Vero cells in high-density vaccine cultures often face nutrient starvation, especially in suspension-adapted Vero cells. Previous studies showed that serum starvation dramatically enhances autophagy and mitophagy in suspension-adapted Vero cells. Transcriptomic profiling also revealed significant upregulation of DDIT3, a marker of endoplasmic reticulum stress (ERS), in suspension-adapted Vero cells compared to adherent cells. To investigate the functional role of DDIT3, an Earle's Balanced Salt Solution (EBSS)-induced starvation model was established in adherent Vero cells, recapitulating key autophagy and ER stress responses observed under suspension conditions. The genetic silencing of DDIT3 by shRNA attenuated autophagy, as evidenced by a reduced LC3-II/LC3-I ratio and impaired autophagosome-lysosome activity. Notably, DDIT3 knockdown enhanced cell proliferation and increased the yield of H1N1 influenza virus under nutrient-deprived conditions. Collectively, these results suggest that DDIT3 may serve as a critical regulator linking ER stress to autophagy in Vero cells, and that the suppression of DDIT3 may represent a promising strategy for developing autophagy-resistant Vero cell lines suitable for high-density suspension culture in vaccine production.

Introduction: Vero cells are extensively used in viral vaccine production, but their adaptation to serum-free suspension culture is hindered by excessive autophagy and strong anchorage dependence. Methods: In this study, we identified Connective Tissue Growth Factor (CTGF/CCN2) as significantly upregulated under starvation-an inducer of autophagy-via RNA-seq screening. A stable CTGF knockdown Vero cell line (knockdown efficiency >50%) was established using lentiviral shRNA. Results: Functional characterization demonstrated that CTGF depletion concurrently attenuated autophagic flux (evidenced by reduced LC3-II/I ratio and lysosomal activity) and impaired cell adhesion (with adhesion rates decreased by 50%-60% on extracellular matrix proteins), while maintaining normal cell proliferation. Discussion: Our findings reveal a new role for CTGF in regulating the environmental adaptation of Vero cells by coordinating autophagy and adhesion functions. The engineered cell line provides a novel strategy to overcome suspension adaptation bottlenecks, offering significant potential for improving vaccine production scalability.

Postmenopausal osteoporosis (PMOP), a common systemic skeletal metabolic disease in postmenopausal women, has attracted considerable attention in terms of prevention and treatment research. As a nutritional supplement, Calcium yak caseinate (CYC) effectively prevented and alleviated osteoporosis in ovariectomized mice. However, the underlying mechanism remains to be elucidated. This study investigated the potential mechanism by which CYC ameliorates osteoporosis in ovariectomized mice by focusing on its regulation of gut microbiota and short-chain fatty acids (SCFAs) to alleviate systemic inflammation. The results showed that compared with the OVX group, CYC increased the Firmicutes/Bacteroidota ratio and significantly increased the abundance of Lactobacillus. PICRUSt analysis indicated that CYC suppressed functional pathways associated with oxidative stress and inflammation. Compared with the OVX group, CYC also significantly increased total SCFAs and acetic acid levels, and markedly increased duodenal goblet cell counts. In terms of biochemistry, compared with the OVX group, CYC significantly enhanced the activity of superoxide dismutase in the liver, while significantly attenuating malondialdehyde and reactive oxygen species accumulation. CYC supplementation was associated with decreased level of in inflammatory cytokines tumor necrosis factor-alpha, interferon-gamma, and interleukin-6, and increased level of the anti-inflammatory cytokine interleukin-10. CYC intervention improved the early bone turnover rate after ovariectomy, mitigating the destruction of cortical microstructure. In summary, CYC regulated gut microbiota dysbiosis in ovariectomized mice, increasing the Firmicutes/Bacteroidota ratio, the relative abundance of beneficial Lactobacillus and the levels of intestinal SCFAs. This was associated with improved intestinal barrier function, reduced systemic inflammation and bone resorption, ultimately ameliorating osteoporosis in ovariectomized mice. These findings support the potential of CYC as a nutritional intervention for PMOP.

