International Journal of Biological Sciences

Japanese Encephalitis Virus (JEV) belongs to the Flavivirus family, and the RNA dependent RNA polymerase (RdRp) domain located at the C terminus of non structural protein 5 (NS5) regulates de novo viral genome synthesis. The conserved priming loop inserted in the thumb domain of RDRP initiates de novo genome synthesis. Interestingly, the reported replication process initiates with an interaction between NS5 (methyltransferase/RDRP) and NS3 (protease/helicase), and inhibiting this interaction directly correlates with the inhibition of viral replication. The function of the priming loop in the context of the NS5 NS3 interaction is not yet known. In this study, we studied the priming loop function in the NS5 NS3 interaction and viral replication. Using a structure based drug design approach, we screened the Maybridge compound library against the RdRp priming loop and identified 15 candidate compounds based on binding energy. Among them, DSHS00151 showed significant dose dependent inhibition of the NS5 NS3 interaction with an IC50 of 3.5 micromolar in the mammalian two hybrid assay, inhibited viral infectivity with an IC50 of 2.54 micromolar, and reduced viral RNA load with an IC50 of 2.9 micromolar, compared to CD10712. Molecular Dynamics (MD) simulations revealed that DSHS00151 exhibits stronger and more stable binding with the priming loop residues (790 to 812) compared to CD10712. The molecular mechanism suggested by the docking of the NS5 and NS3 proteins showed that the priming loop tends to rotate toward the NS3 protein to stabilize the complex, and compound binding restricts this loop rotation, thus compromising the stability of the NS5 and NS3 complex and affecting viral replication. Overall, our study validated the function of the NS5 priming loop as an allosteric site, and compounds binding to this allosteric site belong to the Non Nucleoside Reverse Transcriptase Inhibitors (NNRTI) class of inhibitors, which disrupt the NS5 and NS3 interaction and could be developed as novel anti JEV therapeutics.

Prostate cancer progression is accompanied by dynamic remodeling of malignant cell states, whose metastatic potential and regulatory programs remain poorly defined. Here, we integrated single-cell, spatial, bulk transcriptomic, epigenomic, and experimental data to characterize malignant cell state transitions and identify candidate regulators associated with metastatic progression. A distinct intermediate state exhibits increased copy-number alterations, stemness, proliferation, and MYC activity, together with the highest transcriptional similarity to metastatic tumor cells, and is therefore designated as a metastasis-initiating-like (MIC-like) state. Transcriptional and regulon dynamics along the malignant cell state trajectory highlight FEV as a prominent candidate associated with the MIC-like state, with concordant expression and regulon activity patterns. Functionally, FEV is associated with a hybrid epithelial-mesenchymal phenotype, and its perturbation disrupts this phenotype, accompanied by reduced epithelial and mesenchymal features as well as impaired prostate cancer cell migration and invasion. Beyond this tumor-intrinsic feature, FEV is also associated with reduced cytotoxicity and interferon responses in the tumor microenvironment through its regulation of MIF-family genes. In addition to its potential roles in tumor cell plasticity and immune remodeling, FEV shows a reciprocal association with androgen signaling and distinct expression patterns across treatment response groups, highlighting its potential relevance in therapeutic contexts.

Kaposi's sarcoma (KS) is an angioproliferative tumor associated with infection by human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus (KSHV). Among the viral genes involved in KS pathogenesis, the viral G protein-coupled receptor (vGPCR) plays a key role in oncogenic transformation and angiogenesis, and its sustained activity contributes to tumor development and maintenance. Quercetin (QUE), a naturally occurring flavonoid with well-recognized anticancer properties, has been shown to reduce cell viability and proliferation and to induce apoptosis in vGPCR-transformed endothelial cells. This study further investigated the mechanisms underlying the antineoplastic effects of QUE in vGPCR cells, an in vitro model of Kaposi's sarcoma. Initially, QUE promoted the activation of MAPK signaling pathways in vGPCR cells, increasing the phosphorylation levels of ERK1/2, JNK1/2, and p38, without affecting AKT phosphorylation. In parallel, QUE increased intracellular oxidant levels, followed by lipid peroxidation and DNA damage, supporting a link between oxidative imbalance and genotoxicity. However, increased total glutathione content, and unaltered Gpx4 mRNA expression, suggest the induction of oxidative stress and compensatory antioxidant responses. Furthermore, increased intracellular oxidant levels were associated with MAPK signaling activation, particularly JNK1/2, whereas ERK1/2 and p38 activation may reflect a compensatory response that is insufficient to overcome QUE-induced cytotoxicity. Taken together, these findings indicate that QUE exerts antiproliferative effects in vGPCR cells through a mechanism involving oxidative stress, MAPK activation, and DNA damage, supporting its potential relevance in the study of therapeutic strategies for virus-associated malignancies.

