Deciphering the interaction between PKM2 and the built-in thermodynamic properties of the glycolytic pathway in cancer cells.

Most cancer cells exhibit high glycolysis rates under conditions of abundant oxygen. Maintaining a stable glycolytic rate is critical for cancer cell growth as it ensures sufficient conversion of glucose carbons to energy, biosynthesis, and redox balance. Here we deciphered the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway. Knocking down or knocking out PKM2 induced a thermodynamic equilibration in the glycolytic pathway, characterized by the reciprocal changes of the Gibbs free energy (ΔG) of the reactions catalyzed by PFK1 and PK, leading to a less exergonic PFK1-catalyzed reaction and a more exergonic PK-catalyzed reaction. The changes in the ΔGs of the two reactions cause the accumulation of intermediates, including the substrate PEP (the substrate of PK), in the segment between PFK1 and PK. The increased concentration of PEP in turn increased PK activity in the glycolytic pathway. Thus, the interaction between PKM2 and the thermodynamic properties of the glycolytic pathway maintains the reciprocal relationship between PK concentration and its substrate PEP concentration, by which, PK activity in the glycolytic pathway can be stabilized and effectively counteracts the effect of PKM2 KD or KO on glycolytic rate. In line with our previous reports, this study further validates the roles of the thermodynamics of the glycolytic pathway in stabilizing glycolysis in cancer cells. Deciphering the interaction between glycolytic enzymes and the thermodynamics of the glycolytic pathway will promote a better understanding of the flux control of glycolysis in cancer cells.

Downregulation of malic enzyme 3 facilitates progression of gastric carcinoma via regulating intracellular oxidative stress and hypoxia-inducible factor-1α stabilization.

BACKGROUND: Gastric cancer (GC) is one of the most malignant cancers worldwide. Metabolism disorder is a critical characteristic of malignant tumors related to tumor progression and metastasis. However, the expression and molecular mechanism of malic enzyme 3 (ME3) in GC are rarely reported. In this study, we aim to investigate the molecular mechanism of ME3 in the development of GC and to explore its potential value as a prognostic and therapeutic target in GC. METHOD: ME3 mRNA and protein expression were evaluated in patients with GC using RT-qPCR, WB, and immunohistochemistry, as well as their correlation with clinicopathological indicators. The effect of ME3 on proliferation and metastasis was evaluated using Cell Counting Kit-8 (CCK-8), 5-ethynyl-20-deoxyuridine (EdU) assay, transwell assay, wound healing assay, and subcutaneous injection or tail vein injection of tumor cells in mice model. The effects of ME3 knockdown on the level of metabolites and hypoxia-inducible factor-1α (HIF-1α) protein were determined in GC cells. Oxidative phosphorylation was measured to evaluate adenosine triphosphate (ATP) production. RESULTS: ME3 was downregulated in human GC tissues (P < 0.001). The decreased ME3 mRNA expression was associated with younger age (P = 0.02), pathological staging (P = 0.049), and lymph node metastasis (P = 0.001), while low ME3 expression was associated with tumor size (P = 0.048), tumor invasion depth (P < 0.001), lymph node metastasis (P = 0.018), TNM staging (P < 0.001), and poor prognosis (OS, P = 0.0206; PFS P = 0.0453). ME3 knockdown promoted GC cell malignancy phenotypes. Moreover, α-ketoglutarate (α-KG) and NADPH/NADP+ ratios were reduced while malate was increased in the ME3 knockdown group under normoxia. When cells were incubated under hypoxia, the NADPH/NADP+ ratio and α-KG decreased while intracellular reactive oxygen species (ROS) increased significantly. The ME3 knockdown group exhibited an increase in ATP production and while ME3 overexpression group exhibited oppositely. We discovered that ME3 and HIF-1α expression were negatively correlated in GC cells and tissues, and proposed the hypothesis: downregulation of ME3 promotes GC progression via regulating intracellular oxidative stress and HIF-1α. CONCLUSION: We provide evidence that ME3 downregulation is associated with poor prognosis in GC patients and propose a hypothesis for the ME3 regulatory mechanism in GC progression. The present study is of great scientific significance and clinical value for exploring the prognostic and therapeutic targets of GC, evaluating and improving the clinical efficacy of patients, reducing recurrence and metastasis, and improving the prognosis and quality of life of patients.