BACKGROUND:The aging process is closely related to the accumulation of oxidative stress and imbalance in the gut microbiota. Yak whey powder is rich in various bioactive components; however, whether it can improve the antioxidant status of aging organisms by regulating gut microbiota remains to be elucidated. This study aims to investigate the effects of yak whey powder on d-galactose-induced aging mice by evaluating its impact on antioxidant capacity, gut microbiota, and fecal odor profiles. RESULTS:Intervention with yak whey powder significantly improved the content of superoxide dismutase, thymus index, and weight-gain rate in aged mice, while concurrently reducing malondialdehyde levels. The structure and relative abundance of gut microbiota were regulated, characterized by an increase in the levels of beneficial Lactobacillus and a decrease in the levels of potentially pathogenic Helicobacter. Furthermore, fecal odor varied among different interventions and was effectively discriminated with canonical discriminant analysis. Significant correlations (P < 0.05) were observed among the antioxidant indices, relative abundance of intestinal microbiota, and fecal odor information. CONCLUSION:This study demonstrated that yak whey powder ameliorated oxidative stress and modulated gut dysbiosis in aged mice. The strong correlations between the microbiota, odor profiles, and antioxidant markers suggested that the gut microbiota mediated the antioxidant effects of yak whey powder. These findings established a theoretical foundation for understanding the antioxidant properties of yak whey powder, and provided direction for its development and utilization. © 2026 Society of Chemical Industry.

IntroductionVero cells are the primary host for industrial vaccine production, and high-density suspension culture is the predominant manufacturing approach. However, this method faces major challenges, including low shear tolerance, difficulty adapting to serum-free conditions, metabolic imbalance, prolonged acclimation periods, and batch-to-batch variability. Furthermore, the metabolic reprogramming mechanisms underlying suspension adaptation remain poorly understood.MethodsWe established serum-free suspension adaptation of adherent Vero cells using reduction serum levels strategy. Biological characteristics of suspension-adapted cells were analyzed through morphological observation, cell viability assays, and karyotyping. A recombinant rabies vesicular stomatitis virus infection model was used to assess viral susceptibility and replication capacity. Untargeted liquid chromatography–tandem mass spectrometry metabolomics was then utilized to systematically characterize intracellular metabolic profiles of Vero cells cultured under adherent and suspension conditions. Differential metabolites were identified, and key regulatory pathways were evaluated to elucidate the metabolic mechanisms of suspension adaptation and identify potential optimization targets.ResultsSuspension-adapted Vero cells exhibited uniform morphology, stable genetic characteristics, and robust proliferative capacity, without evidence of degeneration or mutation. Viral sensitivity assays demonstrated efficient viral adsorption and replication, yielding stable viral titers and confirming suitability for vaccine production. Metabolomic analysis identified 119 metabolites (32 upregulated, 47 downregulated, and 40 unchanged), with significant enrichment in alanine/aspartate/glutamate metabolism, glycine/serine/threonine metabolism, and the tricarboxylic acid cycle. Suspension cells displayed Warburg-like metabolic features, including enhanced glycolysis, increased lactate accumulation, and pronounced upregulation of succinate and malate. L-serine and ethanolamine phosphate were strongly upregulated, whereas polyunsaturated fatty acids were significantly reduced. Suspension adaptation involved coordinated remodeling of energy, amino acid, nucleotide, and lipid metabolism; enhancing shear tolerance through membrane lipid remodeling; improving antioxidant capacity via serine metabolism; and supporting high-density proliferation through glycolytic reprogramming.DiscussionThis study established a serum-free suspension adaptation system for Vero cells with stable phenotypic characteristics and high viral susceptibility while elucidating metabolic regulatory mechanisms that underlie suspension adaptation. The findings provide metabolic targets and a theoretical foundation for rational medium design, targeted supplementation strategies, and development of genetically engineered cell lines with enhanced shear resistance.

