Immunometabolism and neutrophil extracellular traps (NETs) play pivotal roles in the pathogenesis of coronavirus disease 2019 (COVID-19) and its postacute sequelae. However, the upstream regulators that reprogram neutrophil lipid metabolism and trigger excessive NET formation remain largely undefined. This study identifies a transfer RNA-derived fragment, tRF-His-GTG-1, enriched in platelet-derived extracellular vesicles, as a key driver of neutrophil lipophagy dysfunction and inflammation in COVID-19. The use on neutrophils from 60 patients and 20 healthy controls, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected hamster model, and multiple in vitro assays shows that severe COVID-19 and long COVID are characterized by increased lipid droplet (LD) accumulation and NET release. Mechanistically, tRF-His-GTG-1 activates Toll-like receptor 8 (TLR8)-mammalian target of rapamycin (mTOR) signaling and suppresses RAB7A expression, changes that impair lipophagic flux. This dual pathway impairs lipophagy and promotes NET formation and proinflammatory cytokine secretion. Importantly, ex vivo treatment with a tRF-His-GTG-1 inhibitor restores lipophagy, reduces LD and NET levels, and suppresses interleukin 1beta (IL-1β)/IL-8 production in patient-derived neutrophils. These findings reveal a novel EV-mediated immunometabolic axis linking platelets to neutrophil dysfunction, and position tRF-His-GTG-1 as a promising RNA-based therapeutic target for COVID-19-associated hyperinflammation.

Gastric cancer (GC) is a leading malignant disease in numerous countries, including Taiwan with limited therapeutic options. Animal viruses including oncolytic avian reovirus (ARV) have the possibility to avoid pre-existing immunity in humans, while being safe and immunostimulatory. Here, we provide a novel insight into oncolytic ARV and UV-ARV-sensitized patient’s peripheral blood mononuclear cells (P-PBMCs) and tumor infiltrating lymphocytes (TILs) killing primary GC (PGC) cells through the surface TLR3 and TRAIL/DR4/DR5 immunogenic apoptosis pathway. We conducted a comprehensive study to reveal whether ARV- or UV-inactivated ARV (UV-ARV)-modulated P-PBMCs or TILs killing ARV- and UV-ARV-sensitized AGS cells and PGC cells derived from clinical patients and to investigate the regulation of surface TLR3 receptor and upstream signaling pathways. Apoptosis analysis by flow cytometry and Western blot, suppression of signal pathway by specific inhibitors, in situ proximity ligation assay (PLA), time-resolved flurometry and lactate dehydrogenase (LDH) cytotoxicity assays, and an in vitro co-culture model were established to study the interplay between ARV- and UV-ARV-sensitized P-PBMCs and TILs to kill PGC cells and their upstream pathways. Our results reveal that increased levels of DR4 and DR5 were observed in ARV and UV-ARV sensitized PGC cells through the TLR3/p38/p53 signaling pathway. Importantly, we found that the σC protein of ARV or UV-ARV interacted with surface TLR3 of CD8+ TILs, thereby triggering the TLR3/NF-κB/IFN-γ/TRAIL signaling pathway which induces immunogenic apoptosis of PGC cells. This study sheds further light on the molecular basis behind ARV oncolysis and facilitates the ARV or UV-ARV as a cancer therapeutic. The study provides novel insights into ARV- or UV-ARV-sensitized P-PBMCs and CD8+ TILs to kill PGC cells through the immunogenic apoptosis pathway. We conclude that P-PBMCs can easily be obtained from GC patients and provide a rich source as TILs to kill PGC cells.