O-GlcNAcylation regulation of RIPK1-dependent apoptosis dictates sensitivity to sunitinib in renal cell carcinoma.

Receptor interacting protein kinase 1 (RIPK1) has emerged as a key regulatory molecule that influences the balance between cell death and cell survival. Under external stress, RIPK1 determines whether a cell undergoes RIPK-dependent apoptosis (RDA) or survives by activating NF-κB signaling. However, the role and mechanisms of RIPK1 on sunitinib sensitivity in renal cell carcinoma (RCC) remain elusive. In this study, we demonstrated that the O-linked ß-N-acetylglucosamine modification (O-GlcNAcylation) of RIPK1 induces sunitinib resistance in RCC by inhibiting RDA. O-GlcNAc transferase (OGT) specifically interacts with RIPK1 through its tetratricopeptide repeats (TPR) domain and facilitates RIPK1 O-GlcNAcylation. The O-GlcNAcylation of RIPK1 at Ser331, Ser440 and Ser669 regulates RIPK1 ubiquitination and the formation of the RIPK1/FADD/Caspase-8 complex, thereby inhibiting sunitinib-induced RDA in RCC. Site-specific depletion of O-GlcNAcylation on RIPK1 affects the formation of the RIPK1/FADD/Caspase 8 complex, leading to increased sunitinib sensitivity in RCC. Our data highlight the significance of aberrant RIPK1 O-GlcNAcylation in the development of sunitinib resistance and indicate that targeting RIPK1 O-GlcNAcylation could be a promising therapeutic strategy for RCC.

CircPPAP2B controls metastasis of clear cell renal cell carcinoma via HNRNPC-dependent alternative splicing and targeting the miR-182-5p/CYP1B1 axis.

BACKGROUND: Renal cell carcinoma (RCC) is one of the most common malignant tumor worldwide. Metastasis is a leading case of cancer-related deaths of RCC. Circular RNAs (circRNAs), a class of noncoding RNAs, have emerged as important regulators in cancer metastasis. However, the functional effects and regulatory mechanisms of circRNAs on RCC metastasis remain largely unknown. METHODS: High-throughput RNA sequencing techniques were performed to analyze the expression profiles of circRNAs and mRNAs in highly and poorly invasive clear cell renal cell carcinoma (ccRCC) cell lines. Functional experiments were performed to unveil the regulatory role of circPPAP2B in the proliferation and metastatic capabilities of ccRCC cells. RNA pulldown, Mass spectrometry analysis, RNA methylation immunoprecipitation (MeRIP), RNA immunoprecipitation (RIP), co-immunoprecipitation (CoIP), next-generation RNA-sequencing and double luciferase experiments were employed to clarify the molecular mechanisms by which circPPAP2B promotes ccRCC metastasis. RESULTS: In this study, we describe a newly identified circular RNA called circPPAP2B, which is overexpressed in highly invasive ccRCC cells, as determined through advanced high-throughput RNA sequencing techniques. Furthermore, we observed elevated circPPAP2B in ccRCC tissues, particularly in metastatic ccRCC tissues, and found it to be associated with poor prognosis. Functional experiments unveiled that circPPAP2B actively stimulates the proliferation and metastatic capabilities of ccRCC cells. Mechanistically, circPPAP2B interacts with HNRNPC in a m6A-dependent manner to facilitate HNRNPC nuclear translocation. Subcellular relocalization was dependent upon nondegradable ubiquitination of HNRNPC and stabilization of an HNRNPC/Vimentin/Importin α7 ternary complex. Moreover, we found that circPPAP2B modulates the interaction between HNRNPC and splicing factors, PTBP1 and HNPNPK, and regulates pre-mRNA alternative splicing. Finally, our studies demonstrate that circPPAP2B functions as a miRNA sponge to directly bind to miR-182-5p and increase CYP1B1 expression in ccRCC. CONCLUSIONS: Collectively, our study provides comprehensive evidence that circPPAP2B promotes proliferation and metastasis of ccRCC via HNRNPC-dependent alternative splicing and miR-182-5p/CYP1B1 axis and highlights circPPAP2B as a potential therapeutic target for ccRCC intervention.

