Journal of Biomolecular Structure and Dynamics

The Na+/K+-ATPase (NKA) regulates ion balance in the kidney and influences cellular processes like proliferation and apoptosis through its signal transduction. The endogenous ligand 20-Hydroxyeicosatetraenoic acid (20-HETE) contributes to inflammation and fibrosis in chronic kidney disease (CKD) and inhibits NKA activity in renal tubules. However, the molecular mechanism of this interaction remains unclear. In this study, we used in-silico approach to investigate the potential interaction between 20-HETE and NKA. Various ligands, including known NKA ligands such as cardiotonic steroids (CTS), 20-HETE, and negative controls, were docked using rigid and Induced Fit Docking to predict the affinity of the ligands toward NKA. Binding free energy calculations with the Prime Molecular mechanics with generalized Born and surface area (Prime MM/GBSA) tools were used to confirm the involvement of key amino acids in ligand-receptor interactions. The docking analyses revealed that 20-HETE exhibited a binding affinity comparable to negative control, with some differences between rigid and induced fit docking. Binding free energy data highlighted key amino acids in the 20-HETE and NKA interaction. Interaction fingerprint and mutations such as Ala330Gly and Val329Ala significantly reduced binding free energy, while Thr804Ala showed a notable decrease, underscoring the potential importance of these amino acids in ligand stabilization. These findings provide computational evidence supporting potential direct interaction between 20-HETE and NKA and identify candidate residues for future experimental validation.

Cholera, caused by Vibrio cholerae, continues to pose a serious global public health challenge, with its impact worsened by rising antibiotic resistance associated with bacterial biofilm formation. This study reveals the role of the hypothetical protein (HP) TYC33605.1 in cyclic-di-GMP (c-di-GMP)-mediated biofilm regulation and identifies natural inhibitors that disrupt this mechanism. Functional annotation revealed TYC33605.1 as a membrane-associated diguanylate cyclase (DGC) with GGDEF and sensory domains, critical for c-di-GMP synthesis and biofilm persistence. Homology modelling and molecular dynamics (MD) simulations validated its stable 3D structure (C-score: -1.22, Ramachandran favoured regions: 91.1%) and dynamic behaviour (average RMSD: 8.55 [A]). Virtual molecular docking screening of 1,092 natural compounds identified Luteolin (CID 5280445) and Sativanone (CID 13886678) as top candidates, exhibiting strong binding affinities (-9.1 and -9.0 kcal/mol, respectively) and forming hydrogen bonds, {pi}-cation, and hydrophobic interactions with key residues (Glu293, Arg364, Ala176). MD simulations (100 ns) confirmed complex stability, with Luteolin and Sativanone showing lower RMSD fluctuations (7.78 [A] and 8.13 [A]) compared to the control and apoprotein. The ADME/Tox profiles highlighted favourable pharmacodynamics (PD), pharmacokinetics (PK), high gastrointestinal absorption, no hepatotoxicity, and drug-likeness (Lipinski compliance). Principal component, probability density function, and binding free energy analyses underscore ligand-induced conformational stability. This study proposes the molecular characterisation of the HP and the bioactive compounds Luteolin and Sativanone as promising inhibitors targeting TYC33605.1, offering a novel strategy to combat biofilm-mediated antibiotic resistance and a framework for analogous antimicrobial discovery in Vibrio cholerae.