Oncolytic viruses (OVs) represent a unique therapeutic platform that combines tumor-selective replication with potent immunomodulatory capacity. In addition to direct oncolysis, OVs can convert tumors into inflammatory niches that support antigen release, dendritic cell activation, and cytotoxic T lymphocyte priming. However, durable responses remain inconsistent across tumor types and patient populations, largely due to tumor immune tolerance mechanisms that restrict both viral propagation and the development of effective anti-tumor immunity. Toll-like receptors (TLRs), as key pattern recognition receptors, play central roles in sensing viral nucleic acids and infection-associated danger signals, orchestrating type I interferon responses, NF-κB-driven inflammation, and downstream adaptive immunity. Notably, TLR signaling is a double-edged sword in virotherapy: it can promote antigen presentation and immune activation while simultaneously accelerating antiviral clearance and limiting intratumoral viral spread. Chemotherapy, a mainstay of cancer treatment, further shapes OV efficacy by altering lymphocyte availability, antigen presentation, myeloid composition, and the balance between immunogenic cell death and immunosuppression. Emerging evidence supports that rational OV-chemotherapy combinations can synergize by enhancing tumor antigen release, reprogramming suppressive myeloid compartments, and creating temporal windows for immune checkpoint blockade. In this review, we summarize the mechanisms underlying tumor immune tolerance, discuss how TLR-mediated innate sensing shapes OV-induced anti-tumor immunity, and evaluate how chemotherapy modulates these interactions. We propose mechanistic frameworks and translational considerations including scheduling, biomarkers, and immune monitoring to guide the development of next-generation OV combination strategies in chemotherapy-treated cancer patients.

Avian reovirus (ARV) is a major poultry pathogen recently recognized for its potential as an oncolytic virus that selectively infects and kills cancer cells without harming healthy human cells. However, the receptors mediating ARV entry into cancer cells remain unclear. Using mouse melanoma B16-F10 cells as a model, this study identified ARV-binding receptor candidates through viral overlay protein binding assay (VOPBA), SDS-PAGE, and LC-MS/MS analysis. Plaque-forming assays (PFAs) evaluated viral replication efficiency, while co-immunoprecipitation (Co-IP) and proximity ligation assay (PLA) confirmed direct interactions between viral σC and host receptor proteins. Functional assays using shRNA knockdown and antibody blocking demonstrated that inhibition of Plg-RKT expression markedly reduced ARV infection. Western blot analysis revealed that ARV binding to Plg-RKT activates Src and p38 MAPK signaling pathways, which promote caveolin-1 phosphorylation and caveolae-mediated endocytosis. These findings identify Plg-RKT as a crucial receptor mediating ARV σC binding and entry into B16-F10 melanoma cells. Furthermore, activation of Src-p38 MAPK signaling was shown to be essential for viral internalization. This study elucidates the molecular mechanism underlying ARV entry into melanoma cells and provides valuable insight for improving the selectivity and therapeutic potential of ARV as an oncolytic virus.

Lidocaine, a local anesthetic, has been shown to modulate immune responses. This study examines its effects on cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating immune cells (TIICs) from gastric cancer patients. PBMCs from healthy donors and TIICs from gastric cancer patients were treated with lidocaine. Cytokine production was assessed using flow cytometry and cytokine assays, with a focus on IFN-γ, IL-12, IL-10, TGF-β, and IL-35 levels. Cytotoxicity against primary gastric cancer cells (PGCCs) was also evaluated. Lidocaine inhibited IFN-γ production in CD8+ PBMCs and IL-12 in CD14+ PBMCs while increasing anti-inflammatory cytokines (IL-10, TGF-β, IL-35) in CD4+CD25+ and CD14+ cells. In TIICs, lidocaine enhanced IFN-γ and IL-12 production in CD8+ and CD14+ cells, while reducing IL-10, TGF-β, and IL-35 levels, promoting an M1-like phenotype in macrophages. Lidocaine also increased IFN-γ production and cytotoxicity in CD8+ TIICs via NF-κB activation. Importantly, lidocaine did not affect the viability of PBMCs, TIICs, or PGCCs at concentrations up to 1.5 mM. Lidocaine reprograms the tumor immune microenvironment, enhancing anti-tumor immune responses, suggesting its potential to modulate immune functions in gastric cancer.