Integrating serum metabolomics analysis and network pharmacology to reveal the potential mechanism of Shengmai Jianghuang San in the treatment of nasopharyngeal carcinoma.

Shengmai Jianghuang San (SMJHS) is a traditional Chinese herbal compound reported to inhibit Nasopharyngeal Carcinoma (NPC) progression and enhance radiosensitivity. However, the specific active ingredients and regulatory mechanisms of SMJHS against NPC, particularly under hypoxic conditions, remain unclear. In this study, Sprague-Dawley (SD) rats were gavaged with Shengmai Jianghuang San (SMJHS), and their blood was collected from the abdominal aorta. UHPLC-Q-Exactive orbitrap MS/MS was used to identify the metabolite profiles of SMJHS drug-containing serum. A molecular network of the active compositions in SMJHS targeting NPC was constructed through network pharmacology and molecular docking. The HIF-1α/VEGF pathway was in key positions. The effects of SMJHS on the proliferation, migration, and radiosensitivity of hypoxic NPC cells were assessed by in vitro experiments. NPC cell lines stably overexpressing HIF-1α were established using a lentivirus to investigate the regulation of HIF-1α/VEGF signaling in hypoxic NPC cells by SMJHS. Through a combination of network pharmacological analysis, cellular biofunctional validation, and molecular biochemical experiments, our study found that SMJHS had an anti-proliferative effect on NPC cells cultured under hypoxic conditions, inhibiting their migration and increasing their radiosensitivity. Additionally, SMJHS suppressed the expression of HIF-1α and VEGFA, exhibiting potential as an effective option for improving NPC treatment.

Lactic acidosis switches cancer cells from dependence on glycolysis to OXPHOS and renders them highly sensitive to OXPHOS inhibitors.

Targeting oxidative phosphorylation (OXPHOS) has emerged as a strategy for cancer treatment. However, most tumor cells exhibit Warburg effect, they primarily rely on glycolysis to generate ATP, and hence they are resistant to OXPHOS inhibitors. Here, we report that lactic acidosis, a ubiquitous factor in the tumor microenvironment, increases the sensitivity of glycolysis-dependent cancer cells to OXPHOS inhibitors by 2-4 orders of magnitude. Lactic acidosis reduces glycolysis by 79-86% and increases OXPHOS by 177-218%, making the latter the main production pathway of ATP. In conclusion, we revealed that lactic acidosis renders cancer cells with typical Warburg effect phenotype highly sensitive to OXPHOS inhibitors, thereby greatly expanding the anti-cancer spectrum of OXPHOS inhibitors. In addition, as lactic acidosis is a ubiquitous factor of TME, it is a potential indicator to predict the efficacy of OXPHOS inhibitors in cancer treatment.

Effect of GADD45G on the radioresistance of nasopharyngeal carcinoma cells.

The development of radioresistance by nasopharyngeal carcinoma (NPC) cells almost always results in tumor recurrence and metastasis, making clinical treatment of the disease difficult. In this study, the mechanism of radioresistance in NPC cells was investigated. First, a gene array and quantitative reverse-transcription-PCR assays were used to screen for genes exhibiting significantly altered expression in the DNA damage signaling pathway. Based on those results, GADD45G was further studied in the context of radioresistance. A GADD45G-knockout NPC cell line (CNE-2R-KO) was constructed using CRISPR-Cas9 technology and used for a comparison of differences in radioresistance with other radiosensitive and radioresistant NPC cells, as evaluated using colony formation assays. Cell cycle changes were observed using flow cytometry. Cell proliferation and migration were measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide and wound healing assays, respectively. The sequencing results revealed the successful construction of the CNE-2R-KO cell line, the radiosensitivity of which was higher than that of its parent radioresistant cell line owing to the GADD45G knockout. This was likely related to the increase in the number of cells in the G1 phase and decrease in those in the S1 phase as well as the increased cell proliferation rate and decreased migratory ability. GADD45G is associated with radioresistance in NPC cells and likely has a role in the occurrence and metastasis of NPC.

