Quantitative Real-time Reverse Transcriptase Research Articles

Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface.

The clinical values of breast cancer resistance protein mRNA levels in plasma microvesicles predicting breast cancer chemotherapy

Objective To investigate the predictive values of peripheral blood microvesicles(MVs) containing breast cancer resistance protein(BCRP) mRNA on chemotherapy effectiveness in breast cancer. Methods We selected 40 cases of breast cancer(BC)chemotherapy,84 cases of palliative chemotherapy in patients with advanced metastatic breast cancer(mBC),and 40 cases of normal volunteers(NF).Immunofluorescence was used to detect mucin 1(MUC1)expression rate and co- expression rate of MUC1 and BCRP in plasma MVs.Real- time quantitative reverse transcriptase- polymerase chain reaction(RT-qPCR) was used to detect BCRP mRNA level in plasma MVs.The correlations between clinicopathologic features and BCRP mRNA level with chemotherapy effectiveness were analyzed by the Kaplan- Meier method and Cox regression. Results MUC1 expression rate and co- expression rate of MUC1 and BCRPin plasma MVs of mBC group were(11.60±0.76)% and(14.06±0.62)%, and those in BC group were (6.67±1.03)% and(1.83±0.39)%(P< 0.05).The BCRP mRNA relative levels of plasma MVs in NF, BCand mBCgroups were- 2.15±0.66,- 2.32±0.75,and- 5.75±0.38 respectively.The BCRP mRNA relative levels of plasma MVs in mBC group was significantly higher than in BC group(P< 0.01). According to Response Evaluation Criteria in solid tumors, mBC group was subdivided into the stable disease(SD)subgroup and disease control(DCR)[complete remission(CR)/partial remission(PR)/SD] subgroup.BCRP mRNA relative levels in plasma MVs of SD subgroup and DCR subgroup were- 4.03± 0.71, and- 6.30±0.44 respectively with the difference being statistically significant(P< 0.01). The median progression- free survival(mPFS)on high and low BCRP mRNA expression groups was 2.80 and 4.00 months respectively(P< 0.01).Cox regression analysis showed BCRP mRNA level in the plasma MVs was an independent prognostic factor in advanced metastatic breast cancer chemotherapy(P<0.05). Conclusion The BCRP mRNA levels in the plasma MVs may predict the response of breast cancer chemotherapy as potential markers. Key words: Breast cancer resistance protein; Chemotherapy; Microvesicles

The experiment study of mesenchymal stem cells modified wtih galectin- 1 gene co-cultured with dermal matrix patch

Objective To explore the feasibility of mesenchymal stem cells(MSCs) modified wtih galectin- 1 gene co- cultured with dermal matrix patch. Methods After the bone marrow MSCs(BMSCs) were transfected with Ad- green fluorescent protein(GFP)- galectin- 1, the fluorescence in BMSCs was observed under fluorescence microscope.Real- time quantitative reverse transcriptase- polymerase chain reaction(RT- qPCR) was used to detect the expression of galectin- 1 gene, and the protein expression of galectin- 1 and desmin was detected by enzyme linked immune sorbent assay(ELISA)and Western blotting, respectively.The growth of BMSCs transfected with Ad- GFP- galectin- 1 and co- cultured with acellular dermal matrix patch(ADM) was observed. Results The green fluoresence was observed and reached to peak at 72 h after the transfection of BMSCs with Ad- GFP- galectin- 1 under fluorescence microscope.The result of RT- qPCR and ELISA confirmed that BMSCs over- expressed galectin- 1.The RT- qPCR result of galectin- 1,which the relative expression folds was 1.00 in phosphate buffer(PBS) group,4.11 in Ad- GFP- galectin- 1 group,1.09 in Ad- GFP group;The result of galectin- 1 measured by ELISA was that 48 h:(197.28±8.78),(25.52±1.56),(24.53±2.96)μg/L; 72 h:(264.33± 5.43),(25.40±2.06),(25.46±1.13)μg/L;96 h:(172.41±3.61),(25.22±1.61),(28.01±1.26) μg/L; 120 h:(155.53±7.25),(24.84±2.35),(26.65±1.54)μg/L ordered by Ad- GFP-galectin- 1 group, Ad- GFP group and PBS group.The expression of desmin which co- cultured with dermal matrix patch(ADM) were well grown. Conclusion Galectin- 1 could induce BMSCs to express desmin.BMSCs transfected with Ad- GFP- galectin- 1 showed good compatibility with ADM. Key words: Galectin-1; Bone mesenchymal marrow cell; Acellular dermal matrix patch; Co-culture

