Fluorescence Quantitative PCR Research Articles

Evaluation of the Efficacy of the Vaccine Production Process in Removing Residual Host Cell DNA from the Vero Cell Rabies Vaccine

Background: The Vero cell rabies vaccine is currently the most widely used human rabies vaccine. However, owing to the presence of residual host cell DNA (HCD) in the final product and the potential tumorigenicity of the DNA of high-passage Vero cells, the WHO not only sets a limit on the number of times cells used in production can be passaged, but also imposes strict requirements on the amount of residual HCD in the final vaccine product. Objectives: To systematically reduce the HCD level in the final vaccine product, multiple purification steps are included in the vaccine production process. This study investigated the effectiveness of key production steps in antigen recovery and DNA removal. Methods: The residual HCD fragment content and size distribution were detected using fluorescence quantitative PCR (qPCR) and capillary gel electrophoresis (CGE), and the rabies virus glycoprotein antigen content was detected using enzyme-linked immunosorbent assay (ELISA). The antigen recovery rate and HCD removal rate in each key process were calculated to evaluate the scientific basis and effectiveness of each production step. Additionally, the stability of the process was studied using multiple commercial batches of the product. Results: The results revealed that the total antigen recovery rate in the production process described in this report was no less than 8.5%, and the effective removal rate of residual HCD was not lower than 99.99%. Moreover, the amount of residual HCD in the final product was far below the quality standard of 2 ng/dose, and most of the residual HCD fragments were smaller than 200 bp. The results of the process stability studies on multiple commercial batches showed that the bulk human rabies vaccine produced by this process had excellent safety and efficacy and that the production process was stable and thus suitable for large-scale batch production. Conclusions: The production process described in this study achieved effective recovery of viral antigens and efficient removal of residual HCD, and the process was stable and controllable, enabling the continuous and stable production of vaccine products that meet WHO recommendations and the relevant requirements of the current edition of the Chinese Pharmacopeia. In addition, this study provides theoretical guidance for optimizing the vaccine production process.

3D printed β-TCP scaffolds loaded with SVVYGLR peptide for promoting revascularization and osteoinduction

It is crucial for the successful transplantation of large segmental bone defects to achieve rapid vascularization within bone scaffolds. However, there are certain limitations including uncontrolled angiogenesis and inadequate vascular function. Therefore, there is an urgent need to develop bone scaffolds with functional vascular networks. In our study, porousβ-tricalcium phosphate (β-TCP) scaffolds with varying pore sizes were prepared by 3D printing technology, loaded with osteopontin derived peptide Ser-Val-Val-Tyr-Gly-Leu-Arg (SVVYGLR) to induce osteoinduction and angiogenesis.In vitro, the proliferation and migration behaviors of human umbilical vein endothelial cell on scaffolds were assessed by Cell Counting Kit-8, confocal laser scanning microscopy and scanning electron microscopy. And the osteogenic ability of bone marrow mesenchymal stem cells was assessed using alkaline phosphatase staining and Alizarin Red S staining. The messenger ribonucleic acid (mRNA) expression levels of cell adhesion molecule (CD31), vascular endothelial growth factor and hypoxia inducible factor-1αin each group were detected by quantitative real-time fluorescence polymerase chain reaction (PCR) analysis.In vivo, cube scaffolds were subcutaneously implanted on the right hips of Sprague-Dawley (SD) rats for 6 weeks. Hematoxylin and Eosin staining, Masson's trichrome staining, and immunohistochemical analysis of osteocalcin and CD31 were performed on slices for every sample with three sections to explore the effect of SVVYGLR-loaded scaffolds on angiogenesis and osteogenic induction for bone reconstruction. The results indicate that 3D printedβ-TCP scaffolds loaded with the SVVYGLR peptide offer superior revascularization and osteoinduction to the scaffolds without the SVVYGLRin situ. Moreover, scaffolds with a pore size of 400 µm demonstrate higher effectiveness compared to those with a 150 µm pore size. The distinct hollow channel scaffolds and the specific SVVYGLR peptide substantially improve cell adhesion, spreading, and proliferation, as well as promote angiogenesis and bone formation. Furthermore, scaffolds with a pore size of 400 µm may exhibit greater efficacy compared to those with a pore size of 150 µm. The results of this study provide an idea for the development of practical applications for tissue-engineered bone scaffolds.

Investigating the role of oviductal mucosa-endometrial co-culture in modulating factors relevant to embryo implantation.

