Canine mammary tumors (CMTs) are the most common neoplasms in intact female dogs. Although the number of publications on CMTs has increased steadily over recent decades, a comprehensive overview of the knowledge structure, research hotspots, and evolutionary trends in this field is still lacking. Publications related to CMTs were retrieved from the Web of Science Core Collection without restrictions on language or document type. Bibliometric and visualization analyses were conducted using CiteSpace (version 6.3.1) and VOSviewer (version 1.6.20). Co-authorship, co-citation, and keyword co-occurrence networks were constructed to identify influential countries, institutions, authors, core journals, research themes, and emerging trends. The annual number of publications on CMTs showed a long-term increasing trend and has remained relatively stable in recent years, indicating a mature research stage. Brazil, the United States, and Italy were the most productive and influential countries, forming the core of the international collaboration network. A limited number of institutions and author groups dominated the field, with strong intra-group collaboration. Keyword and co-citation analyses revealed that histopathology, molecular expression, prognosis constitute the central research themes. Temporal and burst analyses indicated a shift from descriptive pathology toward molecular characterization, prognostic evaluation, reflecting the increasing depth and complexity of research in this field. Research on CMTs has evolved from morphology-based studies to increasingly molecular and clinically oriented approaches. This bibliometric analysis clarifies the intellectual structure and developmental trajectory of the field and provides a systematic reference for future research on CMTs.

Zearalenone is a common mycotoxin that impairs reproductive function, particularly by damaging ovarian granulosa cells. This study investigated the protective effect and underlying mechanism of scutellarin against zearalenone-induced injury in porcine ovarian granulosa cells. Cells were isolated and identified by follicle-stimulating hormone receptor immunofluorescence. Cell viability, cell-cycle distribution, and related molecular changes were evaluated using the MTT assay, flow cytometry, qRT-PCR, and Western blot. Scutellarin showed no obvious cytotoxicity within the tested range and attenuated the zearalenone-induced reduction in cell viability. Flow cytometry analysis demonstrated that scutellarin alleviated zearalenone-induced cell-cycle disturbance. At the molecular level, scutellarin upregulated the expression of cell-cycle-related factors, including CDK1, CDK2, CDK4, and PCNA, and increased the expression of Wnt/β-catenin signaling-related proteins, including WNT5A, β-catenin, c-MYC, and CCND1. WNT5A knockdown further indicated that scutellarin-mediated regulation of PCNA, CDK1, and CDK4 is WNT5A-dependent, whereas its effect on CDK2 may involve a WNT5A-independent mechanism. These findings indicate that scutellarin alleviates zearalenone-induced granulosa cell injury partly through WNT5A-associated modulation of cell-cycle-related proteins, providing mechanistic insights for its protective effects.

OBJECTIVE:The aim of this study is to investigate the association between single nucleotide polymorphisms (SNPs) of porcine complement receptor 1-like (CR1-like) gene and its mRNA expression. METHODS:SNP identification, qRT-PCR absolute quantification, linkage disequilibrium analysis and dual-luciferase reporter assay were performed using 392 forty-day-old Landrace weaned piglets. RESULTS:28 SNPs were identified, 4 loci were significantly correlated with CR1-like expression (p < 0.05). The c.-1372GA mutation led to the loss of GATA-1 binding site, and significantly reduced promoter transcriptional activity (< P0.01). CONCLUSION:CR1-like gene polymorphisms regulate its expression via disrupting transcription factor binding, which provides a theoretical basis for porcine disease-resistant breeding.

