Real-time Fluorescence Quantitative PCR Research Articles

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

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.

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.

Hsa_circ_0007718 facilitates the progression of colorectal cancer by regulating the miR-1299/PSMC2 axis

Colorectal cancer (CRC) represents one of the most prevalent forms of malignant tumors, characterized by a notably high rate of mortality among affected individuals. The primary objective of this investigation is to delve into the functional role of Hsa_circ_0007718 in the context of colorectal cancer and to elucidate its impact on the progression of CRC by modulating the interaction between the miR-1299 microRNA and its target gene, PSMC2. To assess the expression levels of Hsa_circ_0007718, along with miR-1299 and PSMC2, real-time quantitative fluorescent PCR (qRT-PCR) assays were meticulously performed using both CRC cell lines and clinical samples derived from patients. A cellular model was established to investigate the interactions occurring between miR-1299 and Hsa_circ_0007718, as well as the connections to PSMC2, thereby providing a comprehensive understanding of these molecular interactions. The findings of this research revealed a significant upregulation of Hsa_circ_0007718 in both colorectal cancer cell lines and tissue samples. Importantly, the data indicated that the suppression of Hsa_circ_0007718 led to a marked decrease in the proliferation rates, migratory potential, and invasive capabilities of CRC cells. Furthermore, the study confirmed that Hsa_circ_0007718 acts as a downstream target of miR-1299, exerting its regulatory effects by inhibiting miR-1299 and thereby promoting the expression of PSMC2.

Identification of neutrophil extracellular traps genes as potential biomarkers in psoriasis based on bioinformatics analysis

The epidermal infiltration of neutrophils is a hallmark of psoriasis (PSO) and its activation leads to the release of neutrophil extracellular traps (NETs). However, the molecular mechanism of NETs-related genes (NETRGs) has not been extensively studied in PSO. To define NETs-related-biomarkers for PSO. The GSE13355 and GSE78097 datasets, and NETRGs gene set were included in this study. The datasets used in this study were all microarray data. The weighted gene co-expression network analysis (WGCNA) and machine learning algorithms were used to mine key genes. Later on, single-gene gene set enrichment analysis (GSEA) and immune infiltration analysis were implemented. Finally, the expression of key genes was verified using quantitative real-time fluorescence PCR (qRT-PCR). A total of 3 key genes (S100A9, CLEC7A, and CXCR4) were derived, and they all had excellent diagnostic performance. The single-gene GSEA enrichment results indicated that the key genes were mainly enriched in the chemokine signaling pathway and humoral immune response in the high-expression group, while focal adhesion was enriched in the low-expression group. The correlation analysis indicated that all key genes were strongly negatively correlated with resting mast cells and TGF-β family member receptor, while they were strongly positively correlated with activated CD4 memory T cells and antigen processing and presentation. Lastly, the experimental results showed that the expression trends of key genes were consistent with public database. In this study, we successfully screened three potential PSO diagnostic genes (S100A9, CLEC7A and CXCR4) that were closely related to NETs, and these findings not only provided new molecular marker candidates for the precise diagnosis of PSO patients, but also revealed possible future therapeutic targets. However, further in-depth research and validation were necessary.

Ruhao Dashi granules exert therapeutic effects on H1N1 influenza virus infection by altering intestinal microflora composition.

Antiviral medications for influenza could be ineffective due to the emergence of resistant influenza virus strains. Ruhao Dashi (RHDS) granules possess anti-inflammatory and antibacterial effects. The present study aimed to determine the efficacy of RHDS granules in treating influenza-infected mice and the mechanism underlying this treatment as well as its effect on the intestinal flora composition of the infected mice. The HPLC-UV method was used to identify the active components of RHDS granules. ICR mice were infected with influenza A virus (IAV) H1N1 subtype through a nasal drip. After the influenza mice model was successfully established, the pathological changes in the lungs were observed for 5 days after gavage treatment with 0.9% sterile saline and low, medium, and high doses (0.07, 0.14, and 0.28 g/mL, respectively) of RHDS granules. The serum levels of the cytokines IL-6 and TNF-α and sIgA were detected by ELISA. Real-time fluorescence quantitative PCR and western blotting assay were performed to determine the expression levels of the tight junction (TJ) proteins claudin-1, occludin, and zonula occludens-1 (ZO-1) in colon tissues. Furthermore, 16S rRNA gene sequencing of feces samples was conducted to assess the effect of RHDS granules on the gut microbiota. RHDS granules exerted a protective effect on the lung tissues of IAV-infected mice; moreover, the granules reduced the synthesis of proinflammatory cytokines and increased the relative expression levels of claudin-1, occludin, and ZO-1 in colon tissues. Furthermore, RHDS granule treatment increased the relative abundance of Lactobacillus, Akkermansia, and Faecalibaculum and decreased the relative abundance of Muribaculaceae; thus, RHDS granules could stabilize the intestinal microbiota to some extent. RHDS granules exert a therapeutic effect on IAV-infected mice probably by modifying the structural composition of their intestinal microbiota.

