Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with N in PRRSV-Infected PAMs.

One of the most significant diseases in the swine business, porcine reproductive and respiratory syndrome virus (PRRSV) causes respiratory problems in piglets and reproductive failure in sows. The PRRSV nucleocapsid (N) protein is essential for the virus' assembly, replication, and immune evasion. Stages in the viral replication cycle can be impacted by interactions between the PRRSV nucleocapsid protein and the host protein components. Therefore, it is of great significance to explore the interaction between the PRRSV nucleocapsid protein and the host. Nevertheless, no information has been published on the network of interactions between the nucleocapsid protein and the host proteins in primary porcine alveolar macrophages (PAMs). In this study, 349 host proteins interacting with nucleocapsid protein were screened in the PRRSV-infected PAMs through a liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics approach. Bioinformatics analysis, which included gene ontology annotation, Kyoto Encyclopedia of Genes and Genomes database enrichment, and a protein-protein interaction (PPI) network, revealed that the host proteins interacting with PRRSV-N may be involved in protein binding, DNA transcription, metabolism, and innate immune responses. This study confirmed the interaction between the nucleocapsid protein and the natural immune-related proteins. Ultimately, our findings suggest that the nucleocapsid protein plays a pivotal role in facilitating immune evasion during a PRRSV infection. This study contributes to enhancing our understanding of the role played by the nucleocapsid protein in viral pathogenesis and virus-host interaction, thereby offering novel insights for the prevention and control of PRRS as well as the development of vaccines.

SIRT4 Has an Anti-Cancer Role in Cervical Cancer by Inhibiting Glutamine Metabolism via the MEK/ERK/C-Myc Axis.

BACKGROUND/AIM: Glutamine metabolism is crucial in cell proliferation, aging, and apoptosis across various cancer types. Existing research indicates that Sirtuin 4 (SIRT4), primarily located in mitochondria, modulates this process. This study aimed to clarify the regulatory relationship between SIRT4 and glutamine metabolism in cervical cancer. MATERIALS AND METHODS: SIRT4 mRNA levels and their clinical correlation to cervical cancer were analyzed using the UALCAN database. Immunohistochemistry (IHC) was performed to assess SIRT4 protein expression in tissue samples from cervical cancer patients. Transient transfection was employed to create Hela and Siha cell lines with overexpressed SIRT4, mitogen-activated extracellular signal-regulated kinase (MEK), and glutaminase 1 (GLS1). The impact on cellular functions was studied using MTT, soft agar, transwell, and western blotting assays. Glutamate and ATP levels were also measured to evaluate metabolic changes. RESULTS: Low levels of SIRT4 mRNA in cervical cancer tissues correlated with tumor metastasis and poor survival rates. Overexpression of SIRT4 led to suppressed cell proliferation, colony growth, and motility, along with significant down-regulation of GLS expression, a key contributor to glutamine metabolism. Additionally, SIRT4 overexpression resulted in the inactivation of the MEK/ERK/c-myc signaling pathway, while overexpression of MEK reversed these effects. Notably, the inhibitory effects of SIRT4 on cell proliferation, colony formation, migration, and invasion in Hela and Siha cells were significantly attenuated following GLS1 overexpression. CONCLUSION: SIRT4 acts as an anti-cancer agent in cervical cancer by inhibiting glutamine metabolism through the MEK/ERK/c-myc signaling pathway, providing a novel sight for cervical cancer therapy.

PRRSV GP5 inhibits the antivirus effects of chaperone-mediated autophagy by targeting LAMP2A.