The search for effective alternatives or adjuncts to conventional anti-infective strategies in food animals has increased interest in plant-derived bioactive compounds. Matrine, a quinolizidine alkaloid derived from Sophora flavescens, has attracted considerable attention because of its broad bioactivities and potential veterinary relevance. Recent oral pharmacokinetic and intestinal-lumen PBPK studies in pigs have begun to emerge; however, currently available swine data remain largely confined to oral exposure-oriented experiments and do not permit direct route comparison or estimation of absolute oral bioavailability. In the present study, the plasma pharmacokinetics of matrine in pigs were evaluated after single-dose intravenous and oral administration. Twelve healthy piglets received matrine at 8 mg/kg body weight via either the intravenous or oral route, and plasma concentrations were quantified using a validated UPLC-MS/MS method. Pharmacokinetic parameters were estimated by non-compartmental analysis. Compared with intravenous administration, oral dosing produced lower observed exposure based on AUC0-t and a lower peak plasma concentration, with AUC0-t and Cmax values of 418.94 ± 75.52 h·ng/mL and 66.24 ± 8.44 ng/mL, respectively, versus 558.01 ± 59.57 h·ng/mL and 224.64 ± 20.94 ng/mL after intravenous administration. Oral administration was associated with a Tmax of 2.49 ± 0.02 h and a longer apparent terminal half-life and mean residence time than intravenous dosing. These findings provide pharmacokinetic evidence to support future route selection, dosing-regimen design, and PK/PD-based dose optimization of matrine in pigs.

Shuanghuanglian oral liquid (SHL) is widely used in companion animals and poultry, but its molecular mechanism in pneumonia-myocarditis comorbidity and heart-lung inflammatory crosstalk remains largely unclear. This computational study investigated the conserved AKT1/HIF1A-mediated immunoregulatory mechanism of SHL and its cross-species translational potential in veterinary medicine. Network pharmacology was integrated with GO, KEGG, and Reactome enrichment analyses, protein-protein interaction network construction, ADMET evaluation, cross-species sequence homology analysis (human, dog, cattle, and pig), molecular docking, and molecular dynamics simulation. A total of 61 active compounds, 251 putative targets, and 52 common targets associated with pneumonia and myocarditis were identified. These targets were mainly enriched in inflammation- and immune-related pathways, including TNF, IL-17, AGE-RAGE, and PPAR signaling. AKT1 and HIF1A showed high sequence conservation across species (85-98%). Key compounds exhibited favorable binding affinity to AKT1, and molecular dynamics simulation suggested the stability of the Baicalein-AKT1 complex. ADMET analysis suggested favorable pharmacokinetic properties and low predicted toxicity. These findings suggest that SHL may potentially alleviate pneumonia and myocarditis through modulation of the conserved AKT1/HIF1A axis and support its potential as a complementary therapeutic approach for managing heart-lung inflammatory diseases in multiple livestock species. This entirely computational study highlights promising mechanisms that should be further validated in vivo.

In our previous study, maximum non-toxic concentration (MNTC) of recombinant porcine natural killer lysin (rpNK-Lysin) significantly down-regulated Fascin-1, a prognostic and metastatic biomarker, yet the relevant molecular mechanisms that inhibit Fascin1, was not fully understood. In this study, three different types of hepatocellular carcinoma cell lines (SMMC-7721, MHCC 97H and HepG2) were treated with rpNK-Lysin. Scanning electron microscopy was performed to check its effect on filopodia formation, and the expression of ERK, RSK2, CREB1 and Fascin-1 were determined using qPCR and western blot. Our results showed that MNTC rpNK-lysin successfully down-regulated pERK1/2 which further suppressed the phosphorylation of RSK2 and the transcription factor CREB1, leading to inhibit the CREB transcriptional target Fascin-1, which is involved in filopodia formation. This study confirmed that MNTC rpNK-lysin inhibited metastatic biomarker Fascin1 by down regulating ERK1/2 dependent RSK2 and transcription factor, which further suppressed Fascin1.

Objective: To establish a porcine complement receptor type 1-like (CR1-like)-mediated targeted anti-porcine reproductive and respiratory syndrome virus (PRRSV) nanodrug delivery system by investigating interactions between erythrocytes from Landrace piglets (both male and female), PRRSV, and anti-PRRSV nanodrugs. Methods: Optimal conditions for PRRSV sensitization with fresh porcine serum were determined. CR1-like-dependent immune adhesion of porcine erythrocytes to sensitized PRRSV was verified by immunofluorescence, electron microscopy, qPCR, and Western blot. The effect of this adhesion on PRRSV infection of porcine alveolar macrophages (PAMs) was studied using a flow chamber system. Mannose-modified matrine nanoliposomes (MMLNPs) were prepared, characterized, and evaluated for cytotoxicity, targeting ability, and in vitro antiviral activity. Results: PRRSV was optimally sensitized by incubation with fresh porcine serum at 37 °C for 2 h. Porcine erythrocytes specifically adhered to sensitized PRRSV via CR1-like, significantly promoting PRRSV infection of PAMs. Stable, uniform-sized MMLNPs showed no cytotoxicity, targeted PAMs via CR1-like, and exhibited superior antiviral activity to free matrine. Conclusions: CR1-like-mediated immune adhesion is a critical mechanism for PRRSV infection of PAMs. Harnessing this natural pathway enables efficient targeted delivery of matrine nanoliposomes to PAMs, providing a promising translational strategy for PRRSV control.

