Real-time Polymerase Chain Reaction Experiments Research Articles

MiR-195-5p Regulates the Phenotype Switch of CCSM Cells by Targeting Smad7.

IntroductionPhenotype switch refers to the process in which smooth muscle cells change from contractile type to synthetic type and acquire the ability of proliferation. Phenotypic transformation involves many changes of cell function, such as collagen deposition and fibrosis, which affect the normal erectile function of penis.AimTo investigate the role of miR-195-5p in regulating the Phenotype switch of the corpus cavernosum smooth muscle (CCSM) cells.MethodsA small mother against decapentaplegic 7(Smad7) virus vector and a miR-195-5p mimics or an si-Smad7 viral vector and a miR-195-5p inhibitor were transfected into CCSM cells. The cells were obtained by primary culture of rat corpus cavernosum smooth muscle tissue. Real-time polymerase chain reaction (PCR) experiments, Western blotting, hematoxylin-eosin (HE) staining, transwell experiments, MTT assays, and flow cytometry were used to detect miR-195-5p, Smad7, phenotype switch markers of CCSM cells and related protein expression, as well as changes in cell morphology, migration, proliferation and apoptosis.Main Outcome MeasureTo study the regulation of miR-195-5p in CCSM cells by overexpression and silencing strategies.ResultsOverexpressed miR-195-5p promoted the transformation of CCSM cells from a contractile type to a synthetic type. Meanwhile, the migration ability and proliferation ability of CCSM cells increased, and the apoptosis rate decreased. The expression-silencing of miR-195-5p gave rise to the opposite effect. The results of the rescue experiment demonstrated that overexpressed Smad7 rescued the inhibitory of the switch of the CCSM cell phenotype from the contractile type to the synthesis type caused by overexpression of miR-195-5p alone. Moreover, the enhancement effect of the migration ability and proliferation ability of CCSM cells was also eliminated, and the apoptosis rate was increased. Silencing miR-195-5p and Smad7 at the same time resulted in the opposite effect.ConclusionmiR-195-5p may regulate the phenotype switch of CCSM cells by targeting Smad7.Zhang J, Zhang X, Zhang J, et al. miR-195-5p Regulates the Phenotype Switch of CCSM Cells by Targeting Smad7. Sex Med 2021;9:100349.

Anti-thrombotic activity of phenolic acids obtained from Salvia miltiorrhiza f. alba in TNF-α-stimulated endothelial cells via the NF-κB/JNK/p38 MAPK signaling pathway.

Over the past 100years, Salvia miltiorrhiza f. alba (Lamiaceae) (RSMA) roots have been used to cure thromboangiitis obliterans (TAO) in local clinics. This study aimed to confirm the anti-thrombotic efficacy of 12 phenolic acids obtained from RSMA and to clarify the possible underlying mechanisms. The results of quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA) experiments demonstrated that most of the phenolic acids markedly inhibited PAI-1 protein and mRNA levels but increased t-PA protein and mRNA levels in TNF-α-induced EA.hy926 cells (P < 0.05 or 0.001), with lithospermic acid displaying the strongest effect. In vitro anticoagulation and antiplatelet aggregation assays showed that lithospermic acid and salvianolic acid B significantly prolonged prothrombin time (PT), activated partial thromboplastin time (APTT), decreased fibrinogen concentration (FIB), and inhibited platelet aggregation induced by adenosine diphosphate (ADP) in rat blood. Both lithospermic acid and salvianolic acid B markedly down-regulated the expression of factor Xa and factor IIa on the external surface of EA.hy926 cells and demonstrated significant anti-factor IIa and anti-factor Xa activity using chromogenic substrates in vitro. Western blot results revealed that both lithospermic acid and salvianolic acid B also significantly inhibited the expression of TF, p-p65, p-p38, and pJNK proteins induced by TNF-α. These results indicated that all of the phenolic acids appeared to have some anti-thrombotic activity, with salvianolic acid B and lithospermic acid markedly decreasing the chance of thrombosis by regulating the NF-κB/JNK/p38 MAPK signaling pathway in response to TNF-α.