The protozoan parasite Trypanosoma brucei is implicated in deadly African sleeping sickness. Experimental studies show that T. brucei codes for three aquaporin homologs (TbAQP1 to TbAQP3). TbAQP2 has been established as the high affinity drug transporter of drugs pentamidine and melarsoprol. Mutation in TbAQP2 or its loss result in pentamidine-melarsoprol cross-resistance. TbAQP2 is also shown to transport water, glycerol and other solutes to respond to osmoregulation in the infected hosts or glycerol metabolism. Experimentally determined structures of TbAQP2 shows that it adopts the same aquaporin-like hourglass helical fold. However, the so called aromatic/arginine selectivity filter (Ar/R SF) in TbAQP2 has neither arginine nor aromatic residue and all four residues are hydrophobic. Mutation and functional studies have demonstrated the role of Ar/R SF residues in the transport and selectivity of solutes in aquaporin homologs. The intriguing question is how the completely hydrophobic Ar/R SF region enables the transport of water and glycerol molecules. In this study, we used computational approach to elucidate the molecular mechanism of water and glycerol transport. Our equilibrium molecular dynamics simulations showed that the number of water molecules transported by TbAQP2 is almost one order of magnitude higher than that of prototype water channel AQP1. Moreover, the residence time within TbAQP2 channel is much less compared to that found in AQP1. The relatively wider constriction, interactions of water molecules with the selectivity filter residues and the contact duration, all contribute to a large number of water molecules transported through TbAQP2 channel. Our umbrella sampling studies show that when glycerol is transported through TbAQP2, it participates in interactions with channel residues that can be considered as complimentary to that observed in prototype glycerol transporter GlpF. Our studies reveal the molecular mechanism of water and glycerol transport in TbAQP2 and establish that TbAQP2 is an efficient water transporter. Statement of SignificanceTrypanosoma brucei causes African sleeping sickness and a homolog of aquaporin, TbAQP2, is involved in the transport of drugs that are used to treat this disease. Developing anti-parasitic drugs requires the knowledge of molecular mechanism of the proteins function. TbAQP2 has been shown to transport water and glycerol. Permeating solutes have to pass through a narrow constriction region formed by all hydrophobic residues. In the present study, equilibrium molecular dynamics simulations showed that TbAQP2 transports water molecules faster in large quantity in comparison with mammalian AQP1. Higher water transport is due to relatively wider constriction and minimum water interactions with selectivity filter hydrophobic residues. Permeating glycerol molecule is involved in complementary interactions with the channel residues. Our studies reveal how water and glycerol are transported through hydrophobic selectivity filter in TbAQP2.

Magnetic treatment in tomato seeds (Solanum lycopersicum L.) has been studied as a biotechnological technique to induce a reduction in germination times and enhance plant development. However, the modified cellular mechanisms involved in the reduction of germination times or the improvement of development parameters are not yet clearly established. To explore a possible altered cellular mechanism, the effect of homogeneous static magnetic fields on the structure of the cyclic nucleotide-gated channel 6 (CNGC6), the modification in the organization of POPC lipids in the plasma membrane, and changes in calcium ion mobility were evaluated. For this purpose, coarse-grained molecular dynamics simulations were performed using the Martini 3 model in GROMACS, applying five different magnetic flux densities (0.000, 0.001, 0.010, 0.100, 1.000, and 10.000) T over 1 000 ns. The results showed an anisotropic effect in the longitudinal direction of the protein, which generated heterogeneous behavior among the chains of the homotetramer; this altered the conformation of the CNGC6 channel and modified the pore bottleneck. In contrast, no significant changes were observed in the conformational order of the POPC phospholipid chains. As a preliminary, single-replicate exploratory study, these results suggest that homogeneous static magnetic fields may induce specific structural modifications in the CNGC6 ion channel of Solanum lycopersicum L. without compromising the integrity of the lipid bilayer or the dynamics of ion transport within the analyzed timescale; these preliminary findings provide a molecular-level structural basis for future experimental and computational investigations of magnetic field effects on plant cyclic nucleotide-gated channels.

Predicting protein-ligand interactions in the modern drug discovery has revolved from the involvement of artificial intelligence and structural bioinformatics using Graph Neural Networks (GNNs). The limited explainability of GNN models presents an important encumbrance in biomedical research, but it has achieved a high degree of accuracy in determining and identifying binding affinity and active compounds, as evidenced by [1] [2] [3] [4]. Here this research focuses on the interpretation of protein-ligand interactions at a molecular level, a rapidly developing area within Graph Neural Networks (GNNs). Now days modern study handling techniques such as visualization techniques, attention mechanism and model-based feature ascription by model to boost, and make robust and decrease false predictions on binding. Along with some approaches include like graph pooling strategies, message-passing optimization, self-supervised learning, transfer learning and contrastive learning are rapidly utilized to enhance the representative learnings. Furthermore, integration of molecular docking simulations, hybrid deep learning architectures and protein language model gives more reliable & biological predictions of protein-ligand interactions. That focuses on given process that identifies key ligand atoms and binding residues, as well as physicochemical factors influencing affinity, through chemical thought processes. Here this research work identified the challenges of developing biologically significant explanations, transparency, and the corollary dataset biases on interpretability. The research work conducted an in-depth investigation into the consolidation of protein language models to establish more reliable pathways for future research, examining hybrid architectures, transparent and energy-efficient GNNs, and scientifically grounded AI models for drug discovery. My research work highlights that XGNNs establishes a connection between Deep Learning and Biochemical expertise with increased confidence, which will enhance the accuracy of predictive models and computational models.