Why some donors are more willing to donate platelets? ----a qualitative study on 25 regular platelet donors in Guangzhou, China.

BACKGROUND: Since 1998, China has gradually moved toward voluntary uncompensated blood donation. In some cities, the shortage of platelets has been noticeably severe. Mutual assistance that collects blood from one's family and social networks is a potential solution. The measure, however, turned out problematic. There are donors who choose to donate platelets over whole blood without compensations, and donate platelets directly to blood banks instead of via the mutual assistance system. This study explores reasons behind their choices qualitatively. METHODS: This report is based on data conducted from January to February 2018; 25 uncompensated regular platelet donors were interviewed. The blood component donation service team in Guangzhou facilitated the data collection process and referred prospectively eligible blood donors to our research team. The interviews took about 30 min to two hours to complete. The qualitative data were analyzed by using the software ATLAS.ti 8. RESULTS: Platelet donation takes a much long time than whole blood donation and requires complicated processes. It may also cause discomfort as the other blood components are returned to the body, causing physical and psychological distress due to worries about contamination. Thus, platelet donation tends to involve higher time and psychological costs than whole blood donation. Yet, it has short collection intervals that allows for more frequent donations, and urgency of a severer shortage than whole blood. Hence, regular platelet donors may feel higher significance in platelet donation than whole blood donation, with the belief that more lives would be saved. Some whole blood donors thus switched to become platelet donors. Mutual assistance blood donation was not chosen by the participants for platelet donation, because such donations may exert moral pressure to both the donors and recipients. Furthermore, "acquaintance" has been loosely defined; the system has sometimes been manipulated to become profit-making monetary transactions. It hence failed. CONCLUSIONS: The practice of platelet donation reinforces the understanding that blood donation is a gift giving process performed among strangers. A safe and sustainable voluntary blood supply can only be secured in the absence of monetary transactions and moral pressure.

The interaction between DNA methylation and tumor immune microenvironment: from the laboratory to clinical applications.

DNA methylation is a pivotal epigenetic modification that affects gene expression. Tumor immune microenvironment (TIME) comprises diverse immune cells and stromal components, creating a complex landscape that can either promote or inhibit tumor progression. In the TIME, DNA methylation has been shown to play a critical role in influencing immune cell function and tumor immune evasion. DNA methylation regulates immune cell differentiation, immune responses, and TIME composition Targeting DNA methylation in TIME offers various potential avenues for enhancing immune cytotoxicity and reducing immunosuppression. Recent studies have demonstrated that modification of DNA methylation patterns can promote immune cell infiltration and function. However, challenges persist in understanding the precise mechanisms underlying DNA methylation in the TIME, developing selective epigenetic therapies, and effectively integrating these therapies with other antitumor strategies. In conclusion, DNA methylation of both tumor cells and immune cells interacts with the TIME, and thus affects clinical efficacy. The regulation of DNA methylation within the TIME holds significant promise for the advancement of tumor immunotherapy. Addressing these challenges is crucial for harnessing the full potential of epigenetic interventions to enhance antitumor immune responses and improve patient outcomes.

Coupling coordination of the provision of medical services and high-quality economic development in the Yangtze River Economic Belt.