Intrauterine adhesions (IUAs) are a significant clinical challenge, affecting reproductive health and leading to infertility or recurrent pregnancy loss. Understanding the molecular mechanisms underlying IUA prevention is crucial for developing effective treatment strategies. To investigate the interaction between oviductal mucosal cells and endometrial cells and their effects on the expression of key molecules involved in embryo implantation, specifically leukemia inhibitory factor (LIF), avβ3, estrogen receptor (ER), and progesterone receptor (PR). Tubal mucosa and endometrium specimens were collected from 22 patients undergoing surgical interventions. Cells were cultured alone and co-cultured at ratios of 1:1, 1:0.5, and 1:0.1. LIF, avβ3, ER, and PR expression levels were measured using real-time fluorescence quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. Our results demonstrated that LIF expression was significantly augmented in co-culture conditions, particularly in the 1:1 ratio, compared to oviductal mucosa monoculture (P < 0.05). Although LIF expression was also elevated in 1:0.5 and 1:0.1 co-culture ratios, these increases were not statistically significant (P > 0.05). For avβ3, increased expression was observed in the 1:1 co-culture group (P < 0.05), but no significant differences were detected in 1:0.5 and 1:0.1 co-culture groups. ER expression showed a downward trend in co-culture, but without statistical significance (P > 0.05), and PR expression remained stable across all groups. Co-culture modulates key molecules involved in embryo implantation, particularly LIF and avβ3. These findings highlight the potential roles of LIF and avβ3 in IUA prevention strategies and provide important insights for future clinical interventions. Tubal mucosal cells can not only grow in the endometrial cell microenvironment, but also the tolerance of tubal mucosal cells can be improved when they are co-cultured.

Relationship between the risk of intestinal mucosal Epstein–Barr virus and/or cytomegalovirus infection and peripheral blood NK cells numbers in patients with ulcerative colitis: a cross-sectional study in Chinese population

ObjectiveThis study aimed to analyze the relationship between the risk of common opportunistic pathogens Epstein–Barr virus (EBV) and cytomegalovirus (CMV) infection in intestinal mucosal tissues of Ulcerative Colitis (UC) patients and the number of peripheral blood NK cells.MethodsUC patients admitted to a third-grade class-A hospital from January 2018 to December 2023 were selected as research population. Clinical data of the patients were collected from the electronic medical record system. Additionally, samples of intestinal mucosal tissues were obtained for real-time fluorescence quantitative PCR to detect and analyze the viral load of CMV and EBV. Blood samples were collected for lymphocyte subsets analysis. Multivariable logistic regression models analyses was used to determine the odds ratio (OR) and 95% confidence interval (95% CI) for the independent association between NK cells and EBV/CMV infections in UC. We further applied the restricted cubic spline analysis and smooth curve fitting to examine the non-linear relationship between them.Results378 UC patients were enrolled. Of these patients, there were 194 patients (51.32%) with EBV /CMV infection. In multivariable logistic regression analyses NK cells was independently associated with EBV and/or CMV infection after adjusted potential confounders (OR 8.24, 95% CI 3.75–18.13, p &amp;lt; 0.001). A nonlinear relationship was found between NK cells and EBV/CMV infections, which had a threshold around 10.169. The effect sizes and CIs below and above the threshold were 0.535 (0.413–0.692), p &amp;lt; 0.001 and 1.034 (0.904–1.183), p &amp;gt; 0.05, respectively.ConclusionThere was a non-linear relationship between NK cells and EBV/CMV infections. The risk for EBV/CMV infections was not increased with increasing NK cells in individuals with NK cells ≥ 10.169%, whereas the risk for EBV and/or CMV infection was increased with an decreasing NK cells in those with NK cells &amp;lt; 10.169%. The risk of EBV/CMV infections increases when NK cells were below a certain level.

Inhibitory effect of Zhujing Pill on myopia progression: Mechanistic insights based on metabonomics and network pharmacology.

This study endeavored to uncover the mechanisms by which Zhujing pill (ZJP) slows myopia progression. We employed biometric analyses to track diopter and axial length changes in guinea pigs with negative lens-induced myopia (LIM). Through integrating metabonomics and network pharmacology, we aimed to predict the anti-myopic targets and active ingredients of ZJP. Subsequent analysis, including real-time fluorescent quantitative PCR (qPCR) and Western blotting (WB), assessed the expression levels of CHRNA7, LPCAT1, and NOS2 in retinal tissues. Our findings demonstrate that ZJP significantly mitigates diopter increase and axial elongation in LIM guinea pigs. Metabonomic analysis revealed significant changes in 13 serum metabolites, with ZJP reversing the expression of 5 key metabolites. By integrating metabonomics with network pharmacology, we identified core targets of ZJP against myopia and constructed a compound-gene-disease-metabolite network. The expressions of LPCAT1 and CHRNA7 were found to decrease in the LIM group but increase with ZJP treatment, whereas NOS2 expression showed the opposite pattern. This investigation provides the first evidence of ZJP's multifaceted effectiveness in managing myopia, highlighting its impact on multiple components, targets, and pathways, including the novel involvement of LPCAT1 and CHRNA7 in myopia pathogenesis.

Xuebijing Exerts Protective Effects on Myocardial Cells by Upregulating TRIM16 and Inhibiting Oxidative Stress and Apoptosis.

