Real-time Polymerase Chain Reaction Quantification Research Articles

Bioactive Sphene-Based Ceramic Coatings on cpTi Substrates for Dental Implants: An In Vitro Study.

Titanium implant surface modifications have been widely investigated to favor the process of osseointegration. The present work aimed to evaluate the effect of sphene (CaTiSiO5) biocoating, on titanium substrates, on the in vitro osteogenic differentiation of Human Adipose-Derived Stem Cells (hADSCs). Sphene bioceramic coatings were prepared using preceramic polymers and nano-sized active fillers and deposited by spray coating. Scanning Electron Microscopy (SEM) analysis, surface roughness measurements and X-ray diffraction analysis were performed. The chemical stability of the coatings in Tris-HCl solution was investigated. In vitro studies were performed by means of proliferation test of hADSCs seeded on coated and uncoated samples after 21 days. Methyl Thiazolyl-Tetrazolium (MTT) test and immunofluorescent staining with phalloidin confirmed the in vitro biocompatibility of both substrates. In vitro osteogenic differentiation of the cells was evaluated using Alizarin Red S staining and quantification assay and real-time PCR (Polymerase Chain Reaction). When hADSCs were cultured in the presence of Osteogenic Differentiation Medium, a significantly higher accumulation of calcium deposits onto the sphene-coated surfaces than on uncoated controls was detected. Osteogenic differentiation on both samples was confirmed by PCR. The proposed coating seems to be promising for dental and orthopedic implants, in terms of composition and deposition technology.

Protective effect of ginsenoside Rg3 on lung injury in diabetic rats.

Ginsenoside has been used to treat diabetes, while ginsenoside Rg3 is the main active ingredient component of ginseng and is used to study its effects on lung tissue damage in diabetic rats. In this paper, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and flow cytometry were applied to detect the proliferation and apoptosis of BEAS-2B cells treated with different concentrations of Rg3. The inflammatory response and pathological change in the lung tissue of diabetic rats treated with Rg3 were evaluated by enzyme-linked immunosorbent assay, quantative real-time polymerase chain reaction, andhematoxylin and eosin staining immunohistochemistry. Meanwhile, PI3K and MAPK signaling pathway proteins in lung tissue were determined by Western blot analysis. The results showed that ginsenoside Rg3 had no significant influence on the proliferation and apoptosis of BEAS-2B cells. Ginsenoside Rg3 can inhibit inflammatory response and promote the activation of PI3K and MAPK signaling pathways to prevent damages of lung tissues induced by hyperglycemia. The protective effect provided by ginsenoside Rg3 indicates that ginsenoside Rg3 is a potential drug for the treatment of diabetes.

Effects and mechanism of the long-snake moxibustion on ankylosing spondylitis based on Th17/Treg/Th1 immune imbalance

To explore the effects and mechanisms of the long-snake moxibustion on ankylosing spondylitis (AS) based on Th17/Treg/Th1 immune imbalance. A total of 60 AS patients were randomized into an observation group and a control group, 30 cases in each one. In the observation group, the long-snake moxibustion therapy was used on the acupoints of the governor vessel from Dazhui (GV 14) to Yaoshu (GV 2) as well as the bilateral Jiaji (EX-B 2) alternatively. The moxibustion was given once a day, for 7 days continuously as one course. There were 3 days at the interval between the courses and 4 courses were required. In the control group, the routine western medication was provided, the salazosulfapyridine combined with non-steroidal anti-inflammatory drugs were used, for 7 days continuous as one course. A total of 4 courses of medication were required. The enzyme linked immunosorbent assay (ELISA) was adopted to determine the levels of interleukin-6 (IL-6), interleukin-17 (IL-17), interleukin-23 (IL-23) and tumor necrosis factor-α (TNF-α). The real-time quantification polymerase chain reaction (RT-PCR) was used to determine the mRNA expressions of the specific transcription factors, FoxP3 and T-bet of the helper 17 cells (Th17), regulatory T cells (Treg) and T helper 1 cells (Th1). The flow cytometry was applied to determine the rates of Treg, Th1 and Th17, as well as the changes of the inflammatory reaction index, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR). The therapeutic effects were compared between the two groups. After treatment, the total effective rate was 93.3% (28/30) in the observation group, which was better than 86.7% (26/30) in the control group (P<0.05). After treatment, the levels of CRP, ESR, IL-6, IL-17, IL-23 and TNF-α, as well as the rate of Th17 were reduced significantly as compared with those before treatment in the observation group (all P<0.05). The mRNA expressions of FoxP3 and T-bet and the rates of Treg and Th1 were increased as compared with those before treatment (all P<0.05). The change degree in the observation group was significant as compared with the control group (all P<0.05). In the control group, the levels of CRP, ESR, IL-6, IL-17, IL-23 and TNF-α, as well as the rate of Th17 were reduced, and the mRNA expressions of FoxP3 and T-bet and the rates of Treg and Th1 were increased after treatment. But the changes were not significant as compared with those before treatment (all P>0.05). The long-snake moxibustion effectively relieves the clinical symptoms in AS patients and regulates the Th17/Treg/Th1 immune imbalance. Its effect target is probably related to the modulation of the AS immune derangement and the inflammatory responses induced by immune derangement so as to achieve the dual-positive regulatory effect.

