Fluorescence Quantitative PCR Research Articles

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus

Bio-characteristics, tissue expression of miR-375 in hypothalamic-pituitary-ovarian axis and its regulation in reproduction-related diseases

Our study concentrated on the expression of miRNA-375 in the hypothalamic-pituitary-gonadal axis of female Hu sheep. The investigation involved cloning the precursor sequence of miR-NA-375, followed by comparison with database entries and subsequent Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. In our approach, we obtained ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus, and pineal gland from fertile but nonpregnant Hu ewes. MiRNA extraction kit was used to extract miRNA from the above eight tissues. Real-time fluorescent quantitative polymerase chain reaction was used to evaluate the role of miR-375 in the hy-pothalamic-pituitary-gonadal axis. The results of miR-375 precursor sequence cloning were compared with those of Anopheles gambiae, Apis mellifera, Bos taurus, Drosophila melanogaster, Danio rerio, Fugu rubripes, Gallus gallus, Homo sapiens, Monodelphis domestica, Macaca mulatta, Mus musculus, Pan troglodytes, Rattus norvegicus, Tetraodon nigroviridis, Xenopus tropicalis miR-375 in miRBase database. It was found that oar-miR-375 was highly conserved. Notably, miR-375 expression in the pineal gland was significantly higher (p < 0.01) than that in the ovaries, thalamus, cerebellum, brain, uterus, pituitary gland, hypothalamus. The study also involved predicting miR-375 target genes. GO and KEGG enrichment analyses of these predicted target genes revealed that miR-375 is involved in 182 biological processes, affects 186 cellular components, and participates in 184 molecular functions. In terms of pathway enrichment, miR-375 was linked to nine pathways, including the Hippo, Wnt, and mTOR signaling pathways. This study has validated the interaction between miR-375 and its target gene FZD4, which can be recognized and bound to produce effects. These findings lead to the inference that miR-375 may play a crucial regulatory role in sheep reproduction through the Hippo pathway and Wnt pathway, laying a foundation for further exploration of miR-375’s role in this domain.

BRAF deletion in adult patients with Langerhans cell histiocytosis correlates with multisystem disease and poor outcome.

Langerhans cell histiocytosis (LCH) is a rare and highly heterogeneous histiocytosis. There are currently few studies examining the correlation between molecular profiling and clinical phenotype or outcome in adult patients with LCH. The objective of this study was to characterize the genomic landscape of adult LCH and correlate molecular findings with clinical features and patient outcomes. This study included 254 patients aged ≥18 years with biopsy-proven LCH from January 2000 to December 2023. All patients underwent next-generation sequencing (NGS) or fluorescence quantitative PCR (qPCR) for the BRAFV600E mutation. Patient demographics, disease characteristics and treatments were collected through electronic medical records. Patient outcomes were collected through clinical and telephone follow-up. Overall, 254 patients were enrolled. MAPK/PI3K pathway alterations were observed in 77.6%（n=197）of the patients. BRAFV600E mutation was the most common (30.7%, n=78), followed by BRAFindel (18.1%, n=46) andMAP2K1 mutations (12.6%, n=32). The proportion of BRAFindel was much higher in patients with MS involvement than single system disease (24.5% vs 6.6%, p＜0.001). In overall patients, BRAFindel was associated with inferior overall survival (3-year OS 89.6% vs 99.0%, p=0.014) and progression-free survival (3-year PFS 50.0% vs 78.6%, p＜0.001). In MS LCH patients, BRAFindel was associated to worse PFS (3-year PFS 47.8% vs 76.0%, p=0.001). This large study provides molecular and clinical pathologic characterization of adult LCH. BRAFindel was highly correlated with MS LCH, and was associated to worse outcome.

A comprehensive analysis of the defense responses of Odontotermes formosanus (Shiraki) provides insights into the changes during Serratia marcescens infection

BackgroundOdontotermes formosanus (Shiraki) is a highly damaging agroforestry pest. Serratia marcescens is a broad-spectrum insecticidal pathogen and is highly lethal to O. formosanus. However, little is known about the mechanism between them. To improve the biological control of pests, a more in-depth analysis of the interactions between the pests and the pathogens is essential.ResultsWe used RNA-seq, enzyme activity assays and real-time fluorescent quantitative PCR (qPCR) to explore the defense responses of O. formosanus against SM1. RNA-seq results showed that 1,160, 2,531 and 4,536 genes were differentially expressed at 3, 6 and 12 h after SM1 infection, and Kyoto Encyclopedia of Genes and Genomes (KEGG) results indicated that immune response and energy metabolism were involved in the defense of O. formosanus against SM1. Reactive oxygen species (ROS) levels and ROS synthesis genes were significantly elevated, and the antioxidant system were induced in O. formosanus after SM1 infection. In addition, the cellular immune genes were affected, and the Toll, immune deficiency (Imd), Janus kinase/signal transducer and activator of transcription (JAK/STAT), c-Jun N-terminal Kinase (JNK) and melanization pathways were activated. In vitro, Oftermicin, an antimicrobial peptide, had a significantly inhibitory effect on SM1. Furthermore, the expression levels and enzyme activities of phosphofructokinase (PFK), lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and isocitrate dehydrogenase (IDH) in glycolysis and tricarboxylic acid (TCA) cycles were increased.ConclusionsOur results clearly demonstrated that O. formosanus defended against SM1 by activating the antioxidant system, innate immunity and energy metabolism. This study would provide useful information for the development of biological controls of O. formosanus.