Autophagy is an important biological process in host defense against viral infection. However, many viruses have evolved various strategies to disrupt the host antiviral system. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus with a large economic impact on the swine industry. At present, studies on the escape mechanism of PRRSV in the autophagy process, especially through chaperone-mediated autophagy (CMA), are limited. This study confirmed that PRRSV glycoprotein 5 (GP5) could disrupt the formation of the GFAP-LAMP2A complex by inhibiting the MTORC2/PHLPP1/GFAP pathway, promoting the dissociation of the pGFAP-EF1α complex, and blocking the K63-linked polyubiquitination of LAMP2A to inhibit the activity of CMA. Further research demonstrated that CMA plays an anti-PRRSV role by antagonizing nonstructural protein 11 (NSP11)-mediated inhibition of type I interferon (IFN-I) signaling. Taken together, these results indicate that PRRSV GP5 inhibits the antiviral effect of CMA by targeting LAMP2A. This research provides new insight into the escape mechanism of immunosuppressive viruses in CMA. IMPORTANCE: Viruses have evolved sophisticated mechanisms to manipulate autophagy to evade degradation and immune responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus that causes enormous economic losses in the swine industry. However, the mechanism by which PRRSV manipulates autophagy to defend against host antiviral effects remains unclear. In this study, we found that PRRSV GP5 interacts with LAMP2A and disrupts the formation of the GFAP-LAMP2A complex, thus inhibiting the activity of CMA and subsequently enhancing the inhibitory effect of the NSP11-mediated IFN-I signaling pathway, ultimately facilitating PRRSV replication. Our study revealed a novel mechanism by which PRRSV escapes host antiviral effects through CMA, providing a potential host target, LAMP2A, for developing antiviral drugs and contributing to understanding the escape mechanism of immunosuppressive viruses.

Baseline systolic blood pressure, hypertension history, and efficacy of remote ischemic conditioning.

OBJECTIVE: We performed a post hoc exploratory analysis of Remote Ischemic Conditioning for Acute Moderate Ischemic Stroke (RICAMIS) to determine whether hypertension history and baseline systolic blood pressure (SBP) affect the efficacy of remote ischemic conditioning (RIC). METHODS: Based on the full analysis set of RICAMIS, patients were divided into hypertension versus non-hypertension group, or <140 mmHg versus ≥140 mmHg group. Each group was further subdivided into RIC and control subgroups. The primary outcome was modified Rankin Scale (mRS) 0-1 at 90 days. Efficacy of RIC was compared among patients with hypertension versus nonhypertension history and SBP of <140 mmHg versus ≥140 mmHg. Furthermore, the interaction effect of treatment with hypertension and SBP was assessed. RESULTS: Compared with control group, RIC produced a significantly higher proportion of patients with excellent functional outcome in the nonhypertension group (RIC vs. control: 65.7% vs. 57.0%, OR 1.45, 95% CI 1.06-1.98; p = 0.02), but no significant difference was observed in the hypertension group (RIC vs. control: 69.1% vs. 65.2%, p = 0.17). Similar results were observed in SBP ≥140 mmHg group (RIC vs. control: 68.0% vs. 61.2%, p = 0.009) and SBP <140 mmHg group (RIC vs. control: 65.6% vs. 64.7%, p = 0.77). No interaction effect of RIC on primary outcome was identified. INTERPRETATION: Hypertension and baseline SBP did not affect the neuroprotective effect of RIC, but they were associated with higher probability of excellent functional outcome in patients with acute moderate ischemic stroke who received RIC treatment.

Changes in serum inflammatory factors in group B streptococcal infection and their predictive value for premature rupture of membranes complicated by chorioamnionitis.

Objective: The aim of this study as to unveil changes in serum inflammatory factors in pregnant women with genital tract group B Streptococcus (GBS) infection and their predictive value for premature rupture of membranes (PROM) complicated by chorioamnionitis (CS) and adverse pregnancy outcomes. Methods: The value of serum inflammatory factor levels in predicting PROM complicating CS and adverse pregnancy outcomes in GBS-infected pregnant women was evaluated by ELISA. Results: Serum IL-6, TNF-α, PCT and hs-CRP levels were higher in pregnant women with GBS infection. The combined diagnosis of these factors had excellent diagnostic value in PROM complicating CS and adverse pregnancy outcomes. Conclusion: Joint prediction of IL-6, TNF-α, PCT and hs-CRP has the best predictive value for PROM complicating CS and adverse pregnancy outcomes.

[Box: see text].