Alcohol-associated liver disease (ALD) is a leading cause of liver-related morbidity and mortality, yet its pathogenesis remains incompletely understood. While neural regulation is critical for hepatic homeostasis, how autonomic and neurotrophic signaling is regulated in the liver during ALD progression remains largely unexplored. Here, we performed an integrative analysis of bulk and single-cell transcriptomic datasets of human livers across the spectrum of ALD. We identified stage-dependent remodeling and coordinated expression patterns of autonomic and neurotrophic genes. Autonomic signaling exhibited a consistent decline, with reduced expression of sympathetic (DBH, TH) and the parasympathetic (ChAT) markers. In contrast, pan-neuronal and neuroplasticity-associated genes (UCHL1, TUBB3, STMN2) were upregulated, indicating active neural remodeling. In parallel, factors such as MANF, GDNF, and NRG2, with the receptor NTRK1, were upregulated and positively correlated with clinical indicators of disease severity, inflammation and fibrosis. In contrast, trophic factors including NTF3 and CDNF, and receptors such as GFRA1 and GFRA3, were downregulated and inversely associated with these parameters. Single-cell analyses revealed that neurotrophic factors are predominantly expressed by hepatic stellate cells, supporting their role as resident neuroglia-like cells in the liver. In another independent cohort, we observed a coordinated downregulation of NTF3 and sympathetic nerve markers in alcohol-associated hepatitis (AH), suggesting impaired NTF3-mediated neurotrophic support may contribute to sympathetic dysfunction in AH. Collectively, these findings uncover previously unrecognized neuro-regulatory dimensions in ALD and suggest that dysregulated autonomic and neurotrophic signaling may contribute to disease progression. Conflict of interestThe authors have declared that no conflict of interest exists.

Leuprolide, a widely used androgen-deprivation therapy (ADT) for prostate cancer (PC), has been associated with increased cardiovascular events (CVE), although the underlying mechanisms remain unclear. Through integrated spatial transcriptomic analyses of murine heart and immune-stromal profiling in patient blood, we identified immune programs linked to leuprolide-associated CVE risk. Spatial analyses revealed the distinct organization of lymphoid (T+B cells) aggregates and inflammatory macrophages in the plaque following leuprolide exposure that accompanied by ventricular hypertrophy, relative to normal vasculature. Parallel profiling of circulatory soluble and cellular components in patients demonstrated CD8+T cell enriched pattern in ~44% of patients, including memory and type 1 cytotoxic subsets that linked to treatment-induced CVE, such as ventricular dysfunctions, but no contribution of adipocytokines. These translational findings highlight the stratification of leuprolide-associated CVE risk in PC patients based on circulating T cell profiling and could guide the risk mitigation with alternative ADT agents.

Sex-specific differences in COVID-19 outcomes are well documented, with male sex being a key risk factor for severe disease. Multiple factors, including differences in the expression of Interferons (IFNs) and Interferon stimulated genes (ISGs) between the sexes have been identified as contributing to these differences. Adenosine-to-inosine (A-to-I) RNA editing, mediated by ADAR enzymes, is an important component of antiviral innate immune signaling. While previous studies, including us, have identified altered expression and activity of ADARs in response to SARS-CoV-2 infection, it remains unknown whether sex-specific differences in expression and activity of ADARs exist during infection with SARS-CoV-2. We address this question by analyzing a publicly available whole blood RNA sequencing dataset from males and females, matched for age and comorbidity status across pre-, mid-, and post-infection stages of SARS-CoV-2 infection. Our results show distinct patterns of ADAR expression between the sexes across different stages of infection, with significant differences in the expression of ADAR and ADAR2 in pre- and mid- infection that were not observed post-viral clearance. Interestingly, expression of predominantly neural isoform ADAR3 was significantly different and higher in females, mid-infection. Furthermore, these differences in ADAR expression were likely amplified downstream with significant differences in the global editing levels. Correlation analysis revealed that the relationship between ADAR expression and global editing activity differed by sex and stage of infection, indicating that ADAR editing is dynamically regulated and is influenced by factors other than ADAR transcript abundance. Noteworthy, sex-specific differences in ADAR editing were observed within genes that escape X-chromosome inactivation (XCI), a gene set enriched in immune related functions and known to be involved in sex-specific outcomes to viral infections. ADAR editing within these genes differed at baseline and changed across stages of infection in a manner independent of gene expression. Altogether, our findings establish ADAR editing as an additional layer of immune regulation that may be contributing, in part, to the differential clinical manifestations of COVID-19 observed between males and females.