Calcium is a pivotal mineral element in maintaining bone health. Absorption and metabolic balance of calcium are essential for the formation and maintenance of bones. In this study, we investigated the promotional effect of calcium yak caseinate (CYC) on calcium absorption and its mechanism in osteoporotic mice induced by ovariectomy. Here, after ovariectomized (OVX) mice were supplemented with CYC, their apparent calcium absorption rate was significantly improved by regulating serum 25-hydroxyvitamin D, the calcium/phosphorus ratio, and the ratios of phosphorus and calcium to creatinine in urine. Hematoxylin and eosin staining of the duodenum showed that CYC supplementation significantly increased the villus height and to some extent increased the intestinal wall thickness. The gut microbiota and functional prediction studies indicated that CYC altered the gut microbiota composition in OVX mice, characterized by a significant increase in the relative abundance of Lactobacillus. This leads to the upregulation of calcium absorption-related metabolic pathways, including mineral absorption, propanoate metabolism, and synthesis and degradation of ketone bodies. Further analysis showed that CYC promoted calcium absorption by upregulating the expression of TRPV6 protein in the duodenum and TRPV5 and Calbindin-D28k in the kidneys of OVX mice. This study shows that CYC improves the apparent absorption rate of calcium by promoting its absorption in the duodenum and the reabsorption in the kidneys, thereby preventing and improving osteoporosis in OVX mice.

While Gannan yak milk and Gannan cattle-yak milk are unique livestock resources in Gansu Province, China, the specific functional properties of their milk proteins remain to be fully elucidated. To address this, we employed tandem mass tag (TMT) quantitative proteomics to analyze the differentially expressed proteins (DEPs) among Gannan yak milk proteins (YMP), Gannan cattle-yak milk proteins (CYMP), and Holstein milk proteins (HMP). A total of 648 proteins were quantitatively analyzed. Compared with HMP, the DEPs in YMP and CYMP were primarily enriched in immune-related pathways such as the Complement and coagulation cascades, PPAR signaling pathway, and Phagosome. Conversely, the DEPs identified from the CYMP vs. YMP comparison were mainly associated with metabolic pathways, notably the Ribosome pathway. The immunomodulatory effects of these milk proteins were further evaluated in a cyclophosphamide (CTX)-induced immunosuppressed mouse model. Administration of YMP and CYMP significantly ameliorated weight loss, restored thymus/spleen indices, and enhanced serum immunoglobulins (IgG, IgM) and cytokines (IL-4, IL-6, TNF-α). They also reversed the imbalance in splenic T-lymphocyte subsets, increasing CD3+T and CD4+T cells while decreasing CD8+T cells, thereby normalizing the CD4+/CD8+ ratio. Although YMP and CYMP exhibited largely comparable overall efficacy, YMP consistently demonstrated a trend toward greater potency across multiple parameters. In contrast, HMP showed markedly weaker immunomodulatory activity. This research establishes a clear hierarchy in the immunomodulatory properties of these milk proteins, highlighting the potent and similar effects of YMP and CYMP, and provides a scientific basis for the high-value utilization of dairy resources in the Gannan region.

The post-translational modification and stability regulation of RIG-I play critical roles in promoting IFN-I production and maintaining immune homeostasis. In this study, we found that ubiquitin-specific peptidase 1 (USP1) promotes RIG-I protein stability through deubiquitination, which in turn enhances antiviral immunity through the production of inflammatory cytokines, and inhibits the replication of influenza virus in MDCK cells. In contrast, USP1 knockdown inhibited the deubiquitination of RIG-I, decreased the RIG-I protein level, and significantly increased the influenza virus titer. Meanwhile, inhibition of USP1 expression did not have a significant effect on the proliferation of MDCK cells, suggesting that USP1 could be used as a target gene to establish a vaccine-producing MDCK cell line. The above results provide a more comprehensive understanding of the function of USP1 and the antiviral response mechanism, and provide a theoretical and methodological basis for the screening of target genes for the artificial establishment of high-yield MDCK cell lines for vaccine production.

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

Serum-free suspension culture technology for animal cells involves the division and proliferation of cells in serum-free medium as single cells or cell clusters within shaking flasks or bioreactors. This approach enables large-scale cell culture, enhances the yield and quality of biopharmaceuticals, reduces costs, and broadens the applications of animal cells. Serum-free suspension culture of adherent cells (e.g., Madin–Darby canine kidney (MDCK), Chinese hamster ovary (CHO), Vero, baby hamster kidney (BHK-21), and human embryonic kidney (HEK293) cells) has been successfully achieved through direct or indirect adaptation, medium optimization, and genetic engineering. Additionally, novel suspension cell lines, such as duck embryonic stem (EB66) and human retinoblastoma (PER.C6) cells, have been developed as potential new substrates for biopharmaceutical production. This review examines animal cell suspension culture technology and its applications in viral vaccines, recombinant proteins, and monoclonal antibodies, providing insights into the development and utilization of this important technology.