Antimycin A (AA) is widely used as an inhibitor of the mitochondrial respiratory chain, targeting the Qi site of cytochrome bc1 (complex III). In photosynthetic organisms, AA is also well known to inhibit the photosynthetic PROTON GRADIENT REGULATION 5 (PGR5)-dependent cyclic electron flow around photosystem I (CEF-PSI). Although AA is frequently used as a specific inhibitor of PGR5-dependent CEF-PSI in photosynthetic reactions, we recently clarified that some of the major components of AA, which is typically a mixture of closely related compounds, also exert direct inhibitory effects on photosystem II (PSII). Nevertheless, the binding site and binding mode of AA in PSII remain largely unexplored. Structurally, AA consists of a salicylic acid moiety connected via an amide bond to a hydrophobic dilactone ring moiety. To identify important structural factors of AA for exhibiting inhibitory effects on PSII (assessed by QA- reoxidation measurements), we here investigated the relationship between structure and inhibitory potency using 38 AA-like compounds (AALCs), including commercial compounds and a series of synthetic AA analogs. Some AALCs exhibited substantially stronger impacts on PSII than natural AA. High acidity of the phenolic OH and the presence of a free amide NH of the salicylamide moiety were critical for the effects on PSII. In contrast, while the dilactone ring moiety also affected the inhibitory activity, this was replaceable with certain hydrophobic structures. Based on our results, together with the known structure-activity relationship and binding mode of AA in complex III, we propose tentative binding models for AA in PSII. HighlightsO_LIStructure-activity relationship of AA-like compounds on PSII is examined C_LIO_LISeveral AA-like compounds more potent than AA against PSII are identified C_LIO_LIPhenolic OH acidity and free amide NH of salicylamide moiety are key for AA effects C_LIO_LIThe dilactone ring moiety is replaceable with certain hydrophobic structures C_LIO_LITentative binding models for AA in PSII are proposed C_LI

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can use host derived lipids as carbon and energy source for survival. Mammalian cell entry (Mce) associated membrane (Mam) proteins are important for the stability of lipid importing Mce complexes. Mtb has five homologs of Mam proteins referred as orphaned Mam (OmamA-E) proteins. A recent study suggested that OmamC (Rv1363c) is essential for the storage and utilization of lipids under starvation in Mtb. To understand the structure and interactions of OmamC, we generated a truncated soluble variant of OmamC (OmamC129-261). Here, we report on the challenges encountered during the crystallization and structure determination of OmamC129-261 and the strategies applied to overcome them. Despite the AlphaFold2 predicted model proving an initial molecular replacement solution, experimental phasing was necessary to determine the structure of OmamC129-261. Heat treatment of protein prior to crystallization setup removed partially unfolded protein present and played a critical role in enhancing the reproducibility and diffraction quality of OmamC129-261 crystals. Although reported earlier, it is not a widely used method. It is worth to try this method, especially, when faced with poor reproducibility and diffraction of crystals.

Numerous advances in quantum and computational chemistry over the last decades, well as the development of computer science, allowed utilisation of more precise and complex models, which can be now applied to much bigger systems than in the past. The authors used Gaussian, coupled with theoretical methods, to predict a new way of peptide bond formation, which could have taken place in prebiotic conditions. To better tackle this difficult task, the properties of substrates (glycine-derived radicals) were extensively analysed, using the aforementioned tool - Gaussian, paired with taking resonance and hybridisation into account, to better understand the stereochemistry and the very nature of processes taking place. The result is a series of reactions, which without any sophisticated catalysts and with relatively low energy thresholds ({inverted exclamation}20 kcal/mol) can lead to formation of dipeptides (and further, oligopeptides). The authors also hope, the other predicted properties of the investigated molecules can be of use to any researcher, who would like to utilise them in their experiments. Author summaryOur goal was to investigate a way first peptide bonds in prebiotic conditions could have been formed. This is an extremely important step in research into the beginning of life on Earth. We found a very promising series of reactions, which uses atomic hydrogen as its only catalyst and confirmed our expectations with theoretical calculations, using Gaussian. There are two radicals derived from glycine, which perform major roles in the process, so we investigated their properties with Gaussian and verified that the results are in agreement with our own theoretical considerations. This involved checking for possible geometric isomers and conformers and creating models which could explain their properties. We are well aware that such calculations have limitations and there is no model, which is 100% accurate, so our results should be further confirmed by empirical data in the future. However, we still to be as thorough as possible in how we approached the subject.