This study utilized transcriptomic sequencing combined with cellular and animal models to explore the potential mechanisms of Xuebijing in treating sepsis-induced myocardial dysfunction, also known as sepsis-induced myocardial injury. We investigated potential targets and regulatory mechanisms of XBJ injection using network pharmacology and RNA sequencing. The effects of XBJ on oxidative stress and apoptosis levels in human cardiac myocytes (AC16) and C57BL/6 mice exposed to lipopolysaccharide (LPS) were evaluated by Enzyme-Linked Immunosorbent Assay (ELISA), fluorescent probe, Fluorescent Quantitative Polymerase Chain Reaction (qPCR), Western Blot, Transmission Electron Microscopy, oxidative stress-related indicators detection kit, flow cytometry, and Immunohistochemistry (IHC). First, it was verified that XBJ can reduce the deformation of AC16 cardiomyocytes induced by LPS and the production and secretion of ROS (P <0.01). The transcriptome sequencing results showed that the TRIM16 gene was significantly increased after XBJ treatment, and the data of KEGG and GO analyses demonstrated that XBJ could inhibit the pathway expression of oxidative stress damage in AC16 cells, and PCR verified that XBJ could indeed increase the expression level of TRIM16 gene in AC16 cells (P <0.01). Basic animal and cell experiments showed that LPS could inhibit the expression of TRIM16 and NRF2 in cardiomyocytes (P <0.05) and promote the expression of Keap1 (P <0.01), while XBJ could significantly upregulate the expression levels of TRIM16 and NRF2 (P <0.01) and inhibit the expression of Keap1 (P <0.01), thereby affecting the expression levels of downstream proinflammatory cytokines and alleviating LPS-induced oxidative stress damage. In addition, XBJ also inhibited the expression of the pro-apoptotic proteins Bax and c-caspase3 (P <0.01), promoted the expression of the anti-apoptotic protein Bcl2 (P <0.01), and reduced LPS-induced apoptosis by upregulating TRIM16. Our comprehensive data demonstrated that TRIM16 is a key gene in the therapeutic action of Xuebijing in sepsis-induced myocardial dysfunction, protecting myocardial cells from injury through antioxidative stress and anti-apoptotic mechanisms.

Transcriptome-based analysis reveals the toxic effects of perfluorononanoic acid by affecting the development of the cardiovascular system and lipid metabolism in zebrafish.

Perfluorononanoic acid (PFNA) is a perfluoroalkyl acid containing nine carbon chains, with an additional carbon‑fluorine bond that makes it more stable and toxic. Studies have shown that PFNA can harm the reproductive, immune, and nervous systems, as well as many organs, which can increase the risk of cancer. In this study, zebrafish embryos were treated with 0 and 100 μM PFNA for 72 and 96 hpf, and their angiogenesis and haematopoiesis were observed under laser confocal microscopy using Tg (fli1:EGFP) and Tg (gata1:DsRed) transgenic zebrafish. The data showed that PFNA exposure decreased heart rate and slowed blood flow in zebrafish. PFNA was found to inhibit erythropoiesis by O-dianisidine staining. RNA-seq analysis was used to compare gene expression changes in zebrafish from control and 100 μM PFNA-exposed groups at 72 hpf. KEGG results showed significant enrichment of PPAR signaling pathway, fatty acid metabolism, steroid biosynthesis and apoptosis. The RNA-seq results were validated by real-time fluorescence quantitative PCR (RT-qPCR). Oil red O staining and Filipin staining showed increased lipid accumulation after PFNA exposure, and TUNEL staining showed that PFNA exposure led to apoptosis. In conclusion, exposure to PFNA may cause toxic effects in zebrafish by affecting cardiovascular development, causing lipid accumulation and promoting apoptosis.

Study on the Expression and Potential Function of LncRNA in Peripheral Blood of Patients with Ankylosing Spondylitis.

Ankylosing spondylitis (AS) is an autoimmune disease that has the characteristics of difficult early diagnosis and a high disability rate. The objective of this study was to further explore the possible mechanism and potential function of lncRNA in AS. We used lncRNA microarray technology to detect the expression of lncRNA and mRNA in patients with active AS, stable patients, and healthy controls (HC). Afterward, bioinformatics analysis was conducted on differentially expressed genes. Seven differentially expressed lncRNAs were screened out for real-time fluorescent quantitative PCR (RT-qPCR), combined with various clinical indicators for correlation analysis, and the receiver operating characteristic (ROC) curve was used to analyze the potential of lncRNA as a diagnostic marker for AS. The results showed that the expression levels of NR-037662 and ENST00000599316 in the AS subgroups were significantly higher than those in the HC group, while the expression levels of ENST00000577914 and ENST00000579003 were lower than those in the HC group. The expression levels of NR-003542 and ENST00000512051 in the ASA group were significantly higher than those in the ASS and HC groups, while NR-026756 was just the opposite. Spearman's correlation analysis showed that the expression level of NR-003542 was positively correlated with Bath Ankylosing Spondylitis Functional Index (BASFI), Erythrocyte Sedimentation Rate (ESR), and high sensitivity C-Reactive Protein (hsCRP). The expression level of NR-026756 was negatively correlated with the Bath Ankylosing Spine Inflammatory Disease Activity Index (BASDAI), BASFI, ESR, hsCRP, and globulin (GLOB). In addition, it was also found that the ROC curve analysis of the 4 lncRNAs between the AS group (ASA group and ASS group) and the HC group were statistically significant, and the area under the curve (AUC) of NR-037662, ENST00000599316, ENST00000577914, and ENST00000579003 was 0.804, 0.812, 0.706, and 0.698, respectively. It was found that these differentially expressed lncRNAs of AS may be involved in the occurrence and development of the disease. Among them, NR-037662, ENST00000599316, ENST00000577914, and ENST00000579003 might have the potential to become AS diagnostic molecular markers. Moreover, NR -003542, ENST00000512051, and NR-026756 might have the potential to be indicators of disease activity.