Ren-Shen-Bu-Qi decoction alleviates exercise fatigue through activating PI3K/AKT/Nrf2 pathway in mice.

Fatigue is a prevalent issue that can lead individuals to a sub-health condition, impacting their work efficiency and quality of life. There are limited effective treatment options available for fatigue. Ren-Shen-Bu-Qi decoction (RSBQD) is a proprietary herbal remedy that is designed to address fatigue. However, the specific pharmacological mechanisms and basis of RSBQD are not yet fully understood. This study aimed to investigate the pharmacological effects and mechanisms of RSBQD in a mouse model of exercise fatigue. UPLC-Q-Orbitrap HRMS was used to analyze the chemical composition of RSBQD. The pharmacological basis and molecular mechanism of RSBQD on exercise fatigue were predicted using network pharmacology analysis. Subsequently, an exercise fatigue mouse model was established and used to analysis the effects of RSBQD. The potential mechanisms were verified by hematoxylin-eosin (HE) staining, real-time fluorescence quantitative PCR (RT-qPCR), Western blot (WB) and molecular docking. The results showed that 88 main components of RSBQD were identified, which have mainly belonged to flavonoids and carboxylic acid compounds. The network pharmacology analysis indicated that RSBQD ameliorate fatigue through PI3K/AKT signaling pathway. Notably, RSBQD prolonged the swimming time and diminished body weight loss of exercise fatigue mice (P < 0.05). Meanwhile, RSBQD significantly alleviated the injury of liver and kidney induced by exhaustive exercise, and decreasing the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea and BUN levels (P < 0.05). In addition, RSBQD was found could relieve exercise fatigue by decreasing the content of creatine kinase (CK), lactate dehydrogenase (LDH), and lactic acid (LA), but increasing the blood glucose (GLU) and liver glycogen (HG) levels (P < 0.05). RSBQD also significantly increased the hepatic superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) but decreased hepatic malondialdehyde (MDA) levels. Moreover, RSBQD was able to upregulate protein level of activated Nrf2 and PI3K/AKT signaling pathways. RSBQD mitigates exercise fatigue by reversing metabolic changes and reducing oxidative damage through the PI3K/AKT/Nrf2 signaling pathway. This study offers pharmacological support for the utilization of RSBQD in exercise fatigue treatment.

Development and Application of a TaqMan RT-qPCR for the Detection of Foot-and-Mouth Disease Virus in Pigs

The global livestock industry is facing a serious threat from a widespread foot-and-mouth disease virus (FMDV) epidemic. The timely detection of FMDV can significantly mitigate its harmful effects. This study aimed to establish and evaluate a TaqMan fluorescence quantitative PCR assay to assess its sensitivity, specificity, reproducibility, and stability. The standard curve equation range is 6.43 × 109–6.43 × 101 copies/µL, with an R2 value of 0.996 and a standard curve equation of y = −3.586x + 36.245. The method successfully detected 64.3 copies/µL of the target gene for FMDV and exhibited high specificity for FMDV. Repeatability tests demonstrated low coefficients of variation within and between groups (&lt;2%), indicating good reproducibility. The clinical samples analyzed using this method showed results consistent with those of the SYBR Green I RT-qPCR assay, confirming the reliability of the method. Overall, the developed test method displayed high sensitivity, specificity, reproducibility, and stability, making it suitable for the rapid diagnosis of foot-and-mouth disease in clinical settings.

Icariin ameliorates Coxsackievirus B3-induced viral myocarditis by modulating the S100 calcium binding protein A6/β-catenin/c-Myc signaling pathway

