Time Fluorescence Quantitative PCR Research Articles

A nomogram including admission serum glycated albumin/albumin ratio to predict mortality in patients with severe fever with thrombocytopenia syndrome

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a novel tick-borne infectious disease with a high fatality rate. Although several nomograms based on demographic and laboratory data have been reported to predict the prognosis of SFTS in recent studies, baseline serum glycated albumin (GA)/albumin (ALB) ratio included risk model has not been evaluated for the prediction of clinical outcome.MethodsA total of 214 SFTS patients with integral data admitted to our hospital from May, 2020 to November, 2022 were included in this study. SFTS infection was confirmed by real time fluorescent quantitative PCR (qRT-PCR). The demographic characteristics, clinical and laboratory data were collected with in 24 h of admission and 1 to 2 days before discharge and were analysed retrospectively.ResultsFiffty-seven patients (26.6%) died. Multivariate logistic regression analysis showed that age, aspartate aminotransferase (AST), blood glucose (GLU), GA/ALB ratio, neutrophil counts (NEU) and lymphocyte percentage (LYM%) were the independent risk factors for mortality. A nomogram by these factors was created using RMS package in the R program. Area under the receiver operating characteristic (ROC) curve (AUC) of this nomogram was 0.88 (95% CI: 0.83, 0.93). This model showed the excellent net benefit, as revealed by the decision curve analysis. GA/ALB ratios were also independent risk factors for poor out clinical come in subgroups of patients with hyperglycemia on admission and with diabetes history. Nomograms were constructed by the independent risk factors in the subgroups. AUCs of the nomograms in the subgroups showed high predictive values for adverse prognosis.ConclusionsGA/ALB ratios were independent risk factors for mortality in all SFTS patients and subgroups of with hyperglycemia on admission and diabetes history. The nomograms including GA/ALB ratio had high predictive value for adverse clinical outcome.The nomograms provide a basis for clinical decision-making for the treatment of SFTS patients in different clinical settings.

The effect of miR-23b-3p on regulating GH by targeting POU1F1 in Yanbian yellow cattle

This study aimed to evaluate the effect of miR-23b-3p on growth hormone (GH) in pituitary cells of Yanbian yellow cattle. The mRNA and protein levels of GH and miR-23b-3p target genes were measured by real time fluorescence quantitative PCR (qPCR) and Western blot, respectively. The target relationship of miR-23b-3p was validated by double luciferase reporter gene system. The results showed that GH mRNA and protein levels in pituitary cells of Yanbian yellow cattle were significantly lower in the miR-23b-3p-mi group than in the NC group (P<0.01), while GH mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.05). The result of bioinformatics analysis and double luciferase reporter gene system validation proved that miR-23b-3p targeted 3′UTR of pituitary specific transcription factor 1 (POU1F1). POU1F1 mRNA and protein levels were lower miR-23b-3p-mi group than in the NC group (P<0.01), while POU1F1 mRNA and protein levels were higher in the miR-23b-3p-in group than in the iNC group (P<0.01). These results demonstrated that miR-23b-3p could regulate GH expression in pituitary cells by regulating POU1F1 gene.

Pathogen identification and epidemic factor analysis of yellow catfish, Pelteobagrus fulvidraco, red lower jaw disease

Since 2020, another type of epidemic disease, ‘red lower jaw disease’ (RLJD), has broken out in yellow catfish Pelteobagrus fulvidraco every spring in China, causing large-scale deaths and significant economic losses. This new disease has the characteristics of strong infectivity, high mortality and a single host. To prevent and control RLJD, epidemiological investigation, transmission electron microscopy (TEM), virome sequencing, Koch's rule verification and epidemic factor investigation were studied in this article. The clinical symptoms of diseased yellow catfish were pale gill filaments, hyperaemia and swelling of the spleen and kidney. TEM results revealed that a large number of 35–40 nm spherical virus particles without envelope structure were found in the cytoplasm of spleen cells. The liver, spleen and kidney of the diseased fish had serious lesions. After virome sequencing, the calicivirus reads in the unique virus reads accounted for 99.5% of the unique virus reads in the diseased fish, which provided a relevant reference value for identifying the pathogen of RLJD. The full-length sequence of yellow catfish calicivirus (YCCV) obtained at 7405 bp, including the RNA-helicase domain, RdRp-1 domain and d1iqqa domain. At the same time, both PCR and TaqMan fluorescence quantitative PCR detection method of YCCV were established. Through the artificial infection test, YCCV was verified as the pathogen for RLJD. It was also found that YCCV mainly harmed the fish within 1 year, whereas the fish above 2 years had good resistance. The water temperature of the RLJD outbreak was mainly concentrated at 18 °C–24 °C. In the current study, after YCCV was verified as the pathogen of RLJD, epidemic factors including fish age and water temperature were studied, to the best of our knowledge, for the first time to provide a relevant theoretical basis for the pathogenic mechanism and prevention and control measures of yellow catfish RLJD.