Burkholderia mallei, a facultative intracellular Gram-negative pathogen, is the causative agent of glanders that primarily affects solipeds and sporadically transmitted to humans. Current interventions mainly rely on antibiotics; however, increasing resistance and the lack of a licensed vaccine further complicate disease management. In the present study, a consensus-based computational framework was employed on the B. mallei turkey2 proteome. Total 59 proteins - including porins, TonB receptors, autotransporters, and efflux components - were identified as surface exposed outer membrane {beta}-barrel (OMBB) proteins that were used to design a multi-epitope vaccine (MEV) construct. B- and T-cell epitopes were predicted from 59 proteins, and ten epitopes each of cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and B-cell were chosen based on their antigenicity, non-allergenicity, non-toxicity, surface accessibility, and conservation across 32 B. mallei strains. The MEV was included with suitable adjuvants at the N-terminus to enhance its immunogenicity. The 780 amino acid MEV construct was predicted to be antigenic, and soluble upon overexpression with 62.69% random coils, while the rest formed -helices and {beta}-strands. The tertiary structure of the MEV was generated and subsequently validated, indicating good structural quality. Molecular docking of the MEV with toll-like receptor 4 (TLR4) demonstrated strong affinity, and molecular dynamics simulation confirmed the structural stability of the MEV-TLR4 complex. In-silico immune simulation showed the capability of MEV to induce a strong immune response. The study proposes an MEV construct by utilizing surface exposed OMBB proteins which directly interact with the host and serve as effective immunogenic targets against B. mallei infection. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/727591v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@10cd6d8org.highwire.dtl.DTLVardef@1ed3f0borg.highwire.dtl.DTLVardef@c6173forg.highwire.dtl.DTLVardef@1204f73_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen is well known for life-threatening acute infections among the human population. The bacterium can withstand most antibiotics by using their high levels of inherent and acquired resistance mechanisms such as Biofilm-EPS, Persistence, and Quorum sensing (QS). Owing to the importance of adaptive antibiotic multi-drug resistance of P. aeruginosa, the current investigation is aimed to explore the phytochemicals derived from mangrove plants as potential agents to control biofilm and drug resistance mechanisms through a multi-mechanistic computational approach. For identifying potential compounds and target, In-silico drug repurposing technique is implemented by docking/virtual screening of 49 phytochemical compounds against 18 proteins involved in the Persister Cell formation, QS, and EPS synthesis in P. aeruginosa which resulted the proteins RelA and SpoT (persistence), PqsA, and PqSR (QS), and PelA and PelB (EPS synthesis) and compounds Taraxerone and Taraxerol to be potential. The results of docking were well corroborated with MD simulations. These targets and compounds explored through in-silico approach, are found to target potential antimicrobial pathways involving EPS synthesis, persistence genes, and QS, aiming to enhance antibiotic efficacy. Further, this study could be reference for in-vivo and in-vitro investigations to evaluate the further effectiveness of the compounds and potentiality of the proteins for MDR therapeutics of P. aeruginosa.