Mass Spectrometry-Based Proteomic Analysis of Potential Host Proteins Interacting with GP5 in PRRSV-Infected PAMs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a typical immunosuppressive virus causing a large economic impact on the swine industry. The structural protein GP5 of PRRSV plays a pivotal role in its pathogenicity and immune evasion. Virus-host interactions play a crucial part in viral replication and immune escape. Therefore, understanding the interactions between GP5 and host proteins are significant for porcine reproductive and respiratory syndrome (PRRS) control. However, the interaction network between GP5 and host proteins in primary porcine alveolar macrophages (PAMs) has not been reported. In this study, 709 GP5-interacting host proteins were identified in primary PAMs by immunoprecipitation coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Bioinformatics analysis revealed that these proteins were involved in multiple cellular processes, such as translation, protein transport, and protein stabilization. Subsequently, immunoprecipitation and immunofluorescence assay confirmed that GP5 could interact with antigen processing and presentation pathways related proteins. Finally, we found that GP5 may be a key protein that inhibits the antigen processing and presentation pathway during PRRSV infection. The novel host proteins identified in this study will be the candidates for studying the biological functions of GP5, which will provide new insights into PRRS prevention and vaccine development.

YTHDC1 inhibits osteoclast differentiation to alleviate osteoporosis by enhancing PTPN6 messenger RNA stability in an m6A-hUR-dependent manner.

YTHDC1 has been confirmed to mediate osteoporosis (OP) progression by regulating osteogenic differentiation. However, whether YTHDC1 mediates osteoclast differentiation and its molecular mechanism remains unclear. Quantitative real-time polymerase chain reaction and Western blot analysis were performed to detect the levels of YTHDC1, PTPN6, NFATc1, TRAP, RUNX2, alkaline phosphatase, and HUR. YTHDC1 knockout mice was constructed by CRISPR/Cas9 system, and the OP mice model was established by ovariectomy. Hematoxylin and eosin staining and micro-computed tomography were used to evaluate bone formation and bone mass. Mouse primary bone marrow macrophage cells were isolated and induced into osteoclasts. TRAP-positive cells were detected using TRAP staining. MeRIP-qPCR, RIP-qPCR assay, RNA affinity isolation assay, and co-immunoprecipitation assay were used to confirm the interactions among YTHDC1, PTPN6, and HUR. YTHDC1 expression was reduced and positively correlated with lumbar bone mineral density in OP patients. In the ovariectomy model of YTHDC1 knockout mice, bone formation was reduced, bone histomorphology was changed, and osteoclastic-related factor (NFATc1 and TRAP) levels were enhanced. Overexpression YTHDC1 inhibited osteoclast differentiation. YTHDC1 increased PTPN6 messenger RNA stability in an m6A-dependent manner. Moreover, YTHDC1 interacted with HUR to positively regulate PTPN6 expression. PTPN6 knockdown promoted osteoclast differentiation, and this effect was reversed by overexpressing HUR or YTHDC1. YTHDC1 was involved in regulating OP progression through inhibiting osteoclast differentiation by enhancing PTPN6 messenger RNA stability in an m6A-HUR-dependent manner.

Dyslipidemia and Efficacy of Remote Ischemic Conditioning in Acute Moderate Ischemic Stroke: A Post Hoc Analysis of the RICAMIS Study.

BACKGROUND: Ischemic conditioning-induced cardioprotection was attenuated by dyslipidemia in some animal and clinical studies, which is not investigated in patients with stroke. We conducted a post hoc analysis of the RICAMIS (Remote Ischemic Conditioning for Acute Moderate Ischemic Stroke) trial to investigate the association of dyslipidemia on admission with the efficacy of remote ischemic conditioning (RIC). METHODS AND RESULTS: In this analysis, eligible patients were divided into dyslipidemia and normal-lipid groups according to the levels of 4 blood lipid profiles (total cholesterol, triglycerides, low-density lipoprotein cholesterol, and high-density lipoprotein cholesterol), which were further subdivided into RIC and control subgroups. We analyzed the differences in functional outcome between RIC and control subgroups in dyslipidemia and normal-lipid patients, respectively, and the interaction effects of RIC treatment with blood lipid levels were evaluated. Among 1776 patients from intention-to-treat analysis, 1419 patients with data of blood lipid profiles were included in the final analysis. A significantly higher proportion of modified Rankin Scale score 0 to 1 was identified in the RIC versus control subgroup across the normal-total cholesterol group (69.9% versus 63.5%; P=0.04), normal-triglycerides group (68.1% versus 60.5%; P=0.016), high-low-density lipoprotein cholesterol group (65.7% versus 57.7%; P=0.025), and normal-high-density lipoprotein cholesterol group (68.3% versus 60.5%; P=0.005). Similar statistical trends were found in the high-total cholesterol group (62.8% versus 55.5%; P=0.059), high-triglycerides group (67.8% versus 60.1%; P=0.099), normal-low-density lipoprotein cholesterol group (69.8% versus 63.7%; P=0.105), but no statistical significance was found in the low-high-density lipoprotein cholesterol group (63.4% versus 61%; P=0.705). Furthermore, no significant interaction effect of RIC intervention by blood lipid profiles was found. Similar results were obtained for lipids as continuous variables. CONCLUSIONS: Blood lipids on admission was not associated with the neuroprotective effect of RIC.