Expressions of TNF-α, TNFR1 and TNFR2 in different cervical lesions and their relationships with clinicopathological characteristics

Objective To investigate the expressions of tumor necrosis factor-alpha (TNF-α), tumor necrosis factor receptor (TNFR)1 and TNFR2 in different cervical lesions and their relationships with clinicopathological characteristics. Methods Forty-one cases of cervical squamous cell carcinoma (CSCC) patients (CSCC group) treated in 254th Hospital of People′s Liberation Army from January 2015 to December 2017 were selected as the subjects. Forty-nine cases of high grde squmous intrepithelial lesion (HSIL) (HSIL group) and fifty cases of uterine myoma (normal group) were selected as control groups. The expression of TNF-α in cervical tissues of different lesions was detected by immunohistochemistry. The expressions of TNF-α mRNA, TNFR1 mRNA and TNFR2 mRNA were detected by quantificational real-time polymerase chain reaction (qRT-PCR). The expression level of TNFR1 and TNFR2 proteins are measured by Western blotting. The relationships between the expression level of TNF-α mRNA, TNFR1 mRNA and the clinicopathological characteristics of patients were analyzed. Results The positive rates of TNF-α in the normal group, the HSIL group and the CSCC group were 8.0% (4/50), 59.2% (29/49) and 73.2% (30/41). The difference between three groups was statistically significant (χ2=44.786, P<0.001). The positive rates of the HSIL group and the CSCC group were significantly higher than that in the normal group, the difference was statistically significant (χ2=29.175, P<0.001; χ2=40.883, P<0.001), but there was no statistical difference in the positive rates of TNF-α in CSCC group and HSIL group (χ2=1.934, P=0.164). The results of qRT-PCR showed that the expressions of TNF-α mRNA in normal group, HSIL group and CSCC group were 1.32±0.21, 3.64±0.41 and 7.51±1.42. The difference of TNF-α mRNA expression among three groups was statistically significant (F=655.800, P<0.001). The expressions level of TNF-α mRNA in HSIL group and CSCC group were significantly higher than that in normal group (t=31.747, P<0.001; t=51.012, P<0.001), and the expression level of CSCC group was significantly higher than that in HSIL group (t=20.039, P<0.001). The expression levels of TNFR1 mRNA in the normal group, the HSIL group and the CSCC group were 0.42±0.13, 0.89±0.21 and 2.23±0.46. The relative expression of TNFR1 mRNA between the three groups was statistically significant (F=465.900, P<0.001). The expression levels of TNFR1 mRNA in group HSIL and CSCC were significantly higher than that in normal group (t=13.357, P<0.001; t=26.587, P<0.001), and the expression level of CSCC group was significantly higher than that in HSIL group (t=18.407, P<0.001). The expression of TNFR2 mRNA in the normal group, the HSIL group and the CSCC group were 0.38±0.14, 0.41±0.11 and 0.44±0.12. There was no significant difference between three groups (F=2.633, P=0.075). Western blottting showed that the expression intensity of TNFR1 in the normal group, the HSIL group and the CSCC group were 0.84±0.18, 1.95±0.21 and 3.38±0.73, the difference was statistically significant (F=398.000, P<0.001). The expression intensity of TNFR1 in group HSIL and CSCC were significantly higher than that in normal group (t=18.273, P<0.001; t=39.894, P<0.001), and the expression in CSCC group was also significantly higher than that in group HSIL (t=22.357, P<0.001). The expression intensity of TNRF2 in normal group, HSIL group and CSCC group were 0.98±0.15, 1.02±0.17, 1.07±0.21, and the difference was not statistically significant (F=2.938, P=0.056). The results of protein detection were in accordance with the results of mRNA detection. The expression of TNF-α mRNA in the CSCC tissues was related to the size of the tumor (t=-8.868, P<0.001), the degree of differentiation (t=-5.644, P<0.001), the clinical stage (t=-19.329, P<0.001), the depth of infiltration (t=-11.170, P<0.001), and lymph node metastasis (t=-8.339, P<0.001). The expression of TNFR1 mRNA was closely related to the tumor size (t=-13.309, P<0.001), degree of differentiation (t=-13.449, P<0.001), clinical stage (t=-12.949, P<0.001), depth of infiltration (t=-18.124, P<0.001), and lymph node metastasis (t=-20.506, P<0.001). Conclusion In cervical cancer tissues, the expression intensity of TNF-α and TNFR1 increased abnormally, while TNFR2 did not change significantly. The expre-ssions of TNF-α and TNFR1 are positively correlated with the malignancy of cervical cancer. They are potential signals of cervical cancer and are expected to become new therapeutic targets. However, the activation of TNFR2 to downstream signaling pathway is significantly weaker than that of TNFR1. Key words: Uterine cervical neoplasms; Tumor necrosis factor-alpha; Receptors, tumor necrosis factor