PTX3 promotes proliferation, invasion and drug resistance of neuroblastoma cells in children by regulating TLR4/NF-κB signaling pathway

Objective: To investigate the effect of pentraxin 3 (PTX3) on the proliferation, invasion and drug resistance of pediatric neuroblastoma cells and its mechanism. Methods: si-RNA (si-RNA group), si-PTX3 (si-PTX3 group), siRNA+ pcDNA3.1 (siRNA+ pcDNA3.1 group), si-PTX3+ pcDNA3.1 (si-PTX3+ pcDNA3.1 group), siRNA+ pcDNA3.1-Toll-like receptor 4 (siRNA+ pcDNA3.1-TLR4 group) and si-PTX3+ pcDNA3.1-TLR4 (si-PTX3+ pcDNA3.1-TLR4 group) were transfected into SH-SY5Y cells. Collected 32 cases of tumor tissue and cancerous tissue in children with childhood neuromaternal cells who were treated at Zhumadian center hospital from July 2016 to August 2019. Real-time fluorescent quantitative polymerase chain (RT-qPCR) reaction and immunohistochemistry experiments were used to detect the protein expressions of PTX3 in neuroblastoma tissues and normal tissues. 5-Ethynyl-2'-deoxyuridine (EdU) was used to detect the proliferation effect of PTX3 on neuroblastoma cell SH-SY5Y. Western blot experiment was used to detect the protein expression levels of vascular endothelial growth factor (VEGF), resistance-related proteins including P-glycoprotein (P-gp) and multidrug resistance-associated protein 1 (MRP-1), and invasion-related protein matrix metalloproteinase-1 (MMP-1). Results: PTX3 mRNA expressions in neuroblastoma tissues were 0.87±0.07, higher than 0.13±0.06 of normal tissues, and the differences were statistically significant (P<0.05), The expression of the immunohistochemistry test PTX3 protein was consistent with the qRT-PCR results. Compared with the si-RNA group (0.95±0.08; 1.02±0.10), the mRNA and protein expressions of PTX3 in the si-PTX3 group (0.25±0.05; 0.45±0.66) decreased, the differences were statistically significant (all P<0.05). The number of EdU positive cells, invasion rate, VEGF, MMP-1, P-gp and MRP-1 protein expressions in si-RNA group were (31.86±1.86)%, (28.12±2.96)%, (0.58±0.07), (0.44±0.06), (0.46±0.08) and (0.51±0.05), respectively, higher than (19.73±1.22)%, (8.45±1.06)%, (0.25±0.05), (0.19±0.03), (0.19±0.06) and (0.16±0.07) in si-PTX3 group, and the differences were statistically significant (all P<0.05). The Number of EdU positive cells [(19.49±1.68)%], invasion rate [(8.48±1.36)%], VEGF protein expression (0.10±0.15), P-gp (0.18±0.07) , TLR4 (0.45±0.06), p-p65 (0.25±0.05) protein expressions in si-PTX3+ pcDNA3.1 group were relatively lower compared with siRNA+ pcDNA3.1 group [(38.21±2.67)%, (26.39±2.14)%, 0.49±0.05, 0.52±0.06, 0.93±0.14 and 0.82±0.06] (all P<0.05). The number of EdU-positive cells [(62.73±5.18)%], invasion rate [(50.45±3.25)%], VEGF protein expression (2.17±0.17), P-gp (2.15±0.16), TLR4 (2.68±0.16), p-p65 (2.48±0.13) protein expressions in the siRNA+ pcDNA3.1-TLR4 group increased compared with siRNA+ pcDNA3.1 group (all P<0.05). Conclusions: Inhibition of PTX3 can inhibit the proliferation and invasion of neuroblastoma cells SH-SY5Y, and reduce drug resistance. Its mechanism may be achieved by regulating the TLR4/NF-κB signaling pathway. This result can provide a new perspective for pediatric neuroblasts tumor diagnosis and clinical treatment.

Transcriptional Enhancer Factor Domain Family member 4 Exerts an Oncogenic Role in Hepatocellular Carcinoma by Hippo-Independent Regulation of Heat Shock Protein 70 Family Members.

