Dioctyl terephthalate (DOTP), a widely used plasticizer in food packaging and environmental materials, has raised concerns regarding its potential impact on human health. This study aims to investigate the neurotoxicity-related mechanisms of DOTP through an integrated approach combining network toxicology, molecular dynamics simulations, and in vivo validation. We retrieved the DOTP chemical structure from PubChem and predicted potential protein targets using the Similarity Ensemble Approach (SEA), SwissTargetPrediction, and SuperPred. We performed protein-protein interaction (PPI) network analysis using STRING and Cytoscape to identify core neurotoxicity-associated targets. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted to characterize the biological relevance of the 90 intersecting targets. We used AutoDock to assess the binding affinity of DOTP to the core proteins, and employed GROMACS for molecular dynamics (MD) simulations to explore the stability and conformational dynamics of the docked complexes. In vivo experiments, encompassing behavioral assessments, histological examinations, and molecular assays, were conducted to evaluate the effects of DOTP on neurological function, neuronal integrity, and target pathway dysregulation. We identified 90 neurotoxicity-related targets, among which EGFR, BCL2, CASP3, MAPK8, TLR4, NFKB1, and MTOR emerged as core nodes within the PPI network. GO and KEGG analyses revealed the involvement of these targets in diverse biological processes, cellular components, molecular functions, and signaling pathways. Molecular docking indicated favorable binding affinities between DOTP and the identified core targets, a finding further supported by MD simulations. Moreover, DOTP-treated mice exhibited significant neurofunctional deficits and neuronal loss, accompanied by profound oxidative stress, neuroinflammation, and apoptotic activation, substantiating its potential neurotoxicity. Our findings provide a theoretical foundation for understanding the predicted molecular mechanisms of DOTP-induced neurotoxicity. The integration of computational modeling and in vivo phenotypic validation suggests that DOTP may pose neurological risks, highlighting the need for further experimental evaluation of plasticizer alternatives.

Sanqi Qushi formula alleviates podocyte damage in passive Heymann Nephritis rats by inhibiting GPX4 deficiency-mediated ferroptosis via the JNK/FoxO1 signaling pathway.

Hepatitis B (HB) is a major viral infection that endangers millions of people worldwide and traditional interventions such as Tenofovir have proven to be restrictive. Phytomedicine The traditional plant Phytanthus amarus is a wellknown antiviral (especially anti-HBV). It is a molecular docking study that assesses Phyllanthin, a bioactive product obtained in Phyllanthus Marus and tenofovir in the determination of their binding affinities to the core protein of Hepatitis B virus (PDB ID: 5WRE). The findings revealed that Phyllanthin has a MolDock score of -101.92, which is much lower than -86.87 that of Tenofovir, which means that it has a higher binding affinity. The hydrogen bond analysis demonstrated that the weaker bonds of Phyllanthin were compensated with the greater specificity and stability and it is a good candidate to be experimentally validated. These results indicate that Phyllanthin may be a successful alternative/ adjunct to traditional antiviral management of Hepatitis B.

Objectives: IgA nephropathy (IgAN) is a common primary glomerulonephritis with limited treatment options. Gallic acid (GA) has demonstrated renal protective effects, but its precise mechanisms against IgAN remain incompletely elucidated. This study aims to reveal the molecular mechanism by which GA exerts a renal protective effect on IgAN. Methods: Transcriptomics and network pharmacology were combined in an integrative manner. The GSE175759 dataset’s differentially expressed genes (DEGs) were filtered out. SwissTargetPrediction and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) were used to forecast GA’s goals. Core targets and pathways were obtained by functional enrichment analysis. Human mesangial cells (HMCs) were stimulated with polymeric IgA1 (p-IgA1) to create an IgAN model in vitro. Reactive oxygen species (ROS), glutathione/glutathione oxidized (GSH/GSSG), lipid peroxidation, malondialdehyde (MDA), Fe2+, and mitochondrial membrane potential levels were evaluated in relation to GA. Western blot analysis was used in conjunction with gain-of-function (overexpression) and loss-of-function (siRNA) assays to examine the expression of the core protein and the downstream target proteins. Results: Bioinformatic analysis identified 1141 DEGs in IgAN, with mitogen-activated protein kinase (MAPK) signaling being the most significantly enriched pathway. Intersection of 109 GA predicted targets with DEGs yielded 8 candidate genes, including dual-specificity phosphatase 1 (DUSP1), which was the most downregulated gene. In vitro, GA treatment significantly alleviated p-IgA1-induced oxidative stress and ferroptosis in HMCs. These protective effects were dependent on DUSP1. Mechanistically, GA upregulated DUSP1 expression, thereby inhibiting p38 MAPK phosphorylation and subsequently increasing the protein levels of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11). Conclusion: This study demonstrates that GA may attenuate IgAN progression by inhibiting oxidative stress and ferroptosis in HMCs via the DUSP1/p38 MAPK signaling axis.

