Germacrone alleviates okadaic acid-induced neurotoxicity in PC12 cells via M1 muscarinic receptor-mediated Galphaq (Gq)/phospholipase C beta (PLCß)/ protein kinase C (PKC) signaling.

Alzheimer's disease (AD) is a neurodegenerative disorder with prominent individual morbidity and mortality among elderly people. Germacrone (Germ) has been reported to exert dominant protective roles in multiple human diseases, and neurological diseases are also included. The intention of this paper is to determine the impacts of Germ on okadaic acid (OA)-treated PC12 cells and confirm the hidden regulatory mechanism. First, PC12 cells were induced by OA in the absence or presence of Germ. Cell counting kit-8 assay was to monitor cell proliferation. Western blot was to test the protein levels of cholinergic muscarinic M1 receptor (CHRM1), Galphaq (Gq), phospholipase C beta (PLCß) and protein kinase C (PKC). The levels of reactive oxygen species (ROS) and other oxidative stress markers were evaluated using corresponding kits. ELISA was used to estimate the levels of AD markers. RT-qPCR was used to examine the mRNA levels of beta-site amyloid-precursor-protein-cleaving enzyme 1 (BACE-1) and apolipoprotein E (APOE). The results uncovered that Germ enhanced the proliferation of OA-insulted PC12 cells, elevated the protein level of CHRM1 and activated the Gq/PLCß/PKC signaling. Moreover, after OA-induced PC12 cells were administered with Germ, insufficiency of CHRM1 impeded cell proliferation, enhanced oxidative stress and neuron injury and inactivated the Gq/PLCß/PKC signaling. Furthermore, the addition of Gq inhibitor UBO-QIC, PLCß inhibitor U73122 or PKC inhibitor Go6983 reversed the enhanced proliferation, the reduced oxidative stress and neuron injury in OA-treated PC12 cells caused by Germ. Collectively, Germ modulated M1 muscarinic receptor-mediated Gq/PLCß/PKC signaling, thereby alleviating OA-induced PC12 cell injury.

CHP2 Promotes Cell Proliferation in Breast Cancer via Suppression of FOXO3a.

Calcineurin B homologous protein isoform 2 (CHP2), an essential cofactor for Na+/H+ exchanger isoform 1 (NHE1), is identified to be expressed in various malignant cell lines. However, the clinical significance and biological role of CHP2 in breast cancer remain to be established. Here, CHP2 was markedly overexpressed in breast cancer cells and clinical tumor specimens. Immunohistochemical analysis revealed that the expression of CHP2 was significantly correlated with patients' clinicopathologic characteristics like clinical stage, and breast cancer patients with high CHP2 expression had shorter overall survival compared with patients with low CHP2 expression. Moreover, it was demonstrated that overexpressing CHP2 significantly enhanced, whereas silencing endogenous CHP2 inhibited, the proliferation and tumorigenicity of breast cancer cells in vitro and in vivo In addition, overexpression of CHP2 accelerated, whereas inhibition of CHP2 retarded, G1-S phase cell-cycle transition in breast cancer cells. Mechanistically, overexpression of CHP2 activated AKT signaling and suppressed the transactivation of the forkhead box O3 (FOXO3/FOXO3a) transcription factor.Implications: This study discovers a previously unrecognized role of CHP2 in the progression of breast cancer and supports the significance of this gene as a novel prognostic biomarker and a potential therapeutic target for breast cancer. Mol Cancer Res; 16(10); 1512-22. ©2018 AACR.

Association of plasma apolipoprotein CIII, high sensitivity C-reactive protein and tumor necrosis factor-α contributes to the clinical features of coronary heart disease in Li and Han ethnic groups in China.