Transcriptional enhancer factor domain family member 4 (TEAD4) is a downstream effector of the conserved Hippo signaling pathway, regulating the expression of genes involved in cell proliferation and differentiation. It is up‐regulated in several cancer types and is associated with metastasis and poor prognosis. However, its role in hepatocellular carcinoma (HCC) remains largely unexplored. Using data from The Cancer Genome Atlas, we found that TEAD4 was overexpressed in HCC and was associated with aggressive HCC features and worse outcome. Overexpression of TEAD4 significantly increased proliferation and migration rates in HCC cells in vitro as well as tumor growth in vivo. Additionally, RNA sequencing analysis of TEAD4‐overexpressing HCC cells demonstrated that TEAD4 overexpression was associated with the up‐regulation of genes involved in epithelial‐to‐mesenchymal transition, proliferation, and protein‐folding pathways. Among the most up‐regulated genes following TEAD4 overexpression were the 70‐kDa heat shock protein (HSP70) family members HSPA6 and HSPA1A. Chromatin immunoprecipitation–quantitative real‐time polymerase chain reaction experiments demonstrated that TEAD4 regulates HSPA6 and HSPA1A expression by directly binding to their promoter and enhancer regions. The pharmacologic inhibition of HSP70 expression in TEAD4‐overexpressing cells reduced the effect of TEAD4 on cell proliferation. Finally, by overexpressing TEAD4 in yes‐associated protein (YAP)/transcriptional coactivator with PDZ binding motif (TAZ)‐knockdown HCC cells, we showed that the effect of TEAD4 on cell proliferation and its regulation of HSP70 expression does not require YAP and TAZ, the main effectors of the Hippo signaling pathway. Conclusion: A novel Hippo‐independent mechanism for TEAD4 promotes cell proliferation and tumor growth in HCC by directly regulating HSP70 family members.

Modulation of the Extracellular Signal-Regulated Protein Kinase and Tissue Inhibitors of Matrix Metalloproteases-1 Gene in Chronic Neuropathic Pain.

Objectives:The aim of this study is to study the modulation of extracellular signal-regulated protein kinase (ERK) and tissue inhibitors of matrix metalloproteases 1 (TIMP 1) gene in patients with neuropathic pain (NP).Materials and Methods:In the present, cross-sectional, observational study, 2 ml of venous baseline sample was withdrawn from all the patients with neuropathic (NP) or non NP (NNP) soon after their diagnosis or on their first visit to the pain clinic. A real-time quantitative polymerase chain reaction experiment was conducted to measure the mRNA expression of TIMP1 and ERK genes in blood samples. The Delta Ct, Delta Ct, and fold change analysis of both the genes were conducted between patients with NP and NNP.Results:A total of 285 patients with chronic pain were assessed, out of which, 153 patients had NP and 132 had NNP. The average duration of chronic pain was 11 months for 285 patients. The mRNA expression of TIMP1 gene is significantly down regulated (2.65-fold) (P (-f. 01), and the mRNA expression level of ERK is significantly up regulated (2.03-fold) (P (-f. 01) in NP patients when compared with NNP.Conclusion:The mRNA expression of TIMP1 gene is significantly down regulated, and ERK is significantly up regulated in patients with NP. Further, multicentric trials with larger sample size are recommended to confirm this finding.

Are sham-operated mice a valid comparator in studies using a bladder outlet obstruction model? A pitfall reveals a meaningful insight.

To evaluate voiding behavior characteristics in intact and sham mice, and to examine whether intact mice show changes in "normal" micturition with aging. A total of 72 8-week-old mice were divided into two groups - intact and sham - and the latter group was subjected to a sham of partial bladder outlet obstruction surgery. Urination frequency was evaluated (through metabolic cages) at 1, 2, 3, 6 and 12months after the surgery (or at the equivalent time points for the intact mice). To address possible mechanisms for aging and surgical effects on urinary behavior, quantitative real-time polymerase chain reaction assays were carried out. Primary data were evaluated using scatter plots and descriptive statistics. In sham mice, urination frequency showed strong variation at the earlier post-surgical time points (especially at 1month), with variation decreasing with time. Quantitative real-time polymerase chain reaction showed that the serotonin2C receptor-encoding mRNA accumulated to >28-fold higher levels at 24months compared with 3months in intact mice. A major limitation of the quantitative real-time polymerase chain reaction experiments was that we did not separate whole bladder into muscle and mucosa. Although a sham operation is typically used in partial bladder outlet obstruction experiments to provide control animals, the sham group might itself show increased variation in micturition frequency at early times after surgery, compared with intact animals.