The Golgi stress response is a homeostatic mechanism that augments Golgi function when Golgi function becomes insufficient (Golgi stress). Glycosylation of the core proteins of proteoglycans is one of the important functions of the Golgi. If the production of core proteins is increased and the amount of glycosylation enzymes for proteoglycans becomes insufficient (PG-type Golgi stress), the proteoglycan pathway of the Golgi stress response is activated, resulting in the transcriptional induction of glycosylation enzymes, including NDST2, HS6ST1 and GLCE. The transcriptional induction of these glycosylation enzymes is regulated by the enhancer element, PGSE-A; however, transcription factors that induce transcription from PGSE-A have not yet been identified. We herein identified KLF2 and KLF4 as transcription factors that directly bind to PGSE-A, and found that overexpression of KLF2 and KLF4 augments transcriptional induction from PGSE-A during PG-type Golgi stress, whereas their dominant negative mutants suppress the transcriptional induction. Moreover, expression of KLF2 and KLF4 was up-regulated in response to PG-type Golgi stress. Transcriptional induction of human KLF4 gene is regulated by PGSE-A, while that of human KLF2 gene is mainly controlled by a novel enhancer called PGSE-C. These results suggest that KLF2 and KLF4 are important regulators of the proteoglycan pathway of the mammalian Golgi stress response.Key words: Golgi stress, proteoglycan, ER stress, organelle zone, organelle autoregulation, KLF2, KLF4, xyloside.

<p indent="0mm">As a selective autophagy process, mitophagy is crucial for clearing dysfunctional mitochondria and maintaining cellular energy homeostasis and survival. Its core mechanisms are primarily categorized into ubiquitin-dependent pathways, such as the classic PINK1/Parkin pathway, and ubiquitin-independent pathways involving receptors like BNIP3. mTOR, a central kinase that senses nutrients, energy, and growth factors within the cell, forms two functionally distinct complexes, mTORC1 <?A3B2 pi129?>and mTORC2. These complexes work in concert to bidirectionally regulate each stage of mitophagy—from initiation and phagophore formation to final fusion with lysosomes for degradation. The article is primarily divided into two main sections, respectively discussing the topic from the perspectives of core mTOR-related signaling pathways and small molecular proteins. Among these, two representative mTOR-associated pathways are highlighted: the mTOR-AMPK-ULK1 <?A3B2 pi129?>pathway and the PI3K-AKT-mTOR pathway. The former is involved in autophagy initiation and serves as a key mechanism through which cells sense energy status and regulate the onset of autophagy. Under energy-sufficient conditions, it inhibits the formation of the initiation complex, thereby suppressing autophagy. The latter primarily responds to growth factor signals and generally functions to inhibit autophagy, playing an indispensable role in maintaining basal mitochondrial quality. Beyond these classic pathways, mTOR also precisely regulates specific stages of mitophagy by influencing a series of particular proteins. Proteins involved in regulating autophagosome formation and maturation include p300, WIPI2, UVRAG, and Pacer. Primarily under nutrient-rich conditions, these proteins enhance mTORC1 <?A3B2 pi129?>activity, thereby activating the expression of downstream cytokines to inhibit autophagosome formation. Additionally, TFEB, the master regulator of autophagy-lysosome gene expression, is a core protein governing lysosome biogenesis, and its activity is tightly controlled by mTORC1. Studies have shown that activating TFEB can promote mitophagy and exhibits therapeutic potential in disease models such as cancer. Based on the current research on mTOR, the following recommendations can be proposed: First, as an integrative hub within the mitochondrial quality control network, mTOR serves as a dynamic, multi-tiered regulatory nexus capable of achieving spatiotemporally precise regulation of mitophagy initiation, progression, and lysosomal degradation capacity. Future studies should place greater emphasis on the synergistic and antagonistic interplay between mTORC1 <?A3B2 pi129?>and mTORC2 <?A3B2 pi129?>in specific physiological and pathological contexts, as well as their crosstalk with stress signals from other organelles. Second, targeting the mTOR regulatory network represents a potential strategy for treating diseases associated with mitochondrial dysfunction. Given that the mTOR pathway is dysregulated and impacts mitophagy in a variety of disorders—including neurodegenerative diseases, metabolic disorders, and cancers—drug development aimed at precisely modulating this network holds considerable promise. Lastly, efforts should be strengthened to bridge the gap between fundamental mTOR-related mechanisms and clinical research, thereby offering more novel insights for clinical diseases. For example, delving deeper into atypical mitophagy pathways mediated by factors such as OPTN and ZDHHC13, and elucidating how these novel mechanisms interact with mTOR signaling, will deepen our understanding of the plasticity of mitochondrial quality control. This review systematically elucidates recent advances <?A3B2 tlsb=-.025w?>regarding the impact of mTORC2 <?A3B2 pi129?>on mitophagy, while exploring its functional synergy with mTORC1 <?A3B2 pi129?>and its bidirectional regulatory properties under metabolic stress conditions. It aims to provide novel perspectives for an in-depth analysis of the multifaceted regulatory mechanisms of the mTOR signaling network in mitochondrial quality control. Furthermore, it seeks to offer critical theoretical foundations and identify new drug targets for developing precision medicine strategies aimed at restoring mitochondrial quality. <?A3B2 tlsb?>