Effects of microRNA-194 on proliferation, migration, invasion and epithelial- mesenchymal transition of basal- like breast cancer cell line HCC1937

Objective To explore the role of microRNA- 194(miR- 194)in proliferation, migration,invasion and the epithelial- mesenchymal transition(EMT)of human basal- like breast cancer cell line HCC1937. Methods Three groups were set up:experimental group[HCC1937 with lentivirus- miR - 194- enhanced green fluorescent protein(EGFP), MOI= 50], negative control group(HCC1937 with lentivirus- CON157- EGFP), and blank control group(HCC1937 without lentivirus). The miR- 194 expression was detected by real- time quantitative reverse transcriptase- polymerase chain reaction(RT-qPCR). Cell growth ability of HCC1937 was determined by cell counting kit- 8(CCK- 8)assay. Cell migration and invasion ability of HCC1937 were mearsured by a Transwell assay, and the protein of epithelial - mesenchymal transition phenotype was measured by Western blotting. Results The expression of miR-194 in experimental group(432.93±0.01) was higher than in negative control group(0.90±0.02) and blank control group(1)(P< 0.01) The cells in experimental group grew more slowly[(44.81± 0.11)%]than negative control group[(104.29±0.13)%]and blank control group[(100±0)%, P< 0.01].The number of migration and invasion was both less in experimental group than in negative control group and blank control group(P< 0.05).Western blotting revealed that the expression of E- cadherin in experimental group(0.175±0.053) was significantly decreased as compared with negative control group (0.516±0.032)and blank control group(0.433±0.047, P< 0.05), and the expression of N- cadherin in experimental group(0.342±0.012)was significantly increased as compared with negative control group (0.137±0.007)and blank control group(0.142±0.021)(P<0.05) Conclusion miR- 194 can inhibit proliferation, migration and invasion of basal- like breast cancer cells(HCC1937), and also affect EMT process in HCC1937 cells. Key words: Basal- like breast cancer; MicroRNA- 194; Proliferation

Pregnancy-associated microRNAs in plasma as potential molecular markers of ectopic pregnancy

To investigate cell-free pregnancy-associated microRNAs as molecular markers for the diagnosis of ectopic pregnancy. Laboratory study using human plasma samples. Research unit in a university hospital. Plasma samples from 18 women with ectopic pregnancies (EP group), 12 women with spontaneous abortion (SA group), and 26 normal women with singleton pregnancies (NP group). Total RNAs containing small RNA molecules extracted from 1.2mL of plasma. Plasma concentrations of cell-free microRNAs measured by quantitative real-time reverse-transcriptase polymerase chain reaction. Plasma concentrations of cell-free pregnancy-associated microRNAs (miR-323-3p, miR-515-3p, miR-517a, miR-517c, and miR-518b) and serum concentration of human chorionic gonadotropin (hCG) were confirmed to have statistically significantly different plasma or serum concentrations in women with EP, SA, or NP. There was no statistically significant difference in the plasma concentrations of cell-free miR-21 between the three groups. By correlation coefficient analysis, no relationship was detected between serum hCG levels and plasma cell-free miR-517c, miR-515-3p, miR-517a, miR-518b, miR-323-3p, or miR-21 levels. Plasma concentrations of cell-free miR-517a could distinguish EP/SA from NP, yielding an area under the curve of 0.9654 (95% confidence interval, 0.9172-1.0). Plasma concentrations of cell-free miR-323-3p could distinguish EP from SA, yielding an area under the curve of 0.7454 (95% confidence interval, 0.5558-0.9349). Cell-free pregnancy-associated microRNAs have potential as molecular markers of ectopic pregnancy.

