic information obtained from this method against extensive unbiased MD simulations for the dissociation dynamics of two different millimolar fragments from the protein FKBP in explicit water with residence times in nanoseconds to microseconds regime. We find that the metadynamics approach gives the same binding free energy profile, dissociation pathway and ligand residence time as the unbiased MD, albeit using only 6 to 50 times lower computational resources. Furthermore, we demonstrate how the metadynamics approach can self-consistently be used to ascertain whether the reweighted kinetic constants are reliable or not. We thus conclude that the answer to the question posed in the title of this manuscript is: statistically speaking, yes.

biochemistry↗

Multi-dimensional spectral gap optimization of order parameters (SGOOP) through conditional probability factorization

Spectral gap optimization of order parameters (SGOOP) (Tiwary and Berne, Proc. Natl. Acad. Sci. 113 2839 (2016)) is a method for constructing the reaction coordinate (RC) in molecular systems, especially when they are plagued with hard to sample rare events, given a larger dictionary of order parameters or basis functions, and limited static and dynamic information about the system. In its original formulation, SGOOP is designed to construct a 1-dimensional RC. Here we extend its scope by introducing a simple but powerful extension based on the notion of conditional probability factorization where known features are washed out to learn additional and possibly hidden features of the energy landscape. We show how SGOOP can be used to proceed in a sequential and bottom-up manner to (i) systematically probe the need for extending the dimensionality of the RC, and (ii) if such a need is identified, learn additional coordinates of the RC in a computationally efficient manner. We formulate the method and demonstrate its usefulness through three illustrative examples, including the challenging and important problem of calculating the kinetics of benzene unbinding from the protein T4L99A lysozyme, where we obtain excellent agreement in terms of dissociation pathway and kinetics with other sampling methods and experiments. In this last case, starting from a larger dictionary of fairly generic and arbitrarily chosen 11 order parameters, we demonstrate how to automatically learn a 2-dimensional RC, which we then use in the infrequent metadynamics protocol to obtain 16 independent unbinding trajectories. We believe our method will be a big step in increasing the usefulness of SGOOP in performing intuition-free sampling of complex systems. Finally, we believe that the usefulness of our protocol is amplified by its applicability to not just SGOOP but also other generic methods for constructing the RC.

biophysics↗