5-FU promotes HBV replication through oxidative stress-induced autophagy dysfunction.

BACKGROUND & AIMS: Hepatitis B virus (HBV) reactivation is a major problem that must be overcome during chemotherapy for HBV-related hepatocellular carcinoma (HCC). However, the mechanism underlying chemotherapy-associated HBV reactivation is still not fully understood, hindering the development of improved HBV-related HCC treatments. METHODS: A meta-analysis was performed to assess the HBV reactivation risk during transcatheter arterial chemoembolization (TACE). To investigate the regulatory effects and mechanisms of 5-FU on HBV replication, an HBV mouse model was established by pAAV-HBV1.2 hydrodynamic injection followed by intraperitoneal 5-FU injection, and different in vitro models (HepG2.2.15 or Huh7 cells) were established. Realtime RT‒qPCR, western blotting, luciferase assays, and immunofluorescence were used to determine viral parameters. We also explored the underlying mechanisms by RNA-seq, oxidative stress evaluation and autophagy assessment. RESULTS: The pooled estimated rate of HBV reactivation in patients receiving TACE was 30.3 % (95 % CI, 23.1%-37.4 %). 5-FU, which is a chemotherapeutic agent commonly used in TACE, promoted HBV replication in vitro and in vivo. Mechanistically, 5-FU treatment obviously increased autophagosome formation, as shown by increased LC3-II levels. Additionally, 5-FU impaired autophagic degradation, as shown by marked p62 and mCherry-GFP-LC3 upregulation, ultimately promoting HBV replication and secretion. Autophagy inhibition by 3-methyladenine or chloroquine significantly altered 5-FU-induced HBV replication. Furthermore, 5-FU-induced autophagy and HBV replication were markedly attenuated with a reactive oxygen species (ROS) scavenger. CONCLUSIONS: Together, our results indicate that ROS-induced autophagosome formation and autophagic degradation play a critical role in 5-FU-induced HBV reactivation.

The Molecular and Function Characterization of Porcine MID2.

Midline2 (MID2/TRIM1) is a member of the tripartite motif-containing (TRIM) family, which is involved in a wide range of cellular processes. However, fundamental studies on porcine MID2 (pMID2) are still lacking. In this study, we identified and characterized the full length MID2 gene of pig (Sus scrofa). The sequence alignment analysis results showed that pMID2 had an N-terminal RING zinc-finger domain, BBC domain, and C-terminal COS box, FN3 motif, and PRY-SPRY domain that were conserved and similar to those of other vertebrates. Furthermore, pMID2 had the highest expression levels in porcine lung and spleen. Serial deletion and site-directed mutagenesis showed that the putative nuclear factor-κB (NF-κB) binding site may be an essential transcription factor for regulating the transcription expression of pMID2. Furthermore, the immunofluorescence assay indicated that pMID2 presented in the cell membrane and cytoplasm. To further study the functions of pMID2, we identified and determined its potential ability to perceive poly (I:C) and IFN-α stimulation. Stimulation experiments showed pMID2 enhanced poly (I:C)-/IFN-α-induced JAK-STAT signaling pathway, indicating that pMID2 might participate in the immune responses. In conclusion, we systematically and comprehensively analyzed the characterizations and functions of pMID2, which provide valuable information to explore the pMID2 functions in innate immunity. Our findings not only enrich the current knowledge of MID2 in IFN signaling regulation but also offer the basis for future research of pig MID2 gene.

Nano-calcipotriol as a potent anti-hepatic fibrosis agent.