Curculigoside upregulates BMAL1 to decrease nucleus pulposus cell apoptosis by inhibiting the JAK/STAT3 pathway

BackgroundIntervertebral disc degeneration (IVDD) is a natural process that occurs with aging and is the main cause of low back pain (LBP). Basic helix-loop-helix ARNT-like 1 (BMAL1) plays key roles in the pathogenesis of many diseases. The present study investigates the role of curculigoside (CUR), which has been reported to be a potential anti-apoptotic compound in other diseases. MethodsDysregulated genes were identified by RNA sequencing (RNA-seq). Western blotting (WB), immunohistochemistry (IHC), immunofluorescence (IF) staining, and real-time fluorescent quantitative PCR were used to detect BMAL1 expression in 25 human intervertebral disc (IVD) specimens (male: female =13:12), tissues from BMAL1-knockout mice and from an IVDD mouse model. The regulatory effects of CUR and BMAL1 in NP cells after siRNA transfection were examined by flow cytometry, IF staining and WB. The therapeutic effect of intraperitoneal CUR injection was also evaluated in mice. ResultsBMAL1 expression was negatively correlated with IVDD severity and was significantly lower in degenerative NP cells. After BMAL1 knockdown using siRNA, the apoptosis rate of degenerative NP cells was significantly higher, while transfection with a lentivirus overexpressing BMAL1 exerted the opposite effect. Bioinformatics analysis revealed that BMAL1 is regulated by the JAK-STAT3 pathway, and CUR upregulated BMAL1 expression by inhibiting STAT3 phosphorylation, subsequently alleviating NP cell apoptosis and increasing extracellular matrix (ECM) components., thus alleviating IVDD. ConclusionsCUR can inhibit apoptosis and improve the ECM by upregulating BMAL1 expression, which is reduced in IVDD. This study provides a therapeutic strategy to alleviate apoptosis associated with inflammation-induced IVDD.

Etiological analysis of acute respiratory infections in hospitalized children after the relaxation of COVID-19 non-pharmacological interventions in Quzhou, China

BackgroundAcute respiratory infections (ARIs) can cause morbidity and mortality in children. This study was to determine the characteristics of pathogens in hospitalized children with ARIs after the relaxation of COVID-19 non-pharmacological interventions (NPIs) in Quzhou, China.MethodsHospitalized children with ARIs were enrolled between May and October 2023, and thirteen common respiratory pathogens were tested by fluorescent quantitative polymerase chain reaction. Mono- and co-infections were assessed, and the association between pathogens and age was explored using restricted cubic spline analysis.ResultsA total of 1225 children were included, 820 of them detected one pathogen and 238 of them detected two or more pathogens. The dominant pathogen varies monthly. Mycoplasma pneumoniae (Mp) was the most common pathogen in monoinfection, followed by respiratory syncytial virus (RSV) and human rhinovirus (HRV), while influenza virus was detected at a lower rate. Mp + HRV was the most common combination of coinfections. The detection rates of Mp and HRV were higher in coinfections than in monoinfection, but there was no difference in the detection rate of RSV. Children aged 1–3 years had the highest positive detection rates and were more likely to be infected with multiple pathogens, with 40% of respiratory pathogen monoinfection and 47.48% of coinfections (χ2 = 4.245, P = 0.039). In the restricted cubic spline models, a J-shaped association was consistently observed between age and Mp infection, the risk of HRV first increased and then decreased, the risk of RSV was relatively flat until 1.5 years and then decreased rapidly.ConclusionOur study revealed the epidemiological characteristics of ARIs pathogens after the relaxation of NPIs. The positivity rates of Mp, RSV, and HRV are the highest, while those of influenza virus are still low. Additionally, age and season affect the distribution of respiratory pathogens. These findings underscore the importance of ongoing regional pathogen surveillance to guide local public health responses.