The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of ATG9B, WIPI2, LAMP2, OPTN, Rab7a, and DEPTOR and down-regulated expression of ATG4D, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of ATG101, ATG2A, and STX17 and insignificant change in the expression of NBR1, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3Ⅱ/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.

Madin–Darby Canine Kidney (MDCK) cells are a key cell line for influenza vaccine production, due to their high viral yield and low mutation resistance. In our laboratory, we established a tertiary cell bank (called M60) using a standard MDCK cell line imported from American Type Culture Collection (ATCC) in the USA. Due to their controversial tumourigenicity, we domesticated non-tumourigenic MDCK cells (named CL23) for influenza vaccine production via monoclonal screening in the early stage of this study, and the screened CL23 cells were characterised based on their low proliferative capacity, which had certain limitations in terms of expanding their production during cell resuscitation. It was thus our objective to enhance the proliferation efficiency of MDCK cells for influenza vaccine production following cell resuscitation, with a view to improving the production of non-tumourigenic MDCK cells for vaccines and enhancing the production of influenza virus lysate vaccines from MDCK cells through genetic intervention. We concentrated on the protein thrombosponin-1 (THBS1), which was markedly differentiated in the proteomics data of the two MDCK cells. By integrating this finding with related studies, we were able to ascertain that THBS1 exerts a significant influence on the level of cell proliferation and apoptosis. Consequently, our objective was to investigate the impact of THBS1 expression on MDCK cell apoptosis by verifying the differences in THBS1 expression between the two MDCK cells and by interfering with THBS1 expression in the MDCK cells. We found that the knockdown of THBS1 significantly increased the proliferation and apoptosis of CL23 cells without causing significant changes in cell migration and invasion, and its overexpression significantly decreased the proliferation of M60 cells and increased cell migration, invasion, and apoptosis. In addition, the TGF-β/Smad pathway target genes transforming growth factor-β1 (TGF-β1), mothers against decapentaplegic homolog 2 (Smad2), and mothers against decapentaplegic homolog 3 (Smad3), were significantly down-regulated in CL23 cells after THBS1 knockdown and up-regulated in M60 cells after overexpression, with consistent expression identified at both the mRNA and protein levels. The treatment of cells with TGF-β activators and inhibitors further demonstrated that THBS1 regulated MDCK cell proliferation and apoptosis through the TGF-β/Smad signalling pathway. Finally, we found that THBS1 also regulated H1N1 influenza virus replication. These findings enable a comprehensive understanding of the regulatory mechanisms of THBS1 regarding MDCK cell proliferation and apoptosis functions and the effects of influenza virus replication.

The present study aims to investigate the distribution and expression characteristics of HIF-1 alpha, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of plateau yaks and plain yellow cattle and to speculate the possible regulatory role of HIF-1 alpha and its related hypoxia-inducible factors in the adaptation of the yak spleen to the plateau hypoxic environment. Histological features were observed using H&E and PAS stains. Immunohistochemical staining and optical density analysis were applied to investigate the distribution and differences in the expression of HIF-1 alpha, VEGF, VEGFR-2, VCAM-1, and IL-4 in the spleen of yaks and cattle. The results showed that the area of splenic trabeculae and splenic nodules was significantly larger in the yak than in yellow cattle (P<0.05). Glycogen was mainly distributed in splenic arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblasts, and the distribution was significantly higher in the spleen of yaks than in cattle (P<0.05). HIF-1 alpha, VEGF, VEGFR-2, VCAM-1, and IL-4 were mainly expressed in lymphocytes, arterial endothelial cells, vascular smooth muscle cells, splenic blood sinusoidal endothelial cells, and fibroblast cytoplasm, with higher expression in yak spleen (P<0.05). In conclusion, combining the differences in spleen tissue structure, glycogen distribution, and expression distribution of several hypoxia-related factors between yaks and cattle, we suggest that HIF-1 alpha, VEGF, VEGFR-2, VCAM-1, and IL-4 may be important factors in the adaptation of yak spleen to the plateau environment, which provides a theoretical basis for further exploring the adaptation mechanism of plateau hypoxia in yaks.