Calcipotriol (CAL) has been widely studied as a fibrosis inhibitor and used to treat plaque psoriasis via transdermal administration. The clinical application of CAL to treat liver fibrosis is bottlenecked by its unsatisfactory pharmacokinetics, biodistribution, and side effects, such as hypercalcemia in patients. The exploration of CAL as a safe and effective antifibrotic agent remains a major challenge. Therefore, we rationally designed and synthesized a self-assembled drug nanoparticle encapsulating CAL in its internal hydrophobic core for systematic injection (termed NPs/CAL) and further investigated the beneficial effect of the nanomaterial on liver fibrosis. C57BL/6 mice were used as the animal model, and human hepatic stellate cell line LX-2 was used as the cellular model of hepatic fibrogenesis. Immunofluorescence staining, flow cytometry, western blotting, immunohistochemical staining, and in vitro imaging were used for evaluating the efficacy of NPs/CAL treatment. We found NPs/CAL can be quickly internalized in vitro, thus potently deactivating LX-2 cells. In addition, NPs/CAL improved blood circulation and the accumulation of CAL in liver tissue. Importantly, NPs/CAL strongly contributed to the remission of liver fibrosis without inducing hypercalcemia. Overall, our work identifies a promising paradigm for the development of nanomaterial-based agents for liver fibrosis therapy.

The Host E3-Ubiquitin Ligase TRIM28 Impedes Viral Protein GP4 Ubiquitination and Promotes PRRSV Replication.

Porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is a highly pathogenic porcine virus that brings tremendous economic losses to the global swine industry. PRRSVs have evolved multiple elegant strategies to manipulate the host proteins and circumvent against the antiviral responses to establish infection. Therefore, the identification of virus-host interactions is critical for understanding the pathogenesis of PRRSVs. Tripartite motif protein 28 (TRIM28) is a transcriptional co-repressor involved in the regulation of viral and cellular transcriptional programs; however, its precise role in regulating PRRSV infection remains unknown. In this study, we found that the mRNA and protein levels of TRIM28 were up-regulated in PRRSV-infected porcine alveolar macrophages (PAMs) and MARC-145 cells. Ectopic TRIM28 expression dramatically increased viral yields, whereas the siRNA-mediated knockdown of TRIM28 significantly inhibited PRRSV replication. Furthermore, we used a co-immunoprecipitation (co-IP) assay to demonstrate that TRIM28 interacted with envelope glycoprotein 4 (GP4) among PRRSV viral proteins. Intriguingly, TRIM28 inhibited the degradation of PRRSV GP4 by impeding its ubiquitination. Taken together, our work provides evidence that the host E3-ubiquitin ligase TRIM28 suppresses GP4 ubiquitination and is important for efficient virus replication. Therefore, our study identifies a new host factor, TRIM28, as a potential target in the development of anti-viral drugs against PRRSV.

Diabetes, fasting blood glucose and the efficacy of remote ischaemic conditioning: A secondary analysis of the RICAMIS trial.

AIM: To investigate whether diabetes and fasting blood glucose (FBG) levels affect the efficacy of remote ischaemic conditioning (RIC) using the database included in the Remote Ischaemic Conditioning for Acute Moderate Ischaemic Stroke (RICAMIS) trial. METHODS: A total of 1707 patients were enrolled in this post hoc study, including 535 patients with diabetes and 1172 without diabetes. Each group was further divided into RIC and control subgroups. The primary outcome was excellent functional outcome, defined as a modified Rankin Scale (mRS) score of 0 to 1 at 90 days. The difference in the proportion of patients with excellent functional outcome between the RIC subgroup and control subgroup was compared in diabetic and non-diabetic patients, respectively, and the interactions of treatment assignment with diabetes status and FBG were evaluated. RESULTS: Compared with the control group, RIC produced a significantly higher proportion of patients with excellent functional outcome in the non-diabetic group (70.5% vs. 63.2%; odds ratio [OR] 1.487, 95% confidence interval [CI] 1.134-1.949; P = 0.004), while a similar, but not significant difference was observed in the diabetic group (65.3% vs. 59.8%; OR 1.424, 95% CI 0.978-2.073; P = 0.065). Similar results were observed in patients with normal FBG levels (69.3% vs. 63.7%; OR 1.363, 95% CI 1.011-1.836; P = 0.042) and those with high FBG levels (64.2% vs. 58%; OR 1.550, 95% CI 1.070-2.246; P = 0.02). Furthermore, we did not find an interaction effect of intervention (RIC or control) by different diabetes status or FBG levels on clinical outcomes (P > 0.05 for all). However, diabetes (OR 0.741, 95% CI 0.585-0.938; P = 0.013) and high FBG (OR 0.715, 95% CI 0.553-0.925; P = 0.011) were independently associated with functional outcomes in patients overall. CONCLUSION: Diabetes and FBG levels did not influence the neuroprotective effect of RIC in acute moderate ischaemic stroke, although diabetes and high FBG levels were independently associated with functional outcomes.