Identification of Antisense RNA NRAS-AS and Its Preliminary Exploration of the Anticancer Regulatory Mechanism

Objective: To explore the influence of NRAS-AS on the proliferation, apoptosis, cell cycle, migration, and invasion ability of HCC cells, as well as its underlying mechanisms. Methods: A double-stranded cDNA library for liver cancer cells was constructed, and identified NRAS-AS through High-throughput sequencing, bioinformatics, chain-specific fluorescent quantitative PCR, and RACE. NRAS-AS′s effects on HepG2 and SMMC-7721 cells and gene expression were evaluated. Additionally, the study analyzed the influence of NRAS-AS overexpression on tumor formation in nude mice. Immunohistochemistry and Western blotting were used to detect NRAS protein levels in clinical samples. RT-qPCR examined NRAS-AS and NRAS gene expression in HCC and adjacent tissues. Results: NRAS-AS overexpression suppresses HCC cell proliferation and invasion, induces cell cycle alterations in HepG2 and SMMC-7721 cells, and enhances apoptosis. NRAS-AS interference promoted liver cancer invasion, inhibited apoptosis, and influences the cell cycle. Nude mice overexpressing NRAS-AS showed smaller tumors. NRAS-AS expression in liver cancer patients correlated with clinical factors. RT-qPCR revealed an inverse correlation between NRAS-AS and NRAS gene expression in liver cancer and adjacent tissues. IHC analysis revealed reduced NRAS protein expression in HepG2 and SMMC-7721 cells following NRAS-AS overexpression. The impact of AZA treatment on antisense NRAS-AS and sense NRAS gene expression in liver cancer cells was observed, and antisense. Conclusion: Reduced NRAS-AS expression is frequently observed in HCC and is inversely related to NRAS gene expression, suggesting a role in HCC pathogenesis through NRAS regulation. Targeting antisense RNA NRAS-AS could hold promise as a therapeutic target and diagnostic biomarker for HCC.

SCGB1D4 Downregulation Links to Fibrosis in Intrauterine Adhesion Patients and Rat Models.

Intrauterine adhesions (IUA) represent a prevalent uterine endometrial disorder frequently correlated with menstrual irregularities and infertility. This study aims to examine the expression of SCGB1D4 in IUA tissues and assess its potential therapeutic implications by analyzing clinical features and constructing rat and cell models. Clinical characteristics of patients with intrauterine adhesions were compared and analyzed against control subjects. Additionally, a rat uterine adhesion model was successfully established using a combination of mechanical injury and infection. The expression levels of SCGB1D4 in patient tissues and animal models were detected through immunohistochemistry, Western blot, and real-time fluorescence quantitative PCR, and the changes in fibrosis markers COL1A1 and α-SMA were also evaluated. Furthermore, human endometrial stromal cell lines (HESCs) induced by transforming growth factor-β-1 conversion were differentiated into myofibroblasts to establish cell models of intrauterine adhesion. We detected the expression of SCGB1D4 and fibrosis-related factors by real-time fluorescence quantitative PCR and Western blot. Cell proliferation and cell cycle changes were assessed using flow cytometry and CCK8. IUA patients showed increased miscarriage rates and decreased endometrial thickness. Clinical tissue specimens revealed significantly lower expression of SCGB1D4 in the endometrial tissues of IUA patients, accompanied by a notable increase in COL1A1 and α-SMA. The established rat model of intrauterine adhesion exhibited decreased expression of SCGB1D4 and a significant increase in fibrosis. After overexpression of SCGB1D4 on the IUA cell model, SCGB1D4 expression was elevated, while COL1A1 and α-SMA expression was significantly reduced. Cell proliferation was inhibited and cell cycle distribution was altered. This study has confirmed the low expression of SCGB1D4 in patients with intrauterine adhesions (IUA), as well as in animal and cell models. Furthermore, the overexpression of SCGB1D4 in a cell model of IUA demonstrates that it may play a key role in inhibiting fibrosis. SCGB1D4 holds promise as a potential therapeutic target for IUA, providing a new avenue for overcoming fertility issues caused by intrauterine adhesions.

One-Step Multiplex Real-Time Fluorescent Quantitative Reverse Transcription PCR for Simultaneous Detection of Four Waterfowl Viruses

Duck Tembusu virus (DTMUV), duck hepatitis virus (DHV), Muscovy duck reovirus (MDRV), and Muscovy duck parvovirus (MDPV) represent four emergent infectious diseases impacting waterfowl, which can be challenging to differentiate due to overlapping clinical signs. In response to this, we have developed a one-step multiplex real-time fluorescence quantitative reverse transcription PCR (qRT-PCR) assay, capable of simultaneously detecting DTMUV, DHV, MDRV, and MDPV. This method exhibits high specificity, avoiding cross-reactivity with other viruses such as Fowl adenoviruses (FADV), infectious bursal disease virus (IBDV), infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), Haemophilus paragallinarum (Hpg), duck circovirus (DUCV), goose astrovirus (GoAstV), and mycoplasma gallisepticum (MG). The limit of detection (LOD) established for DTMUV, DHV, MDRV, and MDPV was determined to be 27 copies/μL. In the repeatability test, the intra-assay and inter-assay coefficients of variation (CVs) of the recombinant plasmid standard were less than 2%. Utilizing this method, we analyzed 326 clinical specimens sourced from Guangxi over the period spanning October 2021 through December 2023, yielding promising and precise outcomes. The qRT-PCR method established herein exhibits commendable specificity, sensitivity, and repeatability. Furthermore, it boasts a high clinical detection rate, making it a highly effective tool for diagnosing these pathogenic agents in waterfowl.