BackgroundCoxsackievirus B3 (CVB3) is a leading cause of viral myocarditis and is currently lacking specific pharmacological treatments, highlighting the critical need for therapeutic development. Icariin (ICA), a prenylated flavonol glycoside, was previously found to exhibit several pharmacological effects, but its potential to combat CVB3 remains uninvestigated. PurposeThis study aimed to elucidate the anti-CVB3 efficacy of ICA and elucidate its molecular mechanisms. MethodsCVB3-infected HeLa cells, H9C2 cells and neonate rat ventricular cardiomyocytes (NRVCs) were selected as in vitro models, and were treated with ICA at 1 and 10 μM. Additionally, BALB/c mice that were infected with CVB3 via intraperitoneal injection were chosen as in vivo model and were treated with ICA or ribavirin over 3 days. The effect of ICA against CVB3 was determined by Cell Counting Kit-8 (CCK-8) assay, western blot, real-time fluorescence quantitative PCR (RT–qPCR), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC) and flow cytometry. ResultsIn this study, it was found that ICA is capable of reducing CVB3 viral load both in vitro and in vivo. Mechanistic studies suggested that ICA prevents cardiomyocyte apoptosis by attenuating the S100 calcium binding protein A6 (S100A6)/β-catenin/c-Myc signaling pathway. Additionally, ICA inhibits the secretion of proinflammatory cytokines tumor necrosis factor alpha (TNF-α), interleukin-1beta (IL-1β) and CXC motif chemokine ligand 2 (CXCL2) in heart tissue, thereby mitigating CVB3-induced myocarditis. Moreover, ICA also regulates the immune response of CD4+ T, CD8+ T and Treg cells by changing the cells numbers in spleen tissue. Lastly, ICA can reduce the load of other enteroviruses (such as CVA6, CVA16 and EV71) in rhabdomyosarcoma (RD) cells as well. ConclusionOur findings indicate that ICA provides significant protection against CVB3 infection by modulating the S100A6/β-catenin/c-Myc signaling pathway, suggesting its potential use as a novel drug against CVB3 infection in clinical application.

Exploring the mechanism of tetramethylpyrazine in the treatment of osteoarthritis based on network pharmacology

BackgroundOsteoarthritis (OA) is the most common joint disease, which mainly damages articular cartilage and involves the whole joint tissue. It has the characteristics of long course, repeated symptoms and high disability rate, and the incidence trend is gradually increasing. Tetramethylpyrazine (TMP) is the main alkaloid active substance in Ligusticum wallichii, a traditional Chinese medicine, which has the effect of promoting blood circulation and dredging collaterals, and has a good effect on the treatment of early OA, but its molecular mechanism has not been fully clarified so far. Based on network pharmacology, molecular docking simulation and animal experiments, this study explored the target and molecular mechanism of TMP in the treatment of OA.MethodsWe used PubChem, SwissTargetPrediction, and PharmMapper databases to predict the molecular structure and potential targets of TMP. GeneCards and DisGeNET databases were used to predict the relevant targets of OA. Apply UniProt database to convert targets into unified gene names, and proofread and remove duplicate gene names. The intersection targets of TMP and OA obtained on venny2.1.0 website were submitted to the STRING database to construct a PPI network. CytoScape 3.8.2 software was used to analyze the PPI network and obtain the sub-network modules and 10 key targets. The intersection targets of TMP and OA were analyzed by Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment using DAVID 6.8 database. The intersecting targets of TMP and OA, the biological process of GO enrichment, and KEGG signaling pathway were imported into Cytoscape 3.8.2 software to construct the TMP-target-pathway network diagram. Use molecular docking technology to simulate the interaction between TMP molecules and key targets, and predict the binding mode and binding ability. Animal models of rabbit knee osteoarthritis were prepared, and magnetic resonance imager (MRI) and fluorescence quantitative PCR (RT-qPCR) were used to observe the effect of TMP in treating OA as well as the expression of key target genes.Results585 potential targets of TMP, 3,857 potential targets of OA, and 49 intersecting targets of TMP and OA were obtained. The top 10 key target genes were obtained, in order of ranking: ALB, ESR1, IL10, CAT, F2, MPO, C3, CYP3A4, CYP2C9, ANXA1. GO and KEGG analysis implied that the key targets might act on OA by affecting endothelial cell permeability, peri-articular microcirculatory status, NETs production, activation of complement system and coagulation pathway, regulation of immune function of macrophages and T cells, and substance metabolism pathway in vivo, etc. The molecular mechanism might involve the formation of neutrophil extracellular trap, regulation of the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways, etc. Molecular docking simulations showed that the binding energy of IL10 and ANXA1 to TMP was greater than -5Kal/mol, but the other key target proteins showed better binding to TMP, and the binding energy was less than -5 kcal/mol. Animal experiments showed that TMP had a significant therapeutic effect on OA. The TMP group had significantly reduced knee joint effusion and bone marrow damage compared to the OA group (p &amp;lt; 0.05). The qRT-PCR results showed that compared with the OA group, the mRNA expression of ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 in the TMP group increased (p &amp;lt; 0.05), while there was no significant difference in mRNA expression of ALB, IL-10, F2, MPO, etc. (p &amp;gt; 0.05).ConclusionTMP is effective in the treatment of OA, with multi-target and multi-pathway interactions. ESR1, CAT, C3, CYP3A4, CYP2C9, and ANXA1 may be potential targets for TMP treatment of OA. The molecular mechanism mainly involves the formation of neutrophil extracellular trap, regulation of the actin cytoskeleton, complement and coagulation cascades, and T cell receptor signaling pathways, etc.