Melatonin attenuates lung ischemia-reperfusion injury through SIRT3 signaling-dependent mitophagy in type 2 diabetic rats.

Background: Lung ischemia-reperfusion injury (LIRI) remains the major cause of primary lung dysfunction after lung transplantation. Diabetes mellitus (DM) is an independent risk factor for morbidity and mortality following lung transplantation. Mitochondrial dysfunction is recognized as a key mediator in the pathogenesis of diabetic LIRI. Melatonin has been reported to be a safe and potent preserving mitochondrial function agent. This study aimed at investigating the potential therapeutic effect and mechanisms of melatonin on diabetic LIRI. Methods: High-fat-diet-fed streptozotocin-induced type 2 diabetic rats were exposed to melatonin, with or without administration of the SIRT3 short hairpin ribonucleic acid (shRNA) plasmid following a surgical model of ischemia-reperfusion injury of the lung. Lung function, inflammation, oxidative stress, cell apoptosis, and mitochondrial function were examined. Results: The SIRT3 signaling and mitophagy were suppressed following diabetic LIRI. Treatment with melatonin markedly induced mitophagy and restored SIRT3 expression. Melatonin treatment also attenuated subsequent diabetic LIRI by improving lung functional recovery, suppressing inflammation, decreasing oxidative damage, diminishing cell apoptosis, and preserving mitochondrial function. However, either administration of SIRT3 shRNA or an autophagy antagonist 3-methyladenine (3-MA) suppressing mitophagy, and compromised the protective action of melatonin. Conclusion: Data indicated that melatonin attenuates diabetic LIRI through activation of SIRT3 signaling-mediated mitophagy.

Efficacy of remote ischemic conditioning does not differ between men and women: A secondary analysis of the RICAMIS study.

BACKGROUND AND PURPOSE: The present study aimed to determine sex difference in clinical outcomes after Remote Ischemic Conditioning for Acute Moderate Ischemic Stroke (RICAMIS). METHODS: In this secondary analysis of the RICAMIS study, eligible patients aged 18 years or older with acute moderate ischemic stroke who received remote ischemic conditioning (RIC) within 48 h of stroke onset were divided into two groups: men and women. The primary endpoint was an excellent functional outcome, defined as a modified Rankin Scale score of 0-1 at 90 days. Binary logistic regression analyses and generalized linear models were used. RESULTS: Of 1707 eligible patients, 34% (579) were women. Women had a higher burden of hypertension and diabetes, and less alcohol and smoking consumption than men. The mean systolic blood pressure and blood glucose level at randomization were higher in women than in men. Compared with the control group, RIC was associated with an increased rate of primary endpoint in men (unadjusted odds ratio [OR] = 1.277; 95% confidence interval [CI] 0.933-1.644; p = 0.057) and women (unadjusted OR = 1.454; 95% CI 1.040-2.032; p = 0.028). Furthermore, a higher absolute risk difference in primary endpoint between control and RIC groups was found in women (9.2%) than in men (5.7%), but there was no significant interaction effect between sex and intervention on primary outcome (p interaction = 0.545). CONCLUSION: Compared with men, women may have a higher probability of excellent functional outcomes at 90 days in the RIC group than in the control group; however, no interaction effect between sex and intervention was found.