Cryptosporidium parvum is a protozoan parasite responsible for cryptosporidiosis, significantly threatening immunocompromised individuals, particularly HIV/AIDS patients, by causing severe diarrhea and potential mortality. Current treatments are largely ineffective, prompting investigations into new therapeutic options. This study evaluated two antiparasitic drugs: Mebendazole, used for helminth infections, and Artemisinin, used for malaria. The SKSR gene family encodes virulence factors in C. parvum, and Calcium-dependent protein kinase1 (CpCDPK1) regulates the life cycle of C. parvum; targeting these proteins may reduce growth and infection in hosts. In the current study, molecular docking was conducted taking Mebendazole and Artemisinin drugs as ligands, SKSR gene family and CpCDPK1 proteins as drug targets. Results with SKSR showed binding energy of -4.9 kcal/mol, -6.72 kcal/mol for Mebendazole and Artemisinin, respectively. Whereas, with CpCDPK1, the binding energies were -6.44 kcal/mol, -9.18 kcal/mol for Mebendazole and Artemisinin, respectively. Docking of Nitazoxanide (an in-use drug for C. parvum) with SKSR and CpCDPK1 revealed binding energies -4.2 kcal/mol, -4.81 kcal/mol, respectively. The stability of the proteins (targets) upon binding to the ligands was assessed by performing all-atom MD simulations for 100ns using the GROMACS package. No major variations were observed upon binding of Artemisinin and Mebendazole to SKSR and CpCDPK1. The findings of MD simulations imply that both proteins maintain their stability upon binding of Artemisinin and Mebendazole. Molecular Docking and MD simulation studies suggest that Artemisinin and Mebendazole are potential candidates for repurposing in the treatment of C. parvum infections, with recommendations for in vitro studies to validate these findings.

Pasteurella multocida is a facultative anaerobic, Gram-negative coccobacillus that causes pasteurellosis in companion animals (cats and dogs), livestock, and poultry. Close contact with infected animals poses a significant zoonotic risk to humans through bite wounds, scratches, licking and transfer of bodily fluids. Current treatment relies mainly on antibiotics, and the lack of a licensed human vaccine further exacerbates the challenge. In the present study, a consensus-based computational approach was employed on the P. multocida Past 9 proteome. A total of 29 outer membrane {beta}-barrel (OMBB) proteins, including TonB-dependent receptors, porins, autotransporters, adhesins and efflux pumps, were identified and used to design a multi-epitope vaccine (MEV) construct. B-cell and T-cell epitopes were predicted from the identified proteins. Ten epitopes each of cytotoxic T-lymphocyte (CTL) and helper T-lymphocyte (HTL), and three B-cell epitopes were selected based on their antigenicity, non-allergenicity, non-toxicity, surface accessibility, and conservation across eight P. multocida human-infecting strains. The MEV was supplemented with suitable adjuvants at the N-terminus to enhance its immunogenicity. The MEV construct, with a length of 459 amino acids, was predicted to be antigenic, non-allergenic, non-toxic and soluble upon expression. The MEV structural model was generated and subsequently validated, which indicated good structural quality. Molecular docking between MEV and human toll-like receptor 4 (TLR4) demonstrated strong binding affinity, and molecular dynamics simulation confirmed the structural stability of the MEV-TLR4 complex. Immune simulation of the MEV construct elicited a strong immune response. This study proposes a designed MEV candidate against human pasteurellosis and highlights OMBB proteins as potential immunogenic targets for vaccine development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/728361v1_ufig1.gif" ALT="Figure 1"> View larger version (54K): org.highwire.dtl.DTLVardef@320d63org.highwire.dtl.DTLVardef@d0ddeorg.highwire.dtl.DTLVardef@1099802org.highwire.dtl.DTLVardef@dab304_HPS_FORMAT_FIGEXP M_FIG C_FIG