Objective:Swine H1N1 influenza is a critical zoonotic pathogen threatening pig industry economy and public health. The host molecular regulatory network and core genes of H1N1 infection remain unclear, hindering targeted prevention and therapy. Traditional experimental methods fail to efficiently mine high-dimensional transcriptomic data, making precise screening of infection biomarkers difficult. Methods: Transcriptome data (GSE40092) were analyzed to obtain porcine lung DEGs upon H1N1 infection, followed by GO/KEGG functional enrichment. Four machine learning algorithms (LASSO, random forest, SVM-RFE, XGBoost) coupled with stratified nested 5-fold cross-validation screened core genes. Feature stability analysis and external dataset GSE28871 validated biomarker robustness. A gradient-dose H1N1 piglet model and Western blot verified the key gene's in vivo protein expression. Results:A total of 310 H1N1-related DEGs were enriched in immune, inflammatory and viral signaling pathways. All four models accurately discriminated infected and normal lung samples, with SPP1 as the only shared core gene. Cross-validation proved SPP1 screening free of overfitting; external validation yielded an AUC of 0.889, 83.3% sensitivity and 100% specificity. In vivo assays confirmed significant SPP1 protein downregulation under low, medium and high viral doses (p < 0.05). Conclusion:This study combined transcriptomics and multi-machine learning to identify and verify host genes for swine H1N1 infection. SPP1 acts as a stable diagnostic biomarker whose reduced expression correlates with disease progression. Our results reveal new molecular mechanisms of H1N1 pathogenesis and offer a candidate target for swine flu control and zoonotic risk intervention.

IntroductionEncephalomyocarditis virus (EMCV), a highly significant member of the picornaviridae family, infects a broad range of mammalian hosts and causes severe pathological consequences like encephalitis, myocarditis, and neurologic diseases, etc. In our previous research, curcumol demonstrated a significant reduction in EMCV replication in vitro. However, its antiviral effect in vivo needs to be elucidated.MethodsIn this study, the mechanism of curcumol against EMCV replication was demonstrated in vivo by establishing an EMCV (100 TCID50/20 g) infected mice model. At day 3 post EMCV infection, drug treatment groups were continuously treated with curcumol for 5 days.ResultsThe viral load and expression of interleukins such as IL-1β, IL-6, and TNF-α mRNA levels were significantly decreased in the heart and brain tissues at day 9 of EMCV infection (5 days of curcumol treatment), detected by qPCR. Moreover, Western blot analysis revealed that curcumol considerably elevated the protein levels of IκB and P65, while significantly reducing the levels of P-IκB, P-P65, NLRP3 inflammasome, caspase 1, and IL-1β in cardiac and cerebral tissues.DiscussionConclusively, these results revealed that curcumol inhibits the NF-κB/NLRP3 inflammasome pathway and thus alleviates heart and brain damage induced by EMCV infection in Kunming mice. The present findings provide new insights into the therapeutic potential of curcumol in addressing EMCV-induced pathological conditions.