Comparative study of immune responses and intestinal microbiota in the gut-liver axis between wild and farmed pike perch (Sander Lucioperca).

Pike perch (Sander Lucioperca) is a predatory freshwater fish, which is highly popular amongst consumers, owing to its white flesh with a delicate structure and mild flavor. Compared to wild pike perch, the diet of farmed ones has shifted from natural food to artificial feeds. These changes would affect the gut flora of the pike perch. Endogenous metabolites of the intestinal flora are transferred through the gut-liver axis, which affects the physiological functions of the host. By studying wild and farmed individuals of the pike perch, novel insights into the stability of the intestinal flora can be provided. In this study, we measured various immune parameters in the blood, liver and intestine of wild and farmed pike perch using enzyme activity assays and real-time fluorescence quantitative PCR. Gut microbes were also collected for 16S rRNA gene sequencing. Our results showed that the serum low-density lipoprotein cholesterol (LDL-C) levels were twice as high in the wild group as in the farmed group. Furthermore, the activities of glutamate pyruvate transaminase (GPT) and glutamate oxaloacetate transaminase (GOT) in the intestinal tissues of the wild group were 733.91 U/g and 375.35 U/g, which were significantly higher than those of the farmed group. Expression of IL10 in the liver of farmed pike perch was also 4-fold higher than that of wild pike perch. The expression of genes related to the p53-BAX/Bcl2 signaling pathway was higher in both intestinal and liver tissues of wild pike perch compared with farmed. 16S rRNA gene analysis of the gut microflora showed a high relative abundance of Cetobacterium in the gut of farmed pike perch. As a result, our study indicates that dietary differences affect the diversity, composition and relative abundance of the gut flora of the pike perch. Meanwhile, it affects the glycolipid metabolism and immunomodulation of pike perch.

Relationship between serum level of miR-338-3p and miR-105-3p and bone metabolic markers in patients with diabetes nephropathy

Objectives Diabetic nephropathy (DN) is one of the most common and serious complications of diabetes. The purpose of this study was to explore the relationship between serum microRNA-338-3p (miR-338-3p) and miR-105-3p and bone metabolic markers in patients with DN at different stages. Methods A total of 153 patients diagnosed and treated in the Department of Nephrology from July 2020 to October 2021 were selected as the study objects. According to the staging criteria of diabetic nephropathy and 24-h urinary albumin quantitative level, the patients were divided into control group (35 cases), microalbuminuria group (37 cases), clinical stage albuminuria group (27 cases) and renal failure group (54 cases). Gene expressions were measured by real-time fluorescence quantitative PCR. The correlation was analyzed by Spearman. Serum miR-338-3p and miR-150-5p in the prediction of renal failure in DN was analyzed by ROC curve. Results The levels of urinary albumin and serum creatinine were markedly increased with the increase of DN stage (p < 0.05). Compared with the microalbuminuria group, the expression levels of serum miR-383-3p, serum miR-105-3p, 25(OH)-D, BGP and PINP were obviously decreased, but the expression of parathyroid hormone (PTH) and type I collagen (β-CTX) was largely increased in clinical proteinuria group (p < 0.05). Compared with the clinical proteinuria group, the expression levels of serum miR-383-3p, serum miR-105-3p, 25(OH)-D, BGP and PINP were largely decreased, but the expression of PTH and β-CTX was obviously increased in the renal failure group (p < 0.05). Spearman correlation results showed that serum expressions of miR-383-3p and miR-105-3p were negatively correlated with PTH and β-CTX, and positively correlated with 25(OH)-D, BGP and PINP (p < 0.05). ROC curve analysis showed that the AUC of serum miR-338-3p and miR-150-5p was 0.896 with the specificity and sensitivity of 96.66% and 73.47%, which had certain predictive value for the occurrence of renal failure in DN. Conclusions The expression levels of serum miR-383-3p and miR-105-3p were significantly correlated with bone metabolism markers. The combined test can provide new ideas and insights for the clinical treatment of osteoporosis in DN.