Extracellular Signal-regulated Kinase Mitogen-activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt Signaling Are Required for Lipopolysaccharide-mediated Mineralization in Murine Odontoblast-like Cells

IntroductionOdontoblasts play an important role in post-developmental control of mineralization in response to external stimuli in the tooth. The present study investigated whether lipopolysaccharide (LPS), a major bacterial cell wall component, influenced mineralization in a murine odontoblast-like cell (OLC) line and the related intracellular signaling pathways involved. MethodsAlizarin red S staining was used to assess mineralized nodule formation in OLCs in response to LPS. The effects of LPS on gene expression of odontoblastic markers were investigated by using quantitative real-time reverse-transcriptase polymerase chain reaction. The potential involvement of toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), mitogen-activated protein kinase (MAPK), or phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathways in the mineralized nodule formation, and mRNA expression of several odontoblastic markers of OLCs induced by LPS was assessed by using alizarin red S staining and quantitative real-time reverse-transcriptase polymerase chain reaction. Moreover, LPS stimulation resulted in phosphorylation of protein that was determined by Western blot analysis. ResultsOLCs showed reduced mineralized nodule formation and several odontoblastic markers expression in response to LPS exposure. Furthermore, inhibition of TLR4, extracellular signal-regulated kinase (ERK), and PI3K/Akt signaling noticeably antagonized LPS-mediated mineralization in OLCs. However, p38 MAPK, c-Jun N-terminal kinase, and NF-κB signaling inhibitors did not affect LPS-mediated mineralization in OLCs. Notably, LPS treatment resulted in a time-dependent phosphorylation of ERK and PI3K/Akt in OLCs, which was abrogated by their specific inhibitors. ConclusionsLPS decreased mineralization in OLCs via TLR4, ERK MAPK, and PI3K/Akt signaling pathways, but not p38, c-Jun N-terminal kinase, or NF-κB signaling.

γ‐oryzanol reduces caveolin‐1 and PCGEM1 expression, markers of aggressiveness in prostate cancer cell lines

Prostate cancer is a leading cause of death among men due to the limited number of treatment strategies available for advanced disease. γ-oryzanol is a component of rice bran, rich in phytosterols, known for its antioxidant, anti-carcinogenic and endocrinological effects. It is known that γ-oryzanol may affect prostate cancer cells through the down regulation of the antioxidant genes and that phytosterols have anti-proliferative and apoptotic effects. There are evidences showing that some of the components of γ-oryzanol can modulate genes involved in the development and progression of prostate cancer, as caveolin-1 (Cav-1) and prostate specific androgen-regulated gene (PCGEM1). To determine the effects of γ-oryzanol on prostate cancer cell survival we evaluated the cell viability and biomass by MTT and sulforhodamine B assays, respectively. Cell death, cell cycle and pERK1/2 activity were assessed by flow cytometry. The changes in gene expression involved in the survival and progression of prostate cancer cav-1 and PCGEM1 genes were evaluated by quantitative real time reverse transcriptase polymerase chain reaction (RT-PCR) and cav-1 protein by immunofluorescence followed by confocal microscopy analysis. We found that γ-oryzanol decreases cell viability and culture biomass by apoptosis and/or necrosis death in androgen unresponsive (PC3 and DU145) and responsive (LNCaP) cell lines, and signals through pERK1/2 in LNCaP and DU145 cells. γ-oryzanol also appears to block cell cycle progression at the G2/M in PC3 and LNCaP cells and at G0/G1 in DU145 cells. These effects were accompanied by a down regulation in the expression of the cav-1 in both androgen unresponsive cell lines and PCGEM1 gene in DU145 and LNCaP cells. In summary, we used biochemical and genetics approaches to demonstrate that γ-oryzanol show a promising adjuvant role in the treatment of prostate cancer.