Background: Promoting high-level coupling coordination between the provision of medical services (PMS) and high-quality economic development (HED) has emerged as a critical issue in China's pursuit of high-quality development and is now a significant subject of concern in the area of welfare economics. Materials and methods: Based on panel data from 11 provinces and municipalities in the Yangtze River Economic Belt, covering the period from 2010 to 2020, this study conducted an empirical analysis of the coupling coordination between PMS and HED and its influencing factors. Methods combined a comprehensive evaluation model, a coupling coordination model, and a panel Tobit model. Results: The study found that: (1) Regarding the overall situation in the Yangtze River Economic Belt, the overall PMS demonstrates a fluctuating upward trend, while HED fluctuates within the lower range of 0.3 to 0.4. The coupling coordination degree between PMS and HED fluctuates within the moderate range of 0.5 to 0.6. (2) In terms of the spatiotemporal evolution trends, there still exists substantial spatial disparity among provinces and municipalities within the Yangtze River Economic Belt regarding PMS; nonetheless, this gap is gradually narrowing. Significant regional disparities are also observed in HED, with Shanghai, Jiangsu, and Zhejiang leading among the provinces and municipalities in the Yangtze River Economic Belt. The coupling coordination degree between PMS and HED displays notable spatial discrepancies, where downstream areas of the Yangtze River Economic Belt such as Shanghai, Jiangsu, and Zhejiang exhibit a higher coupling coordination degree compared to other provinces and municipalities. However, most provinces and municipalities outside this group remain at a moderately coordinated stage concerning the degree of coupling coordination between PMS and HED. (3) Economic development level and local government competition had a significant negative impact on coupling coordination between PMS and HED, whereas there was a significantly positive impact on the degree of fiscal autonomy and urbanization. Discussion: This study contributes to comprehensively understanding the coupling and coordination relationship between the PMS and HED across provinces and municipalities in the Yangtze River Economic Belt. It provides empirical evidence for the collaborative evolution of PMS and HED.

The structures of two acidic polysaccharides from Gardenia jasminoides and their potential immunomodulatory activities.

This study identified two homogeneous acidic polysaccharides from Gardeniae fructus, GJP50-3 and GJP50-4, which exhibited potential immunomodulatory activities in macrophage activation assays, via liquid-chip technology, and in a zebrafish model. Monosaccharide composition analysis and gel permeation chromatography revealed that GJP50-3 and GJP50-4 were composed of Rha, GalA, Glc, Gal, and Ara in specific ratios and had molecular weights of 91.5 kDa and 140.3 kDa, respectively. Based on FT-IR, GC-MS, and NMR analyses, these polysaccharides were identified as typical pectin polysaccharides with methylation degrees of 24.7 % and 21.4 %, respectively. The primary structures of GJP50-3 and GJP50-4 included linear HG domains and branched RG-I domains with arabinans and AG side chains. In vitro, GJP50-3 and GJP50-4 could stimulate NO release and increase the secretion of TNF-α in a RAW 264.7 macrophage model. Luminex liquid suspension chip detection revealed that GJP50-3 significantly promoted the secretion of multiple interleukins [IL-6, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13], TNF-α, and chemokines (G-CSF, GM-CSF, MCP-1 and RANTES). In vivo, these polysaccharides could also increase NO release and neutrophil count in a zebrafish model. These findings suggested that GJP50-3 and GJP50-4 might have the potential to be used as immunomodulators in the food and pharmaceutical industries.

Integrative transcriptomic profiling of mRNA, miRNA, circRNA, and lncRNA in alveolar macrophages isolated from PRRSV-infected porcine.

Introduction: The porcine reproductive and respiratory syndrome virus (PRRSV) continues to pose a significant threat to the global swine industry, attributed largely to its immunosuppressive properties and the chronic nature of its infection. The absence of effective vaccines and therapeutics amplifies the urgency to deepen our comprehension of PRRSV's intricate pathogenic mechanisms. Previous transcriptomic studies, although informative, are partially constrained by their predominant reliance on in vitro models or lack of long-term infections. Moreover, the role of circular RNAs (circRNAs) during PRRSV invasion is yet to be elucidated. Methods: In this study, we employed an in vivo approach, exposing piglets to a PRRSV challenge over varied durations of 3, 7, or 21 days. Subsequently, porcine alveolar macrophages were isolated for a comprehensive transcriptomic investigation, examining the expression patterns of mRNAs, miRNAs, circRNAs, and long non-coding RNAs (lncRNAs). Results: Differentially expressed RNAs from all four categories were identified, underscoring the dynamic interplay among these RNA species during PRRSV infection. Functional enrichment analyses indicate that these differentially expressed RNAs, as well as their target genes, play a pivotal role in immune related pathways. For the first time, we integrated circRNAs into the lncRNA-miRNA-mRNA relationship, constructing a competitive endogenous RNA (ceRNA) network. Our findings highlight the immune-related genes, CTLA4 and SAMHD1, as well as their associated miRNAs, lncRNAs, and circRNAs, suggesting potential therapeutic targets for PRRS. Importantly, we corroborated the expression patterns of selected RNAs through RT-qPCR, ensuring consistency with our transcriptomic sequencing data. Discussion: This study sheds lights on the intricate RNA interplay during PRRSV infection and provides a solid foundation for future therapeutic strategizing.

