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[en] Highlights: • qPCR remain the gold-standard-method for measuring and validating gene expression levels and alterations. • qPCR reaction volume can be significantly reduced. • 1 μL versus 10 μL total reaction volumes gave comparable results. • The reduction in reaction volume can greatly reduce sample use and reagent cost. We show that the quantitative PCR reaction volume can be reduced significantly compared to the standard procedures, without compromising data quality. By analyzing dilution series (100–10−7) we found that measurement in 1 μl reaction volume indeed gave valid results comparable to 10 μL, when using a routine pipetting robot. This may enable a significant cost reduction through cutback of both biological material as well as chemical reagents.
[en] MicroRNAs (miRNAs) regulate gene expression by binding to mRNA, and can function as oncogenes or tumor suppressors depending on the target. In this study, using qRT-PCR, we examined the expression of six miRNAs (miR-21, miR-31, miR-92a, miR-101, miR-106a and miR-145) in tumors from 193 prospectively recruited patients with colorectal cancer, and associations with clinicopathological parameters and patient outcome were analyzed. The miRNAs were chosen based on previous studies for their biomarker potential and suggested biological relevance in colorectal cancer. The miRNA expression was examined by qRT-PCR. Associations between miRNA expression and clinicopathological variables were explored using Mann–Whitney U and Kruskal-Wallis test while survival was estimated using the Kaplan-Meier method and compared using the log-rank test. MiR-101 was hardly expressed in the tumor samples, while for the other miRNAs, variable expression levels and expression ranges were observed, with miR-21 being most abundantly expressed relative to the reference (RNU44). In our study cohort, major clinical significance was demonstrated only for miR-31, as high expression was associated with advanced tumor stage and poor differentiation. No significant associations were found between expression of the investigated miRNAs and metastasis-free or overall survival. Investigating the expression of six miRNAs previously identified as candidate biomarkers in colorectal cancer, few clinically relevant associations were detected in our patient cohort. Our results emphasize the importance of validating potential tumor markers in independent patient cohorts, and indicate that the role of miRNAs as colorectal cancer biomarkers is still undetermined
[en] During micropropagation of Helleborus ×nigercors, plantlets were observed to be bacterially contaminated. To determine the identity of contaminants, bacteria resistant to surface sterilization were isolated and Gram stained. Polymerase Chain Reaction (PCR) and 16S rRNA sequencing were used to identify bacterial isolates H7G and H7S as belonging to the Paenibacillus and Luteibacteri genera, respectively. Strain H7R had highest sequence similarity to the Pseudomonas, Stenotrophomonas, and Lysobacter genera. Strains H7R and H7S were unable to grow in the absence of plant tissue and other bacterial species. Paenibacillus sp. H7G was screened using combinations of antibiotics including streptomycin sulfate, gentamicin sulfate, and cefotaxime, and was only eradicated by concentrations of gentamicin sulfate above phytotoxic levels. This is the first documented exploration of bacterial endophytes associated with Helleborus species.
[en] Thyroid cancer is the most common endocrine cancer with 1,690 deaths each year. There are four main types of which the papillary and follicular types together account for >90% followed by medullary cancers with 3% to 5% and anaplastic carcinomas making up <3%. Epigenetic events of DNA hypermethylation are emerging as promising molecular targets for cancer detection. Our immediate and long term goal is to identify DNA methylation markers for early detection of thyroid cancer. This pilot study comprised of 21 patients to include 11 papillary thyroid cancers (PTC), 2 follicular thyroid cancers (FTC), 5 normal thyroid cases, and 3 hyperthyroid cases. Aberrant promoter methylation was examined in 24 tumor suppressor genes using the methylation specific multiplex ligation-dependent probe amplification (MS-MLPA) assay and in the NIS gene using methylation-specific PCR (MSP). The frequently methylated genes were CASP8 (17/21), RASSF1 (16/21) and NIS (9/21). In the normal samples, CASP8, RASSF1 and NIS were methylated in 5/5, 4/5 and 1/5 respectively. In the hyperthyroid samples, CASP8, RASSF1 and NIS were methylated in 3/3, 2/3 and 1/3 respectively. In the thyroid cancers, CASP8, RASSF1, and NIS were methylated in 9/13, 10/13, and 7/13 respectively. CASP8, RASSF1 and NIS were also methylated in concurrently present normal thyroid tissue in 3/11, 4/11 and 3/11 matched thyroid cancer cases (matched for presence of both normal thyroid tissue and thyroid cancer), respectively. Our data suggests that aberrant methylation of CASP8, RASSF1, and NIS maybe an early change in thyroid tumorigenesis regardless of cell type