Improving statistical inference on pathogen densities estimated by quantitative molecular methods: malaria gametocytaemia as a case study.

BackgroundQuantitative molecular methods (QMMs) such as quantitative real-time polymerase chain reaction (q-PCR), reverse-transcriptase PCR (qRT-PCR) and quantitative nucleic acid sequence-based amplification (QT-NASBA) are increasingly used to estimate pathogen density in a variety of clinical and epidemiological contexts. These methods are often classified as semi-quantitative, yet estimates of reliability or sensitivity are seldom reported. Here, a statistical framework is developed for assessing the reliability (uncertainty) of pathogen densities estimated using QMMs and the associated diagnostic sensitivity. The method is illustrated with quantification of Plasmodium falciparum gametocytaemia by QT-NASBA.ResultsThe reliability of pathogen (e.g. gametocyte) densities, and the accompanying diagnostic sensitivity, estimated by two contrasting statistical calibration techniques, are compared; a traditional method and a mixed model Bayesian approach. The latter accounts for statistical dependence of QMM assays run under identical laboratory protocols and permits structural modelling of experimental measurements, allowing precision to vary with pathogen density. Traditional calibration cannot account for inter-assay variability arising from imperfect QMMs and generates estimates of pathogen density that have poor reliability, are variable among assays and inaccurately reflect diagnostic sensitivity. The Bayesian mixed model approach assimilates information from replica QMM assays, improving reliability and inter-assay homogeneity, providing an accurate appraisal of quantitative and diagnostic performance.ConclusionsBayesian mixed model statistical calibration supersedes traditional techniques in the context of QMM-derived estimates of pathogen density, offering the potential to improve substantially the depth and quality of clinical and epidemiological inference for a wide variety of pathogens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0402-2) contains supplementary material, which is available to authorized users.

Methods for RNA profiling of gravi-responding plant tissues.

Plant transcriptional responses to gravity stimulation by reorientation are among the fastest measured in any tissue or species. Upon reorientation, changes in abundance of specific mRNAs can be measured within seconds or minutes, for plastid or nuclear encoded genes, respectively. Identifying fast gravity-induced transcripts has been made possible by the development of high-throughput technology for qualitative and quantitative RNA analysis. RNA profiling has undergone further rapid development due to its enormous potential in basic sciences and medical applications. We describe here the current and most widely used methods to profile the changes in an entire transcriptome by high-throughput sequencing of RNA fractions (RNAseq) and single gene transcript analysis using real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR).

Investigation of West Nile virus among healthy blood donors in the western part of Turkey.

THE West Nile virus (WNV) is a mosquito-borne flavivirus causing different forms of infection among humans, varying from asymptomatic illness to fetal central nervous system infection. Turkeylies within an endemic region for WNV. Transfusion of infected blood products is another well-documented major route of transmission. The aim of our study was to investigate the presence of WNV viremia among a healthy donor population from the western part of the country. A total of 438 healthy volunteer blood donors were included in the study. The presence of WNV RNA was investigated by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and anti-WNV IgG was detected by a commercial ELISA test. Ages of volunteer donors were 18-62 years (mean: 34.7) and 34 (7.76%) were women. All samples were negative for WNV RNA by qRT-PCR. Eleven (2.51%) samples, 1 of which was borderline, were positive for anti-WNV IgG. All positive samples were from the western part of the country and 9 of them were from Izmir. Although all donor samples were negative for WNV RNA by qRT-PCR, the risk of WNV transmission via blood products should not be ignored in endemic regions.