Canine mammary tumor (CMT) is the most prevailing neoplasms in female dogs. Matrine demonstrates anti-tumor effects in various organs, its mechanism in CMT treatment remains unclear. This study involved in network pharmacology, molecular docking, dynamics simulations, cell viability, inhibations and migration, to clarify matrine's therapeutic action against CMT. Potential gene/protein targets were identified through PubChem (Matrine), and GeneCards (CMT), with point of intersection targets analyzed via STRING PPI network and Cytoscape. Top 10 hub genes were selected for gene ontology/KEGG pathway analyses (p < 0.05). Molecular docking (JAK2, AR, HDAC2, HDAC6, and NR3C1) revealed strong matrine-JAK2 binding (-7.8 kcal/mol), and dynamics simulations were confirmed by molecular dynamics simulations. Pathway analysis implicated JAK-STAT and PI3K-Akt signaling in matrine's as anti-tumor effects. In vitro results showing that the maximum noncytotoxic concentrations of matrine of canine primary mammary epithelial cells (cPMECs), where the cell viability remained above 90 % at concentrations 280 µM and ≤ 560 µM, respectively, indicating this as the maximum safe concentration for cPMECs, and had a proliferation inhibitory effect time-dependently (12, 24, 48 hrs) on CHMm and CHMp cells within a safe concentration range, and suppression of CMT cells via CCK-8 assays. In 12 h, moderate inhibition was detected, which increased at 24 h, and was most prominent at 48 h. The migration ability of cells decreased at 24 h, respectively. Notably, the 560 µM concentration resulted in over 50 % inhibition in both cell lines after 48 h. Future research should investigate in vivo efficacy to progression matrine as a veterinary oncotherapeutic.

While it is established that complement receptor molecules on the surface of erythrocytes are crucial for the clearance of immune complexes in the body, the molecular mechanisms underlying the interaction between macrophages and erythrocytes in pigs remain inadequately understood. Consequently, we built a detection system with a closed-circulation flow chamber and a constant flow pump. Additionally, we optimized parameters including system flow velocity and fluid shear force. In the circulatory system, our study measured the fluorescence intensity of erythrocyte and pulmonary alveolar macrophages (PAMs) surfaces before and after the blockade of complement receptor 1 (CR1)-like receptors and Fc receptors. The results indicated that porcine erythrocytes and PAMs exhibited a diminished rate of change in fluorescence intensity under the blocked condition. Through transmission electron microscopy, it was observed that PAMs effectively removed sensitized GFP-E. coli adhering immunologically to porcine erythrocytes. The findings indicate that PAMs effectively removed sensitized GFP-E. coli from the surface immunoadhesion of porcine erythrocytes, facilitated by the mediation of surface CR1-like receptors and Fc receptors.

Parvovirus is comprised of a single-stranded DNA structure, encompassing distinct structural and non-structural proteins. Structural proteins are referred as viral proteins, which facilitate for the viral capsid. Among non-structural proteins, NS1 is the most significant, exhibiting substantial characteristics related to viral replication, pathogenicity, and is notably recognized for its remarkable oncolytic properties. NS1 possesses a distinctive structure; however, it differs across different parvovirus species. It is comprised of three fundamental domains: the N-terminal origin binding, helicase domain, and C-terminal domain, all crucial for significant functions. In several parvovirus species, such as CPV, MVM, BPV, and HPV-B19, NS1 halts the cell cycle at distinct stages, including G1, G2, and S phases of the life cycle, and induces cell death. Predominantly, parvovirus NS1 has also been significantly recognized to induce tumor cell death in vitro and in vivo by following different mechanisms, including cytotoxicity, autophagy, immunomodulation, mitochondrial depolarization, and most significantly, apoptosis. This may lead to several intracellular changes, including reactive oxygen species (ROS) level, mitochondria, PARP, caspase, and their subtype activation, ultimately leading to DNA and other cellular level changes, which facilitate apoptotic cell death. These characteristics of NS1 and its combinational therapy revealed a wide range of evidential research that demonstrated its anti-tumor effects through several pathways and can even induce a substantial activation of the immune response. This review mainly aims to elucidate the oncolytic attributes of parvoviral NS1, focusing on its capabilities and the mechanism demonstrated in prior research. It also addresses genetic engineering and combinational therapy aimed at augmenting the oncolytic efficacy of NS1 for more potent application as a tumor therapeutic agent. The increasing focus on virotherapy and precision oncology underscores the necessity for thorough exploration of the molecular mechanisms, delivery techniques, and clinical implications of NS1, thereby facilitating the development of innovative, tumor-selective anticancer approaches.