Oncolytic viruses (OVs) can selectively infect and lyse cancer cells while simultaneously activating the host immune system and making them a prospective class of anticancer immunotherapeutic agents. In particular, mammalian reovirus (MRV) and avian reovirus (ARV) have gained unique attention for their significant properties and clinical potentials. This review explores their mechanisms of action, immunological interactions, and clinical applications in OV therapy. Studies have shown that MRV exploits defective antiviral responses in cancer cells to induce apoptosis and autophagy, whereas ARV modulates mTORC1 and energy metabolism pathways to enhance viral replication. Both OVs elicit robust innate and adaptive immune responses, which are critical for long-term antitumor immunity. Combining OVs with chemotherapy and immunotherapy has shown synergistic effects, enhanced antitumor responses, and overcome treatment resistance. However, challenges around managing the host immune response and the efficient delivery of the virus remain unresolved. Ongoing and future clinical trials are pivotal in validating the therapeutic potential of these OVs and considering them as innovative approaches in the oncology landscape.

Patients with systemic lupus erythematosus (SLE) are highly susceptible to severe infections, with non-typhoidal Salmonella (NTS) often progressing to life-threatening bacteremia. However, the mechanisms underlying this heightened vulnerability remain unclear. Lipid droplets (LDs), key immunometabolic hubs, have been implicated in facilitating Salmonella survival within phagocytes. We identified the tRNA-derived small RNA (tsRNA) tRF-His-GTG-1 as upregulated in SLE and correlated with disease activity. This study investigates the signaling and functional roles of tRF-His-GTG-1 in LD formation and Salmonella persistence in SLE. LDs and tRF-His-GTG-1 expression were analyzed in peripheral blood mononuclear cells (PBMCs) from SLE patients with or without NTS bacteremia and from controls. Bacterial survival, tsRNA function, and signaling pathways were assessed using immunofluorescence, qRT-PCR, colony-forming unit assays, and pharmacological or genetic inhibition. LDs were elevated in SLE PBMCs and further increased by NTS infection, correlating with enhanced bacterial survival. SLE immune complexes (ICs) and IC-primed platelet-derived extracellular vesicles (pEVs) induced LD formation via FcγRIIA-mediated uptake and Toll-like receptor (TLR) 7/8 activation. Both SLE ICs and NTS infection upregulated tRF-His-GTG-1. Mechanistically, tRF-His-GTG-1 directly bound to TLR7/8, activating ERK/p38 signaling to induce PPARδ expression and LD biogenesis. Concurrently, tRF-His-GTG-1 promoted ERK/p38 phosphorylation and IL-10 production. Inhibition of tRF-His-GTG-1 suppressed both LD- and IL-10–dependent pathways, thereby reducing Salmonella survival. tRF-His-GTG-1 enhances Salmonella persistence through a TLR7/8–ERK/p38–PPARδ/IL-10 signaling axis. These findings identify a tsRNA-mediated immunometabolic mechanism linking SLE with bacterial susceptibility and highlight tRF-His-GTG-1 as a potential therapeutic target to improve antibacterial immunity in high-risk patients. Infection is a leading cause of death in patients with systemic lupus erythematosus (SLE), who are particularly susceptible to severe non-typhoidal Salmonella (NTS) infections. In this study, we identified a small RNA, tRF-His-GTG-1, which is elevated in SLE patients and further induced by NTS. This molecule interacts with TLR7/8 in PBMCs to activate the ERK/p38 pathway, subsequently boosting PPARδ activity. This leads to increased lipid droplet (LD) formation and production of the immunosuppressive cytokine IL-10, both of which promote intracellular Salmonella survival. Conversely, inhibiting tRF-His-GTG-1 reduced LD formation, IL-10 production, and bacterial survival. These findings reveal a novel molecular axis in NTS pathogenesis and suggest that targeting tRF-His-GTG-1 may be a therapeutic strategy to prevent severe Salmonella infections in high-risk SLE patients.