Effects of soluble Antarctic krill protein-curcumin complex combined with photodynamic inactivation on the storage quality of shrimp.

A new protein complex (SAKP-Cur) was successfully prepared by combining soluble Antarctic krill protein with curcumin through hydrophobic action. The potency of photodynamic inactivation (PDI) mediated by the complex on preserving the storage quality of shrimp at 4 °C was investigated by microbiological, chemical, physical and histological methods. Results showed that the SAKP-Cur significantly improved the stability of curcumin, and greatly inactivated the native bacteria in shrimp driven by PDI. Meanwhile, the complex-mediated PDI effectively reduced the endogenous enzyme activity, the production of total volatile basic nitrogen (TVB-N) and malondialdehyde (MDA) in shrimp. Moreover, it obviously maintained the integrity and elasticity of the muscle fibers, thereby reducing the loss of water in myofibrils. Notably, the SAKP-Cur enhanced the PDI potency to preserve the freshness of shrimp during 4 °C storage or freeze-thaw cycles treatment. Therefore, the SAKP-Cur coupled with PDI is an effective fresh-keeping technology for aquatic products.

Effect of Shengmai Yin on Epithelial-Mesenchymal Transition of Nasopharyngeal Carcinoma Radioresistant Cells.

OBJECTIVE: To investigate the mechanism by which Chinese medicine Shengmai Yin (SMY) reverses epithelial-mesenchymal transition (EMT) through lipocalin-2 (LCN2) in nasopharyngeal carcinoma (NPC) cells CNE-2R. METHODS: Morphological changes in EMT in CNE-2R cells were observed under a microscope, and the expressions of EMT markers were detected using quantitative real-time PCR (RT-qPCR) and Western blot assays. Through the Gene Expression Omnibus dataset and text mining, LCN2 was found to be highly related to radiation resistance and EMT in NPC. The expressions of LCN2 and EMT markers following SMY treatment (50 and 100 µ g/mL) were detected by RT-qPCR and Western blot assays in vitro. Cell proliferation, migration, and invasion abilities were measured using colony formation, wound healing, and transwell invasion assays, respectively. The inhibitory effect of SMY in vivo was determined by observing a zebrafish xenograft model with a fluorescent label. RESULTS: The CNE-2R cells showed EMT transition and high expression of LCN2, and the use of SMY (5, 10 and 20 µ g/mL) reduced the expression of LCN2 and reversed the EMT in the CNE-2R cells. Compared to that of the CNE-2R group, the proliferation, migration, and invasion abilities of SMY high-concentration group were weakened (P<0.05). Moreover, SMY mediated tumor growth and metastasis in a dose-dependent manner in a zebrafish xenograft model, which was consistent with the in vitro results. CONCLUSIONS: SMY can reverse the EMT process of CNE-2R cells, which may be related to its inhibition of LCN2 expression. Therefore, LCN2 may be a potential diagnostic marker and therapeutic target in patients with NPC.

Transcriptome Profiling of Vero E6 Cells during Original Parental or Cell-Attenuated Porcine Epidemic Diarrhea Virus Infection.