Establishment of multifactor predictive models for the occurrence and progression of cervical intraepithelial neoplasia

BackgroundTo study the risk factors involved in the occurrence and progression of cervical intraepithelial neoplasia (CIN) and to establish predictive models.MethodsGenemania was used to build a gene network. Then, the core gene-related pathways associated with the occurrence and progression of CIN were screened in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) experiments were performed to verify the differential expression of the identified genes in different tissues. R language was used for predictive model establishment.ResultsA total of 10 genes were investigated in this study. A total of 30 cases of cervical squamous cell cancer (SCC), 52 cases of CIN and 38 cases of normal cervix were enrolled. Compared to CIN cases, the age of patients in the SCC group was older, the number of parities was greater, and the percentage of patients diagnosed with CINII+ by TCT was higher. The expression of TGFBR2, CSKN1A1, PRKCI and CTBP2 was significantly higher in the SCC groups. Compared to patients with normal cervix tissue, the percentage of patients who were HPV positive and were diagnosed with CINII+ by TCT was significantly higher. FOXO1 expression was significantly higher in CIN tissue, but TGFBR2 and CTBP2 expression was significantly lower in CIN tissue. The significantly different genes and clinical factors were included in the models.ConclusionsCombination of clinical and significant genes to establish the random forest models can provide references to predict the occurrence and progression of CIN.

Anti-Biofilm Effect of Tea Saponin on a Streptococcus agalactiae Strain Isolated from Bovine Mastitis.

Simple SummaryTea saponin (TS), an inexpensive and easily-available plant extract, exhibited antibacterial activity against a Streptococcus agalactiae strain isolated from a dairy cow with mastitis. In addition, TS can inhibit the biofilm formation ability of this strain by down-regulating the transcript levels of biofilm-associated genes including srtA, fbsC, neuA, and cpsE. Hence, TS might be a potential alternative herbal cure for bovine mastitis.Streptococcus agalactiae (GBS) is a highly contagious pathogen which not only can cause neonatal meningitis, pneumonia, and septicemia but is also considered to be a major cause of bovine mastitis (BM), leading to large economic losses to the dairy industry worldwide. Like many other pathogenic bacteria, GBS also has the capacity to form a biofilm structure in the host to cause persistent infection. Tea saponin (TS), is one of the main active agents extracted from tea ash powder, and it has good antioxidant and antibacterial activities. In this study, we confirmed that TS has a slight antibacterial activity against a Streptococcus agalactiae strain isolated from dairy cow with mastitis and inhibits its biofilm formation. By performing scanning electron microscopy (SEM) experiments, we observed that with addition of TS, the biofilm formed by this GBS strain exhibited looser structure and lower density. In addition, the results of real-time reverse transcription polymerase chain reaction (RT-PCR) experiments showed that TS inhibited biofilm formation by down-regulating the transcription of the biofilm-associated genes including srtA, fbsC, neuA, and cpsE.

Improving the freeze-drying survival rate of Lactobacillus plantarum LIP-1 by increasing biofilm formation based on adjusting the composition of buffer salts in medium

Lactic acid bacteria can improve their resistance to adverse environments through the formation of biofilm. This study found that adding different buffer salts in culture medium had a great impact on the freeze-drying survival rate of the Lactobacillus plantarum LIP-1, which could be linked to biofilm formation. Transcriptome data showed that potassium ions in buffer salt increased the expression of the luxS gene in the LuxS/autoinducer-2 (AI-2) quorum sensing system and increase synthesis of the quorum sensing signal AI-2. The AI-2 signal molecules up-regulated the cysE gene, which helps to promote biofilm formation. By adding a biofilm inhibitor, d-galactose, and performing a real-time quantitative polymerase chain reaction experiment, we found that d-galactose could down-regulated the luxS and cysE genes, reduced biofilm formation, and decreased the freeze-drying survival rate. The results of this study showed that promoting biofilm formation using appropriate buffer salts may lead to better freeze-drying survival rates.

Transcriptome-based selection and validation of optimal house-keeping genes for skin research in goats (Capra hircus)