BACKGROUND: Apolipoprotein CIII (apoCIII) is an independent risk for coronary heart disease (CHD). In this study, we investigated the associations among plasma apoCIII, hs-CRP and TNF-α levels and their roles in the clinical features of CHD in the Li and Han ethnic groups in China. METHODS: A cohort of 474 participants was recruited (238 atherosclerotic patients and 236 healthy controls) from the Li and Han ethnic groups. Blood samples were obtained to evaluate apoCIII, TNF-α, hs-CRP and lipid profiles. Chi-squared, t-tests, and Kruskal-Wallis or Wilcoxon-Mann-Whitney tests, Pearson or Spearman correlation tests and multiple unconditional logistic regression were employed to analyze lipid profiles and variations in plasma apoCIII, TNF-α, hs-CRP in subgroups of CHD and their contributions to CHD using SPSS version 20.0 software. RESULTS: Compared to healthy participants, unfavorable lipid profiles were identified in CHD patients with enhanced systolic pressure, diastolic pressure, fasting blood sugar (FBS), TG, TC, LDL-C, apoB, Lp(a) (P < 0.05, TC and Lp(a); P < 0.01, FBS, TG, LDL-C, apoB); and lower HDL-C and apoAI (P < 0.05). Plasma apoCIII, TNF-α and hs-CRP levels were higher in CHD individuals (16.77 ± 5.98 mg/dL vs. 10.91 ± 4.97 mg/dL; 17.23 ± 6.34 pg/mL vs. 9.49 ± 3.88 pg/mL; 9.55 ± 7.32 mg/L vs. 2.14 ± 1.56 mg/L; P < 0.01 vs. healthy participants). Identical patterns were obtained in the Li and Han groups (16.46 ± 6.08 mg/dL vs. 11.72 ± 5.16 mg/dL; 15.71 ± 5.52 pg/mL vs. 9.74 ± 4.31 pg/mL; 8.21 ± 7.09 mg/L vs. 2.15 ± 1.51 mg/L in Li people; 17.05 ± 5.90 mg/dL vs. 10.07 ± 4.63 mg/dL; 18.59 ± 6.73 pg/mL vs. 9.23 ± 3.38 pg/mL; 10.75 ± 7.44 mg/L vs. 2.12 ± 1.63 mg/L in Han people; P < 0.01). Paired comparisons of subgroups with stable angina, unstable angina, and acute myocardial infarction (AMI) revealed significant variation in plasma levels of apoCIII, TNF-α and hs-CRP (P < 0.01), but not among subgroups with mild, moderate and severe stenosis (P > 0.05). Plasma apoCIII, TNF-α and hs-CRP contributed to the development of CHD (OR = 2.554, 7.252, 6.035, P < 0.01) with paired correlations in CHD patients (apoCIII vs. TNF-α, r = 0.425; apoCIII vs. hs-CRP, r = 0.319; TNF-α vs. hs-CRP, r = 0.400, P < 0.01). CONCLUSIONS: Association among plasma apoCIII, hs-CRP and TNF-α interacts with unfavorable lipid profiles to contribute to the clinical features of CHD with stable angina, unstable angina, and AMI in the Li and Han ethnic groups in China.

Development of noninvasive prenatal diagnosis of trisomy 21 by RT-MLPA with a new set of SNP markers.

INTRODUCTION: Placental mRNA can now be detected in maternal whole blood, raising the possibility of using maternal blood for noninvasive prenatal diagnosis (NIPD) of trisomy 21. We aimed to identify new mRNA-single nucleotide polymorphism (mRNA-SNP) markers suitable for use in reverse-transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) to develop a more reliable diagnostic method for trisomy 21 in Chinese subjects. MATERIALS AND METHODS: Using sequencing, we determined the status of SNPs in genes expressed in the placenta and calculated their heterozygote frequencies to determine which loci were suitable for use in RT-MLPA. Cell-free fetal RNA was extracted from peripheral blood samples of 246 women at 12-24 weeks of pregnancy, and the SNP loci selected were analyzed by RT-MLPA, followed by capillary electrophoresis. Karyotype analyses were used to confirm the diagnosis of trisomy 21. RESULTS: As compared with karyotype analysis, the diagnostic sensitivity and specificity of RT-MLPA were excellent (95 and 100% in different gestational weeks). CONCLUSION: The RT-MLPA technique is a suitable and reliable method for the diagnosis of trisomy 21. Use of RT-MLPA with the SNP markers described here shows good specificity, high sensitivity, and high throughput potential, making this technique suitable for NIPD in clinical practice.