Study on reproductive toxicity of nano-cadmium sulfide with different particle sizes on male mice

Objective: To investigate the reproductive toxicity of cadmium sulfide nanoparticles (Nano-CdS) with different particle sizes on male mice. Methods: In January 2019, 30 SPF grade male mice were randomly divided into a control group, an experimental group[CdS Ⅰ group (particle size approximately 5 nm), and a CdS Ⅱ group (particle size approximately 50 nm) ], with 10 mice in each group. The experimental group was orally gavaged with 100 mg/kg, once a day, while the control group was gavaged with an equal volume of physiological saline for 45 consecutive days. After 45 days, levels of cadmium accumulation in testis were determined directly by AAS, deformity and testicular histopathological changes were also observed. Serum testosterone levels were measured by enzyme-linked immunosorbentassay (ELISA), expression levels of P450scc, 17β-HSD and P450c17 mRNA were determined by real-time PCR. P450c17 protein was determinated by Western Blot. Results: The histopathological results showed that the testes of the experimental group mice showed varying degrees of damage; Ultrastructural observation showed that the ultrastructure of mouse testicular cells in each experimental group showed varying degrees of mitochondrial expansion and disappearance of cristae, as well as irregular nuclear membranes. The degree of damage in CdS Ⅰ group was milder than that in CdS Ⅱ group. Compared with the control group, the cadmium content in the testes of the CdS Ⅰ and CdS Ⅱ groups significantly increased (P=0.001, 0.001), and the CdS Ⅱ group was higher than the CdS Ⅰ group (P=0.001). Compared with the control group, the levels of testosterone in the CdS Ⅰ and CdS Ⅱ groups decreased with statistical significance (P=0.001, 0.001). Real time fluorescence quantitative PCR results showed that compared with the control group, the experimental group's P450scc, 17β-HSD. The expression levels of 17β-HSD and P450c17 mRNA were significantly reduced, with statistically significant differences (P=0.001, 0.001, 0.001), and CdS Ⅱ group 17β-HSD. The expression levels of 17β-HSD and P450c17 mRNA were significantly lower than those of CdS Ⅰ group (P=0.001, 0.036). The Western Blot assay results showed that the expression levels of P450c17 protein in the testes of CdS Ⅰ and CdS Ⅱ groups of mice were significantly reduced, with statistical significance (P=0.001, 0.001) ; And the CdS Ⅱ group was significantly lower than the CdS Ⅰ group (P=0.001). According to Spearman correlation analysis, testosterone levels are correlated with P450scc, P450c17, 17β-HSD mRNA. There is a highly positive correlation between 17β-HSD mRNA levels, with statistically significant differences (r(s)=0.88, 0.80, 0.70, P=0.001, 0.001, 0.004) . Conclusion: Nano cadmium sulfide may induce reproductive toxicity by reducing the expression levels of key enzyme genes and enzyme protein activity in testosterone and its synthesis in mice, and the CdS Ⅱ group has a stronger toxic effect.

Florfenicol can inhibit chick growth and lead to immunosuppression1

Florfenicol (FLO) is a chemically synthesized broad-spectrum antimicrobial agent of amide alcohols for animals, which is one of the most widely used antimicrobials in livestock, poultry, and aquaculture. With the use of FLO, more and more attention has been paid to its hematopoietic toxicity, immunotoxicity, genotoxicity, and embryotoxicity. In this study, SPF chicks at the age of 3 d began to drink water with the FLO at a dose of 100 mg L-1 for six consecutive days, and the growth performance of chicks was monitored, the effect of FLO on immune organs was detected by pathological examination and TUNEL apoptosis staining. In order to evaluate the level of organism immunity, the level of Newcastle disease virus antibody in serum was detected by hemagglutination inhibition test, the content of cytokines (IL-1, IL-2, IL-6, TNF-α, IFN-γ) in serum was detected by ELISA, and the transcription of interferon-related genes (IRF-7, 2’-5’OAS, Mx1) and cytokine genes (IL-6, TNF-α, IFN-γ) in immune organs were detected by real time fluorescence quantitative PCR. The results showed that the early application of FLO could inhibit the growth and development of chicks, and the body weight and immune organ index of the treatment group were lower than those of the control group. Histopathological examination showed that there was a decrease in the number of lymphocytes in the bursa of Fabricius in the treatment group in the early stage of drug withdrawal, and the results of TUNEL apoptosis staining in the bursa of Fabricius showed that obvious lymphocyte apoptosis occurred in the FLO treatment group. Compared with the control group, the transcription levels of interferon-related genes IRF-7, 2’-5’OAS, and cytokine genes IL-6, TNF-α and IFN-γ in FLO treatment group decreased to a certain extent, while the transcription level of Mx1 gene had no significant difference at all time points. The level of serum NDV antibody and the contents of cytokines IL-1, IL-2 and IFN-γ in the FLO treatment group were significantly lower than those in the control group in the early stage of drug withdrawal, but recovered gradually in the later stage. This study showed that FLO has a certain degree of effect on the immune function of chicks, and the results of the study laid the foundation for further research on the mechanism of FLO-induced immunotoxicity.