Oncolytic avian reovirus (ARV) has been identified as a virus capable of selectively infecting and inducing cell death in various cancer cell lines. This study investigates the role of ARV in activating innate immune responses in B16-F10 murine melanoma cells, focusing on the TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways. Our results revealed that the σC protein of ARV interacts with toll-like receptor 3 (TLR3) in the cytoplasm, leading to nuclear translocation of IRF3 and NF-κB as well as the upregulation of IFN-γ, as confirmed by quantitative real-time reverse transcription and polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), proximity ligation assay (PLA), and Western blot. Inhibition assays targeting TLR3, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interferon regulatory factor 3 (IRF3) further validated the involvement of the TLR3-IRF3 and TLR3-NF-κB pathways in IFN-γ activation. Additionally, cells treated with signal transducer and activator of transcription 1 (STAT1) shRNA and Janus kinase (JAK) inhibitor revealed that ARV promotes autophagy via the IFN-γ-JAK-STAT1 pathway. Immunofluorescence staining and LC3-mCherry transfection further confirm ARV's role in triggering autophagy via TLR3-IRF3-IFN-γ-JAK-STAT1 and TLR3-NF-κB-IFN-γ-JAK-STAT1 pathways. Our results revealed that oncolytic ARV induces autophagy and apoptosis in middle to late stages of virus life cycle in murine melanoma cells. These findings highlight the potential of ARV as a novel oncolytic virotherapy through immune pathway activation in cancer cells.

ABSTRACT This study is the first to reveal that oncolytic avian reovirus (ARV) modulates the IKK/NF-κB signaling through the Hsp90-Cdc37, T-complex protein-1 ring complex (TRiC)/Hsc70, and TRiC-phosducin-like protein 1 (PhLP1) chaperone complexes, thereby inducing chaperone-mediated autophagy in mammalian and human cancer lines (referred to as Vero and A549). Specifically, the ARV p17 protein elevates the binding of co-chaperone PhLP1 to TRiC, thereby reducing the complex formation of TRiC-Hsc70 and leading to IκB degradation, suggesting that these two co-chaperones exhibit a competitive relationship. The p17 protein of ARV plays a crucial role in enhancing the formation of the TRiC/PhLP1 complex to protect viral proteins from ubiquitin-proteasome-mediated degradation. Interestingly, we found that ARV p17 enhances the interaction between the Hsp90/Cdc37 chaperone complex and IKK through activation of casein kinase 2 (CK2), which in turn activates NF-κB. Moreover, ARV p17 transcriptionally upregulates Beclin1 and increases the formation of Beclin1-PtdIns3K complex through the CK2-Hsp90/Cdc37 pathway. Immunofluorescence staining reveals that ARV p17 promotes the formation of GFP-LC3 puncta in both Vero and A549 cell lines, while a significant reduction in GFP-LC3 puncta was observed in CK2 and Hsp90 knockdown cells. Interestingly, in situ proximity ligation assay indicated that ARV p17 promotes the interaction between LC3-II and the cytosolic chaperonin complex 2, triggering chaperone-mediated autophagy. This study provides novel insights into ARV-modulated suppression of IκB by regulating co-chaperone PhLP1 and Hsc70 binding to TRiC and activation of the CK2/Hsp90/Cdc37/IKK/NF-κB pathway to induce chaperone-mediated autophagy.IMPORTANCEThis study reveals that the oncolytic avian reovirus (ARV) p17 activates chaperone-mediated autophagy in Vero and A549 cells by modulating the Hsp90/Cdc37, T-complex protein-1 ring complex (TRiC)/Hsc70, and TRiC/phosducin-like protein 1 (PhLP1) chaperone complexes. ARV p17 enhances PhLP1 binding to TRiC, reducing TRiC/Hsc70 complex formation, which promotes IκB degradation and activates the IKK/NF-κB pathway. Additionally, p17 increases casein kinase 2 (CK2)-mediated phosphorylation, strengthening the Hsp90/Cdc37/IKK interaction and transcriptionally upregulating Beclin1, forming the Beclin1/PtdIns3K complex to further induce autophagy. Immunofluorescence shows p17-induced GFP-LC3 puncta, which decreases upon CK2 and Hsp90 knockdown. In situ proximity ligation assay revealed that p17 promotes LC3-II and CCT2 interactions, confirming chaperone-mediated autophagy activation. This study provides novel insights into ARV-modulated suppression of IκB by modulating co-chaperone PhLP1 and Hsc70 binding to TRiC and activation of the CK2/Hsp90/Cdc37/IKK/NF-κB pathway to induce chaperone-mediated autophagy. This work expands our understanding of the role of ARV in regulating host cell autophagy pathways and viral replication. It also provides a new avenue for understanding viral modulation of host cellular processes in the context of oncolytic virotherapy.