Protein tyrosine kinase 7: a hepatocellular carcinoma-related gene detected by triple-combination array

BackgroundHepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. We developed a novel technique to identify cancer-related genes of HCC as follows: triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays, and methylation arrays. Materials and methodsTriple-combination array analysis was performed on one HCC sample from a 68-y-old female patient, and one candidate cancer-related gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in nine HCC cell lines and in samples from 48 HCC patients. Additionally, we evaluated gene expression by immunohistochemistry and Western blotting. ResultsUsing this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate cancer-related gene. PTK7 was revealed to be hypermethylated (methylation value 0.826, range 0–1.0) in cancer tissue, compared with that of adjacent noncancerous tissues (0.047) by methylation array. Of the 48 clinical samples, 30 HCC samples (62.5%) showed PTK7 promoter hypermethylation. Downregulation of PTK7 (expressions in tumor tissues decreased by ≥50% compared with the noncancerous tissues) was significantly associated with age >60 y (P = 0.030) and elevation in serum protein induced by vitamin K absence or antagonists-II (P = 0.033). Moreover, patients with downregulation were significantly inferior in overall survival (P < 0.001) than the others. ConclusionsOur data imply that PTK7 acts as a cancer-related gene and may be a potent prognostic marker for HCC. Triple-combination array analysis was once again found to be useful in identifying cancer-related genes.

Clinical and molecular characterization of rhinoviruses A, B, and C in adult patients with pneumonia

BackgroundHuman rhinoviruses (HRVs) have increasingly been reported to be associated with lower respiratory tract infections. HRV-C has been associated with more severe respiratory illnesses in children. ObjectivesWe investigated the clinical and molecular characteristics of HRV-A, HRV-B, and HRV-C in adults with pneumonia. Study designHRV genotyping and quantitative real-time reverse-transcriptase polymerase chain reaction were performed on 392 adult respiratory specimens consecutively collected from June 2012 to May 2013. Pneumonia was identified by review of medical records and chest radiographs. ResultsAdult patients with pneumonia and identified HRV genotypes (n=165) were included. HRV-A, HRV-B, and HRV-C were identified in 97, 28, and 40 patients, respectively. No differences in underlying diseases, APACHE II score, or frequency of co-infection were observed between the HRV species. Compared with HRV-A, HRV-B was more often associated with neutropenia (21.4% vs. 7.2%, p=0.07), hospital acquisition (32.1% vs. 7.2%, p=0.048), and fever (78.6% vs. 49.3%, p=0.003). Mean viral load (copies/ml) was lower for HRV-B (102.6 vs. 104.1 in HRV-A and 104.3 in HRV-C), and high viral loads (≥104) occurred most frequently with HRV-C (70.0% vs. 57.7% for HRV-A and 21.4% for HRV-B). The incidence of severe pneumonia was similar for HRV-A (18.6%), HRV-B (21.4%), and HRV-C (20.0%), and in-hospital mortality rates did not differ significantly (15.5%, 10.7%, and 12.5%, respectively). ConclusionsIn contrast to previous pediatric studies, no differences were observed in clinical severity or outcomes between the different HRV species in adult patients with pneumonia.

Expression and clinical significance of microRNA-376a in colorectal cancer.

The incidence of colorectal cancer (CRC) is increasing in many Asian countries and microRNAs have already been proven to be associated with tumorigenesis. Currently, microRNA-376a (miR-376a) expression and association with clinical factors in CRC remains unclear. In this study, real-time quantitative reverse transcriptase- polymerase chain reaction (qRT-PCR) was carried out on 53 matched pairs of CRC and adjacent normal mucosa to investigate the expression levels of miR-376a. According to the high or low expression of miR-376a, patients were divided into two groups. The relationship between miR-376a expression and clinicopathological factors of 53 patients was evaluated. Survival analysis of 53 CRC patients was performed with clinical follow- up information and survival curves were assessed by the Kaplan-Meier method. Immunohistochemistry (IHC) staining was performed on sections of paraffin-embedded tissue to investigate the vascular endothelial growth factor (VEGF) expression. MiR-376a showed low expression in cancer tissues compared to the adjacent normal tissues and altered high miR-376a expression tended to be positively correlated with advanced lymph node metastasis and shorter patient survival. VEGF IHC positivity was significantly more common in patients with high expression levels of miR-376a.Those results demonstrated that miR-376a may be a meaningful prognostic biomarker and potential therapeutic target in colorectal cancer.