[en] ATP-binding cassette (ABC) transporters are transmembrane proteins responsible for the efflux of a wide variety of substrates, including steroid metabolites, through the cellular membranes. For better characterization of the role of ABC transporters in prostate cancer (PCa) development, the profile of ABC transporter gene expression was analyzed in PCa and noncancerous prostate tissues (NPT). TaqMan Low Density Array (TLDA) human ABC transporter plates were used for the gene expression profiling in 10 PCa and 6 NPT specimens. ABCB1 transcript level was evaluated in a larger set of PCa cases (N = 78) and NPT (N = 15) by real-time PCR, the same PCa cases were assessed for the gene promoter hypermethylation by methylation-specific PCR. Expression of eight ABC transporter genes (ABCA8, ABCB1, ABCC6, ABCC9, ABCC10, ABCD2, ABCG2, and ABCG4) was significantly down-regulated in PCa as compared to NPT, and only two genes (ABCC4 and ABCG1) were up-regulated. Down-regulation of ABC transporter genes was prevalent in the TMPRSS2-ERG-negative cases. A detailed analysis of ABCB1 expression confirmed TLDA results: a reduced level of the transcript was identified in PCa in comparison to NPT (p = 0.048). Moreover, the TMPRSS2-ERG-negative PCa cases showed significantly lower expression of ABCB1 in comparison to NPT (p = 0.003) or the fusion-positive tumors (p = 0.002). Promoter methylation of ABCB1 predominantly occurred in PCa and was rarely detected in NPT (p < 0.001). The study suggests frequent down-regulation of the ABC transporter genes in PCa, especially in the TMPRSS2-ERG-negative tumors. The online version of this article (doi:10.1186/s12885-015-1689-8) contains supplementary material, which is available to authorized users
[en] Dysregulation of microRNAs (miRNAs) expression is prevalent in human malignancy development and progression. As previously reported, miR-509-5p has been identified as a tumor suppressor in various cancers, but its role and functional mechanism in non-small lung cancer (NSCLC) remains unknown. In the present study, we found that miR-509-5p was significantly downregulated in NSCLC tissues and cell lines using Quantitative real-time PCR (qRT-PCR). In addition, overexpression of miR-509-5p markedly inhibited the proliferation, migration, invasion and phosphorylation of Akt of NSCLC cells. Moreover, we identified tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein gamma (YWHAG) as a direct target of miR-509-5p. Knockdown of YWHAG mimicked the inhibitory effects of miR-509-5p on NSCLC cells for proliferation and motility. Finally, the negative correlation between miR-509-5p and YWHAG expression in NSCLC specimens was demonstrated. Taken together, our present study revealed the tumor suppressive role of miR-509-5p in NSCLC by targeting YWHAG, suggesting that miR-509-5p/YWHAG axis might be considered as a novel and potential target for clinical diagnosis and therapeutics of NSCLC. - Highlights: • The tumor suppressive role of miRNA-509-5p in non-small cell lung cancer is revealed. • MiR-509-5p exerts its influence on NSCLC cells by targeting YWHAG. • MiR-509-5p inhibits the phosphorylation of Akt in NSCLC cells. • MiR-509-5p can be deemed as a potential diagnostic and therapeutic target for NSCLC.
[en] Fipronil is an important member of the phenylpyrazole group of insecticides and is widely used for various crops and vegetables to control insects, thereby exposing birds, animals, and humans to fipronil. Currently, there is limited information on the effects of fipronil exposure in Japanese quail. Therefore, our aim was to assess the reproductive toxicological effects of fipronil in the Japanese quail in a 15-day gavage study and then its recovery over a period of 60 days. Fipronil-administration led to significant losses in both feed intake and body weight. Whereas, the gonadosomatic index was not affected, and histological changes observed in the testes were reversible, particularly by day 45 and day 60 of recovery. Cloacal gland atrophy, reduced foam quantity and a reduction in fertility, sexual and aggressive behaviors, and serum testosterone with elevated estradiol (E2) hormone levels were also observed. All these changes gradually reversed during various recovery periods. Further, alterations in hepatic vitellogenin (Vtg) and estrogen receptor α (ERα) gene expression, assessed by quantitative polymerase chain reaction, were also observed. Specifically, ERα1 was induced after fipronil administration, while the Vtg transcript was elevated during both exposure and recovery periods. Our results showed that fipronil exposure has a profound negative influence on reproductive traits in the male Japanese quail and exhibits an estrogenic activity that can raise the incidence of infertility in males. Nevertheless, most of the changes could be reversed after a recovery period of 30–45 days. - Highlights: • Fipronil exposure has a profound negative influence on reproductive traits in the male Japanese quail. • Fipronil induced alterations in hepatic vitellogenin (Vtg) and estrogen receptor α (ERα) gene expression. • Fipronil exhibited an estrogenic potency that can raise the incidence of infertility in males. • Most of the changes could be reversed after a recovery period of 30–45 days. - Fipronil has a profound negative influence on reproductive traits in the male quail and exhibits an estrogenic activity. Most of the changes could be reversed after a recovery period of 30–45 days.