Expanded Diagnostic Approach to Hepatitis E Virus Detection in Patients with Acute-on-Chronic Liver Failure: A Pilot Study

Introduction: Acute decompensation of pre-existing chronic liver disease (CLD), known as acute-on-chronic liver failure (ACLF), is associated with high mortality. Hepatitis E virus (HEV) as a potential cause was studied. Objectives: The objectives of this study are to evaluate the role of HEV in ACLF patients using an IgM anti-HEV antibody enzyme-linked immunosorbent assay (ELISA), HEV antigen ELISA, and a quantitative HEV polymerase chain reaction (PCR). Materials and Methods: In this prospective cross-sectional study, blood samples were collected from 50 ACLF (cases) as defined by the standard guidelines (APASL, 2014) and 50 patients with stable CLD (controls) from January 2015 to August 2016, after obtaining informed consent. Two IgM ELISAs (MP Diagnostics HEV IgM ELISA 3.0, Singapore and Wantai HEV IgM ELISA, Beijing, China) were compared using plasma from cases and controls. In addition, an HEV antigen detection by ELISA (Wantai, Beijing, China) and a real-time PCR for quantification of HEV RNA in plasma and stool were employed. Results: Ethanol was the leading cause of acute insult in ACLF (54%) cases. HEV infection accounted for 20% of cases. Ten ACLF patients (20%) had 1–3 markers of HEV versus two (4%) among controls (P = 0.0138). Among ACLF cases, one had HEV viraemia (403 IU/ml), faecal shedding (2790 IU/ml) and detectable HEV antigenaemia. Agreement between the two anti-HEV IgM ELISAs was 0.638 (kappa value). Conclusion: This study shows that alcohol is a major contributing factor for both underlying CLD and ACLF while HEV is the most common infectious cause for ACLF, suggesting a need for a vaccination in such patients, whenever made available.

Differences in expression of Mohawk transcription factors and collagen in anterior cruciate ligament grafts between two remodeling outcomes under arthroscopy

Objective To investigate the differences in expression of Mohawk (MKX) transcription factors and collagen of types Ⅰ and Ⅲ in anterior cruciate ligament (ACL) grafts between 2 remodeling outcomes under arthroscopy. Methods Enrolled for this study were 17 patients who had undergone arthroscopic single-bundle ACL reconstruction with autogenous hamstring tendons and secondary arthroscopic exploration 48 to 131 months (average, 83.1 months) after removal of tibial internal fixator at Department of Sports Medicine, The First Affiliated Hospital to Shenzhen University from March 2017 to December 2017. They were divided into a good remodeling group (11 cases) and a fair remodeling group (6 cases) according to the graft quality under arthroscopy (synovial and vascular coverage, and apparent tension, thickness and retear of the grafts). During the secondary arthroscopic procedures, biopsy of the central ACL grafts was performed. Moreover, normal ACL tissues were harvested from 8 contemporary controls of <60 years old who underwent total knee replacement. Immunohistochemical assay and quantificational real-time polymerase chain reaction were conducted to detect the expression of transcription factors and collagen of types Ⅰ and Ⅲ in all the samples. Results In the samples from good remodeling and control groups, there were abundant well-arranged collagen fibers of types Ⅰ and Ⅲ and MKX-positive cells; in the fair remolding group, the collagen fibers of types Ⅰ and Ⅲ and MKX-positive cells were much decreased in number and the fibers were not well arranged. The former 2 groups scored in immunohistochemical assay significantly higher than the latter one (P<0.05). qRT-PCR showed that the expression levels of MKX gene (0.44±0.30), COL1A1 gene (0.52±0.27) and COL3A1 gene (0.60±0.22) in the fair remolding group were significantly lower than in the control group (1.00±0.00, 1.00±0.00 and 1.00±0.00) and than in the good remolding group (0.97±0.67, 0.99±0.38 and 1.00±0.35) (P<0.05). Conclusion Good remodeling ACL grafts with histological maturation under arthroscopy are more similar to normal ACL than fair remodeling ACL grafts in expression of MKX transcription factors and collagen of types Ⅰ and Ⅲ. Key words: Anterior cruciate ligament; Transplantation, autologous; Ligaments, articular; Transcription factors; Collagen