BackgroundIn quantitative real-time polymerase chain reaction (qRT-PCR) experiments, accurate and reliable target gene expression results are dependent on optimal amplification of house-keeping genes (HKGs). RNA-seq technology offers a novel approach to detect new HKGs with improved stability. Goat (Capra hircus) is an economically important livestock species and plays an indispensable role in the world animal fiber and meat industry. Unfortunately, uniform and reliable HKGs for skin research have not been identified in goat. Therefore, this study seeks to identify a set of stable HKGs for the skin tissue of C. hircus using high-throughput sequencing technology.ResultsBased on the transcriptome dataset of 39 goat skin tissue samples, 8 genes (SRP68, NCBP3, RRAGA, EIF4H, CTBP2, PTPRA, CNBP, and EEF2) with relatively stable expression levels were identified and selected as new candidate HKGs. Commonly used HKGs including SDHA and YWHAZ from a previous study, and 2 conventional genes (ACTB and GAPDH) were also examined. Four different experimental variables: (1) different development stages, (2) hair follicle cycle stages, (3) breeds, and (4) sampling sites were used for determination and validation. Four algorithms (geNorm, NormFinder, BestKeeper, and ΔCt method) and a comprehensive algorithm (ComprFinder, developed in-house) were used to assess the stability of each HKG. It was shown that NCBP3 + SDHA + PTPRA were more stably expressed than previously used genes in all conditions analysis, and that this combination was effective at normalizing target gene expression. Moreover, a new algorithm for comprehensive analysis, ComprFinder, was developed and released.ConclusionThis study presents the first list of candidate HKGs for C. hircus skin tissues based on an RNA-seq dataset. We propose that the NCBP3 + SDHA + PTPRA combination could be regarded as a triplet set of HKGs in skin molecular biology experiments in C. hircus and other closely related species. In addition, we also encourage researchers who perform candidate HKG evaluations and who require comprehensive analysis to adopt our new algorithm, ComprFinder.

Chlorogenic Acid Alleviates Allergic Inflammatory Responses Through Regulating Th1/Th2 Balance in Ovalbumin-Induced Allergic Rhinitis Mice.

BackgroundAllergic rhinitis (AR) is a prevalent atopic disorder caused by immune imbalance. Chlorogenic acid (CGA) has antibacterial, antiviral, antioxidative and immunoregulatory effects, but its role in anaphylactic disease remains unclear. The current study aimed to investigate the function of CGA in AR.Material/MethodsAR mice models were induced with ovalbumin (OVA) by orally administrating the mice with 50 mg/kg (L-CGA), 100 mg/kg (M-CGA), or 200 mg/kg (H-CGA) of CGA. The number of nasal rubbings and sneezes was recorded. Afterward, the mice were sacrificed for the collection of blood, nasal lavage fluid (NALF), and nasal tissues. The cells in NALF were counted by hemocytometer and stained by Diff-Quick. Nasal mucosa was observed by H&E staining. ELISA testing was conducted for detecting the levels of anti-OVA antibodies and Th1/Th2-related cytokine. Quantitative real-time polymerase chain reaction experiments were conducted to determine mRNA expressions of Th1/Th2-related cytokines.ResultsIn the OVA-induced AR mice, CGA treatment reduced nasal rubbing and sneezing, and also suppressed the number of total cells, eosinophils, neutrophils, lymphocytes, macrophages, and epithelial cells in NALF. OVA-induced up-regulation of nasal mucosa thickness was inhibited by CGA, and the effects of OVA on IgE, IgG1, and IgG2a were reversed by CGA. The regulatory effects of OVA on mRNA expressions and levels of Th1/Th2-related cytokines were abolished by CGA treatment in AR mice.ConclusionsCGA can alleviate allergic inflammatory responses through regulating Th1/Th2 balance in OVA-induced allergic rhinitis mice.

LncRNA HULC alleviates HUVEC inflammation and improves angiogenesis after myocardial infarction through down-regulating miR-29b.

The aim of this study was to investigate the long non-coding RNA (lncRNA) HULC in promoting angiogenesis after myocardial infarction (MI) and to further investigate its possible mechanism. Twenty-four male Sprague Dawley (SD) rats were randomly divided into two groups, namely, operation group and control group. The rats in the operation group were induced by ligation of the left anterior descending coronary artery, while those in control group received the same surgery without ligating the blood vessels. Seven days after the operation, the myocardial tissues of rats were collected to detect HULC expression by quantitative real-time polymerase chain reaction (RT-PCR). At the same time, the expression of HULC in primary myocardial cells and cardiac microvascular endothelial cells were induced by hypoxia. A hypoxia model was constructed in HUVEC cells, and the effects of HULC were explored by RT-PCR, Western blot Technology (WB), Cell Counting Kit-8 (CCK8) assay, EdU staining, Tube-like structure formation experiments. Thereafter, HULC downstream miRNAs were verified by Luciferase, pull-down, and RNA IP experiments. Similarly, the effects of miR-29b on HUVEC were verified by RT-PCR, WB, CCK8 assay, EdU staining, and tube-like structure formation experiments, respectively. RT-PCR detection results showed that the expression of HULC in myocardial tissues was down-regulated after MI, and the expression of HULC in cardiac microvascular endothelial cells was decreased under hypoxia-induced inflammation. In addition, the overexpression of HULC in HUVEC cells could inhibit the expressions of inflammatory factors (IL-1, IL-6 and IL-8) and promote angiogenesis (increased cell viability, increased tube-like structure formation, and increased cell proliferation). Through Dual-Luciferase reporter gene experiments, it was found that HULC could directly target miR-29b. At the same time, miR-29 overexpression significantly reversed the effects of HULC on cell viability, pro-inflammatory cytokines, and angiogenesis. LncRNA HULC protects HUVEC cells from hypoxia-induced inflammation damage by interacting with miR-29b and inhibiting its expression, and it can also promote angiogenesis.