Porcine epidemic diarrhea virus (PEDV) has led to significant economic losses in the global porcine industry since the emergence of variant strains in 2010. The high mutability of coronaviruses endows PEDV with the ability to evade the host immune response, which impairs the effectiveness of vaccines. In our previous study, we generated a highly cell-passaged PEDV strain, CT-P120, which showed promise as a live attenuated vaccine candidate by providing satisfactory protection against variant PEDV infection in piglets. However, the mechanism by which the attenuated CT-P120 adapts to cells during passage, resulting in increased replication efficiency, remains unclear. To address this question, we conducted a comparative transcriptomic analysis of Vero E6 cells infected with either the original parental strain (CT-P10) or the cell-attenuated strain (CT-P120) of PEDV at 6, 12, and 24 h post-infection. Compared to CT-P10, CT-P120 infection resulted in a significant decrease in the number of differentially expressed genes (DEGs) at each time point. Functional enrichment analysis of genes revealed the activation of various innate immune-related pathways by CT-P10, notably attenuated during CT-P120 infection. To validate these results, we selected eight genes (TRAF3, IRF3, IFNL1, ISG15, NFKB1, MAP2K3, IL1A, and CCL2) involved in antiviral processes and confirmed their mRNA expression patterns using RT-qPCR, in line with the transcriptomic data. Subsequent protein-level analysis of selected genes via Western blotting and enzyme-linked immunosorbent assay corroborated these results, reinforcing the robustness of our findings. Collectively, our research elucidates the strategies underpinning PEDV attenuation and immune evasion, providing invaluable insights for the development of effective PEDV vaccines.

Alkalization of cellular pH leads to cancer cell death by disrupting autophagy and mitochondrial function.

We previously found that lactic acidosis in the tumor environment was permissive to cancer cell surviving under glucose deprivation and demonstrated that neutralizing lactic acidosis restored cancer cell susceptibility to glucose deprivation. We then reported that alternate infusion of bicarbonate and anticancer agent into tumors via tumor feeding artery markedly enhanced the efficacy of transarterial chemoembolization (TACE) in the local control of hepatocellular carcinoma (HCC). Here we sought to further investigate the mechanism by which bicarbonate enhances the anticancer activity of TACE. We propose that interfering cellular pH by bicarbonate could induce a cascade of molecular events leading to cancer cell death. Alkalizing cellular pH by bicarbonate decreased pH gradient (ΔpH), membrane potential (ΔΨm), and proton motive force (Δp) across the inner membrane of mitochondria; disruption of oxidative phosphorylation (OXPHOS) due to collapsed Δp led to a significant increase in adenosine monophosphate (AMP), which activated the classical AMPK-mediated autophagy. Meanwhile, the autophagic flux was ultimately blocked by increased cellular pH, reduced OXPHOS, and inhibition of lysosomal proton pump in alkalized lysosome. Bicarbonate also induced persistent mitochondrial permeability (MPT) and damaged mitochondria. Collectively, this study reveals that interfering cellular pH may provide a valuable approach to treat cancer.

FG7142 combined with restraint stress induces anxiogenic-like effects via downregulation gamma-aminobutyric acid type A receptor subunit alpha1 and 5-hydroxytryptamine 1A receptors expression in the hippocampus.

OBJECTIVES: The existing anxiety animal models are susceptible to interference, and no single animal anxiety model can predict the future anxiolytic potential and profile of new putative anxiolytics. Therefore, to find a better anxiety animal model, we used FG7142, a nonselective benzodiazepine inverse agonist. This anxiety animal model was established by intraperitoneal injection of FG7142 combined with restraint stress. METHODS: Adult male C57BL/6J mice (18-20 g) were randomly classified into five groups (n = 10 per group), namely the control, restraint stress, restraint stress + 10 mg/kg FG7142, restraint stress + 20 mg/kg FG7142, restraint stress +30 mg/kg FG7142. The impact on behavior was explored by elevated plus maze, and marble burying test, followed by immunohistochemistry and quantitative real-time PCR enabled the elucidation of the possible mechanism. RESULTS: Compared with the control group and restraint stress group, intraperitoneal injection of FG7142 combined with restraint stress model group was found to induce anxiogenic-like behavior in elevated plus maze and marble burying test. Moreover, relative to the control group, significantly increased expression of c-fos in the hippocampus and amygdala in the model group was evident, whereas the expression of gamma-aminobutyric acid type A receptor subunit alpha1 and 5-hydroxytryptamine receptor 1A mRNA was significantly decreased in the hippocampus. CONCLUSIONS: These results indicated that FG7142 combined with restraint stress is sufficient to induce anxiety, and its mechanism is associated with downregulation of hippocampal gamma-aminobutyric acid type A receptor subunit alpha1 and 5-hydroxytryptamine 1A receptors.