Detection and Quantification of Genetically Modified Soybean in Some Food and Feed Products. A Case Study on Products Available on Romanian Market

The aim of this paper is to trace genetically modified soybean in food and feed products present on the Romanian market by using molecular extraction, identification and quantification methodologies. Nine samples (3 food samples, 5 soybean samples and 1 soybean meal) were analysed using the classical and real-time polymerase chain reaction (PCR) method. DNA-genetically modified organism (GMO) was not detected in two of the three analysed samples (food products). However, it could be found in four samples ranging below the limit of 0.9%, and in three samples, above the limit of 0.9%. The results obtained through real-time PCR quantification show that DNA-RRS was detectable in different amounts in different samples: ranging between 0.27% and 9.36% in soy beans, and reaching 50.98% in soybean meal. The current research focuses on how products containing GMO above the limit (it is common knowledge that it is necessary to label the products containing more than 0.9% Genetically Modified DNA) are differentiated on the market with a view to labeling food and feed products in terms of the accidental presence of approved genetically modified plants. The benefits brought by genetic engineering in obtaining genetically modified organisms can be balanced with their public acceptance and with certain known or unknown risks that they can bring.

Titration of Lentivirus Vectors.

The titer of a lentivirus vector is often expressed in transducing units per milliliter. This is a functional titer that reflects the lentivirus' ability to transduce a particular cell line under specific conditions. Transduction of other cell lines is likely to be different and will require optimization. 293T cells are used for production of lentivirus stocks, and they can be easily transduced with vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentivirus vectors. Consequently, this cell line is commonly used to determine the functional titer of lentivirus vector stocks produced with this envelope. For lentivirus vectors encoding fluorescent proteins under the control of promoters functional in these cells, titration can be performed using the limiting dilution method or a flow cytometry-based method. For lentivirus vectors lacking a fluorescent marker, or for those carrying promoters that may not be functional in 293T cells, titer can be determined either by real-time polymerase chain reaction (PCR) quantification of viral genomes in genomic DNA from transduced cells or a p24 ELISA-based assay.

Circulating and Urinary miR-210 and miR-16 Increase during Cardiac Surgery Using Cardiopulmonary Bypass – A Pilot Study

A pilot study to measure and compare blood and urine microRNAs miR-210 and miR-16 in patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and off-pump coronary artery bypass grafting surgery. Frequent serial blood and urine samples were taken from patients undergoing cardiac surgery with CPB (n = 10) and undergoing off-pump cardiac surgery (n = 5) before, during, and after surgery. Circulating miR-210 and miR-16 levels were determined by relative quantification real-time polymerase chain reaction. Levels of plasma-free haemoglobin (fHb), troponin-T, creatine kinase, and creatinine were measured. Perioperative serum miR-210 and miR-16 were elevated significantly compared to preoperative levels in patients undergoing cardiac surgery with CPB (CPB vs. Pre Op and Rewarm vs. Pre Op; p &lt; .05 for both). There were increases of greater than 200% in miR-210 levels during rewarming and immediately postoperatively and a 3,000% increase in miR-16 levels immediately postoperatively in urine normalized to urinary creatinine concentration. Serum levels of miR-16 were relatively constant during off-pump surgery. miR-210 levels increased significantly in off-pump patients perioperatively (p &lt; .05 Octopus on vs. Pre Op); however, the release was less marked when compared to cardiac surgery with CPB. A significant association was observed between both miR-16 and miR-210 and plasma fHb when CPB was used (r = −.549, p &lt; .0001 and r = −.463, p &lt; .0001 respectively). Serum and urine concentrations of hypoxically regulated miR-210 and hemolysis-associated miR-16 increased in cardiac surgery using CPB compared to off-pump surgery. These molecules may have utility in indicating severity of cardiac, red cell, and renal injury during cardiac surgery.

Real-time PCR quantification of 9 periodontal pathogens in saliva samples from periodontally healthy Korean young adults.