The tumorigenicity of MDCK cell line is a major concern with respect to its safety for vaccine production, the effect of miRNAs on the tumorigenicity of MDCK cells is poorly understood. In this study, we performed miRNA-Seq on two MDCK cell lines with tumorigenic potential and their derived monoclonal cell lines that lack tumorigenicity. Through bioinformatics analysis, we identified differentially expressed miRNAs and conducted GO and KEGG pathway analyses of their target genes. Our results indicated that miR-2779-x and its target genes exhibited the most significant characteristics associated with tumorigenesis. Injection of live cells overexpressing miR-2779-x into nude mice resulted in a markedly reduced tumorigenesis rate (1/10). Overexpression of miR-2779-x significantly decreased the proliferation and migration capabilities of MDCK cells while enhancing their invasive potential. To identify and localize miR-2779-x target genes, we employed bioinformatics prediction, RT-qPCR, immunofluorescence assays (IFA), fluorescence in situ hybridization (FISH), and dual-luciferase reporter assays. We found that miR-2779-x negatively regulates the expression of PI3KR1 and Caspase 9 at both the gene and protein levels. Additionally, miR-2779-x co-localizes with PI3KR1 in the cytoplasm and directly targets the 3’UTR of PI3KR1. Overexpression of PI3KR1 in miR-2779-x-overexpressing cells restored cellular functions, leading to increased proliferation and migration but decreased invasion. Moreover, miR-2779-x modulates MDCK cell growth and tumorigenesis by influencing the expression and phosphorylation levels of proteins involved in the PI3K/AKT and apoptosis signaling pathways. We proposed the key pathways of miRNA involvement in MDCK cell tumorigenicity and initially revealed the function of miR-2779-x in MDCK cell tumorigenicity.

Hypoxia is one of the factors severely affect renal function, and, in severe cases, it can lead to renal fibrosis. Although much progress has been made in identifying the molecular mediators of fibrosis, the mechanisms that govern renal fibrosis remain unclear, and there have been no effective therapeutic anti-fibrotic strategies to date. Mammals exposed to low oxygen in the plateau environment for a long time are prone to high-altitude disease, while yaks have been living in the plateau for generations do not develop kidney fibrosis caused by low oxygen. It has been suggested that metabolic reprogramming occurs in renal fibrosis and that pyruvate dehydrogenase kinase 1 (PDK1) plays a crucial role in metabolic reprogramming as an important node between glycolysis and the tricarboxylic acid cycle. The aim of this study was to investigate the effects of hypoxia on the renal tissues and renal interstitial fibroblasts of yaks. We found that, at the tissue level, HIF-1α, PDK1, TGF-β1, Smad2, Smad3, and α-SMA were mainly distributed and expressed in tubular epithelial cells but were barely present in the renal mesenchymal fibroblasts of healthy cattle and yak kidneys. Anoptical density analysis showed that in healthy cattle kidneys, TGF-β1, Smad2, and Smad3 expression was significantly higher than in yak kidneys (p < 0.05), and HIF-1α and PDK1 expression was significantly lower than in yak kidneys (p < 0.05). The results at the protein and gene levels showed the same trend. At the cellular level, prolonged hypoxia significantly elevated PDK1 expression in the renal mesangial fibroblasts of cattle and yak kidneys compared with normoxia (p < 0.05) and was proportional to the degree of cellular fibrosis. However, PDK1 expression remained stable in yaks compared with renal interstitial fibroblast-like cells in cattle during the same hypoxic time period. At the same time, prolonged hypoxia also promoted changes in cellular phenotype, promoting the proliferation, activation, glucose consumption, lactate production, and anti-apoptosis in the both of cattle and yaks renal interstitial fibroblasts The differences in kidney structure and expression of PDK1 and HIF-1α in kidney tissue and renal interstitial fibroblasts induced by different oxygen concentrations suggest that there may be a regulatory relationship between yak kidney adaptation and hypoxic environment at high altitude. This provides strong support for the elucidation of the regulatory relationship between PDK1 and HIF-1α, as well as a new direction for the treatment or delay of hypoxic renal fibrosis; additionally, these findings provide a basis for further analysis of the molecular mechanism of hypoxia adaptation-related factors and the adaptation of yaks to plateau hypoxia.