Melatonin promotes osteogenesis of bone marrow mesenchymal stem cells by improving the inflammatory state in ovariectomized rats

To investigate the effects of melatonin (MT) on bone mass and serum inflammatory factors in rats received ovariectomy (OVX) and to investigate the effects of MT on the levels of inflammatory factors in culture medium and osteogenic ability of bone marrow mesenchymal stem cells (BMSCs) stimulated by lipopolysaccharide. Fifteen 12-week-old Sprague Dawley (SD) rats were randomly divided into 3 groups. The rats in Sham group only received bilateral lateral abdominal incision and suture, the rats in OVX group received bilateral OVX, and the rats in OVX+MT group received 100 mg/(kg·d) MT oral intervention after bilateral OVX. After 8 weeks, the levels of serum inflammatory factors [interleukin-1β (IL-1β), IL-6, and tumor necrosis factor α (TNF-α)] were detected using ELISA assay. Besides, the distal femurs were detected by Micro-CT to observe changes in bone mass and microstructure, and quantitatively measured bone volume fraction, trabecular thickness, and trabecular number. The BMSCs were extracted from the femurs of three 3-week-old SD rats using whole bone marrow culture method and passaged. The 3rd-5th passage BMSCs were cultured with different concentrations of MT (0, 1, 10, 100, 1 000 µmol/L), and the cell viability was then detected using cell counting kit 8 (CCK-8) to select the optimal concentration of MT for subsequent experiments. Cells were devided into osteogenic induction group (group A) and osteogenic induction+1/5/10 μg/mL lipopolysaccharide group (group B-D). The levels of inflammatory factors (IL-1β, IL-6 and TNF-α) in cell culture medium were detected using ELISA assay after corresponding intervention. According to the results of CCK-8 method and ELISA detection, the cells were intervened with the most significant concentration of lipopolysaccharide for stimulating inflammation and the optimal concentration of MT with osteogenic induction, defining as group E, and the cell culture medium was collected to detect the levels of inflammatory factors by ELISA assay. After that, alkaline phosphatase (ALP) staining and alizarin red staining were performed respectively in groups A, D, and E, and the expression levels of osteogenic related genes [collagen type Ⅰ alpha 1 chain (Col1a1) and RUNX family transcription factor 2 (Runx2)] were also detected by real time fluorescence quantitative PCR (RT-qPCR). ELISA and Micro-CT assays showed that compared with Sham group, the bone mass of the rats in the OVX group significantly decreased, and the expression levels of serum inflammatory factors (IL-1β, IL-6, and TNF-α) in OVX group significantly increased (P<0.05). Significantly, the above indicators in OVX+MT group were all improved (P<0.05). Rat BMSCs were successfully extracted, and CCK-8 assay showed that 100 µmol/L was the maximum concentration of MT that did not cause a decrease in cell viability, and it was used in subsequent experiments. ELISA assays showed that compared with group A, the expression levels of inflammatory factors (IL-1β, IL-6, and TNF-α) in the cell culture medium of groups B-D were significantly increased after lipopolysaccharide stimulation (P<0.05), and in a concentration-dependent manner. Moreover, the expression levels of inflammatory factors in group D were significantly higher than those in groups B and C (P<0.05). After MT intervention, the expression levels of inflammatory factors in group E were significantly lower than those in group D (P<0.05). ALP staining, alizarin red staining, and RT-qPCR assays showed that compared with group A, the percentage of positive area of ALP and alizarin red and the relative mRNA expressions of Col1a1 and Runx2 in group D significantly decreased, while the above indicators in group E significantly improved after MT intervention (P<0.05). MT may affect the bone mass of postmenopausal osteoporosis by reducing inflammation in rats; MT can reduce the inflammation of BMSCs stimulated by lipopolysaccharide and weaken its inhibition of osteogenic differentiation of BMSCs.