PurposeFew studies have examined periodontal pathogens from saliva samples in periodontally healthy young adults. The purposes of this study were to determine the prevalence of periodontopathic bacteria and to quantify periodontal pathogens in saliva samples using real-time polymerase chain reaction (PCR) assays in periodontally healthy Korean young adults under 35 years of age.MethodsNine major periodontal pathogens were analyzed by real-time PCR in saliva from 94 periodontally healthy young adults. Quantification of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum, Campylobacter rectus, Peptostreptococcus anaerobius, and Eikenella corrodens was performed by DNA copy number measurement.ResultsF. nucleatum and E. corrodens were detected in all subjects; the numbers of positive samples were 87 (92.6%), 91 (96.8%), and 90 (95.7%) for P. gingivalis, P. anaerobius, and C. rectus, respectively. Other pathogens were also detected in periodontally healthy subjects. Analysis of DNA copy numbers revealed that the most abundant periodontal pathogen was F. nucleatum, which was significantly more prevalent than all other bacteria (P<0.001), followed by P. anaerobius, P. gingivalis, E. corrodens, C. rectus, and T. denticola. There was no significant difference in the prevalence of each bacterium between men and women. The DNA copy number of total bacteria was significantly higher in men than in women.ConclusionsMajor periodontal pathogens were prevalent in the saliva of periodontally healthy Korean young adults. Therefore, we suggest that the development of periodontal disease should not be overlooked in periodontally healthy young people, as it can arise due to periodontal pathogen imbalance and host susceptibility.

Metagenomic investigation of gastrointestinal microbiome in cattle.

The gastrointestinal (GI) tract, including the rumen and the other intestinal segments of cattle, harbors a diverse, complex, and dynamic microbiome that drives feed digestion and fermentation in cattle, determining feed efficiency and output of pollutants. This microbiome also plays an important role in affecting host health. Research has been conducted for more than a century to understand the microbiome and its relationship to feed efficiency and host health. The traditional cultivation-based research elucidated some of the major metabolism, but studies using molecular biology techniques conducted from late 1980’s to the late early 2000’s greatly expanded our view of the diversity of the rumen and intestinal microbiome of cattle. Recently, metagenomics has been the primary technology to characterize the GI microbiome and its relationship with host nutrition and health. This review addresses the main methods/techniques in current use, the knowledge gained, and some of the challenges that remain. Most of the primers used in quantitative real-time polymerase chain reaction quantification and diversity analysis using metagenomics of ruminal bacteria, archaea, fungi, and protozoa were also compiled.

Construction of A New Random Short Hairpin RNA Library Based on microRNA Context.

Objective To build an efficient random short hairpin RNA(shRNA)library. Methods shRNA expression vector was constructed with enhanced green fluorescent protein(EGFP)in the upstream of shRNA,driven by pol Ⅱ promoter(CMV).After the constructs were transfected into cells,the proteins were collected.The inhibition efficiency of shRNA was determined by Western blot and dual luciferase reporter system.After the shRNA expression vector was constructed with EGFP in the upstream of shRNA,driven by pol Ⅱ promoter(CMV),shRNA was further embedded into microRNA(miRNA)context.The constructs were transfected into cells,and then the inhibition efficiency of shRNA against target genes was evaluated by quantificational real-time polymerase chain reaction.According to the result of quantificational real-time polymerase chain reaction,a new random shRNA library was constructed based on miRNA context. Results shRNA downstream of a large transcript was transcripted efficiently by pol Ⅱ promoter(CMV).The efficiency of shRNA interference on target gene was improved when shRNA was embedded into miRNA context.Thus,we constructed a new random shRNA library sized 1.8×107 based on miRNA context.Conclusion We successfully constructed a new large random shRNA library.

Effect of ionizing radiation on the NKG2D ligand expression on the tumor cell SCC25 surface and the killing effect on tumor cells

Objective To investigate the effect of ionizing radiation on the expression of NKG2D ligand on the surface of oral squamous cell carcinoma cell line SCC25 and its cytotoxicity to tumor cells. Methods When SCC25 cells were cultured into logarithmic growth phase, they were randomly designed as control (without treatment) and experimental group (2 Gy ionizing radiation treatment) by drawing lots. Flow cytometry was used to detect the expressions of NKG2D ligands major histocompatibility complex class Ⅰ chain-related molecule (MIC)A, MICB, UL16 binding protein (ULBP)1 on the surface of SCC25 in the control group and the experimental group cultured for 24 h. Real-time fluorescence quantification polymerase chain reaction (RT-PCR) was used to detect the changes of NKG2D ligand mRNA expression on SCC25 cell surface after 24 h culture in the experimental group and the control group. The cells were prepared and divided into blank control group (NC), 2 Gy ionizing radiation group (R), NK1 group (target ratio was 5∶1), NK2 group (target ratio was 20∶1), NK1+ R group (target ratio was 5∶1, 2 Gy ionizing radiation), NK2+ R group (target ratio was 20∶1, 2 Gy ionizing radiation). After each group was cultured for 24 h, the killing abilities of ionizing radiation and natural killer (NK) cells to oral squamous cell carcinoma SCC25 cells were detected by CCK8. Results Flow cytometry experiment showed that, among the NKG2D ligands, the MICA fluorescence values of experimental group and control group were respectively 21.04±0.39, 22.90±0.40 (t=2.465, P=0.069), MICB fluorescence values were 27.58±0.50, 29.83±1.05 (t=1.936, P=0.125), and ULBP1 fluorescence values were 21.04±0.40, 21.78±0.50 (t=1.154, P=0.313). This indicated that after ionizing radiation on SCC25, the NKG2D ligand MICA, MICB, ULBP1 expression increased slightly, but the differences were not statistically significant. RT-PCR indicated that mRNA expressions of MICB, ULBP1 were significantly different between the control group and the experimental group (t=18.334, P=0.000; t=6.381, P=0.008). The expressions of the experimental group were respectively 6.49, 1.64 times as those of the control group. The results of CCK8 showed that, there was a significant difference in cell killing ability among NK1 group, NK2 group and NC group (F=344.600, P=0.000), suggesting that NK cells could kill tumor cells, and the higher ratio of NK cells and SCC25, the stronger killing effect. The comparison between R group and NC group showed that the difference in cell killing ability was not statistically significant (P=0.567). NK1+ R group and NK1 group were compared and the difference was not statistically significant (P=0.915). There was no significant difference between NK2+ R group and NK2 group (P=0.678). This showed that the killing effect of ionizing radiation was weak. Conclusion Ionizing radiation can increase the mRNA expression of NKG2D ligands MICB and ULBP1. This may provide a new way for tumor immunotherapy. The killing effect of ionizing radiation on cells is not obvious. It may be related to low radiation dose and only 24 h for cell culture. Key words: Radiation, ionizing; Killer cells, natural; NKG2D ligands