Immune Molecules' mRNA Expression in Porcine Alveolar Macrophages Co-Infected with Porcine Reproductive and Respiratory Syndrome Virus and Porcine Circovirus Type 2.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus 2 (PCV2) are economically important pathogens in swine, and pigs with dual infections of PCV2 and PRRSV consistently have more severe clinical symptoms and interstitial pneumonia. However, the synergistic pathogenesis mechanism induced by PRRSV and PCV2 co-infection has not yet been illuminated. Therefore, the aim of this study was to characterize the kinetic changes of immune regulatory molecules, inflammatory factors and immune checkpoint molecules in porcine alveolar macrophages (PAMs) in individuals infected or co-infected with PRRSV and/or PCV2. The experiment was divided into six groups: a negative control group (mock, no infected virus), a group infected with PCV2 alone (PCV2), a group infected with PRRSV alone (PRRSV), a PCV2-PRRSV co-infected group (PCV2-PRRSV inoculated with PCV2, followed by PRRSV 12 h later), a PRRSV-PCV2 co-infected group (PRRSV-PCV2 inoculated with PRRSV, followed by PCV2 12 h later) and a PCV2 + PRRSV co-infected group (PCV2 + PRRSV, inoculated with PCV2 and PRRSV at the same time). Then, PAM samples from the different infection groups and the mock group were collected at 6, 12, 24, 36 and 48 h post-infection (hpi) to detect the viral loads of PCV2 and PRRSV and the relative quantification of immune regulatory molecules, inflammatory factors and immune checkpoint molecules. The results indicated that PCV2 and PRRSV co-infection, regardless of the order of infection, had no effect on promoting PCV2 replication, while PRRSV and PCV2 co-infection was able to promote PRRSV replication. The immune regulatory molecules (IFN-α and IFN-γ) were significantly down-regulated, while inflammatory factors (TNF-α, IL-1ß, IL-10 and TGF-ß) and immune checkpoint molecules (PD-1, LAG-3, CTLA-4 and TIM-3) were significantly up-regulated in the PRRSV and PCV2 co-infection groups, especially in PAMs with PCV2 inoculation first followed by PRRSV. The dynamic changes in the aforementioned immune molecules were associated with a high viral load, immunosuppression and cell exhaustion, which may explain, at least partially, the underlying mechanism of the enhanced pulmonary lesions by dual infection with PCV2 and PRRSV in PAMs.

Combined effects of vitamin C and cold atmospheric plasma-conditioned media against glioblastoma via hydrogen peroxide.

Glioblastoma is the most lethal intracranial malignant tumor, for which the five-year overall survival rate is approximately 5%. Here we explored the therapeutic combination of vitamin C and plasma-conditioned medium on glioblastoma cells in culture and as subcutaneous or intracranial xenografts in mice. The combination treatment reduced cell viability and proliferation while promoting apoptosis, and the effects were significantly stronger than with either treatment on its own. Similar results were obtained in the two xenograft models. Vitamin C appeared to upregulate aquaporin-3 and enhance the uptake of extracellular H2O2, while the combination treatment increased intracellular levels of reactive oxygen species including H2O2 and activated the JNK signaling pathway. The cytotoxic effects of the combination treatment were partially reversed by the specific JNK signaling inhibitor SP600125. Our results suggest that the combination of vitamin C and plasma-conditioned medium has therapeutic potential against glioblastoma, and they provide mechanistic insights that may help investigate this and other potential therapies in greater depth.

Sulforaphane reduces lipopolysaccharide-induced inflammation and enhances myogenic differentiation of mouse embryonic myoblasts via the toll-like receptor 4 and NLRP3 pathways.

BACKGROUND: Muscle loss and muscle weakness are manifestations of infection-induced sepsis, a condition that can lead to organ failure and death. Toll-like receptor 4 (TLR4) signaling and the NLRP3 inflammasome are involved in the inflammatory storm and the development of sarcopenia during sepsis. They are also potential targets for sepsis treatment. OBJECTIVES: To explore the effects and molecular mechanisms of sulforaphane (SFN) on sepsis-associated inflammation and sarcopenia. MATERIAL AND METHODS: Mouse C2C12 embryonic myoblasts were treated with lipopolysaccharide (LPS) to simulate sepsis-induced sarcopenia. Molecular mechanisms were investigated using quantitative real-time polymerase chain reaction (qRT-PCR), western blot, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). RESULTS: Sulforaphane significantly reduced the secretion of the inflammatory cytokine interleukin-1ß (IL-1ß) by C2C12 cells after LPS treatment, and inhibited the production of intracellular reactive oxygen species (ROS). It also increased the expression of E-myosin heavy chain, myosin ID heavy chain, and myogenin, and induced myogenic differentiation of LPS-treated C2C12 cells. Mechanistically, SFN reduced messenger ribonucleic acid and protein levels of TLR4, NLRP3, apoptosis-associated speck-like protein, and Caspase-1 in C2C12 cells, thereby inhibiting the inflammatory response and promoting myogenic differentiation. In addition, the TLR4 inhibitor TAK-242 induced myogenic differentiation in LPS-pretreated C2C12 cells in a similar manner. CONCLUSIONS: Sulforaphane can reduce sepsis-induced inflammatory responses and enhance myogenic differentiation by regulating the TLR4 and NLRP3 inflammasome pathways.