Copy number variation of EIF4A2 loci related to phenotypic traits in Chinese cattle.

BackgroundGenerally, copy number variation (CNV) is a large‐scale structural variation between 50 bp and 1 kb of the genome. It can affect gene expression and is an important reason for genetic diversity and phenotypic trait diversity. Studies have shown that the eukaryotic translation initiation factor 4A2 (EIF4A2) gene plays an essential role in muscle development in both humans and pigs. However, the influence of bovine EIF4A2’s copy number change on phenotypic traits has not been reported.ObjectivesTo detect the tissue expression profile of the EIF4A2 gene in adult cattle and individuals’ CNV type of variation. Then, we explored the correlation between EIF4A2‐CNV and growth traits in Chinese cattle breeds.MethodsReal‐time fluorescent quantitative reverse transcription PCR (qRT‐qPCR) was used to determine the expression profile of the EIF4A2 gene. Real‐time fluorescent quantitative PCR (qPCR) was used to detect the CNV type of bovine populations. Then, SPSS 26.0 was used for association analysis.ResultsIn this study, a total of 513 individuals in four cattle breeds (Qinchuan cattle [QC], Yunling cattle [YL], Pinan cattle [PN] and Jiaxian cattle [JX]) were detected for EIF4A2 gene's CNV. The results showed that EIF4A2‐CNV has an essential impact on hip width (HW) and rump length (RL) in QC, heart girth (HG), chest depth (CD) and RL in YL and HW in PN. However, it had no significant effect on JX.ConclusionsThe above results suggest that EIF4A2 gene's CNV can be used as a molecular marker for cattle breeding, which is helpful to accelerate the breeding of superior beef cattle breeds.

Exploration of the therapeutic mechanism of Yiqi Jiedu recipe for treatment of primary liver cancer based on network pharmacology and molecular docking

To explore the effective components of Yiqi Jiedu recipe and the main biological processes and signal pathways involved in the therapeutic mechanism of the recipe in treatment of primary liver cancer through network pharmacology and molecular docking approaches. TCMSP, Uniport, Genecards and String databases were searched to obtain the target genes of drugs and disease using Cytoscape 3.8.2 software. GO and KEGG enrichment analyses were performed to identify the common genes in the target genes of the drugs and disease. Using Pubcham, RCSB and Autoduck, the effective components of the drugs were connected with the final core genes. The effects of different concentrations of Yiqi Jiedu recipe on the expressions of the core genes DHX9, HNRNPK, NCL and PABPC1 in HepG2 cells were analyzed with Western blotting and real- time fluorescence quantitative PCR. We finally identified 8 core genes from the drug and disease targets, including DDX5, HNRNPK, PABPC1, DHX9, RPS3A, RPS3, RPL13, and NCL. GO analysis showed that these core genes were involved mainly in the biological processes of adrenaline receptor signal communication, movement of cellular or subcellular components, blood particles, adhesion class and iron ion binding. KEGG analysis showed that the Ras signaling pathway had the greatest gene enrichment. The results of molecular docking suggested that the effective components of the recipe were capable of docking with the core genes under natural conditions, and PABPC1 and stigmasterol had the highest binding energy. In HepG2 cells, treatment with 10% medicated serum for 48 h had the strongest effect on the expression of DHX9, HNRNPK, NCL and PABPC1 (P < 0.05). Yiqi Jiedu recipe is capable of regulating viral expression of primary liver cancer multiple effective components that bind to DHX9, HNRNPK, NCL and PABPC1.

Analysis of transcriptional regulators HpaR1 and Clp regulating the expression of glycoside hydrolase-encoding gene in the Xanthomonas campestris pv. campestris.