Acute toxicity of salicylic acid and its derivatives on the diatom Phaeodactylum tricornutum: Physico-Biochemical and transcriptomic insights

Salicylate pollutants (SAs) poses a serious threat to marine ecosystems as emerging contaminants. However, the toxic effects of SAs on marine phytoplankton, as well as the potential mechanisms and their ecological risks linked with them, are remain largely unknown. In this study, we aimed to evaluate the toxic effects of salicylic acid (SA) and its 5-substituted derivatives (5-sSA) on the marine diatom Phaeodactylum tricornutum, as well as the potential molecular mechanism involved in the toxicity. Physiological assays conducted on P. tricornutum revealed significant changes in photosynthetic pigments, chlorophyll fluorescence parameters, and antioxidant enzyme activities. The results showed that exposure of P. tricornutum to SAs caused a significant decline in chlorophyll contents and damage to the photosystem II (PSII) core resulting in the decline of photosynthesis. Although the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were enhanced, oxidative damage occurred. Transcriptome analysis showed that a large number of differentially expresses genes (DEGs) were significantly enriched in metabolic pathways such as porphyrin metabolism, terpenoid backbone biosynthesis, and carbon fixation in photosynthetic organisms after SA and 5-BrSA treatments. In addition, key genes in transcriptomic metabolic pathways were further analyzed and validated using weighted correlation network analysis (WGCNA) and real-time fluorescence quantitative PCR (qPCR). Considering the above results, SAs mainly inhibit the processes of photosynthesis by repressing the expression of genes involved in secondary metabolite synthesis and photosynthetic carbon sequestration pathways, thus exerting toxic effects on algal cells. The results of the study will provide key data for understanding the ecological risk and toxicity mechanisms of SA pollutants.

Xiangshao Granules Ameliorate Post-Stroke Depression by Inhibiting Activation of Microglia and IDO1 Expression in Hippocampus and Prefrontal Cortex.

To investigate the therapeutic effect of Xiangshao Granules (XSG) on post-stroke depression (PSD) and explore the underlying mechanisms. Forty-three C57BL/6J mice were divided into 3 groups: sham (n=15), PSD+vehicle (n=14), and PSD+XSG (n=14) groups according to a random number table. The PSD models were constructed using chronic unpredictable mild stress (CUMS) after middle cerebral artery occlusion (MCAO). The sham group only experienced the same surgical operation, but without MACO and CUMS stimulation. The XSG group received XSG (60 mg/kg per day) by gavage for 4 weeks. The mice in the sham and vehicle groups were given the same volume of 0.9% saline at the same time. The body weight and behavior tests including open field test, sucrose preference test, tail suspension test, and elevated plus-maze test, were used to validate the PSD mouse model. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and immunofluorescence staining were used to evaluate the anti-inflammatory effects of XSG. The potential molecular mechanisms were explored and verified through network pharmacology analysis, Nissl staining, Western blot, ELISA, and RT-qPCR, respectively. The body weight and behavior tests showed that MCAO combined with CUMS successfully established the PSD models. XSG alleviated neuronal damage, reduced the expressions of pro-apoptotic proteins Caspase-3 and B-cell lymphoma-2 (BCL-2)-associated X (BAX), and increased the expression of anti-apoptotic protein BCL-2 in PSD mice (P<0.05 or P<0.01). XSG inhibited microglial activation and the expressions of pro-inflammatory cytokines including tumor necrosis factor-α, interleukin (IL)-1 β, and IL-6 via the toll-like receptor 4/nuclear factor kappa-B signaling pathway in PSD mice (P<0.05 or P<0.01). Furthermore, XSG decreased the expression of indoleamine 2,3-dioxygenase1 (IDO1) and increased the concentration of 5-hydroxytryptamine in PSD mice (P<0.05 or P<0.01). XSG could reverse the anxiety/depressionlike behaviors and reduce the neuronal injury in the hippocampus and prefrontal cortex of PSD mice, which may be a potential therapeutic agent for PSD.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract Background In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.). Methods We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA). Results The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1). Conclusions The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.

Expression of TLR7/8 in canine sperm and evaluation of the effect of ligand R848 on the sorting of canine X/Y sperm