Acupoint injection inhibiting abnormal secretion of mucin and inflammatory reaction of nasal mucosa in rats with allergic rhinitis through the p38 mitogen-activated protein kinase pathway

To observe the effects of acupoint injection on the expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated (p)-p38 MAPK, mucin 5 subtype AC (MUC5AC) in the nasal mucosa, and serum inflammatory factors of rats with allergic rhinitis, so as to explore the mechanism of acupoint injection in improving inflammatory reactions in the nasal mucosa of rats with allergic rhinitis. SD rats were randomly divided into the normal group, model group, acupoint injection group, and non-acupoint group, with 7 rats in each group. The allergic rhinitis rat model was established using ovalbumin sensitization. Intervention was performed by injecting dexamethasone and 1% lidocaine mixture (0.05 mL) into the bilateral "Yingxiang"(LI20) and "Yintang"(GV24+) acupoints, with injection at non-acupoints for non-acupoint group as the control, once every 3 days for a total of 4 times. Allergic symptoms in the rat nose were evaluated using a cumulative scoring method；histopathological changes in the nasal mucosa were observed after HE staining；serum histamine (HA), interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α) contents were detected using ELISA；real-time fluorescent quantitative PCR was used to detect the expression of MUC5AC mRNA in the nasal mucosa；Western blot was used to detect the expression of p38 MAPK, p-p38 MAPK, and MUC5AC protein in the nasal mucosa. Compared with the normal group, nasal allergic symptom score, serum HA, IL-6, IL-8, and TNF-α contents, nasal mucosal p38 MAPK, p-p38 MAPK proteins expression, MUC5AC mRNA and protein expression were significantly increased(P<0.01) in the model group. Compared with the model group and non-acupoint group, nasal allergic symptom score, serum HA, IL-6, IL-8, and TNF-α contents, nasal mucosal p38 MAPK and p-p38 MAPK proteins expression, MUC5AC mRNA and protein expression were significantly reduced (P<0.01, P<0.05) in the acupoint injection group. Acupoint injection can reduce nasal allergic symptom, and MUC5AC secretion in rats with allergic rhinitis, alleviate nasal mucosal inflammatory reactions, and its mechanism of action may be related to the inhibition of p38 MAPK phosphorylation, thereby reducing the release of inflammatory factors.

Effect of WW-domain transcription regulator 1 on aging regulation of human dental pulp stem cells

Objective: Investigating the changes of phenotype and moleculars associated with aging with the increase of passage times of human dental pulp stem cells (hDPSC), to explore the role of WW-containing transcriptional regulator 1 (WWTR1) in the aging mechanism. Methods: hDPSCs were cultured by tissue block method, and were divided into 4 groups according to the age, algebra, cell knockdown and overexpression of WWTR1 in hDPSCs. Group I: hDPSCs from human teeth were further divided into youth group (15-25 years old) and group middle-aged group (40-50 years old)] according to different ages. Group Ⅱ: according to different passage, hDPSCs were divided into young cells group (hDPSCs were transmitted to P3 generation), and old cells group (hDPSCs were transmitted to P10 generation). Group Ⅲ: hDPSCs were knocked down of WWTR1, which were further divided into knockdown group (sh-WWTR1) and knockdown carrier group (sh-NC). Group Ⅳ: hDPSCs were overexpressed of WWTR1, which were further divided into overexpression group (OE-WWTR1) and overexpression carrier group (OE-NC). Real-time quantitative fluorescent PCR (RT-qPCR) was used to detect the changes of WWTR1 expression in groups Ⅰ and Ⅱ, and cell counting kit-8 (CCK-8) was used for groups Ⅱ, Ⅲ, and Ⅳ. Cell proliferation capacity was detected by CCK-8 assay. The ability of osteogenic differentiation was detected by alizarin red staining. Cell senescence positive rate was detected by age-related β-galactosidase staining. The expression levels of age-related genes p53 and p21 were detected by RT-qPCR. Results: The proportion of senescent cells increased gradually with continuous culture. The proliferation and osteogenic differentiation of hDPSCs in the old group were significantly lower than those in the young group (P<0.001). The expression levels of senescence related genes p53 (2.09±0.24) and p21 (4.91±0.54) in old cell group were higher than those in young cell group respectively [p53: (1.08±0.09) and p21: (1.09±0.08)] (P<0.01, P<0.001). The WWTR1 expression levels of hDPSCs in middle-aged group and old cells group were both decreased compared with those in young group and young cells group (P<0.01). The proportion of senescent cells in knockdown group (44.50±2.42) was higher than that in knockdown carrier group (22.27±0.56) (P<0.001). After knocking down WWTR1 in hDPSCs, the expression levels of age-related genes p53 and p21 were up-regulated (P<0.001), and the abilities of proliferation and osteogenic differentiation in the knockdown group were lower than those in the knockdown carrier group (P<0.001). The proportion of senescent cells in overexpression empty carrier group (20.4±0.79) was higher than that in overexpression group (10.07±0.61) (P<0.001). After WWTR1 overexpression ins hDPSCs, the expression levels of age-related genes p53 and p21 were down-regulated, and the proliferation and osteogenic differentiation ability in overexpression group were higher than those in OE-NC group (P<0.001). Conclusions: WWTR1 can inhibit the expression levels of age-related genes p53 and p21, thus delaying the aging process as well as promoting the proliferation and osteogenic differentiation of hDPSCs.