Xanthomonas campestris pv. campestris (Xcc) is a vascular pathogen that causes black rot in host. It is an important model strain for studying the interaction between the phytopathogen and plants. In Xcc, global transcription regulator HpaR1 that belongs to the GntR family regulates many cellular processes such as the movement and synthesis of extracellular polysaccharides and extracellular enzymes, and is associated with hypersensitive response (HR) and pathogenicity. On the other hand, the global transcriptional regulator Clp regulates the secretion and synthesis of extracellular enzymes and extracellular polysaccharides, and is associated with the pathogenicity of Xanthomonas. Previous studies have shown that both HpaR1 and Clp bind to the promoter region of the glycoside hydrolase encoding gene (named ghy gene). This study investigates the molecular mechanism of the co-regulation of HpaR1 and Clp on the expression of ghy gene. Through electrophoresis mobility shift assay (EMSA), we found that both HpaR1 and Clp bind to the promoter regions of gene ghy in vitro. Both HpaR1 and Clp also bind to the promoter regions of gene ghy in vivo by chromatin immunoprecipitation (ChIP) assays. DNase I footprinting and 5'-RACE assays showed that both HpaR1 and Clp bind to the -35 region upstream of the ghy promoter. The HpaR1 binding site was located upstream of the Clp binding site. RT-qPCR and in vitro transcription assays showed that HpaR1 negatively while Clp positively regulates the transcription of gene ghy. Furthermore, HpaR1 inhibits the activation of Clp on the transcription of gene ghy in vitro. Our findings indicate that HpaR1 and Clp exhibit opposite effect on the transcription of gene ghy. It is speculated that HpaR1 may regulate the expression of gene ghy by inhibiting the activity of RNA polymerase.

Analysis of m6A methylation in skin tissues of different sex Liaoning cashmere goats

N6-methyladenosine (m6A) is the most frequent internal modification of mRNA and lncRNA in eukaryotes. We used two high-throughput sequencing method, m6A-seq and RNA-seq to identify pivotal m6A-modified genes in cashmere fineness and fiber growth. 8062 m6A peaks were detected by m6A-seq, including 2157 upregulated and 6445 downregulated. Furthermore, by comparing m6A-modified genes of the male Liaoning Cashmere Goat (M-LCG) and female Liaoning Cashmere Goat (F-LCG) skin tissues, we get 862 differentially expressed m6A-modified genes. To identify differently expressed m6A genes associated with cashmere fineness, 11 genes were selected for validation using real time fluorescent quantitative PCR in M-LCG and F-LCG. This study provides an acadamic basis on the molecular regulation mechanism of m6A modification in cashmere growth process.

Selection and validation of internal reference genes for qPCR in Polygonatum cyrtonema tubers at different development stages and in response to abiotic stress

In order to analyze the expression of genes involved in steroidal saponin biosynthesis pathway in Polygonatum cyrtonema tubers, it is very important to select internal reference genes that are stably expressed at different development stages and in response to abiotic stress. According to the previously established P. cyrtonema transcriptome database and reported internal reference genes in plant, this study systematically analyzed eight candidate internal reference genes including histone H2 A, glyceraldehyde-3-phosphate dehydrogenase, ACTIN, β-tubulin, ubiquitin-conjugating enzyme-E2-10, elongation factor 1-alpha isoform, 18 S rRNA and α-tubulin 4 for expression stability in P. cyrtonema tubers at different development stages and in response to methyl jasmonate(MeJA) stress by using Real time fluorescence quantitative PCR(qPCR). Based on the statistical analysis of qPCR results by using GeNorm, NormFinder and BestKeeper softwares, the expression of ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform are the most stable in P. cyrtonema tubes at different development stages and in response to MeJA stress. The two internal reference genes were further validated by analyzing the expression of 4 genes involved in steroidal saponin biosynthesis pathways. In conclusion, ubiquitin-conjugating enzyme-E2-10 and elongation factor 1-alpha isoform can be used as the most appropriate internal reference genes for qPCR analysis in P. cyrtonema. This study also provide a foundation for future investigate the molecular mechanism of steroidal saponin biosynthesis pathways in P. cyrtonema.

The diagnosis value of real time fluorescence quantitative PCR for tuberculosis in paraffin embedded tissue

目的： 探讨即时荧光定量PCR（qPCR）法在检测石蜡包埋组织中结核杆菌的应用价值。 方法： 回顾性分析湖北省十堰市太和医院病理科2017—2019年间1 213例用qPCR法检测过结核杆菌核酸的病例，将检测结果与石蜡切片抗酸染色及临床随访结果进行比较，验证其诊断价值。 结果： 1 213例样本中，627例qPCR法检测结果为阳性，586例为阴性。临床随访证实其中876例为结核病，337例为其他疾病。经计算得出qPCR法的灵敏度为71.3%，特异度为99.4%，准确率为79.1%。这1 213例中892例有抗酸染色结果，其中阳性280例，阴性612例。结合临床诊断，抗酸染色的灵敏度为41.5%，特异度为99.5%，准确率为55.7%。 结论： 与抗酸染色相比，qPCR法对石蜡包埋组织中结核分枝杆菌的检测有较高的灵敏度，有助于作出正确诊断。.