VEGFB ameliorates insulin resistance in NAFLD via the PI3K/AKT signal pathway

BackgroundNon-alcoholic fatty liver disease (NAFLD) is one of the most universal liver diseases with complicated pathogenesis throughout the world. Insulin resistance is a leading risk factor that contributes to the development of NAFLD. Vascular endothelial growth factor B (VEGFB) was described by researchers as contributing to regulating lipid metabolic disorders. Here, we investigated VEGFB as a main target to regulate insulin resistance and metabolic syndrome.MethodsIn this study, bioinformatics, transcriptomics, morphological experiments, and molecular biology were used to explore the role of VEGFB in regulating insulin resistance in NAFLD and its molecular mechanism based on human samples, animal models, and cell models. RNA-seq was performed to analyze the signal pathways associated with VEGFB and NAFLD; Palmitic acid and High-fat diet were used to induce insulin-resistant HepG2 cells model and NAFLD animal model. Intracellular glucolipid contents, glucose uptake, hepatic and serum glucose and lipid levels were examined by Microassay and Elisa. Hematoxylin-eosin staining, Oil Red O staining, and Periodic acid-schiff staining were used to analyze the hepatic steatosis, lipid droplet, and glycogen content in the liver. Western blot and quantitative real-time fluorescent PCR were used to verify the expression levels of the VEGFB and insulin resistance-related signals PI3K/AKT pathway.ResultsWe observed that VEGFB is genetically associated with NAFLD and the PI3K/AKT signal pathway. After VEGFB knockout, glucolipids levels were increased, and glucose uptake ability was decreased in insulin-resistant HepG2 cells. Meanwhile, body weight, blood glucose, blood lipids, and hepatic glucose of NAFLD mice were increased, and hepatic glycogen, glucose tolerance, and insulin sensitivity were decreased. Moreover, VEGFB overexpression reduced glucolipids and insulin resistance levels in HepG2 cells. Specifically, VEGFB/VEGFR1 activates the PI3K/AKT signals by activating p-IRS1Ser307 expression, inhibiting p-FOXO1pS256 and p-GSK3Ser9 expressions to reduce gluconeogenesis and glycogen synthesis in the liver. Moreover, VEGFB could also enhance the expression level of GLUT2 to accelerate glucose transport and reduce blood glucose levels, maintaining glucose homeostasis.ConclusionsOur studies suggest that VEGFB could present a novel strategy for treating NAFLD as a positive factor.Graphical

Comparative analysis of the NF-Y transcription factor family identifies VaNF-YA6 as a positive regulator of salt and drought tolerance in grapevine

Nuclear factor Y transcription factors (NF-Y TFs) play crucial roles in plant responses to abiotic stress. However, there is a lack of research on the comparative analysis of evolutionary relationships, real-time quantitative fluorescence PCR (RT-qPCR), and functions of NF-Y TFs to screen key NF-Y TFs that are resistant to salt and drought stresses between Vitis vinifera (V. vinifera) and Vitis amurensis (V. amurensis). In this study, 27 and 26 NF-Y TFs were identified in V. vinifera and V. amurensis, respectively, and were divided into three subgroups. Subcellular localization prediction revealed that NF-Ys TFs were mainly located in the nucleus. Interestingly, the NF-YA protein sequence of ‘NTKKLDWEFWGCCDDCEKWFGGCC’ was lost in the V. vinifera compared to V. amurensis, whereas the sequence ‘SSVYSQPWWGHSIVCVA’ was gained, thus, these sequences might be closely related to the functions performed. RT-qPCR analysis of ‘Pinot Noir’ (cultivated variety) and ‘Zuoyouhong’ (wild variety) plantlets demonstrated that the expression levels of VaNF-YA6, VaNF-YB5, VvNF-YA3, VvNF-YA5, and VvNF-YC2 were significantly upregulated under 400 mmol·L-1 NaCl and 10% PEG treatments for 24 h Subcellular localization showed that the VaNF-YA6-GFP fusion protein was functioned primarily in the nucleus. Overexpression of VaNF-YA6 in grapevine leaves and Arabidopsis thaliana (Arabidopsis) could significantly enhance tolerance to salt and drought stresses by improving VvSOS2, VvSOS3, VvABF3, VvCPK6 expression levels, enzyme activities, and other protective substances. In summary, our study provides a theoretical basis for the further use of VaNF-YA6 to improve salt and drought resistance in grapevines.