Peroxisomes are dynamic organelles vital for lipid metabolism and redox homeostasis. In Saccharomyces cerevisiae, the expression of peroxisomal proteins is tightly regulated in response to metabolic conditions. Here, we provide the first absolute quantification of the yeast peroxisomal proteome under peroxisome-inducing (oleate) and fermentative (glucose) conditions using a label-free mass spectrometry approach. We determined protein copy numbers for ~ 4500 proteins, including 99 peroxisomal and peroxisome-associated proteins. Our data reveal that the overall peroxisomal proteome is approximately threefold more abundant in oleate-grown cells, constituting 2.8% (2.01 × 106 protein copies) of the total proteome compared to 0.8% (6.67 × 105 protein copies) in glucose. Considering only peroxisomal core proteins, i.e., proteins exclusively or predominantly localized in peroxisomes, total copy numbers for peroxisomal proteins were even ninefold higher on oleate (0.9%, 6.29 × 105 protein copies) compared to glucose (0.1%, 7.78 × 104 protein copies), reflecting the necessity for peroxisomal functions such as fatty acid beta-oxidation. Enzymes of the beta-oxidation and glyoxylate cycle showed up to > 500-fold higher abundance in oleate. In contrast, core components of the peroxisomal protein import machinery (e.g., Pex5, Pex14) exhibited only moderate changes (~ 2- to 8-fold). In addition to metabolic enzymes and components of the peroxisomal protein import pathways, we provide copy number data for proteins involved in cellular stress response, peroxisome proliferation, division and organization, peroxisome-associated membrane contact sites, and metabolite transporter. Taken together, our dataset offers a quantitative framework of peroxisomal remodeling under different metabolic conditions and highlights the organelle’s adaptive flexibility, providing a valuable resource for future studies on peroxisome biology.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00418-026-02458-w.

Computational network toxicology of non-nutritive sweeteners in ulcerative colitis: From Aspartame-MMP9 interaction to mechanism-guided intervention.

Identification and screening of bioactive peptides against nephropathy derived from Mantidis Oötheca based on complement C3 inhibition.

Photosystem II (PSII) is highly sensitive to light-induced damage. Photoinhibition, the light-dependent inactivation of PSII, is associated with an increase of excitation-energy quenching. Recovery from photoinhibition involves migration of PSII complexes from the appressed to the non-appressed region of the thylakoid membrane, where D1 (the PSII core protein most sensitive to photodamage) is degraded and repair occurs. However, it remains unclear whether damaged PSII core complexes accumulate in the stroma lamellae when repair is blocked. Here, we combined confocal Fluorescence Lifetime Imaging Microscopy (FLIM) with biochemical fractionation of the thylakoid membrane to investigate the localization of damaged PSII following photoinhibition in the presence of lincomycin, an inhibitor of D1 synthesis. This condition mimics natural stress scenarios such as heat, where D1 synthesis is impaired. FLIM analysis of structured, intact thylakoid membranes, segmented into grana- and stroma-lamellae-enriched regions, revealed a decrease in PSII fluorescence lifetime upon photoinhibition, consistent with increased excitation-energy quenching. Surprisingly, no significant difference in fluorescence lifetime components was observed between membrane domains, suggesting that damaged, quenched PSII does not accumulate in the stroma lamellae under these conditions. Western blot analysis of biochemically isolated membrane fractions confirmed a uniform decrease in D1 levels across grana and stroma lamellae upon photoinhibition. Our results indicate that when D1 synthesis is blocked, the relocation and degradation of photodamaged PSII proceed efficiently enough to prevent its accumulation in the stroma lamellae. This reveals new aspects of PSII repair and demonstrates the strength of FLIM for spatially resolved analysis of the thylakoid membrane.