The aim of this study was to investigate the effects of hypoxia-induced phenotype, glucose metabolism, ROS levels, and the PDK1-mediated regulation of TGF-β/Smad signaling in yellow cattles, yaks, and those overexpressing PDK1 PASMCs using growth curves, flow cytometry, scratch experiments, glucose and lactic acid assays, RT-qPCR, and Western blotting. The results showed that hypoxia significantly promoted proliferation, migration, antiapoptosis, ROS levels, glucose consumption, and lactate production in yellow cattle PASMCs (p < 0.05), and the cells were dedifferentiated from the contractile phenotype; conversely, hypoxia had no significant effect on yak PASMCs (p > 0.05). PDK1 overexpression significantly promoted proliferation, antiapoptosis, glucose consumption, and lactate production in yak PASMCs under normoxia and hypoxia (p < 0.05), decreased their migration levels under hypoxia (p < 0.05), and dedifferentiated the contractile phenotype of the cells. Overexpression of PDK1 in yak PASMCs is detrimental to their adaptation to hypoxic environments. Yak PASMCs adapted to the effects of hypoxia on lung tissue by downregulating the expression of genes related to the PDK1 and TGF-β/Smad signaling pathways. Taken together, the regulation of PDK1-mediated TGF-β/Smad signaling may be involved in the process of yaks’ adaptation to the hypoxic environment of the plateau, reflecting the good adaptive ability of yaks. The present study provides basic information to further elucidate the mechanism of PDK1-mediated TGF-β/Smad signaling induced by hypoxia in the lungs of yaks, as well as target genes for the treatment of plateau diseases in humans and animals.

The brain is an important part of the mammalian nervous system, is highly sensitive to hypoxia, and plays an important role in the adaptation of the body to hypoxic environments. This study was conducted to study the distribution and expression of hypoxia-related factors (hypoxia-inducible factor 1α, HIF-1α; erythropoietin, EPO; vascular endothelial growth factor, VEGF; vascular cell adhesion molecule, VCAM) in the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina in yaks of different ages (4d, 6-months-old and adult). Paraffin sections were obtained from the cerebellum, cerebrum, medulla oblongata, and corpora quadrigemina of healthy yak for 4-day-old, 6-months-old and adult yaks. Histological characteristics were assessed by haematoxylin staining. Immunohistochemical staining was performed to detect the distribution and expression of HIF-1α, EPO, VEGF and VCAM proteins. Immunohistochemical results showed that HIF-1α, EPO, VEGF, and VCAM were expressed in the pyramidal cell layer of the yak cerebrum, and distributed in the cerebellum granulose cell layer, Purkinje cell layer and medulla layer, and were mainly positive in Purkinje cells and medulla. It is expressed in the cell bodies of the medulla oblongata and the quadrimatous neurons. The expression level in the medulla oblongata was higher, indicating may play a crucial role in functional cohesion. The expression of HIF-1α in 4 d cerebellar tissues was higher than that in other age groups, and the expression of HIF-1α in the medulla oblongata increased with age. In addition, the expression levels of EPO and VEGF in the 6-month-old group were slightly higher than those in the other age groups. It is speculated that EPO and VEGF have obvious protective effects on brain tissue in the 6-month-old age group; VCAM showed no significant differences in the cerebrum, cerebellum, medulla oblongata, or corpora quadrigemina of the yaks. This study provides basic data for further exploration of the adaptive mechanism of plateau yak brain tissue.

