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Highlights • A one-step, quantitative method for CMNV was developed. • The assay had a detection limit of 5.7 viral copies. • The assay was no cross-reactivity observed with six common shrimp viruses. • This is a useful tool for CMNV detection and quantification. Abstract Covert mortality nodavirus (CMNV), an emerging RNA virus, is the pathogen of viral covert mortality disease (VCMD), which has emerged as a cause of serious losses in shrimp aquaculture in China. To improve VCMD diagnosis, a one-step, real-time TaqMan probe-based reverse transcription quantitative PCR (RT-qPCR) was developed in this study. The TaqMan RT-qPCR was optimized firstly, whereby the best results were obtained with 0.2 μM of each primer, 0.2 μM probe, and 0.5 μL Enzyme Mix II. The optimal reaction program was determined as 15 min at 51ºC for reverse transcription and 5 min at 95 ºC, followed by 40 cycles of denaturation at 94 ºC for 10 s, and annealing and extension at 52.7 ºC for 30 s. The optimized assay detected as little as 9.6 pg total RNA from CMNV-infected shrimp and 5.7 copies of the target plasmid. The RT-qPCR assay for CMNV with a high correlation coefficient (r2 = 0.996) was developed basing on the standard curve generated by plotting the threshold cycle values (y) against the common logarithmic copies (log 10 n c as x; n c is copy number) of pMD20-CMNV. The diagnostic sensitivity and specificity of this assay versus the previously reported RT-qPCR was 96.2% and 98.0%, respectively. This method is highly specific to CMNV, as it showed no cross-reactivity with other common shrimp viruses. It is anticipated that the newly developed and optimized RT-qPCR assay will be instrumental for the rapid diagnosis and quantitation of CMNV. [ABSTRACT FROM AUTHOR]

Omega-3 fatty acids, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have been shown to possess definitively suppressive effects on the growth of epithelial ovarian cancer cells. This study investigated the differential effects of pure EPA and DHA on the growth of epithelial ovarian cancer cells and the potential molecular mechanisms that may be involved. There were significant time- and dose-dependent inhibitory effects of both EPA and DHA on cellular proliferation of the epithelial ovarian cancer cell line TOV-21G (P< 0.05). TOV-21G cells pretreated with peroxisome proliferator receptor activator gamma (PPARγ) antagonist, GW9662, markedly suppressed EPA/DHA-induced apoptosis as determined by TUNEL assay, Annexin V-FITC/PI staining, and caspase-3 activity. EPA/DHA significantly induced PPARγ and p53 overexpression as observed in immunoblotting assay and the induction of p53 by EPA/DHA was abolished by GW9662. In all cases, the effect of DHA was significantly more potent than that of EPA (P< 0.05). Our findings suggested that DHA may be more effective than EPA in growth suppression of TOV-21G cells and the biologic effects may be partly mediated by PPARγ and p53 activation. Further research is required to elucidate additional divergent mechanisms to account for apparent differences between EPA and DHA. [ABSTRACT FROM PUBLISHER]

Aim: The BRCA1 promoter is hypermethylated in ovarian cancer patients. We postulated that this hypermethylation might be involved in ovarian cancer progression. Methods: To confirm our hypothesis, tissue and serum samples were collected from ovarian carcinoma patients and categorized according to tumor stage. Healthy or benign ovarian disease tissue samples and corresponding serum samples were used as controls. Breast and ovarian cancer susceptibility gene 1 (BRCA1) promoter methylation levels were detected by real-time polymerase chain reaction (PCR). Real-time PCR was also used to evaluate BRCA1 gene expression, and Western blot was performed to assay the expression of BRCA1 protein. Results: BRCA1 showed hypomethylation in 30 normal ovarian and 30 benign ovarian tumors, but showed hypermethylation or methylation in ovarian cancer patients. There was also a significant difference in the BRCA1 promoter methylation levels between different ovarian cancer stages. Compared to stage I and the control groups, there were higher BRCA1 promoter methylation frequencies in stage II and III ovarian cancers. BRCA1 methylation correlated with the loss of BRCA1 expression. BRCA1 promoter in stage I tumors showed hypomethylated. Conclusion: Promoter hypermethylation may act as a biomarker for sporadic ovarian cancer progression, but is unlikely to be helpful in the early diagnosis of ovarian cancer. [ABSTRACT FROM AUTHOR]