Introduction:This study aimed to elucidate the mechanism of zinc sulfate against porcine reproductive and respiratory syndrome virus (PRRSV) through transcriptomic data and experimental validation. Methods:Initially, the expression of PRRSV N gene and protein were quantified using qPCR and immunofluorescence, respectively. High-throughput RNA sequencing was performed to analyze global gene expression changes in PRRSV-infected Marc-145 cells treated with zinc sulfate. Transcriptomic data were subjected to bioinformatic analyses, including Venn diagram assessments, protein-protein interaction network construction using the STRING database, and identification of hub genes via Cytoscape 3.10.0. Functional enrichment analysis of Gene Ontology and KEGG pathways were performed using R (v4.4.3). Subsequently, oxidative stress parameters in PRRSV-infected Marc-145 cells treated with zinc sulfate were detected using biochemical assays. The modulatory effects of zinc sulfate on inflammatory response and apoptosis were evaluated through qPCR and western blot, measuring the expression of cytokines (IL-6, IL-8, TNF-α, IL-10), apoptosis-related proteins (Caspase-3, Bax, Bcl-2), and key components of the NF-κB pathway. Finally, flow cytometry was employed to assess cellular apoptosis rates. Results:The result demonstrated that zinc sulfate significantly suppressed PRRSV replication. The transcriptomic analysis revealed that compared to the PRRSV-infected group, there were 14 upregulated and 50 downregulated targets in zinc sulfate treatment group. Among these, ten core downregulated and upregulated targets were well enriched in the inflammation and apoptosis pathways, respectively. The experimental verification results demonstrated that compared to the PRRSV-infected group, zinc sulfate treatment significantly diminished intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) levels, while elevating the enzymatic activities of superoxide dismutase (SOD) and catalase (CAT) (p < 0.05). It also suppressed the expression of IL-6, IL-8, TNF-α, while upregulating IL-10 (p < 0.05). In addition to, it also upregulated the phosphorylation levels of IκBα and p65 (p < 0.05), and decreased the expression of Caspase-3, cleaved-Caspase-3, Bax, while enhancing Bcl-2 (p < 0.05). Simultaneously, flow cytometry analysis further confirmed that zinc sulfate substantially attenuated late-stage and overall apoptosis rates in PRRSV-infected cells (p < 0.05). Conclusion:These results implied that zinc sulfate restricted oxidative stress, diminished inflammatory response and induced apoptotic suppression to confer protection against PRRSV infection.

Introduction:Previous studies have suggested that matrine may improve animal production performance, but its role and underlying mechanisms remain unclear. Methods:Following the determination of the LD50 of matrine in ICR mice, the long-term toxic effects of matrine on SD-weaned rats were evaluated. 0.375, 0.75, 1.5, and 3 mg/kg matrine were added to the feed of weaned piglets, respectively. The feed intake and body weight of piglets were recorded to evaluate the growth-promoting effect of matrine. The feces and blood of weaned piglets were collected to explore the mechanism of matrine improving the growth performance of piglets. Results:Our findings imply that the LD50 of matrine in mice was 202.54 mg/kg, and matrine did not cause any hazardous effects when administered to rats within the range of 24.5-50 mg/kg for 180 days. Furthermore, supplementation of 0.375, 0.75, and 1.5 mg/kg matrine can increase ADG (average daily weight gain), and ADFI (average daily feed intake), and decrease the FCR (food conversion rate) of piglets. Additionally, 0.375 and 0.75 mg/kg matrine could increase the positive rate of porcine circovirus type 2 (PCV2) vaccine antibody in serum of piglets. We analyzed the correlation between intestinal flora, fecal metabolites, and growth performance through Mothur software and found that the impact of matrine on ADG, ADFI, and FCR might be associated to Gemmiger formicilis and thiamine. Conclusion:These findings revealed that matrine can improve the growth performance of weaned piglets by increasing the abundance of Gemmiger formicilis and thiamine content in feces.