Dengue Virus Serotype 2 is a human pathogenic flavivirus that encodes a non-structural protein 3 (DEN2-NS3) containing a helicase domain essential for viral replication. DEN2-NS3 utilizes energy derived from NTP hydrolysis to unwind dsRNA and dsDNA. A galloylated catechin, (-)-epigallocatechin gallate (EGCG), was previously reported to be highly potent against the Zika Virus NS3 helicase, with an IC50 value observed at 295.7 nM. This prompted an investigation to determine if three catechins, namely, (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECG), and EGCG, would act as potent inhibitors of DEN2-NS3. Enzyme-inhibition assays revealed that the helicase catalytic domain, DEN2-NS3(S171-K618), is strongly inhibited by these galloylated catechins. We observed Ki values of 400 {+/-} 86.6 nM for EGCG (mixed-mode inhibition with respect to ATP) and 550 {+/-} 250 nM for ECG (uncompetitive inhibition with respect to ATP). Furthermore, using a computational workflow starting with SiteMap, we provide evidence that a highly druggable pocket exists within the RNA-binding cavity, involving residues ASP290, ARG387, ASP409, MET429, HIS487, ASP541, ARG599, and ASP603. These catechins were each analyzed through 200-ns molecular dynamics (MD) simulations to evaluate the binding stability within the target DEN2-NS3 binding pocket. Computational results revealed that EGCG and ECG maintained high stability, forming shared, highly persistent amino acid contacts (>45% occupancy) with ASP603, ARG599, ASP541, and ARG387. In conclusion, we have demonstrated that EGCG and ECG achieve strong binding and allosteric disruption of the critical RNA-binding channel. We suggest that future structural optimization of these compounds into stable prodrug derivatives could yield promising antiviral therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/733882v1_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@2db363org.highwire.dtl.DTLVardef@5c2fdaorg.highwire.dtl.DTLVardef@49bf8eorg.highwire.dtl.DTLVardef@1bf31f1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Cutaneous melanoma is the most aggressive malignant skin tumor, and metastasis represents the primary cause of patient mortality. Bisphenol S (BPS) has an unclear influence on melanoma metastasis and its underlying molecular mechanisms. Methods: Potential BPS targets were predicted using the SEA, SwissTargetPrediction, and SuperPred databases. Based on TCGA-SKCM transcriptomic data, differential expression analysis was performed, and Weighted Gene Co-expression Network Analysis (WGCNA) was employed to construct a gene co-expression network. Candidate genes were obtained by integrating BPS-related targets, differentially expressed genes (DEGs), module genes, and univariate Cox regression genes, followed by Gene Ontology (GO)/Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network construction. Least Absolute Shrinkage and Selection Operator (LASSO)-Cox regression was applied to screen core prognostic genes and construct a risk prediction model. Further analyses included network construction, molecular docking, and 100 ns molecular dynamics (MD) simulation. Results: Integration of BPS-related targets, DEGs, WGCNA module genes, and Cox regression results yielded 13 candidate genes enriched in kinase activity regulation and melanoma-related pathways. LASSO-Cox regression ultimately identified three core prognostic genes--ABCB1, PIM2, and TSHR--all significantly upregulated in metastatic tissues, with area under the curve (AUC) values of approximately 0.7. High-expression patients exhibited significantly better overall survival than low-expression patients (P < 0.05). A nomogram incorporating the three genes and clinical parameters demonstrated good calibration performance. Within the ceRNA network, MALAT1 and hsa-miR-155-5p were identified as key regulatory molecules, and 37 potential transcription factors were predicted, including CEBPA, JUN, and STAT3. Molecular docking revealed strong binding affinities of BPS toward ABCB1 , PIM2, and TSHR, and MD simulations confirmed the structural stability of all three complexes. Conclusion: ABCB1, PIM2, and TSHR are the core target genes through which BPS influences melanoma metastasis via multidrug resistance, kinase signaling, and receptor-mediated signal transduction. The prognostic model based on these three genes demonstrates good clinical applicability, and the ceRNA and transcription factor regulatory networks provide a systematic molecular basis for understanding the association between BPS exposure and melanoma metastasis.

Ovarian and colorectal cancers have the highest incidence and mortality in the world, after breast cancer. Despite the initial response to Pt-drugs or 5-fluorouracil (5-FU), many cancer cells develop resistance to these drugs. For this reason, new therapeutic strategies represent an important medical need, in particular for drugs that are being studied in combination with methods that promote their entry into the cell. Among these strategies, electrochemotherapy (ECT), the combination of drugs with electroporation (EP), a physical method that uses high-frequency electrical pulses to create pores into which chemotherapy drugs can permeate, is gaining interest. In this study, we have evaluated the effect of ECT on the growth of both ovarian (A2780 and A2780/CP) and colorectal (HCT116) cancer cell lines using platinum derivatives (Cisplatin, Carboplatin and Oxaliplatin), as DNA alkylating agents, and human thymidylate synthase (hTS) inhibitors, both traditional (5FU) and novel TS destabilizers (compounds E3 and E7). To this aim, synergism quotient-like analysis to determine whether electroporation gives an advantage in terms of cytotoxicity was applied to the relative IC20 and IC50 concentrations of each drug. Results showed that two of the three Pt-drugs have greater efficacy when combined with EP. 5-FU and the new TS inhibitors E3 and E7 also take advantage of ECT because EP increases drug uptake into the cell, even in resistant cells. In conclusion, ECT appears to be a viable strategy to obviate the problem of resistance in ovarian and colorectal cancers, to deliver compounds inside cells overcoming uptake limits, especially for the low lipophilic compounds whose cytotoxic efficacy is hampered by the obstacle of biological membranes.