Shrimp hemocyte iridescent virus (SHIV) is a recently reported shrimp virus, which threats the cultured white-leg shrimp Litopenaeus vannamei and can cause huge economic loss in shrimp farming industry in China. A quantitative real time polymerase chain reactio (qPCR) assay was developed using a TaqMan probe to detect and quantify SHIV. A pair of qPCR primers, which amplify a 188 bp DNA fragment, and a TaqMan probe were selected from ATPase gene (ORF114R) of the SHIV genome. The primers and TaqMan probe used in this assay were shown to be specific for SHIV and did not react with White spot syndrome virus (WSSV), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Hepatopancreatic parvovirus (HPV), Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease (VP AHPND ), and Enterocytozoon hepatopenaei (EHP), or healthy shrimp DNA. The detection limit of the qPCR method was as low as 4 copies per reaction. The diagnostic sensitivity and the diagnostic specificity of the qPCR compared with nested PCR were 95.3% and 99.2%, respectively. The resulting standard curves showed high correlation coefficient values (R 2  = 0.998) in the range of 4 × 10 9 to 4 × 10 0 DNA copies/reaction. Test of farm samples showed that SHIV was detected in L. vannamei , Fenneropenaeus chinensis and Macrobrachium rosenbergii contained SHIV ranged from 1.21E+02 to 7.95E+07 copies (μg DNA) −1 . Quantitative detection of different tissues in artificial infected shrimp showed that haemolymph contained the highest SHIV load and muscle contained lowest SHIV load. [ABSTRACT FROM AUTHOR]

A TaqMan probe and a pair of specific primers were selected from the small subunit ribosomal DNA (SSU rDNA) sequence of Enterocytozoon hepatopenaei (EHP); this real-time PCR assay was developed and optimized. It showed a good linearity in detecting standards of EHP SSU rDNA fragments from 4 × 10 2 to 4 × 10 8 copies/reaction using the established method. The detection limit of the qPCR method was as low as 4 × 10 1 copies per reaction, which was higher than the conventional PCR and SYBR Green I-based EHP qPCR reported. Using the qPCR assay, EHP was detected in four batches of slow-growing Penaeus vannamei specimens collected from Tianjin and Zhejiang Province in China was detected using qPCR. The results showed that all the hepatopancreas from the slow-growing P. vannamei specimens were detected as EHP-positive. EHP copies of hepatopancreas in some batches had a negative correlation with the body mass index (BMI) of shrimps; however, not all batches of specimens had this negative correlation between EHP copies of hepatopancreas and BMI. This qPCR technique is sensitive, specific and easy to perform (96 tests in <3 h), which provides technical support for the detection and prevention of EHP. [ABSTRACT FROM AUTHOR]

Purpose: To elucidate the role of tumor-associated macrophage (TAM) in the loss of ERα in endometrial cancer (EC) and the underlying mechanism.Materials and Methods: Tissue microarrays and immunohistochemistry assays were performed using endometrial cancer tissue along with coculture, immunofluorescence, invasion assays and ChIP-qPCR using a human endometrial cancer cell line.Results: Compared with normal tissue, an increased number of TAM was found in EC tissue (34.0 ± 2.6 vs. 8.3 ± 1.1, respectively; p < 0.001), which may downregulate ERα (27.4%, p < 0.05 for HEC-1A and 16.9%, p < 0.05 for Ishikawa) and promote EC cell invasion (1.8-fold, p < 0.001 for HEC-1A and 2.0-fold, p < 0.001 for Ishikawa). Furthermore, we found that TAM-derived CXCL8 mediated the loss of ERα and cancer invasion via HOXB13. HOXB13 was highly expressed in the ERα-negative subtype (r = -0.204, p = 0.002) and low expression of ESR1 was associated with a poor prognosis for EC patients (log-rank p < 0.05).Conclusion: TAM-secreted CXCL8 downregulated the ERα expression of EC cells via HOXB13, which may be associated with cancer invasion, metastasis and poor prognosis. [ABSTRACT FROM AUTHOR]