Preventive effect of Lactobacillus plantarum HFY09 on HCl/ethanol-induced gastric injury in mice

The aim of this study was to investigate the effect of Lactobacillus plantarum HFY09 on gastric injury induced by HCl/ethanol in Kunming mice. The results showed that HFY09-H inhibited any increases in gastric juice volume, maintained the normal pH value of gastric acid, and reduced the damage caused to the gastric mucosa and gastric wall, the inhibition rate on the injury area reaches 63.70%. Compared with the negative control group, HFY09 increased the levels of serum somatostatin (SS) and vasoactive intestinal peptide (VIP), and also decreased the levels of substance P (SP), endothelin-1 (ET-1), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). In addition, real time fluorescent quantitative PCR (Q-PCR) also confirmed that high-dose HFY09 (109 CFU/kg/day) upregulated the mRNA expression of copper/zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), catalase (CAT), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS), and downregulated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). At the same time, the results of the HFY09 treatment group were similar to those of the ranitidine treatment group. These results indicate that HFY09 can prevent gastric injury induced by HCl/ethanol in vivo. Therefore, HFY09 may play a potential role in the treatment of gastric diseases.

Cardiac injury after acute carbon monoxide poisoning and its clinical treatment scheme.

This study was designed to investigate cardiac injury after acute carbon monoxide poisoning and its clinical treatment scheme. Seventy patients with moderate and severe acute carbon monoxide poisoning (ACOP) admitted from January 2017 to December 2018 into The Affiliated Hospital of Qingdao University were regarded as a research group (RG), and another 30 healthy adults undergoing physical examination in the hospital during the same period were selected as a control group (CG). Thirty-five patients in the RG who received hyperbaric oxygen therapy were considered as group A, and 35 patients who received extracorporeal membrane oxygenation therapy were considered as group B. The effective rates and complications of the two groups after treatment were compared. The concentrations of creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH) of myocardial enzymes at different time points before and after treatment were detected. Expression of miR-30a in the blood of experimental subjects was detected by time-fluorescence quantitative PCR, and the relationship between miR-30a expression and ACOP patients was analyzed. Patients in groups A and B achieved obvious efficacy, but the effective rate and incidence rate of complications in the extracorporeal membrane oxygenation (ECMO) group were better than those in the hyperbaric oxygen group. The concentrations of CK-MB and LDH in group A and group B were significantly higher than those in control group (P<0.01). The expression level of miR-30a in the RG was significantly higher than that in the control group (P<0.05). Both hyperbaric oxygen therapy and ECMO therapy have obvious efficacy on ACOP patients, but the latter is better than the former. The expression level of miR-30a in blood of ACOP patients increased significantly, which is positively correlated with myocardial injury, and it decreased after treatment. It is believed that miR-30a can provide a reference index for early diagnosis and prediction of disease progression and prognosis in cardiac injury of ACOP.

EXPRESSION AND CHARACTERISATION OF CUCUMBER FRUIT FLESH THICKNESS-RELATED GENE CSA2M058670.1

Bulked segregant analysis combined with specific length amplified fragment sequencing techniques have been applied to determine the fine genetic mapping of fruit flesh thickness-related genes in cucumber. Herein, the Csa2M058670.1 gene was subjected to real time fluorescence quantitative PCR (qRT-PCR) and sequence analysis, indicating a strong correlation with cucumber fruit flesh thickness. Expression and characterisation of the Csa2M058670.1 gene were performed based on previous studies. The results of the fluorescence-based quantitative PCR showed that Csa2M058670.1 was expressed in all organs, but levels were highest in fruit peel, fruit flesh, and female flowers. Furthermore, Csa2M058670.1 expression was induced by abiotic stresses including drought, low temperature, and high salt. Domain analysis revealed that the protein encoded by Csa2M058670.1 possesses an SET (Su(var), Enhancern of zeste CE(z), and Trithorax) domain that may control cell division and differentiation. Therefore, we speculated that Csa2M058670.1 might affect fruit flesh thickness in cucumbers by influencing cell division.

Enhancement of bleomycin production in Streptomyces verticillus through global metabolic regulation of N-acetylglucosamine and assisted metabolic profiling analysis