[en] Improved insight into the molecular characteristics of the different ovarian cancer subgroups is needed for developing a more individualized and optimized treatment regimen. The aim of this study was to a) identify differentially expressed miRNAs in high-grade serous ovarian carcinoma (HGSC), clear cell ovarian carcinoma (CCC) and ovarian surface epithelium (OSE), b) evaluate selected miRNAs for association with clinical parameters including survival and c) map miRNA-mRNA interactions. Differences in miRNA expression between HGSC, CCC and OSE were analyzed by global miRNA expression profiling (Affymetrix GeneChip miRNA 2.0 Arrays, n = 12, 9 and 9, respectively), validated by RT-qPCR (n = 35, 19 and 9, respectively), and evaluated for associations with clinical parameters. For HGSC, differentially expressed miRNAs were linked to differentially expressed mRNAs identified previously. Differentially expressed miRNAs (n = 78) between HGSC, CCC and OSE were identified (FDR < 0.01%), of which 18 were validated (p < 0.01) using RT-qPCR in an extended cohort. Compared with OSE, miR-205-5p was the most overexpressed miRNA in HGSC. miR-200 family members and miR-182-5p were the most overexpressed in HGSC and CCC compared with OSE, whereas miR-383 was the most underexpressed. miR-205-5p and miR-200 members target epithelial-mesenchymal transition (EMT) regulators, apparently being important in tumor progression. miR-509-3-5p, miR-509-5p, miR-509-3p and miR-510 were among the strongest differentiators between HGSC and CCC, all being significantly overexpressed in CCC compared with HGSC. High miR-200c-3p expression was associated with poor progression-free (p = 0.031) and overall (p = 0.026) survival in HGSC patients. Interacting miRNA and mRNA targets, including those of a TP53-related pathway presented previously, were identified in HGSC. Several miRNAs differentially expressed between HGSC, CCC and OSE have been identified, suggesting a carcinogenetic role for these miRNAs. miR-200 family members, targeting EMT drivers, were mostly overexpressed in both subgroups, among which miR-200c-3p was associated with survival in HGSC patients. A set of miRNAs differentiates CCC from HGSC, of which miR-509-3-5p and miR-509-5p are the strongest classifiers. Several interactions between miRNAs and mRNAs in HGSC were mapped
[en] Erythropoietin (EPO) has been well characterized as a renal glycoprotein hormone regulating red blood cell production by inhibiting apoptosis of erythrocyte progenitors in hematopoietic tissues. EPO exerts regulatory effects in cardiac and skeletal muscles. Duchenne muscular dystrophy is a lethal degenerative disorder of skeletal and cardiac muscle. In this study, we tested the possible therapeutic beneficial effect of recombinant EPO (rhEPO) in dystrophic muscles in mdx mice. Total strength was measured using a force transducer coupled to a computer. Gene expression for myostatin, transforming growth factor-β1 (TGF-β1), and tumor necrosis factor-α (TNF-α) was determined by quantitative real time polymerase chain reaction. Myostatin expression was significantly decreased in quadriceps from mdx mice treated with rhEPO (rhEPO=0.60±0.11, control=1.07±0.11). On the other hand, rhEPO had no significant effect on the expression of TGF-β1 (rhEPO=0.95±0.14, control=1.05±0.16) and TNF-α (rhEPO=0.73±0.20, control=1.01±0.09). These results may help to clarify some of the direct actions of EPO on skeletal muscle
[en] O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins. In recent years, the roles of O-GlcNAcylation in several human malignant tumors have been investigated, and O-GlcNAcylation was found to be linked to cellular features relevant to metastasis. In this study, we modeled four diverse ovarian cancer cells and investigated the effects of O-GlcNAcylation on ovarian cancer cell migration. We found that total O-GlcNAcylation level was elevated in HO-8910PM cells compared to OVCAR3 cells. Additionally, through altering the total O-GlcNAcylation level by OGT silencing or OGA inhibition, we found that the migration of OVCAR3 cells was dramatically enhanced by PUGNAc and Thiamet G treatment, and the migration ability of HO-8910PM cells was significantly inhibited by OGT silencing. Furthermore, we also found that the expression of E-cadherin, an O-GlcNAcylated protein in ovarian cancer cells, was reduced by OGA inhibition in OVCAR3 cells and elevated by OGT silencing in HO-8910PM cells. These results indicate that O-GlcNAcylation could enhance ovarian cancer cell migration and decrease the expression of E-cadherin. Our studies also suggest that O-GlcNAcylation might become another potential target for the therapy of ovarian cancer. -- Highlights: • We examine the migration potential of diverse ovarian cancer cells. • We examine the total O-GlcNAcylation level of diverse ovarian cancer cells. • Increasing O-GlcNAcylation level will enhance the migration of ovarian cancer cells. • Reducing O-GlcNAcylation level will inhibit the migration of ovarian cancer cells. • The mechanism explains O-GlcNAcylation enhance ovarian cancer cell migration