Study on the antihypertensive mechanism of acupuncture at "Renying" (ST9) and "Zusanli" (ST36) via gastrin/cholecystokinin B receptors

To observe the effect of acupuncture on serum gastrin content and urinary sodium excretion in spontaneously hypertensive rat (SHR), so as to explore its potential mechanism in the treatment of hypertension. Thirty-two male SHRs were randomly divided into model, hydrochlorothiazide, acupuncture and sham acupuncture groups, with 8 rats in each group, and 8 male Wistar-kyoto rats were taken as the control group. Rats in the hydrochlorothiazide group received gavage of hydrochlorothiazide solution (10 mg·kg-1·d-1), once daily for 4 weeks. Acupuncture was applied to bilateral "Renying" (ST9) and "Zusanli" (ST36) or non-acupoint on both sides for rats in the acupuncture and sham acupuncture groups, with manual stimulation every 10 minutes for a total of 20 minutes, once a day for a total of 4 weeks. The systolic blood pressure of the tail-artery was measured before and 1, 2, 3, and 4 weeks after the intervention. HE staining was used to observe the pathological changes in renal tissue. Serum gastrin contents were detected using enzyme-linked immunosorbent assay (ELISA). Urinary sodium content was measured by atomic absorption spectrometry. The expression levels of cholecystokinin B receptor (CCKBR) and Na+/K+-ATPase proteins in renal tissue were detected by Western blot, and the mRNA expression levels of CCKBR and the α1 subunit of Na+/K+- ATPase (ATP1A1) were detected by fluorescence quantitative PCR. Compared with the control group, the systolic blood pressure of the tail artery in the model group were increased significantly before intervention and at the 1st, 2nd, 3rd and 4th weeks of intervention (P<0.05). Before intervention, the 24 h urine volume of the model, hydrochlorothiazide, acupuncture and sham acupuncture groups were decreased significantly (P<0.05). After intervention, the 24 h urine volume and urinary sodium excretion in the model group were significantly decreased (P<0.05)；the expression levels of CCKBR protein and mRNA in renal tissue were significantly decreased (P<0.05)；the expression levels of Na+/K+-ATPase protein and ATP1A1 mRNA were significantly increased (P<0.05)；the glomerulus was mildly congested with a small amount of lymphocyte infiltration. Compared with the model group, the systolic blood pressure of the tail artery in the hydrochlorothiazide and the acupuncture groups were decreased significantly at the 2nd, 3rd and 4th weeks of intervention (P<0.05)；the 24 h urine volume and urinary sodium excretion in the hydrochlorothiazide and the acupuncture groups were significantly increased (P<0.05)；the serum gastrin content and the expression levels of CCKBR protein and mRNA in renal tissue were significantly increased (P<0.05)；the expression levels of Na+/K+-ATPase protein and ATP1A1 mRNA were significantly decreased (P<0.05)；there were no obvious pathological changes in renal tissue. A small number of lymphocyte focal infiltration around blood vessels was observed in the kidney tissue of the sham acupuncture group. Acupuncture can significantly reduce the tail artery systolic blood pressure of SHR, which may be related to its effect in increasing serum gastrin content and CCKBR expression, inhibiting sodium pump reabsorption, thus promoting urinary sodium excretion.

Expression and localization of Uhrf1 in the major reproductive organs of yaks during different reproductive cycles

Uhrf1 is a multi-domain and multifunctional epigenetic regulator playing key roles in DNA methylation, cell metabolism, and cell proliferation. To investigate the role of Uhrf1 in the reproductive physiology of female yaks, we collected three reproductive organs (ovaries, oviducts, and uteri) from healthy yaks during three reproductive phases (follicular, luteal, and gestational phases), with a total of nine groups. Real-time fluorescence quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry (IHC) were employed to determine the expression levels of Uhrf1 and the subcellular localization of this protein. RT-qPCR and Western blotting results showed that Uhrf1 was expressed highest in the oviduct during the follicular phase, moderate expression in the uterus during the gestational phase, and the lowest expression in the uterus during the luteal phase (P < 0.05). IHC results showed that Uhrf1 was mainly expressed in the ovarian germinal epithelium, theca follicular, follicular granulosa, luteal cells, oviduct mucosal epithelial cells, and uterine glands (UG) of yaks. In conclusion, Uhrf1 was differentially expressed in the major reproductive organs during the reproductive cycle of female yaks, indicating its important regulatory role in the reproductive physiology of yaks.

NF-κB transcription factors regulate the expression of immunity-related genes in different cell lines of silkworm (Bombyx mori)

The Toll and immune deficiency (IMD) signaling pathways in insects are highly conserved in evolution and regulate the expression of antimicrobial peptides (AMPs) and other immune-related genes mainly through nuclear factor-kappa B (NF-κB) transcription factors. However, the differences of NF-κB transcription factors Rels and Relish in the expression regulation of AMPs and other immune-related genes in silkworm (Bombyx mori) have not been systematically reported. In this study, the BmRelA, BmRelB and BmRelish1 genes were cloned and their eukaryotic cell overexpression vectors were constructed. After the recombinant vectors were transfected into BmE and BmN cells, the expression of AMPs and immune-related genes was detected by real-time fluorescence quantitative PCR. The results showed that the expression of AMP genes Defensin2 and Gloverin2 was mainly regulated by Relish, the expression of Moricin was mainly regulated by RelA and RelB, and the expression of other AMP genes was jointly regulated by both. In addition, the expression levels of peptidoglycan recognition proteins (PGRPs), β-1, 3-glucan recognition proteins (βGRPs), lysozymes (Lys) and lysozyme-like proteins (LLPs), and nitric oxide synthase (NOS) were up-regulated to varying degrees in different cell lines in response to RelA, RelB and Relish1, suggesting that the expression of these immune-molecules was also regulated by Toll or IMD pathways in silkworm. Compared with the regulatory specificity of transcription factors in Drosophila Toll and IMD signaling pathways on the expression of AMPs, this study found that the regulatory patterns of Rels and Relish1 on the expression of AMPs in silkworm are more complex, which provides an experimental basis for further analysis of the effect mechanism and feedback mechanism of Toll and IMD pathways in insects.