BackgroundAvian reovirus (ARV) is an oncolytic virus that induces autophagy and apoptosis in cancer cells, modulates the immune response, and exposes tumor-associated antigens to the immune system, making it a promising candidate for cancer therapy. Cancer cell migration and invadopodia formation are essential processes in metastasis, and targeting these mechanisms could be beneficial in limiting cancer progression.MethodsThis study investigated the effects of ARV p17 protein on cancer cell migration and invadopodia formation in HeLa and A549 cell lines. Molecular assays were conducted to examine the expression and interactions of key signaling molecules, including nucleoporin Tpr, p53, PTEN, FAK, Src, Rab40b, PI3K, Akt, TKs5, and Nck1. Analysis of TKs5, Nck1, and Rab40b mRNA levels by quantitative real-time RT-PCR. Furthermore, invadopodia detection, gelatin degradation assay, and Fluorescence imaging was performed to visualize invadopodia structures and assess extracellular matrix degradation. Additionally, rescue experiments were performed by co-transfecting cells with mutant PTEN (C124A), TKs5, or Rab40b plasmids to confirm their roles in mediating the effects of p17.Resultsp17 suppressed nucleoporin Tpr, resulting in the activation of p53 and upregulation of PTEN. This blocked the formation of the FAK-Src complex and inhibited the Rab40b-PI3K-Akt signaling pathway. p17 also transcriptionally downregulated TKs5, Nck1, and Rab40b, thereby reducing the formation of TKs5-Nck1 and TKs5-Rab40b complexes, which are critical for invadopodia formation. Fluorescence imaging confirmed a marked reduction in invadopodia formation and matrix degradation in cells expressing p17. Restoration of invadopodia formation upon co-transfection with mutant PTEN, TKs5, or Rab40b confirmed that these molecules are key mediators of p17’s inhibitory effects.ConclusionARV p17 inhibits cancer cell migration and invadopodia formation by activating the p53-PTEN pathway and suppressing essential signaling and scaffolding complexes (FAK-Src, Rab40b-PI3K-Akt, TKs5-Nck1, and TKs5-Rab40b). These findings suggest that p17 plays a crucial anti-metastatic role and may serve as a novel therapeutic agent for targeting invasive cancer cells.

The release mechanism of avian reovirus (ARV) from host cells is orchestrated by several pathways and many of these mechanisms remained elusive. Here, we report that inhibition of exosome proteins CD81 and CD63 significantly reduced the relative release of the virus. We observed that ARV induced exosome protein expression over time and found that p17 protein play a pivotal role in virus release. Immunofluorescence assays revealed that ARV virions are coated with autophagosome and are then transported to the extracellular vesicles for release. Suppression of autophagosome maturation with Thapsigargin (TG), bafilomycin A1, or Rab7a shRNA disrupts fusion with lysosomes, resulting in a substantial drop in both the viral release ratio and virus titers. To further identify whether the virus uses autophagy to transfer nascent virus to exosomes as mechanism to avoid degradation caused by bone marrow stromal cell antigen-2 (BST-2), depletion of BST-2 by the shRNA increased virus release and virus titer. Inhibition of autophagosome maturation with TG resulted in a decrease in viral protein levels and virus release, confirming a crucial role of autolysosome formation in virus release. Furthermore, knockdown of BST-2 moderately reversed TG-modulated inhibition of virus release. Taken together, this study provides novel insights into ARV-induced autolysosome and suppression of BST-2 enhancing progeny viruses to extracellular vesicles (EVs) for release.