Effects of 2D-Shear Wave Elastography on Brain-Derived Neurotrophic Factor (BDNF) in the Brains of Neonatal Mice and Exploration of the Mechanism.

BackgroundThe aim of this study was to explore the effect and duration of 2-dimensional shear wave elastography (2D-SWE) irradiation on the expression of brain-derived neurotrophic factor (BDNF) in the brains of neonatal mice and to preliminarily investigate whether its mechanism is neuronal apoptosis.Material/MethodsNeonatal mice (within 48 hours of birth) were subjected to 2D-SWE irradiation of the brain for 10 minutes (group S1), 20 minutes (group S2), and 30 minutes (group S3). The mice were sacrificed immediately after irradiation or 24 hours after irradiation. Brains were collected for real-time polymerase chain reaction (RT-PCR) and western blot experiments to determine the expression of BDNF in each group. TdT-mediated dUTP nick-end labeling (TUNEL) was performed to observe neuronal apoptosis in the brain.ResultsThe results of PCR and western blots from the brains of neonatal mice that were sacrificed immediately after irradiation show that S1, S2, and S3 were significantly different from those in the control group. The PCR and western blot results of brain tissues from neonatal mice sacrificed at 24 hours after irradiation showed that there was no significant difference between the S1, S2, S3, and control groups. The results of TUNEL experiments showed that there was no statistically significant difference in the number of apoptotic neurons between the S1, S2, S3, and control groups.Conclusions2D-SWE irradiation of neonatal mice for more than 10 minutes downregulated the expression of BDNF. This effect disappeared within 24 hours after the irradiation, and the 2D-SWE scan seemed not to induce neuronal apoptosis.

Genome-Wide Analysis of WRKY Genes and Their Response to Salt Stress in the Wild Progenitor of Asian Cultivated Rice, Oryza rufipogon

WRKY gene family is widespread in plants, which is of significance in determining plant development and stress response. Although WRKY transcription factors have been widely characterized in many plants, a genome-wide analysis of the WRKY gene family is still lacking in Oryza rufipogon. In this study, we identified 101 O. rufipogon WRKY (OrWRKY) transcription factors, which were further classified into eight subgroups. Phylogenetic analysis showed that OrWRKY transcription factors were supported by highly conserved motifs and gene structures. Chromosomal distribution of OrWRKYs indicated that most genes were dispersed on all twelve chromosomes, especially enriched on Chromosome 1. Syntenic analysis revealed that 69 (68.3%) genes were derived from either segmental (49) or tandem duplication events (20), suggesting a essential role of segmental duplications. We characterized a total of 39 orthologous gene pairs between O. sativa ssp. japonica WRKY (OsjWRKY) and OrWRKY. We then performed quantitative real-time polymerase chain reaction (qPCR) experiments to validate tissue-specific and differential expression of the OrWRKYs. We also investigated corresponsive expression of the OrWRKYs in response to salt stresses in leaves and roots. This study gains a new insight on the evolution of the OrWRKYs and will help further functional characterization of candidate genes towards wild rice germplasm exploration for rice genetic improvement.

Promoter Genotyping and mRNA Expression - Based Analysis of the PTGDR Gene in Allergy.