The aim of this study was to analyze the expression pattern of Toll receptor 7/8 (TLR7/8) in canine sperm, and explore the feasibility of using TLR7/8 ligand resiquimod(R848)to separate canine X and Y sperm. In this study, cellular immunofluorescence was used to analyze the expression of TLR7/8 in canine sperm, real-time fluorescence quantitative PCR was used to calculate the proportion of X sperm in the lower layer of the incubation solution with R848 to evaluate the sorting effect of R848 on canine X/Y sperm, and sperm quality detection system was used to analyze the effect of R848 on the motility of canine sperm. The mechanism of effect of R848 on canine sperm motility was analyzed by Western blot. The results showed that TLR8 was not expressed in all canine sperm, while TLR7 was expressed in all canine sperm and was localized in the head and tail of sperm. When 0.4 μM R848 was incubated with canine sperm for 1 h, the total motility, average path velocity (VAP), average straight-line velocity (VSL), and average curved-line velocity (VCL) of canine sperm were significantly decreased(P<0.05). There was no significant difference between the lower and upper layers of the R848 treatment group and the control group(P>0.05), and the proportion of X sperm was nearly half. The levels of NF-κB and GSK3α/β phosphorylation of sperm in R848 treatment group were significantly increased compared with control group(P<0.05). The above results showed that TLR7/8 was not differentially expressed in canine X and Y sperm. R848 could decrease the motility of canine spermatozoa and inhibit sperm motility by the GSK3α/ β-hexokinase pathway through the phosphorylation of NFκB and GSK3α/β, while could not separate X and Y spermatozoa. The method of sorting X/Y sperm based on TLR7/8 is not feasible for dogs.

Screening and analysis of GULP1 downstream target genes based on transcriptomic sequencing.

GULP1 is an engulfment adaptor protein containing a phosphotyrosine-binding (PTB) domain, and existing studies have shown that it can promote glucose uptake in 3T3-L1 adipocytes. To further explore key metabolically related differential genes downstream of GULP1, this study conducted transcriptome analysis on adipocytes and skeletal muscle cells overexpressing GULP1. Subsequently, abnormally expressed genes were subjected to bioinformatic analysis, and real-time fluorescent quantitative PCR (qRT-PCR) was used for mutual validation with transcriptome sequencing. The results indicated that, with a threshold of P < 0.05 and |Log2FoldChange| ≥ 1 for screening differentially expressed genes, compared with control cells, there were 278 upregulated and 263 downregulated genes in adipocytes overexpressing GULP1. Metabolism-related GO (Gene Ontology) terms included cholesterol biosynthetic process, cholesterol metabolic process, response to lipopolysaccharide, lipid metabolic process, etc. A total of 52 metabolically related differentially expressed genes were enriched in 10 KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways, with lipid metabolism being highly enriched. In skeletal muscle cells overexpressing GULP1, there were 280 upregulated and 302 downregulated genes, with metabolism-related GO terms including hormone metabolic process, response to lipopolysaccharide, one-carbon metabolic process, etc. A total of 86 metabolically related differentially expressed genes were enriched in 10 KEGG pathways, with amino acid metabolism, lipid metabolism, and carbohydrate metabolism being highly enriched. GULP1's biological functions are extensive, including lipid metabolism and oncology. This study, through transcriptomics and bioinformatic analysis, identified key metabolically related differential genes downstream of GULP1, obtained metabolically related differential genes and signaling pathways after GULP1 overexpression, providing important theoretical basis for future research on GULP1 downstream target genes.

Detection of Porcine Circovirus (PCV) Using CRISPR-Cas12a/13a Coupled with Isothermal Amplification.

The impact of porcine circovirus (PCV) on the worldwide pig industry is profound, leading to notable economic losses. Early and prompt identification of PCV is essential in managing and controlling this disease effectively. A range of detection techniques for PCV have been developed and primarily divided into two categories focusing on nucleic acid or serum antibody identification. The methodologies encompass conventional polymerase chain reaction (PCR), real-time fluorescence quantitative PCR (qPCR), fluorescence in situ hybridization (FISH), loop-mediated isothermal amplification (LAMP), immunofluorescence assay (IFA), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA). Despite their efficacy, these techniques are often impeded by the necessity for substantial investment in equipment, specialized knowledge, and intricate procedural steps, which complicate their application in real-time field detections. To surmount these challenges, a sensitive, rapid, and specific PCV detection method using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas12a/13a coupled with isothermal amplification, such as enzymatic recombinase amplification (ERA), recombinase polymerase amplification (RPA), and loop-mediated isothermal amplification (LAMP), has been developed. This novel method has undergone meticulous optimization for detecting PCV types 2, 3, and 4, boasting a remarkable sensitivity to identify a single copy per microliter. The specificity of this technique is exemplary, with no observable interaction with other porcine viruses such as PEDV, PRRSV, PRV, and CSFV. Its reliability has been validated with clinical samples, where it produced a perfect alignment with qPCR findings, showcasing a 100% coincidence rate. The elegance of merging CRISPR-Cas technology with isothermal amplification assays lies in its on-site testing without the need for expensive tools or trained personnel, rendering it exceptionally suitable for on-site applications, especially in resource-constrained swine farming environments. This review assesses and compares the process and characteristics inherent in the utilization of ERA/LAMP/RPA-CRISPR-Cas12a/Cas13a methodologies for the detection of PCV, providing critical insights into their practicality and effectiveness.