Overexpression of MicroRNA-145 Promotes Ascending Aortic Aneurysm Media Remodeling through TGF-β1

In this study the role of microRNA-145 expression in ascending aortic aneurysm wall media remodeling was examined. Aortic wall samples were obtained from 10 patients who had undergone surgery for ascending aortic aneurysm. Control aortic tissue samples were obtained from 10 patients who had undergone coronary artery bypass graft. The levels of microRNA-145 were analyzed using real time quantitative reverse transcriptase polymerase chain reaction, and western blots were used to determine the expressions of osteopontin (OPN) and collagen. In vitro cultures of vascular smooth muscle cells (VSMCs) were established from both patient groups. The VSMCs were transfected with either microRNA-145 mimics or microRNA-145 inhibitors to determine the effect of microRNA-145 on the expression of OPN and collagen. Furthermore, cells were co-transfected with microRNA-145 mimics and TGF-b1 siRNA to investigate whether TGF-b1 is involved in the process that microRNA-145 increases the expression of OPN and collagen. Aortic microRNA-145, OPN and collagen III was increased in ascending aortic aneurysm patients compared with controls (p < .05). VSMCs transfected with microRNA-145 mimics increased the expression of both OPN (average of 1.59-fold, p < .05) and collagen III (mean of 1.71-fold, p < .05). Cells transfected with microRNA-145 inhibitors decreased expression of both OPN and collagen III compared with negative controls. In addition, the inhibition of TGF-b1 decreased the positive effect of microRNA-145 on the expression of OPN and collagen III. The increased expression of microRNA-145 promotes media remodeling through TGF-b1 in the aortic aneurysm wall.

Heat Stress Upregulates the Expression of TLR4 and Its Alternative Splicing Variant in Bama Miniature Pigs

Alternative splicing is a cellular mechanism in eukaryotes that results in considerable diversity of gene products. It plays an important role in several diseases and cellular signal regulation. Heat stress is a major factor that induces immunosuppression in pigs. Little is known about the correlation between alternative splicing and heat stress in pigs. Therefore, this study aimed to clone, sequence and quantify the alternative splicing variant of toll-like receptor 4 (TLR4) in Bama miniature pigs (Sus scrofa domestica) following exposure to heat stress. The results showed that the second exon of TLR4 was spliced and 167 bp shorter in the alternative splicing variant, and the protein was putatively identified as a type of truncated membrane protein consisting of extramembrane, transmembrane and intramembrane regions lacking a signal peptide. Further, it was not a nonclassical secretory protein. Five potential reference genes were screened for their potential as reliable standards to quantify the expression of TLR4 alternative spliced variants by real-time quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). The stability of these reference genes was ranked using the geNorm and NormFinder programs, and ribosomal protein L4 (RPL4) and TATA box-binding protein (TBP) were found to be the two genes showing the most stable expression in the in vitro cultured peripheral blood mononuclear cells (PBMCs) during heat shock. The mRNA level of the TLR4 gene (both classical and spliced) in stressed pigs increased significantly (P<0.05). Further, the expression levels of the alternative spliced variant of TLR4 (TLR4-ASV) showed a 2–3 folds increase in heat-stressed PBMCs as compared to control pigs. The results of the present study suggested that heat shock might modulate the host immune response by regulating the expressions of TLR4 and its alternative splicing variant.