Effect of acupuncture on AMPK/ULK1 signaling pathway of hippocampus in chronic stress-induced depression rats

To observe the effect of manual acupuncture stimulation on changes of behavior and hippocampal signaling pathways of AMP-activated kinase (AMPK)/UNC-51-like kinase 1 (ULK1) in rats with chronic unpredictable mild stress (CUMS)-induced depression, so as to explore its molecular mechanism underlying improvement of depression. Thirty-two male SD rats were randomly divided into normal control, model, acupuncture and medication (fluoxetine) groups, with 8 rats in each group. The depression model was established by using CUMS (fasting, water depredation, restraint, swimming in cold water, crowding, stroboscope stimulation, and tail clipping, each of which was used once a week) for 6 weeks. Manual acupuncture stimulation was applied to "Baihui"(GV20) and "Yintang"(EX-HN3) for 10 min, once daily, 6 times a week for 6 weeks before every-day modeling. Rats of the medication group received gavage of fluoxetine (10 mg/kg) once a day for 6 weeks before every-day modeling. The percentage of sucrose preference, and the percentages of open arm times and open arm time in the elevated plus maze experiments were detected. The expression levels of AMPK, phosphorylated (p)-AMPK, ULK1, p-ULK1, mammalian target of rapamycin (mTOR) and p-mTOR proteins in the hippocampus tissue were detected by Western blot, and the expression of autophagy effecting protein (Beclin-1) and the selective autophagy receptors (p62) mRNA in the hippocampal tissue was detected using real-time fluorescence quantitative PCR, respectively. Compared with the normal group, the percentage of sucrose preference, percentage of times of entering the open arm and percentage of open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1 /ULK1 ratio of proteins, and Beclin-1 mRNA were significantly decreased (P<0.01, P<0.05), and the expression levels of p-mTOR /mTOR ratio and p62 mRNA were significantly increased (P<0.01, P<0.05) in the model group. In comparison with the model group, the percentage of sucrose preference, percentage of open arm times and open arm time, the expression levels of p-AMPK /AMPK ratio, p-ULK1/ULK1 ratio of proteins, and Beclin-1 mRNA were significantly increased in both acupuncture and medication groups (P<0.01, P<0.05), while the expression levels of p-mTOR /mTOR ratio of proteins, and p62 mRNA were markedly decreased only in the medication group (P<0.05). Acupuncture treatment can alleviate depression-like behavior in CUMS rats, which may be related to its functions in up-regulating AMPK/ULK1 signaling pathway to induce cellular autophagy in the hippocampus.

Banxia xiexin decoction prevents the development of gastric cancer.

In China banxia xiexin decoction (BXD) has been used in treating gastric cancer (GC) for thousands of years and BXD has a good role in reversing GC histopathology, but its chemical composition and action mechanism are still unknown. To investigate the mechanism of action of BXD against GC based on transcriptomics, network pharmacology, in vivo and in vitro experiments. The transplanted tumor model was prepared, and the nude mouse were pathologically examined after administration, and hematoxylin-eosin staining was performed. The active ingredients of BXD were quality controlled and identified using ultra-performance liquid chromatography tandem quadrupole electrostatic field orbitrap mass spectrometry (UPLC-Q-Orbitrap MS/MS), and traditional Chinese medicines systems pharmacology platform, drug bank and the Swiss target prediction platform to predict the relevant targets, the differentially expressed genes (DEGs) of GC were screened by RNA-seq sequencing, and the overlapping targets were analyzed to obtain the key targets and pathways. Cell Counting Kit-8, apoptosis assay, cell migration and Realtime fluorescence quantitative polymerase chain reaction were used for in vitro experiments. All dosing groups inhibited the growth of transplanted tumors in laboratory-bred strain nude, with the capecitabine group and the BXD medium-dose group being the best. A total of 29 compounds and 859 potential targets in BXD were identified by UPLC-Q-Orbitrap MS/MS and network pharmacology, RNA-seq sequencing found 4767 GC DEGs, which were combined with network pharmacology and analyzed 246 potential therapeutic targets were obtained and pathway results showed that BXD may against GC through the Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKt) signaling pathway. In vitro cellular experiments confirmed that BXD-containing serum and LY294002 could inhibit the proliferation of GC cells, promote apoptosis, and inhibit the migration of GC cells by decreasing the expression of EGFR, PIK3CA, IL6, BCL2 and AKT1 in the PI3K-Akt pathway in MGC-803 expression. BXD has the effect of inhibiting tumor growth rate and delaying the development of GC. Its mechanism of action may be related to the regulation of PI3K-Akt signaling pathway.