Distinctiveness of the cagA genotype in children and adults with peptic symptoms in South China.

BACKGROUND: Helicobacter pylori infection is different between children and adults, not only in infection rate but also in virulence genotypes. However, the 3' region of CagA, important in stomach carcinogenesis, still remains unclear in children. The present study aims to compare the frequency of cagA and the distribution of its subtypes between children and adults in South China. MATERIALS AND METHODS: One hundred and twenty-eight children and 99 adults with peptic symptoms were enrolled in our research. Histology, rapid urease test, and real-time polymerase chain reaction (PCR) assay were used to diagnose H. pylori infection. vacA s1 was detected by real-time PCR, and EPIYA motifs in the 3' region of CagA by conventional PCR and DNA sequencing. RESULTS: H. pylori infection was diagnosed in 53 children and 62 adults. vacA s1 was identified in 90.6% and 91.9% of infected children and adults, respectively. Furthermore, cagA was identified in 73.6% and 82.3% of infected children and adults, respectively. No patient with multiple cagA subtypes was observed. A higher prevalence of more virulent cagA genotype was found in children compared to adults (p < .05). Thirty-eight of 39 (97.4%) cagA-positive children were found to have EPIYA-ABD and only one (2.6%) with EPIYA-ABC. In adults, four types of EPIYA motifs--ABC (29.4%), ABD (64.7%), ABAB (2%), and AAD (3.9%)--were identified, and the ABD type was found more commonly in severe diseases, such as atrophic gastritis (53.3%) and gastric cancer (71.4%). CONCLUSION: cagA genotypes in children and in adults are different, and EPIYA-ABD may have potential clinical implication in the development of gastric cancer in South China.

Simultaneous detection of different respiratory virus by a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting assay.

Cell culture and immunofluorescence (IF) assays have been traditionally used for the laboratory diagnosis of respiratory viral infections, but these assays have a low sensitivity and are time consuming. We developed a multiplex reverse transcription polymerase chain reaction combined with flow-through reverse dot blotting (mRT-PCR-FT-RDB) assay for the simultaneous detection of influenza virus type A including H5 subtype and H9 subtype, influenza virus type B, parainfluenza virus types 1 and 3, respiratory syncytial virus, human rhinovirus, and human coxsackievirus. In comparison with viral culture and IF assay as the gold standard method, the mRT-PCR-FT-RDB assay gave a sensitivity and a specificity of 100% and 98%. The high sensitivity and specificity, the rapid result turnaround time, and the reduced expense of the mRT-PCR-FT-RDB assay compared with viral culture and IF assay suggest that this assay would be a significant improvement over traditional ones for the detection of respiratory viruses in a clinical laboratory.

Development of a multiplex quantitative fluorescent PCR assay for identification of rearrangements in the AZFb and AZFc regions.

The azoospermia factor b (AZFb) and azoospermia factor c (AZFc) regions in the human Y chromosome consist of five palindromes constructed from six distinct families of amplicons and are prone to rearrangement. Partial deletion and duplication in the region can cause azoospermia or oligozoospermia and male infertility. The aim of the study was to establish a quantitative fluorescent PCR (QF-PCR) assay to classify AZFb and AZFc rearrangements. A single pair of fluorescent primers was designed to amplify simultaneously the amplicon in AZFc and the length-variant homologous sequences outside of the region as control. Since the copy number of the control sequences is fixed in the human genome, dosage of the target could be easily obtained through comparing the height of the fluorescent peaks between the target and the control after amplification with limited PCR cycles. Most types of rearrangements in AZFb and AZFc regions could be classified with QF-PCR containing four such primer pairs. Eleven types of rearrangement in AZFb and AZFc regions were well discriminated with QF-PCR. In conclusion, QF-PCR is a simple and reliable method to detect rearrangements in AZFb and AZFc.

Development of a multiplex real-time polymerase chain reaction for the detection of influenza virus type A including H5 and H9 subtypes.