Raman spectroscopy (RS) has been shown to differentiate various disease states in tissue samples, yet the impact of sample substrate on spectral quality and downstream machine-learning (ML) performance is under-explored. We collected 960 spectra from 36 formalin-fixed, paraffin-embedded colonic specimens mounted in parallel on calcium fluoride (CaF2) and mirrored stainless-steel (SS) slides. Raw SS spectra were more intense than CaF2, and routine dewaxing left prominent paraffin peaks on CaF2 that were largely absent on SS. Extending CaF2 xylene immersion to 48 h reduced key paraffin peaks by 11 to 23%, aligning its biochemical profile with SS. Nested cross-validated classifiers were built to distinguish true disease (T1) from epithelial misplacement (EM). The highest accuracies were observed on SS (0.65 - 0.66), while CaF2 improved from 0.41 - 0.57 to 0.65 - 0.69 after extended dewaxing, chiefly via gains in recall. Although optimal dewaxing renders CaF2 diagnostically non-inferior, SS still offers superior accuracy, faster preparation, at approximately 4% of the cost, supporting its integration into scalable, AI-enabled RS pathology workflows.

Psoriasis is a chronic skin disease characterized by red, scaly plaques mainly driven by dysregulated T cells that induce keratinocytes (KCs) proliferation and inflammation. Despite significant advancements in disease management through biologics and topical therapies, these treatments offer only temporary relief and fail to prevent disease recurrence. Considering the current therapeutic needs, we described herein a novel small molecule TEM1657, derived from 3,5-dicaffeoylquinic acid with anti-inflammatory potency in imiquimod-induced psoriasis mouse model as well as in Th17-stimulated KCs. Mechanistically, its action is mediated through modulation of mitochondrial dynamics, specifically via OPA1, an inner mitochondrial membrane protein that alters mitochondrial calcium influx, and further impacts cellular bioenergetics. Moreover, TEM1657 rapidly suppresses transcription of NFKBIZ and various other psoriasis-associated genes. These observations highlighted a pathway that represents an innovative target in psoriasis and other NFKBIZ-related disorders.

Chikungunya virus (CHIKV) infection causes acute febrile illness that frequently progresses to chronic arthralgia. Although monocytes are CHIKV targets, whether viral exposure drives their differentiation into macrophages to sustain persistent inflammation remains incompletely understood. To address this, we integrated transcriptomic profiling of pediatric CHIKV patients during acute and convalescent phases, in vitro differentiation of primary human monocytes into macrophages under CHIKV infection, and single-cell RNA-sequencing (scRNA-seq) of joint-associated macrophages from an immunocompetent mouse model of chronic CHIKV arthritis at 28 days post-infection. In acute patients, we observed a marked upregulation of monocyte/macrophage markers alongside robust, IFN-dependent JAK-STAT signaling. In vitro, CHIKV-infected monocytes differentiated into persistently infected macrophages (MDM-CHIKV) that closely aligned with an M1-polarized transcriptional profile. These cells exhibited STAT1/NF-{kappa}B-dependent inflammation, ISG induction, and enhanced antigen presentation capacity. Remarkably, MDM-CHIKV uniquely produced IFN-{pi}/IL-27, revealing an alternative non-canonical axis where JAK-STAT activation persists despite the absence of classical type I IFNs. Functionally, MDM-CHIKV displayed an enhanced respiratory burst upon immune complex uptake, pointing to Fc{gamma}R-mediated pathology, as well as complement activation and T-cell stimulatory capacity. However, upon TLR4 challenge, these cells retained partial regulatory responses via IL-10 and TGF-{beta}; this hybrid immunophenotype offers a potential mechanistic explanation for relapsing arthralgia. Additionally, scRNA-seq of murine joints confirmed that persistent CHIKV replication was restricted to a distinct macrophage subset that resembles MDM-CHIKV, establishing these cells as the primary in vivo viral reservoir. Mirroring our human findings, joint-associated murine macrophage subsets exhibited elevated IFN-{pi}/IL-27 expression alongside robust NF-{kappa}B/STAT1-dependent inflammatory signatures (Tnf, Il1{beta}, Il6, Ccl2, Ccl5, Cxcl9, Cxcl10, Cxcl1-3, and/or Ptgs2), underscoring their critical role in sustaining chronic arthritis. Collectively, our findings demonstrate that CHIKV actively drives monocyte-to-macrophage differentiation into a persistently infected, M1-skewed hybrid phenotype that serves as a viral reservoir and fuels pro-arthritic inflammation. Consequently, macrophage reprogramming, the non-canonical IFN-{pi}/IL-27-STAT1 axis, Fc{gamma}R-mediated phagocytosis, and complement activation emerge as central pathogenic nodes and promising therapeutic targets for mitigating chronic post-CHIKV arthropathy.