TRAF4 promotes lung cancer development by activating tyrosine kinase of EGFR

Objective: To explore the role of tumor necrosis factor receptor-associated factor 4 (TRAF4) in promoting the abnormal activation of epidermal growth factor receptor (EGFR) and its effect on lung cancer cell proliferation, migration and invasion. Methods: Tumor tissues from patients who underwent lung adenocarcinoma resection at The First Affiliated Hospital of Second Military Medical University, from January 2015 to May 2017 were collected, and the expressions of TRAF4 and Ki-67 in lung cancer tissues were detected by immunohistochemistry, the mRNA levels of Cyclin D and Vimentin were detected by real-time fluorescence quantitative PCR (qRT-PCR). The effect of TRAF4 on the tumor growth ability of lung cancer A549 cells was investigated by the xenograft model, the effect of TRAF4 or EGFR on the tumor proliferation ability was detected by using cell counting kit 8 (CCK8) and BrdU assay, and the migration and invasion abilities of tumor cells were detected by Transwell assay. Different structural domain deletion expression vectors of TRAF4 and EGFR were constructed to transfect cells, and the interaction mode of TRAF4 and EGFR was investigated by immunoprecipitation assay. Results: The expression of TRAF4 in non-small cell lung cancer (NSCLC) tissues was positively correlated with the expressions of Ki-67, cyclin D, and vimentin (r2: 0.438, 0.695, and 0.736, respectively, all P＜0.01). Immunohistochemical assay of tumor tissues from NSCLC patients showed that tissues with high expression of TRAF4 were also high in Ki-67. Patients with high TRAF4 expression (TRAF4 positivity >30%) had a shorter progression-free survival (PFS) time than that of patients with low TRAF4 expression (TRAF4 positivity ≤30%) (median PFS of 12 and 19 months, respectively; P=0.034). Traf4-/- cells had a weakened proliferative capacity than traf4+/+ cells and formed tumors with smaller size (P＜0.05). The expression level of Ki-67 in the tumor tissues formed by traf4-/- cells [(45.6±8.7)%] was lower than that in the tumor tissues formed by traf4+/+ cells [(62.3±10.3)%, P=0.015], the mRNA levels of cyclin D (1.01±0.15) and vimentin (1.01±0.12) in the traf4-/- cells were lower than those of the traf4+/+ cells (3.41±0.32 and 3.12±0.18, respectively, both P＜0.05).The western blot results showed that, with the elevated intracellular expression level of TRAF4, phosphorylation level of EGFR was significantly increased in both wild-type EGFR and activation mutant EGFR-expression cells. The capacities of proliferation, migration and invasion of A549 cells was weakened after EGFR knockdown (all P＜0.01). Immunoprecipitation experiments showed that TRAF4 binds to the peptide segment of the near-membrane region of EGFR through the TRAF structural domain, and the mutual binding between EGFR molecules was enhanced under TRAF4 overexpression conditions. Increasing TRAF4 expression promoted EGFR molecular phosphorylation and activation of downstream signaling. Conclusions: TRAF4 expression is elevated in NSCLC tissues and tumor cells, which promotes tumor proliferation, migration and invasion. TRAF4 directly binds to EGFR molecules, enhances its own phosphorylation and activates the downstream signaling pathway by promoting the interaction between EGFR molecules.

Berberine Ameliorates High-fat-induced Insulin Resistance in HepG2 Cells by Modulating PPARs Signaling Pathway.

Berberine (BBR), also known as berberine hydrochloride, was isolated from the rhizomes of the Coptis chinensis. Studies have reported that BBR plays an important role in glycolipid metabolism, including insulin (IR). The targets, and molecular mechanisms of BBR against hyperlipid-induced IR is worthy to be further studied. The related targets of BBR were identified via Pharmmapper database and relevant targets of diabetes were obtained through GeneCards and Online Mendelian Inheritance in Man (OMIM) database. The common targets were employed with the STRING database and visualized with the protein-protein interactions (PPI) network. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to explore the biological progress and pathways. In vitro, human hepatocellular carcinomas (HepG2) cell was used as experimental cell line, and an insulin resistant HepG2 cell model (IR-HepG2) was constructed using free fatty acid induction. After intervention with BBR, glucose consumption and uptake in HepG2 cells were observed. Molecular docking was used to test the interaction between BBR and key targets, and real-time fluorescence quantitative PCR was used to detect the regulatory effect of BBR on related targets. 262 overlapped targets were extracted from BBR and diabetes. In the KEGG enrichment analysis, the peroxisome proliferator activated receptor (PPAR) signaling pathway was included. In vitro experiments, BBR can significantly increase sugar consumption and uptake in IR HepG2 cells, while PPAR inhibitors can weaken the effect of BBR on IR-HepG2. The PPAR signaling pathway is one of the important pathways for BBR to improve high-fat-induced insulin resistance in HepG2 cells.