MicroRNA Changes in Cerebrospinal Fluid After Subarachnoid Hemorrhage.

Delayed cerebral ischemia (DCI) accounts for a major part of the morbidity and mortality after aneurysmal subarachnoid hemorrhage (SAH). MicroRNAs (miRNAs) are pathophysiologically involved in acute cerebral ischemia. This study compared miRNA profiles in cerebrospinal fluid from neurologically healthy patients, as well as SAH patients with and without subsequent development of DCI. In a prospective case-control study of SAH patients treated with external ventricular drainage and neurologically healthy patients, miRNA profiles in cerebrospinal fluid were screened and validated using 2 different high-throughput real-time quantification polymerase chain reaction techniques. The occurrence of DCI was documented in patient charts and subsequently reviewed independently by 2 physicians. MiRNA profiles from 27 SAH patients and 10 neurologically healthy patients passed quality control. In the validation, 66 miRNAs showed a relative increase in cerebrospinal fluid from SAH patients compared with neurologically healthy patients (P<0.001); 2 (miR-21 and miR-221) showed a relative increase in SAH patients with DCI compared with those without (P<0.05) in both the screening and validation. SAH is associated with marked changes in the cerebrospinal fluid miRNA profile. These changes could be associated to the development of DCI. URL: http://www.clinicaltrials.gov. Unique identifier: NCT01791257.

MiR-34a and miR-206 act as novel prognostic and therapy biomarkers in cervical cancer

BackgroundRecent evidence indicated that the aberrant expression of microRNA plays a crucial role in the development of cervical cancer. The overall shorter survival was strongly related to the abnormal expression of microRNA-34a (miR-34a) and microRNA-206 (miR-206), which target B cell lymphoma-2(Bcl2) and c-Met. Hepatocyte growth factor (HGF)/c-Met pathway is related to the occurrence, development and prognosis of cervical cancer, and c-Met is significantly overexpressed in cervical squamous cell carcinoma. Bcl2 is also considered to be a promising target for developing novel anticancer treatments.MethodsIn this study, we detect the expression of miR-34a and miR-206 in the cervical cancer tissue through quantificational real-time polymerase chain reaction (qRT-PCR) assay, and the expression of Bcl2 and c-Met from cervical cancer tissue were detected by immunohistochemistry.ResultsThe expression of miR-34a and miR-206 were down-regulated in the cervical cancer tissue through qRT-PCR assay. As target genes of miR-34a and miR-206, Bcl2 and c-Met were up-regulated in cervical cancer tissues through qRT-PCR assay and immunohistochemistry. Kaplan–Meier and log-rank analysis revealed that down-regulated expression of miR-34a and miR-206 were strongly related to shorter overall survival. Multivariate Cox proportional hazards model for all variables that were statistically significant in the univariate analysis demonstrated that miR-34a (P = 0.038) and miR-206 (P = 0.008) might be independent prognostic factors for overall survival of patients suffering from cervical cancer.ConclusionsThe up-regulation of Bcl2 and c-Met promotes the cervical cancer’s progress, and the expression of miR-34a and miR-206 significantly correlated with the progression and prognosis in cervical cancer. All of these suggested that miR-34a and miR-206 might be the novel prognostic and therapy tools in cervical cancer.

EFFECT OF ASTRAGALOSIDE ON VITAMIN D-RECEPTOR EXPRESSION AFTER ENDOTHELIN-1-INDUCED CARDIOMYOCYTE INJURY.