Avian influenza viruses (AIVs) are endemic in wild birds and, if transmitted to poultry, can cause serious economic losses. The aim of this study was to develop a multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) for rapid detection of influenza virus type A, including H5 and H9 subtypes. The selected primers and various labeled TaqMan reporter probes corresponding to matrix, H5, and H9 genes were used in a multiplex real-time RT-PCR to simultaneously detect triple fluorescent signals. The results showed that the multiplex real-time RT-PCR assay can be applied to detect RNA of influenza virus type A including H5 and H9 subtypes with a high specificity and a sensitivity of 10 copies per reaction. As a result of its short turnaround times and a high specificity and sensitivity, the assay is very suitable for large-scale screening during AIV outbreaks.

Determination of hepatitis B virus genotype by flow-through reverse dot blot.

BACKGROUND: The clinical outcome and response to therapy of hepatitis B virus infection differ depending upon viral genotype. Most methods of determining the viral genotype are relatively time-consuming and costly. Moreover, the results of some methods are influenced by single nucleotide mutations. OBJECTIVES: To develop a novel HBV genotyping process insensitive to single nucleotide mutations using an improved reverse dot blot method employing the principle of "flow-through hybridization". STUDY DESIGN: The flow through reverse dot blot (FT-RDB) method was developed using DNA from different HBV genotypes. HBV sequences from Genebank were used to design primers and probes. Specificity and sensitivity of the method were evaluated with clinical samples in which the HBV viral load was quantified by real-time PCR. Results were compared to those of multiplex PCR and sequencing. Another 59 clinical samples were used to test the clinical applicability of the method. RESULTS: We showed that FT-RDB could be made insensitive to single nucleotide mismatch by adjusting the hybridization temperature. All HBV-negative samples showed no signals in the assay. The detection sensitivity of the method was found to be between 10(3) and 10(4) DNA copies/ml. The results of FT-RDB were 84% concordant with those of multiplex PCR, and 96% concordant with sequencing results in 101 cases. The genotype all 59 clinical samples was accurately identified. CONCLUSIONS: We demonstrated that the FT-RDB method was rapid, reliable, accurate and inexpensive. It appears to be useful for routine clinical HBV genotyping even in non-specialized hospital laboratories.

Cyp2c19 genotype and omeprazole hydroxylation phenotype in Chinese Li population.

1. CYP2C19 is a polymorphism of cytochrome P450, which is responsible for the metabolism of many drugs. The genetic polymorphism shows interethnic variation and it has been demonstrated that the frequency of poor metabolizers (PM) and the distribution of alleles of CYP2C19 vary among Chinese ethnic nationalities. The aim of the present study was to investigate the incidence of CYP2C19 polymorphism in the Chinese Li population. 2. One hundred and sixty-five unrelated healthy Li subjects were identified with respect to CYP2C19 by genotype and phenotype analysis. A polymerase chain reaction-restriction fragment length polymorphism method was performed for genotyping. The plasma concentrations of omeprazole and 5-hydroxyomeprazole were assayed by reversed-phase high-performance liquid chromatography and the omeprazole hydroxylation index (HI) was determined. 3. The frequency distribution of omeprazole HI is bimodal and the antimode for HI was estimated to be 5.6. The prevalence of phenotypic PM in the Li population was 16.6% (13.7-19.5; 95% CI). Genotype analysis revealed that the frequencies of the CYP2C19*1, *2 and *3 alleles in the Li population were 0.617 (0.590-0.644; 95% CI), 0.353 (0.327-0.379; 95% CI) and 0.031 (0.021-0.041; 95% CI), respectively. The frequency of genotypic PM was 14.7% (11.9-17.5; 95% CI), which almost agreed with the frequency of phenotypic PM. Omeprazole HI was significantly different among the different genotype groups (P < 0.05). 4. The present study revealed that the incidence of the CYP2C19*1, *2 and *3 alleles in Chinese Li population is different to that in other ethnic populations of China. There was an obvious relationship between CYP2C19 genotype and omeprazole hydroxylation phenotype, and about 90% of phenotypic PM can be explained by the CYP2C19*2 and *3 alleles.