Abstract Background Hyperactive neutrophil extracellular traps (NETs) formation plays a crucial role in active severe systemic lupus erythematosus (SLE). However, what triggers the imbalance in dysregulated NETs formation in SLE is elusive. Transfer RNA-derived small RNAs (tsRNAs) are novel non-coding RNAs, which participate in various cellular processes. We explore the role of tsRNAs on NETs formation in SLE. Methods We analyzed the levels of NETs DNA and platelet-derived extracellular vesicles (pEVs) from 50 SLE patients and 20 healthy control subjects. The effects of pEVs on NETs formation were evaluated by using immunofluorescence assay and myeloperoxidase-DNA PicoGreen assay. The regulatory mechanism of pEVs on NETs formation and inflammatory cytokines production were investigated using an in vitro cell-based assay. Results Increased circulating NETs DNA and pEVs were shown in SLE patients and were associated with disease activity (P < 0.005). We demonstrated that SLE patient-derived immune complexes (ICs) induced platelet activation, followed by pEVs release. ICs-triggered NETs formation was significantly enhanced in the presence of pEVs through Toll-like receptor (TLR) 8 activation. Increased levels of tRF-His-GTG-1 in pEVs and neutrophils of SLE patients were associated with disease activity. tRF-His-GTG-1 interacted with TLR8 to prime p47phox phosphorylation in neutrophils, resulting in reactive oxygen species production and NETs formation. Additionally, tRF-His-GTG-1 modulated NF-κB and IRF7 activation in neutrophils upon TLR8 engagement, resulting IL-1β, IL-8, and interferon-α upregulation, respectively. Conclusions The level of tRF-His-GTG-1 was positively correlated with NETs formation in SLE patients; tRF-His-GTG-1 inhibitor could efficiently suppress ICs-triggered NETs formation/hyperactivation, which may become a potential therapeutic target.

Mango ( Mangifera indica ) is favored for its flavor and nutritional benefits, yet it also poses significant allergenic risks, potentially triggering severe anaphylactic reactions in sensitive individuals. Addressing the urgent need for accurate allergen detection, this study introduces a novel approach utilizing immunomagnetic nanoparticles (IMNPs) combined with ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to identify the 40-kDa major mango allergen in processed foods. By employing 100 nm IMNPs, the method effectively isolates and concentrates the allergens from complex food matrices, facilitating precise UPLC-MS/MS analysis. Demonstrating exceptional specificity, the technique accurately distinguishes mango allergens from those in nine other fruits and vegetables. It achieves low limits of detection (LODs) of 0.032 mu g/mL in juice and 0.165 mu g/g in cookies. When tested on 34 commercial products and compared with a real-time PCR method approved by the Taiwan FDA, the method validated its high specificity (100%) and sensitivity (92.9%), based on the ingredient labeling of these products. These advancements mark a significant leap in allergen detection technology, offering a sensitive, specific, and practical solution for identifying mango allergen residues in processed foods, thereby enhancing food safety protocols and protecting public health.

This study demonstrates for the first time that the matrix (M) protein of BEFV is a nuclear targeting protein that shuttles between the nucleus and the cytoplasm in a transcription-, carrier-, and energy-dependent manner. Experiments performed in both intact cells and digitonin-permeabilized cells revealed that M protein targets the nucleolus and requires carrier, cytosolic factors or energy input. By employing sequence and mutagenesis analyses, we have determined both nuclear localization signal (NLS) 6KKGKSK11 and nuclear export signal (NES) 98LIITSYL TI106 of M protein that are important for the nucleocytoplasmic shuttling of M protein. Furthermore, we found that both lamin A/C and chromosome maintenance region 1 (CRM-1) proteins could be coimmunoprecipitated and colocalized with the BEFV M protein. Knockdown of lamin A/C by shRNA and inhibition of CRM-1 by leptomycin B significantly reduced virus yield. Collectively, this study provides novel insights into nucleocytoplasmic shuttling of the BEFV M protein modulated by lamin A/C and CRM-1 and by a transcription- and carrier- and energy-dependent pathway.