Metabolomic analysis of porcine intestinal epithelial cells during swine acute diarrhea syndrome coronavirus infection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, positive single-stranded RNA virus belonging to Coronaviridae family, Orthocoronavirinae subfamily, Alphacoronavirus genus. As one of the main causes of swine diarrhea, SADS-CoV has brought huge losses to the pig industry. Although we have a basic understanding of SADS-CoV, the research on the pathogenicity and interactions between host and virus are still limited, especially the metabolic changes induced by SADS-CoV infection. Here, we utilized a combination of untargeted metabolomics and lipomics to analyze the metabolic alteration in SADS-CoV infected cells. Significant changes were observed in 1257 of 2225 metabolites identified in untargeted metabolomics, while the number of lipomics was 435 out of 868. Metabolic pathway enrichment analysis showed that amino acid metabolism, tricarboxylic acid (TCA) cycle and ferroptosis were disrupted during viral infection, suggesting that these metabolic pathways may partake in pathological processes related to SADS-CoV pathogenesis. Collectively, our findings gain insights into the cellular metabolic disorder during SADS-CoV infection, offer a valuable resource for further exploration of the relationship between virus and host metabolic activities, and provide potential targets for the development of antiviral drugs.

Swine acute diarrhea syndrome coronavirus induces autophagy to promote its replication via the Akt/mTOR pathway.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, single-stranded, positive-sense RNA virus belonging to the Coronaviridae family. Increasingly studies have demonstrated that viruses could utilize autophagy to promote their own replication. However, the relationship between SADS-CoV and autophagy remains unknown. Here, we reported that SADS-CoV infection-induced autophagy and pharmacologically increased autophagy were conducive to viral proliferation. Conversely, suppression of autophagy by pharmacological inhibitors or knockdown of autophagy-related protein impeded viral replication. Furthermore, we demonstrated the underlying mechanism by which SADS-CoV triggered autophagy through the inactivation of the Akt/mTOR pathway. Importantly, we identified integrin α3 (ITGA3) as a potential antiviral target upstream of Akt/mTOR and autophagy pathways. Knockdown of ITGA3 enhanced autophagy and consequently increased the replication of SADS-CoV. Collectively, our studies revealed a novel mechanism that SADS-CoV-induced autophagy to facilitate its proliferation via Akt/mTOR pathway and found that ITGA3 was an effective antiviral factor for suppressing viral infection.

Porcine TRIM21 RING-finger E3 ubiquitin ligase is essential for anti-PRRSV activity.

Porcine reproductive and respiratory syndrome (PRRS) causes substantial economic losses to the global pig industry. Members of the tripartite motif (TRIM) family are the important effectors of the innate immune response against viral infections. We have previously characterized the entire porcine TRIM (pTRIM) family, and predicted pTRIM5, 14, 21, 25 and 38 as host restriction factors against PRRSV infection. However, little is known about whether and how pTRIMs restrict the infection of PRRSV. In this study, we firstly performed the amino acid alignments of the RING domain of pTRIM5, 21, 25 and 38, and found that pTRIM proteins contained the characteristic consensus C3HC4 type zinc-binding motif which is important for the ubiquitination function. Then we detected the mRNA changes of pTRIMs in porcine alveolar macrophages (PAMs) by transcriptome sequencing after PRRSV infection in piglets. Transcriptional profiles showed that the expression of pTRIM5, 21 and 26 was significantly (P < 0.05) up-regulated, consistent with their expression in vitro. Finally, as the most up-regulated gene after PRRSV infection both in vivo and in vitro, pTRIM21 was investigated for its anti-PRRSV activity in immortalized PAMs (iPAMs) in two aspects: knockdown and overexpression of pTRIM21. Knockdown of endogenic pTRIM21 could significantly promote PRRSV replication at 12 and 24 h post infection in iPAMs. Meanwhile, overexpression of pTRIM21 could significantly suppress PRRSV replication but not affect its attachment and endocytosis. Moreover, pTRIM21 RING-finger E3 ubiquitin ligase was essential for anti-PRRSV activity. Our data enhance our understanding of the pTRIMs against PRRSV infection, which may help us develop novel therapeutic tools to control PRRSV.