This study aims to identify potential inhibitors of the Ag85C enzyme from Mycobacterium tuberculosis through primary docking, pharmacophore modelling, and ADME/pharmacokinetics analysis. Delamanid and Pretomanid emerged as the top hits in primary docking, demonstrating strong binding affinities and interactions with key active site residues. A robust pharmacophore model was developed, identifying essential features for binding and used to screen 4254 antituberculotic compounds, yielding three promising candidates: F0314-0040, F0398-0505, and F0349-2716. These compounds exhibited high binding affinities in major docking studies. ADME and pharmacokinetics analysis revealed favorable Drug likeness and absorption profiles, supporting their potential as effective antituberculotic agents. The integration of computational methods provided a comprehensive approach for rational drug design.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behavior and impaired social interactions. Recent reports from human postmortem brain samples of ASD show transcriptome-wide dysregulations, including changes in differential gene expression and alternative splicing. An important mechanism regulating transcriptome function is the editing of double-stranded RNA by adenosine deaminases acting on RNA (ADARs), which play regulatory roles in neurodevelopment and innate immunity. We explore the ADAR editing changes associated with the use of Selective Serotonin Reuptake Inhibitors (SSRIs) in Arid1b haploinsufficient mouse models. Our results show dynamic changes in site-specific editing rates in autistic mice. We further demonstrate the transcriptome-wide effects of differential ADAR editing in terms of changes in miRNA-mRNA interactions. Moreover, our findings highlight a site-specific change in editing rates for evolutionarily conserved sites in mammals and ASD associated risk genes, such as Gria2. Our analysis of significant biological processes shows the association of differentially edited genes with processes vital for neurotransmission. Overall, our findings imply that the use of SSRIs can induce distinct ADAR editing signatures, potentially contributing to ASD pathology.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

Despite the advent of immunotherapy in the treatment of human-papilloma-virus (HPV)-negative head and neck squamous cell carcinoma, the overall prognosis still remains dismal. Novel therapeutic approaches are thus urgently needed. We recently reported that SET and MYND containing 3 (SMYD3), a protein methyltransferase that is known to activate the transcription of its target genes through H3K4me3, is overexpressed in ~60% of HPV-negative HNSCC tumors, it drives proliferation and invasion, and induces resistance to immunotherapy in HPV-negative HNSCC. These phenotypes are mediated partially through transcriptional regulation of cell cycle, EMT and immune-related target genes by SMYD3. Despite its enzymatic inhibitory potency in HPV-negative HNSC cell lines, the commercially available SMYD3 inhibitors EPZ031686 and BAY-6035 demonstrated no in vitro phenotypic efficacy in these cell lines, supporting that SMYD3 exerts its oncogenic functions predominantly through catalytically-independent mechanisms. Thus, to effectively target SMYD3, we designed a series of SMYD3 proteolysis targeting chimeras (PROTACs). Treatment of HPV-negative HNSCC cell lines with SMYD3 PROTACs at nanomolar concentrations degraded SMYD3 effectively and induced a drastic decrease in the proliferative and invasive potential of HPV-negative HNSCC cells. One of the designed compounds, IAP-08, showed a promising DC50 at ~177nM with Dmax of ~90%, and effectively induced the formation of ternary complexes with SMYD3 and the XIAP E3 ligase. Furthermore, IAP-08 showed a favorable pharmacokinetic profile in vivo, underscoring the feasibility for further clinical development of this compound. Mass spectrometry analysis of IAP-08 supported its high specificity towards SMYD3. SMYD3 depletion by IAP-08 in HNSCC cells revealed transcriptional regulation of cell cycle, EMT- and type I IFN response genes, in contrast to SMYD3 inhibition which had only a weak transcriptional effect. In summary, we show that inhibition of the enzymatic activity of SMYD3 is not sufficient to repress its oncogenic activity and provide evidence that depletion through PROTACs effectively targets the oncogenic functions of SMYD3 in HPV-negative HNSCC.