Migraine headaches affect over one billion people internationally and can be defined as episodes of acute severe pain wrapping around the head and are normally accompanied by nausea, blurry vision, and sensitivity to light and sound. While triggers that cause migraines may vary among patients, evidence shows they are involved with the trigeminovascular system (a network of blood vessels in the brain in conjunction with the trigeminal nerve). Activation of the trigeminal neurons triggers the release of vasoactive neuropeptides, such as calcitonin gene-related peptide (CGRP), leading to neurogenic inflammation and vasodilation of cranial blood vessels. The development of 5-HT1B serotonin agonist drugs, commonly known as triptans, have been an effective measure of migraine relief. The drugs created in this research were found to have improved docking scores within the 5-HT1B binding site compared to that of naratriptan. The two drugs proposed in this paper would need to undergo further investigation to determine the feasibility of laboratory synthesis and clinical trials.

Emerin (EMD), an inner nuclear membrane protein essential for nuclear architecture integrity, gene expression, cellular signaling, and chromatin stability, interacts with the LINC complex and participates in cytoskeleton-nucleoskeleton communication by binding to nuclear actin filaments. EMD is implicated in migration, invasion, and metastasis in some tumors, but its role in glioblastoma (GBM) remains unclear. This study evaluated the effects of EMD knockdown and overexpression in GBM cell lines following genotoxic treatment with cisplatin. In both wild-type p53 (U87-MG) and mutant p53 (U138-MG) GBM cells, EMD expression is high, and cisplatin treatment did not affect these protein levels. EMD knockdown in U87-MG cells significantly increased cisplatin IC50, viability, and proliferation. Conversely, stable overexpression of EMD in U87-MG cells led to reduced cisplatin IC50, viability, proliferation, and migration. EMD knockdown or overexpression did not affect any U138-MG phenotypes, with or without cisplatin treatment. Modulation of EMD levels causes morphological changes in stress fiber cytoskeleton, whereas overexpression of EMD in U87-MG cells promotes an increase and a decrease in nuclear and cytoplasmic actin levels, respectively. These biological responses of U87-MG cells overexpressing EMD were coincidentally associated with alterations in the levels of pH2AX(Ser139), p-p53(Ser15), p53, and p21Kip1 proteins after cisplatin exposure. In sum, modulation of EMD levels affects the viability, migration, and proliferation of wild-type p53 GBM cells treated with cisplatin, suggesting unknown roles in the DNA damage response and repair. This work highlights EMD as a potential regulator of GBM chemoresistance and a target for therapeutic intervention.

BackgroundReactive Oxygen Species (ROS) are the small, unstable and highly reactive species, having DNA oxidizing ability. Oxidation of the DNAs purine and pyrimidine bases can lead to single or double strands in this macromolecule. In this situation, the ATM molecule, a serine-threonine kinase, targets several proteins for phosphorylation, which causes the cell cycle to stop and the DNA damage repair begins. It has previously been proven that natural polyphenols have the cancer inhibiting properties due to their high efficacy and low side effects. Silibinin is the main herbal and medical ingredient in Milk Thistle (Silybum marianum) is a polyphenol flavonolignan, which has been widely considered as an antioxidant and anticancer agent. The purpose of the present study was to investigate the ATM gene expression and measurement of reactive oxygen species (ROS) in SKBR3 cell line, treated with Silibinin. Materials and MethodsAt first, the SKBR3 cell line was cultured in RPMI1640 culture medium and MTT assay was carried out to evaluate the Silibinin cytotoxicity. Flow Cytometry was carried out for cell cycle analysis, apoptotic induction, and ROS detection. While, Real Time PCR was used to evaluate the ATM gene expression in the Silibinin-treated and un-treated SKBR3 cells. ResultsPresent results have shown that 150 {micro}M Silibinin had the most significant cytotoxicity and apoptotic induction influence after the treatment period of 48 h. Flow cytometry data have shown that Silibinin induced considerable amount of apoptosis and caused cell cycle arrest at G1/S phase and induced production of ROS. Real-time PCR results have revealed that Silibinin increased the ATM expression in SKBR3 cell line. ConclusionSilibinin causes increased ATM gene expression by inducing ROS production, which initiates cell cycle arrest and apoptotic induction in SKBR3 cells line.