Prostaglandin D2 receptors are acquiring a relevant role as potential therapeutic targets in allergy. PTGDR has been described as a candidate gene in allergic disease, although functional studies on this gene are lacking. Objective: The objective of this case-control study was to investigate the potential role of PTGDR in allergy. The study population comprised 195 allergic patients and 112 healthy controls. The PTGDR promoter polymorphisms -1289G>A, -1122T>C, -881C>T, -834C>T, -613C>T, -549T>C, -441C>T, -197T>C, and -95G>T were amplified by polymerase chain reaction (PCR) and sequenced. PTGDR expression levels were analyzed using quantitative PCR and normalized to GAPDH and TBP mRNA levels. All procedures were performed following the Minimum Information for Publication of Quantitative Real-Time PCR Experiment guidelines. PTGDR expression levels were significantly higher in allergic patients than in controls (P<.001). Receiver operating characteristic analysis for expression of PTGDR showed a sensitivity of 81.4% compared with 67% for IgE levels. In addition, differences in the genotypic distribution of the polymorphisms -1289G>A and -1122T>C were found in allergic patients (P=.009). The results indicate that PTGDR overexpression is associated with allergy. The polymorphisms -1289G>A and -1122T>C partly explain the variation in expression we observed. PTGDR expression could have a potential role as a biomarker and pharmacogenetic factor in allergy.

Expression of melatonin receptors and CD4 in the ovine thymus, lymph node, spleen and liver during early pregnancy.

As a pineal gland hormone, melatonin acts through its receptors to modulate the immune system. The immune system is composed of primary and secondary organs, and immune organs are adapted to the presence of the fetal alloantigen during pregnancy. However, it is unclear whether melatonin affects maternal immune organs during early pregnancy in sheep. In this study, the ovine thymus, lymph node, spleen and liver were sampled at day 16 of the oestrous cycle, and at days 13, 16 and 25 of pregnancy. The expression of melatonin receptor 1A (MT1), melatonin receptor 1B (MT2) and cluster of differentiation 4 (CD4) was detected by quantitative real-time polymerase chain reaction, Western blot and immunohistochemistry experiments. Our results showed that during early pregnancy there was an upregulation of MT1 mRNA and protein in the thymus, lymph node and liver, and there was a downregulation in the spleen. The expression of MT2 mRNA and protein was increased in the thymus but decreased in the spleen and liver, and there was no significant change in the lymph node during early pregnancy. CD4 protein was upregulated in the thymus, lymph node and liver, but there were no significant changes in the spleen during early pregnancy. In conclusion, early pregnancy induces tissue-specific expression of MT1, MT2 and CD4, which may be due to the different functions of the thymus, lymph node, spleen and liver. Further, melatonin is involved in immune regulation of the maternal thymus, lymph node, spleen and liver during early pregnancy in sheep.

The MIQE Guidelines' tenth anniversary: The good and bad students

The Polymerase Chain Reaction (PCR) is the most valuable tool that marked the history of molecular biology since the discovery of the DNA structure by Watson and Crick. Its impact on human health over the last 30 years had lead researchers in the field to put strict rules referred to as the Minimum Information for publication of Quantitative real-time PCR Experiments (MIQE) guidelines since 2009. The aim of the current analysis is to shed light on the practice of applying and citing the original MIQE in the published articles over the last decade.We showed that qPCR is a global technique, but the usage of the MIQE is still lagging in the emerging economies around the globe. We have shown that researchers following the MIQE have better chances of publishing highly cited papers. The MIQE represent the laws for this technique: they enslave us, molecular biologists, into a strict path with financial burdens, but they free us from the “human-errors” a machine would impose on us. As science seeks perfection especially when dealing with human problems, the MIQE, as assessed by the publications' quality over the years, did indeed achieve this step forward towards making the PCR an infallible tool.

Selection and Validation of Appropriate Reference Genes for Real-Time Quantitative PCR Analysis in Needles of Larix olgensis under Abiotic Stresses

Larix olgensis Henry is an important afforestation species in northeastern China because of its fast juvenile growth, high-quality timber, and significant economic and ecological values. The selection of appropriate reference genes is necessary for the normalization of gene expression determination during quantitative real-time polymerase chain reaction (qRT-PCR) experiments. In this study, qRT-PCR was used to study gene expression. Three software packages geNorm, NormFinder, BestKeeper were used, and a comprehensive ranking of candidate reference genes was produced based on their output to evaluate the expression stability of 16 candidate reference genes from L. olgensis under drought, salt, cold, and heat stress. PP2A-1 and GAPDH ranked as the most stable reference genes under drought and cold stress, PP2A-1 and UBQ10 were most stable under salt stress, and TIP41 and ACT2 were most stable under heat stress. The least stable gene was ADP, which ranked the last under all treatments. Expression profile analysis of the antioxidant gene CAT using the two most stable and the single least stable reference genes under each stress further verified that the selected reference genes were suitable for gene expression normalization. This study provides an important foundation for the selection of suitable reference genes for the normalization and quantification of L. olgensis gene expression under abiotic stress conditions.