Background Non-alcoholic fatty liver disease (NAFLD) is a multifaceted metabolic disorder, whose global prevalence is rapidly increasing. Acetyl CoA carboxylases 1 (ACACA) is the key enzyme that controls the rate of fatty acid synthesis. Hence, it is crucial to investigate the function of ACACA in regulating lipid metabolism during the progress of NAFLD. Methods Firstly, a fatty liver mouse model was established by high-fat diet at 2nd, 12th, and 20th week, respectively. Then, transcriptome analysis was performed on liver samples to investigate the underlying mechanisms and identify the target gene of the occurrence and development of NAFLD. Afterwards, lipid accumulation cell model was induced by palmitic acid and oleic acid (PA ∶ OA molar ratio = 1∶2). Next, we silenced the target gene ACACA using small interfering RNAs (siRNAs) or the CMS-121 inhibitor. Subsequently, experiments were performed comprehensively the effects of inhibiting ACACA on mitochondrial function and lipid metabolism, as well as on AMPK- PPARα- CPT1A pathway. Results This data indicated that the pathways significantly affected by high-fat diet include lipid metabolism and mitochondrial function. Then, we focus on the target gene ACACA. In addition, the in vitro results suggested that inhibiting of ACACA in vitro reduces intracellular lipid accumulation, specifically the content of TG and TC. Furthermore, ACACA ameliorated mitochondrial dysfunction and alleviate oxidative stress, including MMP complete, ATP and ROS production, as well as the expression of mitochondria respiratory chain complex (MRC) and AMPK proteins. Meanwhile, ACACA inhibition enhances lipid metabolism through activation of PPARα/CPT1A, leading to a decrease in intracellular lipid accumulation. Conclusion Targeting ACACA can reduce lipid accumulation by mediating the AMPK- PPARα- CPT1A pathway, which regulates lipid metabolism and alleviates mitochondrial dysfunction.

Circular RNAs (circRNAs) are non-coding RNAs discovered in recent years, which are produced by back-splicing involving the 3' and 5' ends of RNA molecules. There is increasing evidence that circRNAs have important roles in cancer, neurological diseases, cardiovascular and cerebrovascular diseases, and other diseases. In addition, host circRNAs and virus-encoded circRNAs participate in the body's immune response, with antiviral roles. This review summarizes the mechanisms by which host and viral circRNAs interact during the host immune response. Comprehensive investigations have revealed that host circRNAs function as miRNA sponges in a particular manner, primarily by inhibiting viral replication. Viral circRNAs have more diverse functions, which generally involve promoting viral replication. In addition, in contrast to circRNAs from RNA viruses, circRNAs from DNA viruses can influence host cell migration, proliferation, and apoptosis, along with their effects on viral replication. In summary, circRNAs have potential as diagnostic and therapeutic targets, offering a foundation for the diagnosis and treatment of viral diseases.

The MDCK cell line is perceived as better than the embryos of hen eggs for the production of influenza vaccines, but the tumorigenicity of these cells is concerning. Epidermal growth factor receptor (EGFR) is likely to be a crucial target that contributes to the tumorigenicity of MDCK cells. In this study, EGFR-knockdown and EGFR-overexpression cell lines were established. EGFR's influence on cell growth, migration, clonogenic ability, and flu virus susceptibility was evaluated in vitro, and its role in cell tumorigenicity was examined in nude mice. GST pull-down coupled with mass spectrometry (MS) and bioinformatics analysis identified EGFR-interacting proteins. The expression levels of these proteins, as well as those of PI3K-AKT- and MAPK-ERK-signaling-pathway-related molecules, were confirmed at both gene and protein levels. The result indicates that EGFR overexpression can enhance cell proliferation, migration, and clonal formation; EGFR knockdown could effectively curtail tumorigenesis and amplify the titers of influenza viruses in MDCK cells. An analysis of the underlying mechanism identified a total of 21 interacting proteins implicated in tumor formation, and among these, AKT1, CDK4, GNB2, and MAPK8 were confirmed at both gene and protein levels. EGFR can activate key factors of the PI3K-AKT signaling pathway, AKT and PI3K, and promote their phosphorylation levels. Consequently, we concluded that EGFR interacts with GNB2, facilitating transmembrane signal transduction, activating the PI3K-AKT signaling cascade, controlling cell cycle alterations, stimulating cell proliferation, and promoting tumorigenesis.