Ribosomes are ribonucleoproteins that are responsible for protein synthesis. They consist of ribosomal proteins and ribosomal RNAs (rRNAs). Pre-rRNAs are co-transcriptionally processed and chemically modified. The 2'-O-methylation of rRNAs is guided by box C/D small nucleolar ribonucleoprotein particles (snoRNPs), which are composed of a box C/D snoRNA and the core proteins NOP56, NOP58, SNU13, and the methyltransferase fibrillarin. Catalytically active box C/D snoRNPs function in nucleoli. We performed trio whole-exome sequencing in a proband with a severe neurodevelopmental disorder including global developmental delay, microcephaly, seizures, and ophthalmological and brain abnormalities and his healthy parents and identified the homozygous synonymous variant c.516G>A; p.Leu172= in NOP58. In fibroblasts of the proband, we demonstrated skipping of exon 7 in most NOP58 mRNAs, while ∼20% canonically spliced NOP58 transcripts were detected in the proband compared with control cells. NOP58 protein levels were reduced to ∼12% in proband cells that concomitantly reduced fibrillarin levels. Analysis of nucleoli in proband-derived fibroblasts revealed changes in the number of nucleolar condensates and in nucleolar morphology. We found reduced levels of three box C/D snoRNAs required for 2'-O-methylation and of one box C/D snoRNA important for 2'-O-methylation and pre-rRNA processing. Analysis of pre-rRNA maturation by RT-qPCR revealed increased 45S and 21S pre-rRNA levels, whereas the amplification signal for the 47S, 32S, and 26S pre-rRNAs was substantially decreased in proband compared with control cells. Together, our data unveil that the homozygous NOP58 variant c.516G>A represents a hypomorphic allele and underlies the neurodevelopmental phenotype in the proband, likely by impairing pre-rRNA maturation.

Background and objectiveMyeloid-derived suppressor cells (MDSCs) constitute a population of cells with immunosuppressive functions, potentially playing a pivotal role in the progression of chronic hepatitis C (CHC) to liver fibrosis. This study aimed to elucidate the molecular mechanisms by which HCVc-induced MDSCs interact with hepatic stellate cells to influence the onset and progression of liver fibrosis.MethodsCD14+ monocytes were isolated and purified from healthy human peripheral blood. These cells were stimulated in vitro with hepatitis C virus core protein (HCVc) to induce differentiation into MDSCs. CD14+ monocytes or HCVc-induced MDSCs were co-cultured with the human hepatic stellate cell line LX2 to establish an in vitro co-culture system. The TGF-β signaling pathway was blocked using a neutralizing antibody. LX2 proliferation was assessed via the MTT assay, LX2 activation via ELISA, and LX2 apoptosis via flow cytometry.ResultsHCVc induced CD14+ monocytes to differentiate into MDSCs. HCVc-induced MDSCs promoted LX2 proliferation and type I collagen (Col-1) synthesis while inhibiting LX2 apoptosis, thereby driving the onset and progression of liver fibrosis. This effect was suppressed by TGF-β neutralizing antibodies.ConclusionHCVc-induced MDSCs mediate the regulation of LX2 proliferation, activation, and apoptosis via TGF-β signaling, thereby promoting hepatic fibrosis. This “HCVc–MDSCs–TGF-β–LX2” axis establishes, for the first time, a direct link between viral infection, immunosuppressive myeloid cells, and the hepatic fibrosis process, providing potential targets for developing novel therapeutic strategies for hepatitis C-associated liver fibrosis.