OVGP1 is expressed in the canine oviduct at the time and place of oocyte maturation and fertilization.

In the dog, oocyte maturation, fertilization, and early embryo development take place within the oviduct in the presence of increasing circulating progesterone (P4) levels. Expression of the oviduct-specific glycoprotein 1 (OVGP1), known in other species to be estrogen-dependent, was explored by real-time quantitative reverse-transcriptase PCR, Western blotting, and immunohistochemistry in oviducts from adult Beagle bitches during anestrus and at five specific time periods around ovulation: during pro-estrus before the luteinizing hormone (LH) peak (Pre-LH); after the LH peak and before ovulation (Pre-ov); and at Days 1, 4, and 7 after ovulation (n = 6 bitches per stage). Plasma estradiol-17β (E2) and P4 were assayed at all stages. The expression of canine OVGP1 (cOVGP1) was undetectable during anestrus, increased significantly from Pre-LH to Day 1 in parallel with a decrease in plasma E2-to-P4 levels, remained high at Day 4, then decreased at Day 7 in parallel with an increase in plasma P4 levels. In contrast to other mammals, the expression of cOVGP1 was higher in the isthmus than in the ampulla at all stages. In order to explore the potential regulation of cOVGP1 expression by steroids, the 5'-flanking region of the corresponding gene was analyzed for the presence of estrogen- (ERE) and P4-response-element (PRE). An imperfect ERE and three half-ERE were found in this region, but no PREs. In conclusion, cOVGP1 is highly expressed at the time and site of oocyte maturation and fertilization, and is probably under E2 regulation. Further studies are needed to identify the potential roles of cOVGP1 in each process.

Reference gene selection for quantitative real-time reverse-transcriptase PCR in orchardgrass subjected to various abiotic stresses

Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is a powerful tool for the measurement of gene expression; however, the accuracy of this approach depends on the stability of reference genes. The objective of the present study was to identify the stable reference genes in orchardgrass (Dactylis glomerata L.), a principal cool-season forage grass in the world. Ten candidate reference genes were selected in this study including ATP-binding [ABC], actin [ACTIN], cyclophilin [CYP2], glyceraldehyde 3-phosphate dehydrogenase [GAPDH], beta-amylase 4 [BAM4], zeitlupe [ZTL], MAP Kinase 4 [MPK4], ubiquitin-conjugating enzyme [UBC], S-adenosylmethionine decarboxylase [SAMDC], and translationally controlled tumor protein [TCTP]. The candidate genes were assessed in orchardgrass leaves and roots under conditions of drought, high salinity, heat, waterlogging, and abscisic acid (ABA) treatments. We used GeNorm, BestKeeper, NormFinder, and RefFinder for qRT-PCR normalization and validation to determine that the expression of these reference genes was stress-dependent. ACTIN, CYP2, and ABC were found to be the most stably expressed genes for drought stress while ACTIN, TCTP, and ABC were the most stable under salt stress. ACTIN, CYP2, and ABC were all found to be good reference genes for studying heat stress. Likewise, CYP2, MPK4, and ABC were most suitable to study waterlogging, and ACTIN, CYP2, and MPK4 were determined as the three best reference genes for ABA studies. Our study identified and validated the possible reference genes in orchardgrass that may be used for quantification of target gene expression under various abiotic stresses.

