Amresh Prakash

1. TAF15 (TATA-box binding protein-associated factor 15) is a multifunctional RNA- and DNA-binding protein belongs to the FET family, which also includes FUS and EWSR1. These family members contrib...

The most prevalent form of non-small cell lung cancer (NSCLC), lung adenocarcinoma (LUAD), was closely associated with cigarette smoking. The discovery of smoking-specific biomarkers is crucial to carcinogenesis induced by smoking and designing specific therapies. The purpose of this study was to differentiate between smoking -related LUAD and non-smoking ones based on integrative transcriptomic. Annotated smoking status (smoker vs. non-smoker) and sufficient sample size were used to choose publicly available mRNA (2,266) and miRNAss (45) expression datasets of LUAD patients. GEO2R was used to identify differentially expressed genes (DEGs) and microRNAs (DEMs), and cross-platform meta-analysis. Inverse correlation and target prediction were used in constructing a smoking-specific miRNA-mRNA regulatory network. The identification of hub genes was based on protein-protein interaction (PPI) network and confirmed by survival data in TCGA-LUAD. The integrative meta-analyses lead to the finding of a group of DEGs that are constantly related to cigarette smoking in LUAD utilizing various independent datasets. Out of these 12 major genes such as AURKA, SOCS3, RARA, ELK1 and CAV1 showed significant changes in their expression levels between the smoker and non-smoker groups which highlights their potential as smoking-specific molecular signatures. Functional enrichment analysis indicated that out of the 40 pathways that were identified, 14 of them are majorly found in the major cancer-related pathways that are determined by tobacco exposure such as PI3K-AKT signalling, cytokine-cytokine receptor interactions and cell cycle regulation. Together, the cigarette smoking-related biomarkers discovered in LUAD via such a thorough meta-analysis strategy show the possibility of becoming diagnostic biomarkers, prognostic biomarkers, and therapeutic targets, and need further experimental research to determine their clinical utility.

Interferon-β is a β-helical cytokine that plays a critical role in modulating neuroinflammatory pathways. We measured the stability of IFN-β, associated with equilibrium, in terms of change in Gibbs free energy (ΔG_D⁰) between the folded (N) and denatured (D) states in the native buffer (i.e., during the absence of strong chemical denaturants, Guanidinium chloride (GdmCl) and urea are known to destroy noncovalent interactions responsible for maintaining the folded state). We have deliberated on the denaturation of IFN-β at 25 °C and pH 7.5 using two different spectroscopic probes, namely, fluorescence emission maxima (λ_max, nm) and circular dichroism (ellipticity, θ in millidegrees) at 222 nm. From these in vitro studies, we reached the following conclusions: (i) Both denaturants induce a single step (“N” to “D”) transition. (ii) Denaturation of IFN-β protein is irreversible. (iii) Protein stability in terms of ΔG_D⁰ is very low (ΔG_D⁰ = ± 3.12 kcal·mol^–1), which is likely due to the presence of hydrophobic patches (nonpolar side chains) on the surface of the native protein as shown by ANS (8-anilinonaphthalene-1-sulfonic acid) steady-state measurements. Atomistic simulations were conducted to obtain detailed molecular insights into the structural changes occurring with increased denaturant concentration (6 M GdmCl and 8 M urea) for a period of 500 ns in water at different temperatures (300–500 K). The analysis of results obtained from simulations corroborates those obtained from in vitro measurements. This investigation is the first comprehensive analysis of the unfolding trajectories of IFN-β under different denaturants.

In Candida species, including Candidozyma auris (formerly Candida auris), overexpression of efflux pumps is a well-established mechanism of antifungal resistance. However, accumulating evidence indicates that impaired drug import may also significantly contribute to reduced antifungal susceptibility. Sugar importers, historically viewed solely as hexose transporters (HGTs), are now emerging as potential indirect modulators of antifungal uptake. Here, we performed a comprehensive inventory and functional analysis of the HGT family in C. auris to assess its contribution to antifungal import. Phylogenetic analyses revealed that C. auris HGTs are more closely related to those of Candida albicans (C. albicans) than Saccharomyces cerevisiae (S. cerevisiae). All HGT genes showed basal expression, with several significantly downregulated upon fluconazole (FLC) exposure. To establish functional relevance, we generated a mini-library of HGT deletion mutants. Notably, the Δhgt13 strain exhibited markedly increased FLC resistance, concomitant with reduced intracellular FLC accumulation and decreased membrane permeability. Consistently, molecular docking and molecular dynamics simulations demonstrated strong and stable interactions between FLC and Hgt13p. Together, these findings implicate Hgt13p as a key determinant of FLC import and membrane permeability, revealing reduced FLC import could also contribute to antifungal resistance in C. auris.