Integrated proteomic and metabolomic analyses reveal testicular metabolic mechanisms underlying sperm quality in drakes

Chronic hepatitis B virus (HBV) infection remains a global public health challenge, and the currently approved medications can not achieve a cure. Synthetic triterpenoids have shown promising therapeutic potential for liver pathologies. In our search for novel antiviral agents against HBV, we found that two triterpenoids, 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) and CDDO-ethyl amide (CDDO-EA), significantly inhibited HBV DNA replication. Further mechanistic investigation indicated that these two compounds did not significantly alter the levels of total HBV pgRNA, but dramatically reduced extracellular pgRNA and intracellular encapsidated pgRNA in a dose-dependent manner. Western blot analysis indicated minimal effects on core protein expression. Interestingly, using a particle gel assay, we observed that CDDO and CDDO-EA promoted the formation of empty capsids with no alteration in electrophoretic mobility. Moreover, we demonstrated that both compounds modulated the phosphorylation status of the core protein. Further cellular thermal shift assay (CETSA), surface plasmon resonance (SPR) assay, and molecular docking analyses collectively suggested that CDDO and CDDO-EA could bind directly to the dimer–dimer interfaces of HBV core protein. Finally, a synergistic effect was observed between CDDO-EA and lamivudine in reducing intracellular and extracellular HBV DNA levels. Our findings indicate that triterpenoids CDDO and CDDO-EA are new mechanistically type of HBV capsid assembly modulators and warranted for further development as lead compounds against HBV.

Chondroitin sulfate proteoglycan-4 (CSPG4) is a transmembrane cell surface proteoglycan that promotes malignant progression in melanoma. Elevated CSPG4 expression in melanoma cells is associated with several malignant phenotypic properties, including increased tumor cell invasion, tumorigenic potential, and metastasis. Magnetically aligned collagen gels with entrapped cells were used to model the aligned extracellular matrix in the tumor microenvironment and to identify the key role of CSPG4 in sensing contact guidance. The data show that CSPG4-expressing WM1552C Radial Growth Phase (RGP) melanoma cells exhibit enhanced contact guidance along with increased migration speed in contrast to paired counterparts that lack CSPG4. This required the presence of a pERK 1,2 phospho-acceptor site on the cytoplasmic tail of the core protein. Furthermore, short-term treatment of CSPG4-expressing cells with the clinically used mutant active BRAF inhibitor vemurafenib reduced both guidance and speed. These findings support the role of CSPG4 overexpression and mutant active BRAF-in promoting increased contact guidance. The results are discussed in terms of expanding what is known about the potential tumor biology and clinical implications of CSPG4-related impact on malignant invasion during early phases of melanoma progression. The online version contains supplementary material available at 10.1007/s12195-025-00882-x.

Carbofuran, a widely used carbamate pesticide, is an endocrine disruptor with documented reproductive toxicity, yet the mechanisms underlying its ovarian toxicity remain incompletely understood. This study employed integrated network toxicology and untargeted metabolomics to investigate these mechanisms in female C57BL/6J mice that had been chronically exposed to carbofuran (0.5 or 2.0 mg/kg for 45 days, once daily). Methods included histopathological evaluation, serum hormone ELISA, network prediction of toxicity targets, molecular docking, and metabolomics profiling. Results demonstrated that carbofuran exposure induced dose-dependent ovarian damage, including reduced follicular reserve, increased atresia, abnormal corpus luteum, and disrupted hormone levels. Network toxicology identified 38 common targets, with EGFR, GSK3B, APP, and JAK2 as core proteins, indicating potential high affinity. Metabolomics suggests significant alterations in pathways such as phenylalanine, tyrosine, tryptophan biosynthesis and arginine/proline metabolism. Our collective evidence indicates that carbofuran may induce ovarian toxicity through multifaceted mechanisms involving endocrine disruption, oxidative stress, inflammatory activation, and metabolic disturbance. This study provides novel experimental insights into the reproductive toxicity mechanisms of carbofuran, offering a theoretical basis for health risk assessment and intervention strategies.

Acclimation of Photosynthesis to Environment 1 (APE1) has been identified as a protein involved in the adaptation of plants to high illumination. Several studies indicate its association with PSII, but its specific function in photosynthesis has not yet been determined. In this study, we used biochemical and molecular biology approaches to identify the function of APE1 in Arabidopsis (Arabidopsis thaliana) and the cyanobacterium Synechocystis sp. PCC 6803. APE1 elimination in the plant altered various, seemingly unrelated, photosynthetic processes, such as maximum quantum efficiency of PSII, non-photochemical quenching (NPQ), and state transitions. The levels of PSII core proteins were reduced in the ape1 mutant. Our results indicated that APE1 is involved in the formation or stabilization of the RC47 complex of PSII, in both plants and cyanobacteria. The alterations in NPQ and state transitions of the plant were a consequence of the lesion in PSII. These alterations were observed only when the mutant was stressed by high-light (HL) treatment, confirming that APE1 is necessary for optimal PSII performance under HL stress.