Immunosuppression increases disease risk, and the natural compound polydatin (PD) has been reported to modulate immune-related disorders. In cyclophosphamide-induced immunosuppressed mice, PD was evaluated for its immunomodulatory effects. Immune organ indices were measured, while H&E staining and ELISA assessed spleen pathology and serum cytokine levels. The proliferation of splenic lymphocytes, both total and subpopulation, was determined using concanavalin A or lipopolysaccharide stimulation, with flow cytometry analyzing peripheral blood and splenic lymphocytes, thymic T cell subtypes, cell cycling, and bromodeoxyuridine incorporation. Western blotting was used to assess Ki67, PCNA expression, and MAPK activation. PD significantly alleviated cyclophosphamide-induced reductions in spleen and thymus indices, improved the organization of red and white pulp in the spleen, and restored TNF-α and IFN-γ levels. It reversed cyclophosphamide-induced cell cycle arrest, characterized by increased PCNA and decreased Ki67, and corrected the diminished numbers of B and T cells and the reduced CD4+/CD8+ ratio in the thymus. In vitro, PD directly promoted splenic lymphocyte proliferation and cell cycling via MAPK activation. Overall, our findings demonstrated that PD alleviated mouse immunosuppression by activating splenic lymphocyte proliferation and re-organizing thymic T cell development and differentiation.

Microfluidics is an emerging technology for buffer exchange in bioprocessing applications. However, achieving buffer exchange with simplicity of operation and high throughput in a straightforward channel design remains a challenge. This study presents a novel semicircular microchannel design that allows for the deterministic regulation of helical and Dean vortices through geometric confinement. By incorporating micro-obstacles into semicircular microchannels with large dimensions (900 μm wide and 100 μm high), we observe a substantial enhancement in secondary flows, leading to a unique fluid distribution across a wide range of flow rates. This design enables a high particle separation efficiency (>96.27%) coupled with a low fluorescein purity (<4.46%) at a high throughput of 3 × 106 particles/min. The proposed methodology, characterized by ease of production (simple semicircular microchannels with large dimensions), user-friendly operation (uniform flow rates in both sheath and sample inlets), and efficient buffer exchange capabilities (typically 3 mL min-1), demonstrates significant potential for advancing microfluidic systems in biological and biomedical research.

Immortalized cell lines constructed through transfecting genes such as the hTERT and the SV40-LT provide stable cellular resources for both scientific exploration and industrial implementation. Although advancements have been documented in the establishment of immortalized cell types, research on immortalization of specialized animal cell types remains an underexplored domain. To explore the applicable value of the dzo, a yak-cattle hybrid endemic to northwestern China, and develop potential cell substrates that can be used for the production of BVDV vaccines, this study adopts an immortalization strategy with the hTERT and SV40-LT genes to construct an immortalized dzo kidney cell line. This study employed a lentiviral vector system to stably integrate SV40-LT into dzo renal cells, successfully generating the immortalized NBLS cell line. Compared to liposome-mediated transfection, lentiviral delivery demonstrated superior gene transfer efficiency through high integration capacity and broad tropism. NBLS cells maintained robust proliferation (viability > 90%), normal cell cycle distribution, and diploid karyotype (2n = 60) through 50 passages, whereas hTERT-only transfectants exhibited viability decline below 70% after passage 10. Functional validation revealed NBLS cells displayed enhanced BVDV susceptibility (lgTCID50 = 10^- 6.59/0.1 mL), with tenfold increased sensitivity compared to primary counterparts. The results will provide potential materials for BVDV vaccine production and species-specific cellular models for investigating plateau-adapted disease resistance mechanisms and screening novel vaccine antigens.

The complement system is crucial for immune defense, linking innate and adaptive immunity. In the classical and lectin pathways, C4 is split into C4b, triggering opsonization, lysis, and the removal of pathogens and damaged cells. Dysregulated activation of C4 and other components of the classical pathway can lead to tissue damage and heightened inflammation, whereas appropriate regulation of C4b activity serves to mitigate excessive inflammation and prevent injury. ELISA analysis demonstrated C4 activation and cleavage during the co-incubation of PRRSV with fresh porcine serum. Immunoelectron microscopy revealed that porcine red blood cells could immunologically adhere to PRRSV, and C4b was involved in this adhesion process. BLAST (NCBI BLAST+ 2.14.1) analysis revealed that porcine CR1-like CCPs 1-3, CR1-like CCPs 12-14, and CR1-like CCPs 19-21 share high similarity with the CCP 1-3 region of human CR1, which mediates C4b binding. Yeast two-hybrid assays confirmed that all three CR1-like fragments bind C4b. To elucidate the interaction mechanism, homology models of C4b and CR1-like fragments were constructed, followed by molecular docking and dynamics simulations, identifying 18 key amino acids in porcine CR1-like involved in C4b binding. Surface plasmon resonance further validated the binding affinity of CR1-like CCPs 1-3, its mutant 118I, and C4b. These results enhance our understanding of complement regulation and provide a foundation for developing therapeutic strategies targeting complement-related diseases.