Porcine Circovirus type (PCV) 2 is an important pathogen that has been circulating worldwide and has cuased serious economic loss in pig industry. However, both PCV3 and PCV4 are newly emerging viruses. In Taiwan, PCV2 has been one of the critical pathogens in pig frams and PCV3 has been detected since 2016; however, the epidemiolog of PCV3 in Taiwan remains unclear and PCV4 has yet to be identified. Therefore, in order to detect the positive rate of PCV2, to investigate the epidemiolog of PCV3 in the pig farms, and to examine whether pigs were infected with PCV4 in Taiwan, a total of 128 samples from 46 clinical cases of pigs were collected from September 2020 to December 2021. The case detection rates were 54.3 % for PCV2, 43.5 % for PCV3, and 2.2 % for PCV4. The results suggested that the positivity rates for both PCV2 and PCV3 were still high in Taiwan. In addition, PCV3 was detected among cases from all 7 sampled counties and in 11 of the 16 sampling months, suggesting that PCV3 may lead to endemic pig disease in Taiwan. Surprisingly, the PCV4 was also detected, suggesting the first PCV4 case in Taiwan. The complete genomes derived from the identified PCV3 and PCV4 strains were subsequently sequenced followed by phylogenetic analysis. The results suggested that the 17 identified PCV3 strains could be divided into Taiwanese-like and Japanese-like strains. In addition, the amino acid residues at positions 27, 80, and 212 in the identified PCV4 cap protein were asparagine, isoleucine, and methionine, respectively, and thus the identified PCV4 was catalorized into clade PCV4b. Consequently, it is concluded that (i) the prevalence of PCV2 and PCV3 is still high in Taiwanese pigs, (ii) PCV3 has may be an endemic infection in Taiwan and can be classified into Japanese-like and Taiwanese-like strains, (iii) PCV4 was detected for the first time in Taiwanese pigs and can be classified into PCV4b. It remains unclear how PCV2, PCV3, and PCV4 were introduced to Taiwan, and thus continuous investigation of emerging pathogens in pigs is needed.

Lidocaine, a commonly used local anesthetic, has demonstrated modulatory effects in various cells. This study investigates the impact of lidocaine on cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating immune cells (TIICs) from gastric cancer patients. Flow cytometry and cytokine assays revealed that lidocaine inhibited IFN-γ and IL-12 production in CD8+ and CD14+ PBMCs, respectively, while enhancing anti-inflammatory cytokines IL-10, TGF-β, and IL-35 in CD4+CD25+ and CD14+ PBMCs. In contrast, lidocaine promoted the secretion of pro-inflammatory IFN-γ and IL-12 in CD8+ and CD14+ TIICs, while reducing IL-10, TGF-β, and IL-35 levels, thus shifting CD14+ macrophages toward an M1-like phenotype. Additionally, lidocaine decreased PD-1 expression and increased IFN-γ production in CD8+ TIICs via NF-κB activation, enhancing their cytotoxicity against primary gastric cancer cells (PGCCs). Importantly, lidocaine did not affect the viability of normal PBMCs, TIICs, or PGCCs at concentrations up to 1.5 mM. These findings suggest that lidocaine reprograms the tumor immune microenvironment, supporting its potential as an adjunct therapy in gastric cancer by enhancing anti-tumor immunity.

Coronaviruses (CoVs) are significant animal and human pathogens, characterized by being enveloped RNA viruses with positive-sense single-stranded RNA. The Coronaviridae family encompasses four genera, among which gammacoronaviruses pose a major threat to the poultry industry, which infectious bronchitis virus (IBV) being the most prominent of these threats. Particularly, IBV adversely affects broiler growth and egg production, causing substantial losses. The IBV strains currently circulating in Taiwan include the IBV Taiwan-I (TW-I) serotype, IBV Taiwan-II (TW-II) serotype, and vaccine strains. Therefore, ongoing efforts have focused on developing novel vaccines and discovering antiviral agents. The envelope (E) proteins of CoVs accumulate in the endoplasmic reticulum-Golgi intermediate compartment prior to virus budding. These E proteins assemble into viroporins, exhibiting ion channel activity that leads to cell membrane disruption, making them attractive targets for antiviral therapy.In this study, we investigated the E proteins of IBV H-120, as well as IBV serotypes TW-I and TW-II. E protein expression resulted in inhibited bacteria growth, increased permeability of bacteria to β-galactosidase substrates, and blocked protein synthesis of bacteria by hygromycin B (HygB). Furthermore, in the presence of E proteins, HygB also impeded protein translation in DF-1 cells and damaged their membrane integrity. Collectively, these findings confirm the viroporin activity of the E proteins from IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. Next, the viroporin inhibitors, 5-(N,N-hexamethylene) amiloride (HMA) and 4,4’-diisothiocyano stilbene-2,2’-disulphonic acid (DIDS) were used to inhibit the viroporin activities of the E proteins of IBV H-120, IBV serotype TW-I, and IBV serotype TW-II. In chicken embryos and chickens infected with IBV serotypes TW-I and IBV TW-II, no survivors were observed at 6 and 11 days post-infection (dpi), respectively. However, treatments with both DIDS and HMA increased the survival rates in infected chicken embryos and chickens and mitigated histopathological lesions in the trachea and kidney. Additionally, a 3D pentameric structure of the IBV E protein was constructed via homology modeling. As expected, both inhibitors were found to bind to the lipid-facing surface within the transmembrane domain of the E protein, inhibiting ion conduction. Taken together, our findings provide comprehensive evidence supporting the use of viroporin inhibitors as promising antiviral agents against IBV Taiwan isolates.