Sishen Pill & Tongxieyaofang ameliorated ulcerative colitis through the activation of HIF-1α acetylation by gut microbiota-derived propionate and butyrate

BackgroundUlcerative colitis (UC) is a chronic inflammatory bowel disease closely related to gut microbiota dysbiosis and intestinal homeostasis imbalance. Sishen Pill&Tongxieyaofang (SSP-TXYF) has a long history of application in traditional Chinese medicine and is widely used in UC clinics. However, its mechanism of action is still unclear. PurposeThis study aimed to explore the potential regulatory role of SSP-TXYF in protecting against UC through metabolites produced by the intestinal microbiota, and elucidate its underlying molecular mechanism. Study design and Methods16S rRNA and UPLC-QE-Orbitrap-MS were used to assess the microbiota and short-chain fatty acids (SCFAs). A rat model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced gut microbiota dysbiosis was used to study the effects of SSP-TXYF on UC in vivo. Intestinal epithelial cells-6 (IEC-6) were treated with lipopolysaccharide (LPS). The intestinal mucosal barrier (IMB) functions were investigated by alcian blue staining and western blot analysis. The mechanism of SSP-TXYF influenced the HIF-1α acetylation pathway was examined by real-time fluorescence quantitative PCR (qPCR), Western blotting, and Co-immunoprecipitation. ResultsUsing 16S rRNA gene-based microbiota analysis, we found that SSP-TXYF ameliorated TNBS-induced gut microbiota dysbiosis. We found that SSP-TXYF significantly inhibited the decreased abundance of Firmicutes in UC rats, in addition, the abundance of Actinobacteria was also improved. The mechanism of SSP-TXYF-treated TNBS-induced UC resulted from improved IMB functions via the activation of hypoxia-inducible factor-1 (HIF-1α) acetylation. Notably, SSP-TXYF Enriched microbiota-derived metabolites propionate and butyrate, which could activate HIF-1α acetylation in IEC. Furthermore, exogenous treatment of propionate and butyrate reproduced similar protective effects as SSP-TXYF to UC through improving HIF-1α-dependent IMB functions. ConclusionsOverall, our findings suggest that the gut microbiota-propionate/butyrate-HIF-1α-IMB axis plays an important role in SSP-TXYF-maintaining intestinal homeostasis, which may represent a novel approach for UC prevention via the intervention of any link in this axis.

Study on serum proteomic characteristics of workers with rare earth samarium oxide pneumoconiosis

Objective: To screen differential proteins in the serum of workers with rare earth samarium oxide pneumoconiosis, in order to provide new ideas for finding its early diagnostic biomarkers. Methods: In April 2019, three male workers diagnosed with samarium oxide pneumoconiosis at a rare earth factory in Baotou City, Inner Mongolia Autonomous Region were selected as the observation group, and three male workers who were not exposed to dust were selected as the control group. The serum was sequenced using the Label-free proteomic method to screen for differentially expressed proteins, followed by cluster of orthologous groups of proteins (COG) annotation, gene ontology (GO) enrichment analysis, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. The interaction gene library retrieval tool database and Cytoscape 3.9.1 software were used to draw protein-protein interaction networks. CytoHubba plugin was used to screen for differentially expressed proteins with high scores, and real-time fluorescence quantitative polymerase chain reaction (RT/q-PCR) was used to validate the proteomic sequencing results. Results: A total of 45 up-regulated differentially expressed proteins and 5 down-regulated differentially expressed proteins were screened out in the serum of workers with rare earth samarium oxide pneumoconiosis. In the COG functional classification, post-translational modifications, protein turnover, and chaperones were the most numerous. GO enrichment included 25 entries for biological processes such as complement activation (classical pathways), 15 entries for cellular components such as extracellular recombinants, and 10 entries for molecular functions such as protein binding. The pathways identified by KEGG enrichment analysis mainly included infectious diseases, immune system, signal transduction, and immune related diseases. The top 10 scoring proteins were haptoglobin, complement C1r subcomponent, complement C1s subcomponent, apolipoprotein C-Ⅲ, apolipoprotein A-Ⅱ, prothrombin, afamin, complement component C8 gamma chain, complement component C6, complement component C7. The RT/q-PCR validation results showed that the mRNA expression levels of haptoglobin, prothrombin and complement C1s subcomponent in the observation group were significantly higher than those in the control group, and the differences were statistically significant (P<0.05) . Conclusion: Ten differentially expressed proteins in the serum of workers with rare earth samarium oxide pneumoconiosis are screened, which provides a good idea for the screening of biomarkers for early diagnosis of samarium oxide pneumoconiosis.