Abstract: Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, but little was known about CADs in tea plants (Camellia sinensis). A full-length cDNA sequence (CsCAD2) was isolated by suppressive subtractive hybridization (SSH) in Ectropis oblique feeding-induced tea plants, and another two full-length cDNA sequences (CsCAD1 and CsCAD3) were obtained from a transcriptome obtained by deep sequencing. However, they showed only 20–54% identities. Phylogenetic analysis revealed that they belonged to three different families. DNA gel blotting analysis revealed that two copies of CsCAD1 and CsCAD2 genes existed in tea genome, but CsCAD3 likely had only one copy. Recombinant proteins of these CsCADs were produced in Escherichia coli. The activity of purified recombinant CsCAD2 protein was up to 0.43μmolmin−1 mg−1. However, the other two recombinant proteins had lower activities, probably due to incomplete refolding. qRT-PCR analysis indicated that while CsCAD3 was strongly up-regulated in tea plants after E. oblique attack and mechanical damage, CsCAD1 and CsCAD2 showed only moderate or no changes in transcript levels. Treatment of defence-related hormones methyl jasmonate (MeJA) and salicylic acid (SA) elevated the expression of CsCAD1 and CsCAD2, but decreased the transcript abundance of CsCAD3. The transcript levels of CsCAD2 did not change after applying abscisic acid (ABA), whereas CsCAD1 and CsCAD3 were induced. These results suggested that these three CsCAD genes in tea plants may play a role in defense against insects and pathogens and adaptation to abiotic stresses and these genes likely have divergant functions. [Copyright &y& Elsevier]

Abstract: Objective: Current biomarkers cannot completely distinguish sepsis from systemic inflammatory response syndrome (SIRS) caused by other non-infectious diseases. Circulating microRNAs (miRNAs) are promising biomarkers for several diseases, but their correlation with sepsis is not totally clarified. Methods: Seven miRNAs related to inflammation or infection were included in the present study. Serum miRNA expression was investigated in 50 patients diagnosed with sepsis, 30 patients with SIRS and 20 healthy controls to evaluate the diagnostic and prognostic value. Expression levels of serum miRNAs were determined by quantitative PCR using the Qiagen miScript system. Serum CRP and IL-6 levels were determined by enzyme linked immunosorbent assay. Results: Serum miR-146a and miR-223 were significantly reduced in septic patients compared with SIRS patients and healthy controls. The areas under the receiver operating characteristic curve of miR-146a, miR-223 and IL-6 were 0.858, 0.804 and 0.785, respectively. Conclusion: Serum miR-146a and miR-223 might serve as new biomarkers for sepsis with high specificity and sensitivity. (ClinicalTrials.gov number, NCT00862290.) [Copyright &y& Elsevier]

Lately, long non-coding RNA (lncRNA) is recognized as a key regulator of gastric cancer (GC) which has aroused great interest in the fields of medicine, toxicology, and functional food. Studies related to LncRNA expression microarray data indicate that BX357664 is down-regulated in GC specimens. However, the expression pattern and molecular mechanism of BX357664 in GC have not been studied so far. The purpose of this study was to investigate the expression of lncRNA BX357664 in GC and its function in GC cell lines. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the level of BX357664 in 50 pairs of cancer tissues and adjacent non-cancer tissues collected from GC patients. It was found that BX357664 level was lowered in cancer specimens than adjacent non-cancer tissues and correlated with tumor size and TNM stage. Also, we used cell counting kit 8 (CCK8), cell clone formation assay and transwell assay, which affirmed that up-regulation of BX357664 inhibited the proliferation, migration, and invasion of GC cells, but promoted apoptosis. In the dual-luciferase report analysis, BX357664 acted as a miR-183–3p ceRNA to target and regulate the expression of PTEN and affect the PI3K/AKT pathway. These results indicate that BX357664 can inhibit the proliferation and metastasis of GC through the miR-183–3p/PTEN/PI3K/AKT pathway, which may serve as potential targets for the treatment of GC in the future. • LncRNA BX357664 is down-regulated in GC tissues. • LncRNA BX357664 can inhibit the proliferation, migration in GC cells. • LncRNA BX357664 can target and regulate the expression of PTEN. [ABSTRACT FROM AUTHOR]