Neuropathic pain involves spinal microglial activation and metabolic changes.Forsythoside B (FB) has anti-inflammatory effects, but its role in neuropathic pain and microglial glycolysis remains unclear. We investigated the effects and mechanisms of FB in mice with chronic constriction injury (CCI) and in LPS-stimulated BV2 microglia.FB reduced mechanical allodynia and thermal hyperalgesia in CCI mice. FB also reduced the spinal levels of HIF-1, HK2, PKM2, LDHA, and CD86. It decreased the proportion of IBA1+/CD86+ cells and the levels of IL-1{beta}, IL-6, and TNF-. In LPS20 stimulated BV2 microglia, FB reduced glycolysis-related proteins, proinflammatory markers, and cytokine release. FB also reduced glycolytic activity and partly restored basal mitochondrial respiration. FB did not change Hif1a mRNA levels. However, it Manuscript Click here to access/download;Manuscript;Manuscript.docx increased HIF-1 ubiquitination and promoted its proteasome-dependent degradation. HIF-1 overexpression partly reversed the effects of FB on glycolytic enzymes, CD86 expression, and proinflammatory cytokine production. Immunoprecipitation followed by mass spectrometry and UbiBrowser analysis identified USP7 as a candidate HIF27 1-associated deubiquitinase. Molecular docking and cellular thermal shift assays supported a possible interaction between FB and USP7. FB reduced the association between USP7 and HIF-1. USP7 knockdown produced effects similar to those of FB on HIF-1 ubiquitination and downstream glycolytic and inflammatory responses.Overall, FB alleviated neuropathic pain and reduced proinflammatory microglial responses. These effects may involve the promotion of HIF-1 degradation through modulation of the USP7-HIF-1 regulatory axis.

Cisplatin remains as standard chemotherapy for patients with HNSCC, but rapid development of drug resistance has limited patient benefit. The p53 tumor suppressor plays a central role in the cellular response to DNA damage in cancer, triggering apoptosis to prevent propagation of damaged cells in tumor development. TP53 gene mutations occur in 65-86% of HNSCC. Small molecule PG3 induces the integrated stress response (ISR), leading to apoptosis via the HRI-eIF2-ATF4-PUMA axis. We hypothesized that a combination of PG3 plus cisplatin could increase apoptosis in TP53-mutated HNSCC cells through enhanced induction of the ISR and ATF4. PG3 synergized with cisplatin to inhibit cell viability, leading to potent apoptosis in TP53-deficient cells. The effect was regulated through the HRI-ATF4-NOXA pathway. Furthermore, we identified that cisplatin activates HRI and leads to the degradation of CReP (constitutive repressor of eIF2 phosphorylation) via E3 ligase {beta}-TrCP, contributing to the induction of the ISR. We noted decreased ATF4 levels after treatment with cisplatin, CPT, or PG3 and cisplatin. Thus, combined therapy of PG3 plus cisplatin likely results in adaptation and acquired resistance via degradation of ATF4. We targeted the degradation mechanism of ATF4 by inhibiting {beta}-TrCP1, CK1{delta}, or CK2, respectively. Each approach successfully blocked ATF4 degradation induced by cisplatin or PG3 plus cisplatin and enhanced apoptosis. Our results provide a rational strategy for triple treatments, involving an ISR inducer, a DNA damaging drug, and a {beta}-TrCP inhibitor/CK1{delta} inhibitor/CK2 inhibitor, to achieve potent and prolonged anti-tumor effects and overcome chemoresistance in HNSCC.