Screening of key genes related to M6A methylation in patients with heart failure

ObjectiveThis study aims to identify m6A methylation-related and immune cell-related key genes with diagnostic potential for heart failure (HF) by leveraging various bioinformatics techniques.MethodsThe GSE116250 and GSE141910 datasets were sourced from the Gene Expression Omnibus (GEO) database. Correlation analysis was conducted between differentially expressed genes (DEGs) in HF and control groups, alongside differential m6A regulatory factors, to identify m6A-related DEGs (m6A-DEGs). Subsequently, candidate genes were narrowed down by intersecting key module genes derived from weighted gene co-expression network analysis (WGCNA) with m6A-DEGs. Key genes were then identified through the Least Absolute Shrinkage and Selection Operator (LASSO) analysis. Correlation analyses between key genes and differentially expressed immune cells were performed, followed by the validation of key gene expression levels in public datasets. To ensure clinical applicability, five pairs of blood samples were collected for quantitative real-time fluorescence PCR (qRT-PCR) validation.ResultsA total of 93 m6A-DEGs were identified (|COR| > 0.6, P < 0.05), and five key genes (LACTB2, NAMPT, SCAMP5, HBA1, and PRKAR2A) were selected for further analysis. Correlation analysis revealed that differential immune cells were negatively associated with the expression of LACTB2, NAMPT, and PRKAR2A (P < 0.05), while positively correlated with SCAMP5 and HBA1 (P < 0.05). Subsequent expression validation confirmed significant differences in key gene expression between the HF and control groups, with consistent expression trends observed across both training and validation sets. The expression trends of LACTB2, PRKAR2A, and HBA1 in blood samples from the qRT-PCR assay aligned with the results derived from public databases.ConclusionThis study successfully identified five m6A methylation-related key genes with diagnostic significance, providing a theoretical foundation for further exploration of m6A methylation’s molecular mechanisms in HF.

Multi-omics reveals lactylation-driven regulatory mechanisms promoting tumor progression in oral squamous cell carcinoma

BackgroundLactylation, a post-translational modification, is increasingly recognized for its role in cancer progression. This study investigates its prevalence and impact in oral squamous cell carcinoma (OSCC).ResultsImmunohistochemical staining of 81 OSCC cases shows lactylation levels correlate with malignancy grading. Proteomic analyses of six OSCC tissue pairs reveal 2765 lactylation sites on 1033 proteins, highlighting its extensive presence. These modifications influence metabolic processes, molecular synthesis, and transport. CAL27 cells are subjected to cleavage under targets and tagmentation assay for accessible-chromatin with high-throughput sequencing, and transcriptomic sequencing pre- and post-lactate treatment, with 217 genes upregulated due to lactylation. Chromatin immunoprecipitation-quantitative PCR and real-time fluorescence quantitative PCR confirm the regulatory role of lactylation at the K146 site of dexh-box helicase 9 (DHX9), a key factor in OSCC progression. CCK8, colony formation, scratch healing, and Transwell assays demonstrate that lactylation mitigates the inhibitory effect of DHX9 on OSCC, thereby promoting its occurrence and development.ConclusionsLactylation actively modulates gene expression in OSCC, with significant effects on chromatin structure and cellular processes. This study provides a foundation for developing targeted therapies against OSCC, leveraging the role of lactylation in disease pathogenesis.

Differential circRNA Screening and Identification of Subcutaneous Fat Tissue in Plateau-type Yaks under Different Feeding Methods

Background: In order to explore the differential expression of circular RNA (circRNA) in the subcutaneous fat tissue of Qinghai yaks under different feeding methods during the dry grass period and its regulatory mechanism. Methods: The present study adopted Illumina sequencing technology to sequence the transcriptome of subcutaneous fat tissue of Qinghai yaks naturally grazed and fattened by housed feeding during the dry grass period and used bioinformatics software to screen out the differentially expressed. The circRNAs were screened for differential expression using bioinformatics software. circRNAs related to fat metabolism were screened by GO function and KEGG pathway enrichment analysis of their source genes and three differentially expressed circRNAs were randomly screened and the expression of the circRNAs was verified by real-time fluorescence quantitative PCR (qRT-PCR). Result: Transcriptome sequencing results showed that a total of 628 differentially expressed circRNAs were obtained, of which 219 were up-regulated and 409 were down-regulated. GO functional enrichment analysis showed that the circRNAs in the free-grazing group originated from genes for major molecular constructs, cellular parts, organs, bioregulation and cellular processes, etc. KEGG pathway enrichment analysis showed that the circRNAs could be participate in signaling pathways such as phagosomes, protein digestion and absorption. qRT-PCR results showed that the expression levels of the three circRNAs were consistent with the sequencing results and the sequencing results were accurate and reliable. This experiment provides a molecular basis for further understanding of fat metabolism in yaks.