Background:Astragaloside, which is one of the main components of Astragalus membranaceus, has been widely used in the treatment of congestive heart failure in China, and it can protect cardiomyocytes. Its mechanism of action remains unclear. Therefore, the present study was carried out to investigate the influence of astragaloside on rat cardiomyocytes stimulated with endothelin-1 (ET-1), and explored the underlying mechanism.Materials and Methods:ET-1 was used to stimulate primary rat cardiomyocytes and establish a cardiomyocyte hypertrophy model. Different astragaloside doses were administered in combination with ET-1. Cardiomyocyte hypertrophy and apoptosis were examined using transmission electron microscopy (TEM) and flow cytometry, respectively. The molecular mechanism was explored by analyzing the mRNA of the vitamin D receptor (VDR), cytochrome P450 family 27 subfamily B member 1(CYP27B), cytochrome P450 family 24 subfamily A member 1(CYP24A) and renin mRNA levels by quantificational real-time polymerase chain reaction(qRT-PCR).Results:Rat cardiomyocyte hypertrophy model was established successfully. Astragaloside administration significantly affected cell apoptosis and significantly inhibited ET-1-induced cardiomyocyte hypertrophy in a dose-dependent manner. Astragaloside treatment affected the expression of signaling molecules in the vitamin D axis.Conclusion:Astragaloside inhibits ET-1-induced cardiomyocyte hypertrophy. This effect can be reversed by regulating the levels of the relevant factors in the vitamin D axis.

B-Myb Is Up-Regulated and Promotes Cell Growth and Motility in Non-Small Cell Lung Cancer.

B-Myb is a transcription factor that is overexpressed and plays an oncogenic role in several types of human cancers. However, its potential implication in lung cancer remains elusive. In the present study, we have for the first time investigated the expression profile of B-Myb and its functional impact in lung cancer. Expression analysis by quantificational real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry demonstrated that B-Myb expression is aberrantly overexpressed in non-small cell lung cancer (NSCLC), and positively correlated with pathologic grade and clinical stage of NSCLC. A gain-of-function study revealed that overexpression of B-Myb significantly increases lung cancer cell growth, colony formation, migration, and invasion. Conversely, a loss-of-function study showed that knockdown of B-Myb decreases cell growth, migration, and invasion. B-Myb overexpression also promoted tumor growth in vivo in a NSCLC xenograft nude mouse model. A molecular mechanistic study by RNA-sequencing (RNA-seq) analysis showed that B-Myb overexpression causes up-regulation of various downstream genes (e.g., COL11A1, COL6A1, FN1, MMP2, NID1, FLT4, INSR, and CCNA1) and activation of multiple critical pathways (e.g., extracellular signal-regulated kinases (ERK) and phosphorylated-protein kinase B (Akt) signaling pathways) involved in cell proliferation, tumorigenesis, and metastasis. Collectively, our results indicate a tumor-promoting role for B-Myb in NSCLC and thus imply its potential as a target for the diagnosis and/or treatment of NSCLC.