BackgroundBleomycin is a broad-spectrum glycopeptide antitumor antibiotic produced by Streptomyces verticillus. Clinically, the mixture of bleomycin A2 and bleomycin B2 is widely used in combination with other drugs for the treatment of various cancers. As a secondary metabolite, the biosynthesis of bleomycin is precisely controlled by the complex extra-/intracellular regulation mechanisms, it is imperative to investigate the global metabolic and regulatory system involved in bleomycin biosynthesis for increasing bleomycin production.ResultsN-acetylglucosamine (GlcNAc), the vital signaling molecule controlling the onset of development and antibiotic synthesis in Streptomyces, was found to increase the yields of bleomycins significantly in chemically defined medium. To mine the gene information relevant to GlcNAc metabolism, the DNA sequences of dasR-dasA-dasBCD-nagB and nagKA in S. verticillus were determined by chromosome walking. From the results of Real time fluorescence quantitative PCR (RT-qPCR) and electrophoretic mobility shift assays (EMSAs), the repression of the expression of nagB and nagKA by the global regulator DasR was released under induction with GlcNAc. The relief of blmT expression repression by BlmR was the main reason for increased bleomycin production. DasR, however, could not directly affect the expression of the pathway-specific repressor BlmR in the bleomycins gene cluster. With at the beginning of bleomycin synthesis, the supply of the specific precursor GDP-mannose played the key role in bleomycin production. Genetic engineering of the GDP-mannose synthesis pathway indicated that phosphomannose isomerase (ManA) and phosphomannomutase (ManB) were key enzymes for bleomycins synthesis. Here, the blmT, manA and manB co-expression strain OBlmT/ManAB was constructed. Based on GlcNAc regulation and assisted metabolic profiling analysis, the yields of bleomycin A2 and B2 were ultimately increased to 61.79 and 36.9 mg/L, respectively.ConclusionsUnder GlcNAc induction, the elevated production of bleomycins was mainly associated with the alleviation of the inhibition of BlmT, so blmT and specific precursor synthesis pathways were genetically engineered for bleomycins production improvement. Combination with subsequent metabolomics analysis not only effectively increased the bleomycin yield, but also extended the utilization of chitin-derived substrates in microbial-based antibiotic production.

Low expression of microRNA-125b enhances the expression of STAT3 and contributes to cholesteatoma growth.

MicroRNA-125b has been found to be down-regulated in many types of malignant tumours and diseases with excessive proliferation of keratinocytes, such as cutaneous squamous cell carcinoma and psoriasis. Cholesteatoma, which is mainly composed of keratinocytes, also has characteristics of abnormal proliferation similar to a malignant tumour. However, the expression and regulatory mechanisms of miR-125b and its downstream genes in cholesteatoma have not been clarified. Real time fluorescence quantitative PCR was applied to detect the expression of miR-125b in the cholesteatoma and corresponding retroauricular skin. Immunohistochemical staining and western blot were used to detect signal transducers and activators of transcription 3 (STAT3) and the downstream gene cyclin D1, survivin, and vascular endothelial growth factor (VEGF) in the cholesteatoma and corresponding retroauricular skin. The targeted regulatory relationship between miR-125b and STAT3 was confirmed by dual luciferase reporter assay. Proliferation and apoptosis of transfected HaCaT cells were detected by MTS, cell cycle, and apoptosis assays. We observed down-regulation of miR-125b and up-regulation of STAT3, cyclin D1, survivin, and VEGF in cholesteatoma tissues. STAT3 was a direct target gene of miR-125b. Inhibition of miR-125b enhanced STAT3 and its downstream genes expression, promoted HaCaT cell proliferation, and inhibited apoptosis. The results of this study demonstrate that miR-125b can influence the growth of cholesteatoma by targeting STAT3 and its downstream genes, including cyclin D1, survivin, and VEGF, thus providing an opportunity to establish new medical therapy strategies and facilitating further study of the pathogenesis of cholesteatoma.

GPX2 suppression of H2O2 stress regulates cervical cancer metastasis and apoptosis via activation of the β-catenin-WNT pathway.

BackgroundIncreasing evidence suggests that glutathione peroxidase 2 (GPX2) plays important roles in the tumorigenesis and progression of various human cancers, such as colorectal carcinomas and lung adenocarcinomas. However, the role of GPX2 in cervical cancer is unclear. In this study, we identified the role of GPX2 in cervical cancer tissues and cell lines.Materials and methodsThe basal mRNA and protein expression of GPX2 in cervical cancer cells and a series of key molecules in the epithelial to mesenchymal transition (EMT) and WNT/β-catenin pathways were examined via real time fluorescence quantitative PCR (qRT-PCR) and Western blot assays. The biological phenotype of the cervical cancer cell lines was detected by the cloning formation and transwell assays, and intracellular reactive oxygen species (ROS) levels were detected by flow cytometry. Finally, the GPX2 expression level in 100 clinical cervical tissues was examined by immunohistochemistry.ResultsWe found that GPX2 was highly expressed in cervical cancer tissues compared to normal individuals and promoted the proliferation and metastasis of cervical cancer cells, and this promotion correlated with the activation of EMT and WNT/β-catenin signaling in vitro. GPX2 was determined to reduce apoptotic damage by reducing hydroperoxides. According to the characteristics and verification of GPX2, this series of phenotypes is clearly related to oxidative stress in cells. Furthermore, we verified that GPX2 was highly expressed in cervical cancer tissues and promoted the metastasis of cervical cancer.ConclusionIn summary, we found that GPX2 was highly expressed in cervical cancer cells and promoted the proliferation and metastasis of cervical cancer by affecting oxidative stress. Our study provides a new target for the clinical treatment of cervical cancer.