PurposeThe West Nile Virus (WNV) remains a global health problem, necessitating the identification of effective antiviral strategies. This study aimed to identify potential druggable targets for WNV and assess the repurposing of three FDA-approved antivirals – remdesivir, rilpivirine, and doravirine – through comprehensive in silico evaluations.Patients and MethodsThrough molecular docking, molecular dynamics simulations (MDs), and Molecular Mechanics Generalized Born/Surface Area (MM-GBSA) free energy calculations, we assessed the stability, binding affinity, and thermodynamic profiles of the drug-protein complexes, focusing on the core protein, nonstructural protein NS3 serine protease, and two domains of nonstructural protein NS5: RNA-dependent RNA polymerase (RdRp) and methyltransferase (MTase).ResultsDoravirine showed the most favorable and stable interactions across multiple targets. Specifically, it exhibited strong and persistent binding within the C-terminal tunnel and N-terminal hydrophobic pocket of the core protein, as well as at the KDKE motif and SAH-binding site of the NS5 MTase domain. Triplicate MD simulations and residue-level fluctuation analyses further confirm doravirine’s stability and consistent interaction patterns in all binding sites, highlighting its potential as a promising candidate for WNV inhibition with multitarget activity.ConclusionThese findings provide in silico evidence supporting doravirine as a promising multitarget inhibitor of WNV, warranting further investigation for its repurposing for WNV treatment.

Malignant lymphoma (ML) is a heterogeneous group of cancers originating from lymphocytes and lymphoid tissues. Despite advances in treatment, refractory and relapsed cases remain a significant challenge, leading to poor prognosis and highlighting the need for novel therapies. This study aimed to use data mining and experimental validation to identify potential traditional Chinese medicine (TCM) therapeutic agents for ML and explore the underlying mechanisms. A comprehensive search on TCM prescriptions for ML treatment was conducted across multiple databases. Association rule mining was applied to identify commonly used herb combinations. In vivo experiments were performed to evaluate the effects of the core combination on tumor growth in the ML xenograft tumor model. Network pharmacology was used to explore the involved mechanisms. Molecular docking was used to assess the binding affinity of the key active ingredients to core proteins. Western blot analysis was conducted to evaluate the expression of proteins associated with the involved pathway. A total of 118 eligible TCM prescriptions for ML treatment were identified across various databases, including 213 herbs. Association rule analysis identified "Glycyrrhiza uralensis, Prepared Rehmannia Root, and Astragalus membranaceus" as the core herb combination for ML treatment. In vivo experiments showed that the identified combination inhibited tumor formation in the ML model. Functional analysis showed significant enrichment in the PI3K-Akt signaling pathway. Molecular docking revealed strong binding affinities between the key ingredients and the core proteins of this pathway. Western blot analysis showed a significant reduction in the ratios of p-PI3K/PI3K and p-AKT/AKT in the herb-treated group compared to the control. This study used data mining to explore the herbal characteristics in the treatment of ML and identify a core herb combination. In vivo experiments showed that the combination inhibited tumor growth in ML, which was associated with the suppression of PI3K-Akt signaling pathway activation. These findings support the potential of TCM-based therapies for ML and lay a foundation for further research and clinical application for the disease.

Bovine viral diarrhea (BVD) is a highly infectious disease with a global distribution caused by the bovine viral diarrhea virus (BVDV), primarily affecting cattle. Dairy farms play a central role in the persistence and spread of BVDV in Italy, making control strategies and genetic studies essential to reduce its circulation. This work aimed to identify and characterize the genotype and subgenotype of BVDV infecting cattle in a specific area of Sardinia. Ten BVDV Sardinian strains were isolated and sequenced from the blood of infected cattle collected into EDTA tubes during outbreaks between 2018 and 2024. Then, to characterize the isolates, phylogenetic and variant analyses were performed on the entire collection of BVDV genomes available to date in GenBank. All Sardinian isolates were assigned to the BVDV-1b subgenotype. Except for two divergent strains, the isolates clustered into a distinct monophyletic clade characterized by 61 exclusive variants absent in all other analyzed sequences. These findings point to the existence of a distinct Sardinian genomic signature. Moreover, among these mutations, 19 missenses distributed on genes encoding the E1, E2, Core, NS3, NS4B and NS5A proteins could have a relevant functional impact, given the role these proteins play in the virus life cycle and in interaction with the host.