To explore whether the p17 protein of oncolytic avian reovirus (ARV) mediates cell migration and invadopodia formation, we applied several molecular biological approaches for studying the involved cellular factors and signal pathways. We found that ARV p17 activates the p53/phosphatase and tensin homolog (PTEN) pathway to suppress the focal adhesion kinase (FAK)/Src signaling and downstream signal molecules, thus inhibiting cell migration and the formation of invadopodia in murine melanoma cancer cell line (B16-F10). Importantly, p17-induced formation of invadopodia could be reversed in cells transfected with the mutant PTENC124A. p17 protein was found to significantly reduce the expression levels of tyrosine kinase substrate 5 (TKs5), Rab40b, non-catalytic region of tyrosine kinase adaptor protein 1 (NCK1), and matrix metalloproteinases (MMP9), suggesting that TKs5 and Rab40b were transcriptionally downregulated by p17. Furthermore, we found that p17 suppresses the formation of the TKs5/NCK1 complex. Coexpression of TKs5 and Rab40b in B16-F10 cancer cells reversed p17-modulated suppression of the formation of invadopodia. This work provides new insights into p17-modulated suppression of invadopodia formation by activating the p53/PTEN pathway, suppressing the FAK/Src pathway, and inhibiting the formation of the TKs5/NCK1 complex.

This work presents an immunomagnetic nanoparticle (IMNP)-based amperometric immunosensor designed for the detection of 40-kDa major mango allergens, known to potentially induce fatal anaphylactic shock in susceptible individuals. By utilizing IMNPs, the assay selectively isolates and concentrates 40-kDa major mango allergens from food samples, followed by their specific capture using monoclonal antibodies immobilized on gold nanoparticles (AuNPs)-modified screen-printed carbon electrodes. Detection is achieved through a peroxidaseconjugated secondary antibody utilizing an HQ/H2O2 system. With optimized conditions, the immunosensor exhibits exceptional specificity, effectively distinguishing mango from nine other fruits and vegetables, and achieving low limits of detection (LOD) for 40-kDa major mango allergens at 1.45 mu g/mL in buffer, 2.25 mu g/mL in juice, and 1.45 mu g/mL in lactic acid beverage. Validation of the assay was performed using 24 commercial food products, which were analyzed sequentially by Western blot to confirm the immunosensor's results. This validation demonstrated the assay's outstanding specificity at 100 % and high sensitivity at 91.7 %. These results emphasize the immunosensor's capability for rapid and sensitive detection of 40-kDa major mango allergens in processed foods, offering a significant improvement in allergen detection technology.

By analyzing results from VOPBA and LC-MS/MS, we have determined that cell-surface AnxA2 and ADGRL2 modulate ARV entry. After ARV binding to receptors, Src and p38 MAPK signaling were triggered and, in turn, increased the phosphorylation of caveolin-1 (Tyr14) and upregulated dynamin 2 expression to facilitate caveolin-1–mediated and dynamin 2-dependent endocytosis.