The prognostic value of circulating microRNA-29b in left ventricular hypertrophy in patients with hypertension

Objective: To analyze the relationship between the level of microRNA-29b in circulation and left ventricular hypertrophy in hypertensive patients. Methods: A total of 240 subjects from Henan Province People's Hospital from June 2015 to June 2018 were included in the present study. Among them, 160 were hospitalized patients, and were divided into two groups. Patients with simple hypertension and had no left ventricular hypertrophy (80 cases) were in the simple hypertension group (HBP-NLVH), and patients with hypertension combined with left ventricular hypertrophy (80 cases) were in the high blood pressure with left ventricular hypertrophy group (HBP-LVH). Normal control subjects (80 cases) were those with no hypertension and randomly selected from the medical center of Henan Province People's Hospital. Serum microRNA-29b expressions were detected by real time fluorescence quantitative PCR. The thickness of interventricular septum (IVSD) and left ventricular posterior wall thickness (LVPWD) were measured by echocardiography. Results: Compared with the normal control group (1.95±0.79), the relative expression of microRNA-29b in the patients both in the HBP-NLVH group (2.67±0.92) and the HBP-LVH group (5.12±1.23) was up-regulated, and the difference between normal control and patients was statistically significant (P<0.05). In patients, the microRNA-29b level in the HBP-LVH group was significantly higher than that in the HBP-NLVH group (P<0.05). The expression level of microRNA-29b was positively correlated with IVSD (r=0.71, P<0.05) and LVPWD (r=0.74, P<0.05), respectively. The sensitivity and specificity of serum microRNA-29b levels in the diagnosis of left ventricular hypertrophy in hypertension patients were 96.8% and 91.3%, respectively. Conclusion: Serum microRNA-29b level is elevated in hypertensive patients with left ventricular hypertrophy, and is positively correlated with left ventricular hypertrophy. The circulation microRNA-29b might be a useful biomarker with prognostic value in left ventricular hypertrophy in hypertension patients.

Expression and significance of CD47, PD1 and PDL1 in T-cell acute lymphoblastic lymphoma/leukemia

Although dose intensification strategies achieve a favorable prognosis for pediatric patients of T-lmphoblastic lymphoma/leukemia (T-LBL/ALL), numerous side effects have been followed. Molecular targeted therapies will be needed to optimize the current treatment strategy for T-LBL/ALL. The aim of this study was to analyse expression and significance of CD47, PD1 and PDL1 in. T-LBL/ALL. We performed immunohistochemistry staining and real time fluorescence quantitative PCR (qRT-PCR) on FFPE tissues. Immunohistochemistry results showed that the high expression rate of CD47 protein was 46.4% (26/56) and the positive expression rate of PDL1 protein was 37.5% (21/56). PD1 expression was observed in tumor infiltrating lymphocytes in approximately 20% of T-LBL/ALL patients, but not expressed on tumor cells of T-LBL/ALL. And the results of qRT-PCR showed that the relative expression levels of CD47, PDL1 and PD1 mRNA in 56 cases of T LBL/ALL were significantly higher than those in control group (6.915 vs 4.050, 12.255 vs 2.575, 37.990 vs 3.615), and the differences were all statistically significant (p all <0.05). Univariate analysis showed that age, CD47 protein, CD47 mRNA,PDL1 protein and PDL1 mRNA expression were closely correlated with prognosis (P all <0.05). We found that the overall one-year survival rates of patients with a high expression (≥M) of CD47 and PDL1 mRNA were higher than in patients with low expression (<M). However, the overall one-year survival rate of patients with a high expression (≥M) of CD47 and PDL1 protein were lower than in patients with low expression (<M). And patients with ≤25 years old had a worse prognosis than with >25 years old. Multivariate Cox regression analysis showed that the high expression of CD47 and PDL1 protein were independent prognostic factors (both p < 0.05). In a word, PD1/PDL1 and CD47 may be involved in the disease progression and prognosis of T-LBL/ALL, and detection and targeting of CD47 and PD1/PDL1 may provide a rational basis to for treatment of T-LBL/ALL.