Long non-coding RNA small nucleolar RNA host gene 7 expression level in prostate cancer tissues predicts the prognosis of patients with prostate cancer.

Long non-coding small nucleolar RNA host gene 7 (lncRNA SNHG7) is located on chromosome 9q34.3 in length of 984 bp. SNHG7 has been found to play the role of oncogene in varieties of cancers, and its dysregulation has been found to be associated with carcinogenesis and progression. In the present study, we examined the expression of SNHG7 in prostate cancer tissues and in paired adjacent normal prostate tissues, and we further explored the clinical significance and prognostic value of SNHG7 in prostate cancer patients.A total of 127 prostate cancer tissues were collected from prostate cancer patients who underwent radical prostatectomy between April 2011 and March 2019 at the department of urology, Pudong New Area People's Hospital. Real-time quantitative polymerase chain reaction experiment was performed to detect the relative expressions of SNHG7 in the prostate cancer tissues and normal prostate tissues. The Kaplan–Meier method was used to create survival curves and the log-rank test was used to determine statistical significance. A Cox proportional hazard analysis was used to evaluate the prognostic factors in univariate and multivariate analyses.Compared with paired adjacent normal prostatic tissues, SNHG7 expression was increased in prostate cancer tissues (P < .001). Increased SNHG7 expression correlated with Gleason score (P = .021), bone metastasis (P = .013), pelvic lymph node metastasis (P = .008), and TNM stage (P = .007). Multivariate Cox regression analyses revealed increased SNHG7 expression was independently associated with a poor prognosis of prostate cancer patients (hazard ratio [HR] = 2.839, 95% confidence interval [CI] = 1.921–8.382, P = .038).This study showed that lncRNA-SNHG7 was overexpressed in prostate cancer tissues, and it might contributes to the development and progression of prostate cancer. Furthermore, the SNHG7 expression was associated with the prognosis of prostate cancer, suggesting a potential target for the treatment and prognosis of prostate cancer. Nevertheless, the underlying modulatory mechanism by which SNHG7 aggravates prostate cancer progression need to be further studied.

Bladder Regeneration Using a Polycaprolactone Scaffold with a Gradient Structure and Growth Factors in a Partially Cystectomized Rat Model.

BackgroundTissue engineering can be used for bladder augmentation. However, conventional scaffolds result in fibrosis and graft shrinkage. This study applied an alternative polycaprolactone (PCL)-based scaffold (diameter = 5 mm) with a noble gradient structure and growth factors (GFs) (epidermal growth factor, vascular endothelial growth factor, and basic fibroblast growth factor) to enhance bladder tissue regeneration in a rat model.MethodsPartially excised urinary bladders of 5-week-old male Slc:SD rats were reconstructed with the scaffold (scaffold group) or the scaffold combined with GFs (GF group) and compared with sham-operated (control group) and untreated rats (partial cystectomy group). Evaluations of bladder volume, histology, immunohistochemistry (IHC), and molecular markers were performed at 4, 8, and 12 weeks after operation.ResultsThe bladder volumes of the scaffold and GF group recovered to the normal range, and those of the GF group showed more enhanced augmentation. Histological evaluations revealed that the GF group showed more organized urothelial lining, dense extracellular matrix, frequent angiogenesis, and enhanced smooth muscle bundle regeneration than the scaffold group. IHC for α-smooth muscle actin, pan-cytokeratin, α-bungarotoxin, and CD8 revealed that the GF group showed high formation of smooth muscle, blood vessel, urothelium, neuromuscular junction and low immunogenicity. Concordantly, real-time polymerase chain reaction experiments revealed that the GF group showed a higher expression of transcripts associated with smooth muscle and urothelial differentiation. In a 6-month in vivo safety analysis, the GF group showed normal histology.ConclusionThis study showed that a PCL scaffold with a gradient structure incorporating GFs improved bladder regeneration functionally and histologically.