Cancer metastasis often presents the main reason for mortality in cancer patients. As such, metastasis prevention may facilitate long-term survival of individuals affected by this disease. Previous studies carried out by our laboratory demonstrated that the prNK-lysin inhibits the growth and metastasis of cancer cells in vitro. However, the intrinsic properties of cationic oncolytic peptides severely limit prNK-lysin's clinical applicability, as no dedicated delivery system for it has been reported. In an effort to overcome these limitations and enhance the anticancer efficacy of prNK-lysin, we designed low-modulus soft hydrogels as a new strategy for prNK-lysin delivery. This pH-responsive, HA-modified dextran-based hydrogel achieves precise controlled release of prNK-lysin within solid tumors, offering an innovative approach for efficient local delivery of protein/peptide drugs. Our results demonstrated that the system features favorable injectability and biocompatibility, along with sustained drug release capability, while mitigating prNK-lysin's systemic adverse reactions. Moreover, prNK-lysin@DEX/HA Gel exhibits growth and metastasis inhibitory effects on breast cancer cells both in vitro and in vivo. Significantly, its inhibitory effects were augmented in vivo compared to free prNK-lysin, consistent with improved drug retention. Additionally, preliminary findings indicate the potential of prNK-lysin to stimulate anti-tumor immunity. Overall, this soft hydrogel system could enhance the inhibitory effect of prNK-lysin on orthotopic breast cancer growth and lung metastasis. Therefore, it may represent a valuable technology for future breast cancer treatment.

IntroductionCellular machinery is built upon proteins and their functional interrelationships. Their network evaluation is essential for a comprehensive insight into biological processes and may establish a foundation for predicting antivirulence. Antiviral peptides (AVPs) have robust, broad-spectrum anti-virulence capabilities. Nevertheless, the existing predicted AVPs database is insufficient and necessitates more precise, reliable annotations. This study aimed to screen differentially expressed proteins and peptides of healthy and porcine reproductive and respiratory syndrome virus (PRRSV)-infected tissues and to predict AVP’s using Machine learning and Deep learning based computational methods.MethodsLungs, small intestine and large intestine samples were collected to validate and quantify proteins and peptides through proteomics, and followed by predicting AVPs by employing machine learning (ML) and deep learning (DL). Models were developed exploiting significant features based on physicochemical characteristics, encompassing amino acid composition (AAC), secondary structure, and hydrophilicity. Proteomics analysis facilitated peptide qualification through GO, KEGG, COG, and PPI analysis. To predict AVPs, we employed a DL graph neural network (GNN) by making its inaugural implication in this domain and benchmarked its efficacy against conventional ML random forest (RF) and support vector machine (SVM) models.ResultsFindings demonstrated that lysine, arginine, and leucine were ranked nearly 0.1, highlighting their significant importance in prediction. Additionally, the correlation heatmap showed that lysine and glutamate exhibited the strongest positive association (0.57). RF model achieved an area under the curve (AUC) of 0.95 ± 2, verified via 5-fold cross-validation. In contrast, GNN and SVM models yielded 0.94 ± 1 AUC, demonstrating comparable performance across models, and revealed that the RF model outperformed compared to the others.DiscussionIntegrating proteomics with computational modeling revealed peptides with antiviral potential against PRRSV. The RF model demonstrated the best discriminative power, and amino acid composition played a key predictive role. Consequently, these comparative predictive results may serve as revolutionized and distinctive resources for the experimental validation and identification of PRRSV AVPs as prospective therapeutics.