MP94-01 PROTECTIVE EFFECT OF PLATELET-RICH PLASMA ON URETHRAL STRICTURE MODEL OF MALE RATS

You have accessJournal of UrologyBladder & Urethra: Anatomy, Physiology & Pharmacology II1 Apr 2017MP94-01 PROTECTIVE EFFECT OF PLATELET-RICH PLASMA ON URETHRAL STRICTURE MODEL OF MALE RATS HASAN TAVUKCU, OMER AYTAÇ, FATIH ATUG, BURÇIN ALEV, OZGE CEVIK, AYSEN YARAT, SULE CETINEL, GÖKSEL SENER, and HALUK KULAKSIZOGLU HASAN TAVUKCUHASAN TAVUKCU More articles by this author , OMER AYTAÇOMER AYTAÇ More articles by this author , FATIH ATUGFATIH ATUG More articles by this author , BURÇIN ALEVBURÇIN ALEV More articles by this author , OZGE CEVIKOZGE CEVIK More articles by this author , AYSEN YARATAYSEN YARAT More articles by this author , SULE CETINELSULE CETINEL More articles by this author , GÖKSEL SENERGÖKSEL SENER More articles by this author , and HALUK KULAKSIZOGLUHALUK KULAKSIZOGLU More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2904AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urethral stricture formation is caused by fibrosis after excessive collagen formation to the any injury or trauma to the urethra. We evaluated the affects of platelet-rich plasma (PRP) on urethral stricture model of male rats. METHODS Urethral stricture model was performed by coagulation current to the male urethra. There were 4 groups of 6 rats in each one: control (C), C+PRP applied, urethral stricture (US), US+PRP applied. PRP was applied to the urethra after coagulation current induced injury as soon as possible. On 14th day all rats were sacrified and urethral tissues investigated for collagen type I (col I), collagen type III (col III), platelet-derived growth factor-A (PDGF-A), platelet-derived growth factor-B (PDGF-B) and transforming growth factor-B (TGF-B) with quantative real-time polymerase chain reaction (RT-PCR) and Western-Blot analysis. The efffect of urethral damage and healing was investigated for col I/col III ratio. RESULTS Collagen type I increase in fibrosis process in US is well defined. Collagen type I/ collagen type III ratio was significantly high in US group (*, p=0,000) than others while US+PRP group had comparable results with the control group (p=0,999). (Graphic 1) RT-PCR analysis of col I, col III, PDGF-A, PDGF-B and TGF-B was shown in table 1. Graphic 1: Col I/Col III type ratio of each group. Table 1: Results of RT-PCR analysis of col I, col III, PDGF-A, PDGF-B and TGF-B.(* Significant between US and US+PRP groups; & not significant between C and US+PRP groups; ** significant between C and US+PRP groups) CONCLUSIONS Our results show that PRP has a preventive effect on stricture formation in US rat model shown by its effect on collagen synthesis, especially in recurrent cases. Further studies that eventually show the effects of PRP on human tissues is necessary and promising. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1245 Advertisement Copyright & Permissions© 2017MetricsAuthor Information HASAN TAVUKCU More articles by this author OMER AYTAÇ More articles by this author FATIH ATUG More articles by this author BURÇIN ALEV More articles by this author OZGE CEVIK More articles by this author AYSEN YARAT More articles by this author SULE CETINEL More articles by this author GÖKSEL SENER More articles by this author HALUK KULAKSIZOGLU More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Microarray Analysis of Differential Gene Expression Profile Between Human Fetal and Adult Heart

Although many changes have been discovered during heart maturation, the genetic mechanisms involved in the changes between immature and mature myocardium have only been partially elucidated. Here, gene expression profile changed between the human fetal and adult heart was characterized. A human microarray was applied to define the gene expression signatures of the fetal (13-17weeks of gestation, n = 4) and adult hearts (30-40years old, n = 4). Gene ontology analyses, pathway analyses, gene set enrichment analyses, and signal transduction network were performed to predict the function of the differentially expressed genes. Ten mRNAs were confirmed by quantificational real-time polymerase chain reaction. 5547 mRNAs were found to be significantly differentially expressed. "Cell cycle" was the most enriched pathway in the down-regulated genes. EFGR, IGF1R, and ITGB1 play a central role in the regulation of heart development. EGFR, IGF1R, and FGFR2 were the core genes regulating cardiac cell proliferation. The quantificational real-time polymerase chain reaction results were concordant with the microarray data. Our data identified the transcriptional regulation of heart development in the second trimester and the potential regulators that play a prominent role in the regulation of heart development and cardiac cells proliferation.

Human Cytomegalovirus-Encoded Receptor US28 Is Expressed in Renal Allografts and Facilitates Viral Spreading In Vitro.

Renal transplantation is the preferred treatment for patients with end-stage renal disease. Human cytomegalovirus (HCMV) activation is associated with decreased renal graft function and survival. Human cytomegalovirus encodes several immune modulatory proteins, including the G protein-coupled receptor US28, which scavenges human chemokines and modulates intracellular signaling. Our aim was to identify the expression and localization of US28 in renal allograft biopsies by immunohistochemistry and determine its role in viral spreading in vitro. Immunohistochemistry revealed US28 in 31 of 34 renal transplant biopsies from HCMV-seropositive donors. Expression was independent of HCMV viremia or IgG serostatus. US28 was predominantly expressed in the cytoplasm of vascular smooth muscle cells (VSMCs) and tubular epithelial cells, with a median positivity of 20% and 40%, respectively. Also, US28-positive cells were present within arterial neointima. In contrast to US28, HCMV-encoded immediate early antigen was detected in less than 5% of VSMCs, tubular epithelial cells, interstitial endothelium, interstitial inflammatory infiltrates, and glomerular cells.Primary VSMCs were infected with green fluorescent protein-tagged wild type or US28-deficient HCMV. The viral spreading of US28-deficient HCMV, via culture medium or cell-to-cell transmission, was significantly impeded as shown by green fluorescent protein (ie, infected) cell quantification and quantitative real-time polymerase chain reaction. Additionally, the number and size of foci was smaller. In summary, HCMV-encoded US28 was detected in renal allografts from HCMV-positive donors independent of viremia and serostatus. Also, US28 facilitates HCMV spreading in VSMCs in vitro. Because the vasculature is affected in chronic renal transplant dysfunction, US28 may provide a potential target for therapeutic intervention.