BackgroundTuberculosis remains a leading global infectious killer, with BCG offering inconsistent adult protection and rising drug-resistant strains demanding novel vaccine strategies. We report the first multi-epitope vaccine construct simultaneously targeting three previously unexplored Mycobacterium tuberculosis virulence proteins; EccB3, MycP, and polyketide synthase which collectively govern nutrient acquisition, ESX secretion integrity, and innate immune evasion. MethodsUsing a reverse vaccinology pipeline, B-cell, CTL, and HTL epitopes were predicted, filtered for allergenicity, toxicity, and IFN-{gamma} induction, then assembled into an 823-residue chimeric construct incorporating beta-defensin and PADRE adjuvants with AAY/GPGPG linkers, covering [~]90% global HLA diversity. The construct underwent AlphaFold structure prediction, 3DRefine refinement, disulfide engineering, PROCHECK/ProSA validation, ClusPro 2.0 docking against TLR1/TLR2, and C-IMMSIM immune simulation. ResultsThe construct (82.3 kDa, instability index 32.48) showed strong structural quality (94.7% favoured Ramachandran residues), stable TLR1/TLR2 binding (weighted energy: -1,371.0 kcal/mol), and robust in silico immune responses and durable memory cell formation following booster simulation. ConclusionThis computationally validated construct represents a promising multi-target TB vaccine candidate warranting experimental advancement.

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become a global concern over the last two decades. Bovine TB primarily affects cattle, but other domestic livestock are also affected and it is more common in less developed and developing countries. The significant loss of livestock leads to trade restrictions and economic crises. Zoonotic potential of bovine TB raises health concerns for the public. Currently, no effective treatment is available and animal slaughtering is usually undertaken to reduce the burden of it in the environment. Antibiotic therapy can be used on animals living in captivity, but it is not reliable for herd or free-grazing animals. The BCG vaccine is another option available for treating the disease, but it shows limited efficacy in cattle. The prevention of bovine TB is a long-term goal that can only be accomplished by developing a more effective vaccine than BCG and designing new drugs. In this research, we propose therapeutic drug targets and vaccine for treating bovine TB. The conceptual framework for vaccine developed in this study uses a number of bioinformatics approaches to identify potential vaccine candidates and construct an in-silico epitope-based vaccine. Our holistic framework identified potential therapeutic candidates by directly analysing the proteome of TB bacterial strains. Specifically, we performed a comparative proteomic analysis of 11 Mycobacterium bovis strains to cover the diversity and identify conserved proteins among those strains for developing the bovine TB vaccine. An extensive reverse vaccinology and immunoinformatics analysis provided 26 highly immunogenic, non-toxic and non-allergenic epitopes (CTL epitopes-8, HTL epitopes-2 and B-cell epitopes-16) for Mycobacterium bovis required for three-dimensional structure construction of TB vaccine. The constructed epitope-based vaccine showed a potent interaction inside the host, thus generating efficient cell-mediated and humoral immune responses. Next, a framework based on a novel subtractive proteomic approach was developed for identifying bovine TB drug targets. We performed this approach on the 11 Mycobacterium bovis strains and identified nine drug targets that are conserved, essential, antigenic and have unique metabolic pathways in Mycobacterium bovis. These drug targets could further help investigate therapeutic drugs for the treatment of bovine TB. Several bioinformatics prediction tools were used together to ensure checks and balances, aiming to reduce the chance of errors and provide accurate results. The vaccine and drug targets developed in this study can be tested experimentally with confidence for further validation as therapeutics with the potential to eradicate bovine TB globally. The strategies implemented in the study are generic and can be used for other zoonotic infectious diseases. This study would be a game changer in the field of bovine tuberculosis treatment.