Abstract 2870: ZGPAT gene expression in non-tumor hepatocellular carcinoma tissue is a likely biomarker for survival risk

Abstract [Background &amp; Aim] Hepatocellular carcinoma (HCC) often recurs after surgery and multicentric occurrence is more common than intrahepatic metastasis. We therefore examined factors in non-tumor liver tissue in HCC. [Material &amp; Methods] Control samples, termed super normal liver tissues (SN), were taken from 11 cases of metastatic liver cancer. One of corresponding samples of normal tissues (CN) was selected for comparison from a typical specimen with chronic hepatitis C and HCC. We performed expression profiling and methylation arrays on these samples, identified genes that differed between SN and CN. We analyzed the identified genes using quantitative real-time reverse-transcriptase polymerase chain reaction on SN from 11 cases of metastatic liver cancer and CN from 109 cases of HCC. [Results] We found the zinc finger, CCCH-type with G patch domain (ZGPAT) gene to have expression decreased 4.198-fold in CN, and higher methylation status in CN (0.718), than in SN (0.360). The average expression value of the ZGPAT gene (ZGPAT value ×103 / GAPDH) decreased in CN (9.94 ± 10.68) relative to SN (28.55 ± 22.05). Compared with other patients, those with strongly reduced ZGPAT expression (&amp;lt; 1.5) in their non-tumor liver samples had significantly shorter overall survival (P = 0.024), but not recurrence-free survival (P = 0.515). [Discussion &amp; Conclusion] Our results indicate that expression of the ZGPAT gene in non-tumor liver tissue is a good biomarker for the risk of HCC development. ZGPAT gene is reportedly a transcription repressor that negatively regulates expression of epidermal growth factor receptor, which in turn, mediates cell proliferation, survival, migration. In treating HCC, detection of both prognostic factors from non-tumor liver tissue and malignant factors of the primary cancer lesion is important. Citation Format: Mitsuhiro Hishida, Shuji Nomoto, Yoshikuni Inokawa, Nao Takano, Mitsuro Kanda, Naoki Iwata, Chie Tanaka, Daisuke Kobayashi, Yoko Nishikawa, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. ZGPAT gene expression in non-tumor hepatocellular carcinoma tissue is a likely biomarker for survival risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2870. doi:10.1158/1538-7445.AM2014-2870

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

BackgroundXenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltransferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance.MethodsThe porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced, and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining.ResultsThe porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes, respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells.ConclusionThe functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response detected after pig-to-baboon cardiac xenotransplantation.

The effects of LPS on adhesion and migration of human dental pulp stem cells in vitro

ObjectivesThe aim of the present study was to investigate the effects of lipopolysaccharide (LPS) on the migration and adhesion of human dental pulp stem cells (hDPSCs) and the associated intracellular signalling pathways. MethodshDPSCs obtained from impacted third molars were exposed to LPS and in vitro cell adhesion and migration were evaluated. The effects of LPS on gene expression of adhesion molecules and chemotactic factors were investigated using quantitative real-time reverse-transcriptase polymerase chain (qRT-PCR). The potential involvement of nuclear factor NF-kappa-B (NF-κB) or mitogen-activated protein kinase (MAPK) signalling pathways in the migration and adhesion of hDPSCs induced by LPS was assessed using a transwell cell migration assay and qRT-PCR. ResultsLPS promoted the adhesion of hDPSCs at 1μg/mL and 10μg/mL concentrations, 1μg/mL LPS showing the greater effect. Transwell cell migration assay demonstrated that LPS increased migration of hDPSCs at 1μg/mL concentration while decreasing it significantly at 10μg/mL. The mRNA expressions of adhesion molecules and chemotactic factors were enhanced significantly after stimulation with 1μg/mL LPS. Specific inhibitors for NF-κB and extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK), and P38, markedly antagonised LPS-induced adhesion and migration of hDPSCs and also significantly abrogated LPS-induced up-regulation of adhesion molecules and chemotactic factors. In addition, specific inhibitors of SDF-1/CXCR4, AMD3100 significantly diminished LPS-induced migration of hDPSCs. ConclusionsLPS at specific concentrations can promote cell adhesion and migration in hDPSCs via the NF-κB and MAPK pathways by up-regulating the expression of adhesion molecules and chemotactic factors. Clinical significanceLPS may influence pulp healing through enhancing the adhesion and migration of human dental pulp stem cells when it enters into pulp during pulp exposure or deep caries.