Computational safety evaluation of phytochemical libraries commonly relies on models trained on experimentally tested compounds and applied to much larger and chemically diverse collections. An important but often overlooked question is whether predicted compounds are adequately represented within the experimental chemical space underlying these models. Here, we examined this question for the Indian Medicinal Plants, Phytochemistry and Therapeutics (IMPPAT) database using experimentally characterized datasets for three anti-target endpoints: hERG blockade, cytochrome P450 (CYP) inhibition, and pregnane X receptor (PXR) induction. After structure standardization, 14,956 unique IMPPAT compounds were compared with 8,877 hERG, 4,905 CYP, and 4,138 PXR reference compounds using consensus nearest-neighbor similarity based on Morgan and atom-pair fingerprints. At a Tanimoto similarity threshold of 0.80, only 0.92%, 0.30%, and 0.46% of IMPPAT compounds had close measured analogs in the hERG, CYP, and PXR datasets, respectively. Relative to the internal nearest-neighbor distributions of the reference datasets, a typical IMPPAT compound lay near the 1st percentile, indicating substantial chemical-space separation. Compounds with close experimental analogs also showed distinct molecular characteristics, with descriptor-based logistic regression yielding cross-validated AUC values of 0.77-0.80. Plant-level enrichment was markedly reduced after excluding widely distributed metabolites and was further restricted when the nearest measured analog was required to have pIC50 or pEC50 [≥] 5. The remaining signals were concentrated in a limited number of taxa, including Rauvolfia, Sinopodophyllum/Dysosma, Cinchona, and Catharanthus roseus. Overall, most IMPPAT phytochemicals lack close structural representation among currently measured anti-target compounds. Computational safety predictions for these molecules should therefore be interpreted as extrapolations and accompanied by explicit measures of structural support. Absence of a close experimental analog should be regarded as a gap in available evidence rather than evidence of safety.

Background: Biopsy-measurable biomarkers that stratify colorectal cancer (CRC) by therapeutic responsiveness remain scarce. We asked whether dual MDM2 promoter (P1/P2) usage acts as a molecular switch separating two opposed CRC phenotypes: a chromosomal instability type and an environment-adaptive type (microsatellite instability-like/serrated pathway with gastric metaplasia). Methods: Sixty three organoid samples from 22 CRC patients were classified by deep-learning morphology (VGG16) and an array based MDM2 Splicing Index. P1 and P2 signatures were evaluated in TCGA-COAD/READ (n = 624) and GSE39582 (n = 519), and drug responses were examined in cell lines (GDSC2, DepMap) and 65 independent CRC organoid lines. Statistics used Wilcoxon, Kruskal Wallis and Spearman tests (Benjamini Hochberg-corrected) and patient-level mixed models. Results: Deep earning classification reached 98.5% test accuracy; morphology corresponded to isoform usage: Type1 (compact glandular) morphology predominated in P1-dominant samples (median Type1 fraction 0.826 versus

Although therapeutic advancements have dramatically improved outcomes for cancer patients in recent decades, advanced cancer remains a significant challenge. There is still an urgent need for new and more effective strategies that minimize toxicity and improve survival outcomes in cancer patients. Natural products from microorganisms are a rich and promising source of bioactive compounds with substantial therapeutic potential in cancer treatment. Recently, B. proteolyticus strain OSUB18, isolated from switchgrass, has been demonstrated to promote plant growth and regulate reactive oxygen species (ROS) production. Here, we investigated the anticancer potential of this plant-associated microbe and demonstrated that its crude extracts exhibited broad-spectrum cytostatic activity across diverse solid tumor cell lines. Importantly, OSUB18 extracts displayed limited cytotoxicity toward normal human mammary epithelial cells and mouse fibroblasts. This potentially selective cytostatic activity was retained within a water soluble, heat-labile, and protease-resistant fraction. The extracts primarily suppressed cell proliferation, accompanied by increased lipid peroxidation, with preliminary evidence suggesting the potential involvement of ferroptosis. These findings suggest that the bioactive component may be a selective cytostatic agent with multifaceted mechanisms of action.

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

Aims: Premature coronary artery disease (PCAD) lacks specific molecular drivers, and the role of immunosenescence is unclear. We investigated whether aging-related gene dysregulation in T cells contributes to PCAD. Methods: We combined bulk transcriptomics of PBMCs from 12 PCAD patients and 21 controls, single-cell RNA sequencing of PBMCs and human atherosclerotic plaques, weighted gene co-expression network analysis, gene perturbation network analysis, and molecular docking. Results: KRAS was identified as a hub gene intersecting PCAD-associated genes and aging-related genes. Single-cell analysis showed KRAS upregulation predominantly in effector CD8+ T cells, which exhibited the highest senescence scores that were further elevated in disease. Network perturbation of KRAS strongly impacted the cell killing pathway. KRAS-high effector CD8+ T cells were detected in coronary and carotid plaques, displaying enhanced cytotoxicity, exhaustion, and senescence features. Additionally, a candidate small molecule was computationally predicted to bind inactive KRAS. Conclusions: Elevated KRAS expression in senescent, cytotoxic CD8+ T cells is associated with PCAD, bridging immunosenescence and premature atherosclerosis. This finding provides a novel biomarker candidate and potential therapeutic entry point, awaiting further functional validation.