In Candida species, including Candidozyma auris (formerly Candida auris), overexpression of efflux pumps is a well-established mechanism of antifungal resistance. However, accumulating evidence indicates that impaired drug import may also significantly contribute to reduced antifungal susceptibility. Sugar importers, historically viewed solely as hexose transporters (HGTs), are now emerging as potential indirect modulators of antifungal uptake. Here, we performed a comprehensive inventory and functional analysis of the HGT family in C. auris to assess its contribution to antifungal import. Phylogenetic analyses revealed that C. auris HGTs are more closely related to those of Candida albicans (C. albicans) than Saccharomyces cerevisiae (S. cerevisiae). All HGT genes showed basal expression, with several significantly downregulated upon fluconazole (FLC) exposure. To establish functional relevance, we generated a mini-library of HGT deletion mutants. Notably, the Δhgt13 strain exhibited markedly increased FLC resistance, concomitant with reduced intracellular FLC accumulation and decreased membrane permeability. Consistently, molecular docking and molecular dynamics simulations demonstrated strong and stable interactions between FLC and Hgt13p. Together, these findings implicate Hgt13p as a key determinant of FLC import and membrane permeability, revealing reduced FLC import could also contribute to antifungal resistance in C. auris.

The RNA Recognition Motif (RRM) domain of the Ewing sarcoma (EWS) protein plays a pivotal role in RNA binding and gene regulation, being crucial for its function. However, its structural dynamics are yet to be revealed. Herein, we performed 5.5 μs cumulative molecular dynamics (MD) simulations to investigate the unfolding pathways of the EWS-RRM domain in urea and DMSO across 300–500 K. The unfolding process was characterized by using free-energy landscape (FEL) analysis, hydrogen-bond occupancy, and Gaussian Mixture Model (GMM) clustering. At lower temperatures (300–350 K), the RRM largely retained its native conformation, while extensive unfolding occurred between 400 and 450 K. Results revealed multiple conformational ensembles: native (N), native-like intermediate (I_N), intermediate (I), and unfolded (U) states, underlying the unfolding pathway of RRM. In urea at 400 K, a long-lived I-state dominated, with transient N and I_N-populations, whereas in DMSO, the I_N-state appeared more stable, that transitioned into tightly packed I-states, reflecting a stepwise unfolding via compact intermediates. At 450 K, the protein reached the U-state in both solvents, though unfolding occurred more readily in urea. This study highlights the solvent-dependent unfolding mechanisms and heterogeneous I-states of EWS-RRM, providing insight into its stability, misfolding, and potential relevance to Ewing sarcoma pathogenesis.

The ATP-binding cassette transporter superfamily plays a pivotal role in cellular detoxification and drug efflux. ATP-binding cassette subfamily G member 2 (ABCG2) referred to as the Breast cancer resistance protein has emerged as a key member involved in multidrug resistance displayed by cancer cells. Understanding the molecular basis of substrate and inhibitor recognition, and binding within the transmembrane domain of ABCG2 is crucial for the development of effective therapeutic strategies. Herein, utilizing state-of-the-art molecular docking algorithms and molecular dynamic (MD) simulations, molecular binding of substrates and inhibitors with ABCG2 are defined, distinctly. We performed extensive virtual screening of Drugbank to identify the potential candidates, and MD simulations of docked complexes were carried out in POPC lipid bilayer. Further, the binding affinities of compounds were estimated by free binding energy employing MM-GBSA. To gain deeper insight into the binding affinities and molecular characteristics contributing to inhibitory potential of certain substrates, we included some well-known inhibitors, like Imatinib, Tariquidar, and Ko 143, in our analysis. Docking results show three compounds, Docetaxel > Tariquidar > Tezacaftor having the highest binding affinities (≤ 12.00 kcal/mol) for ABCG2. Remarkably, MM-GBSA results suggest the most stable binding of Tariquidar with ABCG2 as compared to the other inhibitors. Furthermore, our results suggested that Docetaxel, Ozanimod, Pitavastatin, and Tezacaftor have the strongest affinity for the drug-binding site(s) of ABCG2. These results provide valuable insights into the key residues that may govern substrate/inhibitor recognition, shedding light on the molecular determinants influencing substrate specificity, transport kinetics, and ABCG2-mediated drug efflux. Graphical abstract