Ischemic Preconditioning Alleviates Cerebral Ischemia-Reperfusion Injury by Interfering With Glycocalyx.

Ischemic preconditioning (IPC) could protect the blood-brain barrier (BBB), but the underlying mechanism is not well understood. This preclinical study aimed to investigate whether glycocalyx could be involved in the neuroprotective effect of IPC on cerebral ischemia-reperfusion injury (IRI) and the possible mechanism in rat middle cerebral artery occlusion/reperfusion (MCAO/R) model. Neurological deficit scores, infarct volume, and brain edema were measured to assess the neuroprotection of IPC. Several serum biomarkers related to glycocalyx damage, such as hyaluronic acid (HA), heparan sulfate (HS), and syndecan-1 (SYND1), were evaluated, and their changes were normalized to the ratio of postoperative/preoperative concentration. Western blot and immunofluorescence were used to evaluate the content and cellular location of HA-related metabolic enzymes. This study found that (1) IPC improved brain infarction and edema, neurological impairment, and BBB disruption in IRI rats; (2) IPC significantly up-regulated HA ratio and down-regulated HS ratio, but did not affect SYND1 ratio compared with the IRI group. Moreover, the increased HA ratio was negatively related to brain edema and neurological deficit score. (3) IPC affected HA metabolism by up-regulating hyaluronate synthase-1 and matrix metalloproteinase-2, and down-regulating hyaluronidase-1 in brain tissue. Together, this is the first report that the neuroprotective effect of IPC on IRI may be mediated through interfering with glycocalyx in the MCAO/R model.

Activating Fc Gamma Receptors and Viral Receptors Are Required for Antibody-Dependent Enhancement of Porcine Reproductive and Respiratory Syndrome Virus Infection.

Antibody-dependent enhancement (ADE) is an event in preexisting sub-, or non-neutralizing antibodies increasing the viral replication in its target cells. ADE is one crucial factor that intensifies porcine reproductive and respiratory syndrome virus (PRRSV) infection and results in PRRSV-persistent infection. Nevertheless, the exact mechanisms of PRRSV-ADE infection are poorly understood. In the current research, the results of the ADE assay showed that porcine immunoglobulin G (IgG) specific for the PRRSV significantly enhanced PRRSV proliferation in porcine alveolar macrophages (PAMs), suggesting that the ADE activity of PRRSV infection existed in pig anti-PRRSV IgG. The results of the RNA interference assay showed that knockdown of the Fc gamma receptor I (FcγRI) or FcγRIII gene significantly suppressed the ADE activity of PRRSV infection in PAMs, suggesting that FcγRI and FcγRIII were responsible for mediating PRRSV-ADE infection. In addition, the results of the antibody blocking assay showed that specific blocking of the Sn1, 2, 3, 4, 5, or 6 extracellular domain of the sialoadhesin (Sn) protein or selective blockade of the scavenger receptor cysteine-rich (SRCR) 5 domain of the CD163 molecule significantly repressed the ADE activity of PRRSV infection in PAMs, suggesting that Sn and CD163 were involved in FcγR-mediated PRRSV-ADE infection. The Sn1-6 domains of porcine Sn protein and the SRCR 5 domain of porcine CD163 molecule might play central roles in the ADE of PRRSV infection. In summary, our studies indicated that activating FcγRs (FcγRI and FcγRIII) and viral receptors (Sn and CD163) were required for ADE of PRRSV infection. Our findings provided a new insight into PRRSV infection that could be enhanced by FcγRs and PRRSV receptors-mediated PRRSV-antibody immune complexes (ICs), which would deepen our understanding of the mechanisms of PRRSV-persistent infection via the ADE pathway.