Effect and mechanism of carvacrol on putrescine production by Obesumbacterium proteus in Xinjiang sausage

Here, we explored the effect of carvacrol on growth, gene expression, and putrescine accumulation in O. proteus isolated from Xinjiang sausages. Scanning electron microscopy was used to analyze the cell morphology of the test bacteria. Reverse transcription real-time fluorescence quantitative polymerase chain reaction was used to analyze the gene expression of arginine decarboxylase and ornithine decarboxylase in the test bacteria at different carvacrol concentrations, and single-molecule real-time sequencing was used to identify the species composition in sausage. Moreover, relevant parameters like putrescine accumulation and microbial growth were evaluated. Results showed that the production of putrescine was not only dependent on putrescine-producing strains, but also on the expression of key genes in the putrescine production pathway. Carvacrol was found to inhibit the growth of putrescine-producing strains and decrease putrescine accumulation by releasing O. proteus content, while also reducing putrescine levels through the downregulation of arginine decarboxylase (adiA) and ornithine decarboxylase (speF) genes involved in the putrescine biosynthesis pathway. Moreover, carvacrol significantly reduced the abundance of harmful bacteria (Enterobacteria and Pseudomonas) and the putrescine content in the sausage. Consequently, adding carvacrol extracted from spices to sausages is an effective means to inhibit putrescine content.

Development and application of a quadruplex TaqMan fluorescence quantitative PCR typing method for Streptococcus suis generalis, type 2, type 7 and type 9.

Streptococcus suis (SS) is one of the most important pathogens causing major economic losses in the global pig farming industry and is a serious threat to public health safety. It has multiple serotypes, with poor cross-protection between serotypes, and effective typing methods are lacking. In this study, a quadruplex TaqMan fluorescence quantitative PCR assay that can differentiate between Streptococcus suis types 2, 7 and 9 was developed using the gdh gene, a generic gene for Streptococcus suis, and cps2J, cps7H and cps9J, genes encoding podocarp-associated genes for types 2, 7 and 9, respectively, as targets. The method is specific enough to accurately type Streptococcus suis pigmentosus without detecting non-target pathogens (Escherichia coli, Pasteurella multocida, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and etal). The sensitivity was high, with a minimum lower detection line of 10 copies for P-SS and P-SS9, and 100 copies for P-SS2 and P-SS7. The standard curves generated showed good linearity with R2 of 0.999, 0.999, 0.997 and 0.998 respectively. The repeatability was good, with coefficients of variation between batch to batch and batch to batch tests ranging from 0.21% to 1.10%. Testing of 156 samples yielded 68 positive and 88 negative samples, of which the positive rate of SS was 5.77% (9/156), SS2 was 20.51% (32/156), SS7 was 8.33% (13/156) and SS9 was 9.6% (15/156), which was in line with the existing fluorescent quantitative PCR assay of 93.75%~100%, which was higher than the detection rate of conventional PCR. The quadruplex TaqMan fluorescence quantitative PCR method of Streptococcus suis generic, type 2, 7 and 9 established in this study can accurately differentiate the three serotypes of Streptococcus suis that currently have high prevalence and pathogenicity, which is of great importance for accurate clinical prevention and treatment, epidemiological investigation and vaccine development.

Genome-Wide Identification of the ClpB Gene Family in Tomato and Expression Analysis Under Heat Stress.

Tomato is a widely grown horticultural crop, and its growth process is often affected by high temperatures. Caseinolytic Protease B (ClpB), a homologous protein to heat shock protein 101 (HSP101), plays a vital role in plant heat adaptation and development. In this study, we identified six SlClpB genes in tomatoes, distributed across four chromosomes. Collinearity analysis revealed that the gene pairs SlClpB-2 and SlClpB-3A, as well as SlClpB-3C and SlClpB-12, resulted from segmental duplication events. Phylogenetic and motif analyses showed that ClpB proteins possess highly conserved domains across different species. We used RNA-seq data to analyze the expression patterns of the ClpB family. Among them, SlClpB-3A and SlClpB-12 exhibited increased expression in multiple tissues under heat stress. Specifically, SlClpB-2, SlClpB-3A, and SlClpB-3C were highly expressed in the fruit orange stage and in flower buds under heat treatment, while in seedlings, SlClpB-2 and SlClpB-3A exhibited heat-induced expression. Real-time quantitative fluorescent PCR (qRT-PCR) results showed that the expression of SlClpB-2 and SlClpB-3A was significantly increased under heat stress in the leaves and buds of Ailsa Craig, Micro-Tom, and M82. Overall, our findings provide valuable insights into the regulatory mechanisms of SlClpB genes in response to heat stress.