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The discovery of cell‑free DNA fetal (cff DNA) in maternal plasma during pregnancy provides a novel perspective for the development of non‑invasive prenatal diagnosis (NIPD). Against the background of maternal DNA, the use of the relatively low concentration of cff DNA is limited in NIPD. Therefore, in order to overcome the complication of the background of maternal DNA and expand the scope of cff DNA application in clinical practice, it is necessary to identify novel universal fetal‑specific DNA markers. The GeneChip Human Promoter 1.0R Array set was used in the present study to analyze the methylation status of 12 placental tissue and maternal peripheral blood whole‑genome DNA samples. In total, 5 fetus differential hypermethylation regions and 6 fetus differential hypomethylation regions were identified. In order to verify the 11 selected methylation regions and detect the differential CpG sites in these regions, a bisulfate direct sequencing strategy was used. In total, 87 fetal differential methylation CpG sites were identified from 123 CpG sites. The detection of fetal differential methylation DNA regions and CpG sites may be instrumental in the development of efficient NIPD and in the expansion of its application in other disorders. [ABSTRACT FROM AUTHOR]

The present study aimed to identify the mutation causing an atypical syndrome. High-resolution single nucleotide polymorphism (SNP) arrays are considered to be a major detection method for submicroscopic chromosomal rearrangements smaller than 5 Mb in size. Genomic DNA samples of the patient and his parents were converted to a final concentration of 50 ng/ml. The Illumina BeadScan genotyping system and the HumanOmni1-Quad Chip were employed to obtain the signal intensities of SNP probes. The patient presented with congenital heart disease, autism, mental retardation, growth retardation, hypercalcemia, nephroliths and cleft palate. The karyotypes of the patient and his parents were normal. The present study employed high-resolution SNP arrays to analyze the whole genome for copy number variations (CNVs). A total of 309 CNVs were discovered. A de novo 1.5 Mb gain of chromosome 7q11.23 (Chr7: 72,357,322-73,856,472) was identified following exclusion of CNVs presented in the Database of Genomic Variants. In conclusion, to the best of our knowledge, the current study describes the first case of a patient presenting with Williams-Beuren syndrome alongside supravalvular aortic stenosis, autism and cleft palate, and identifies an atypical deletion at 7q11.23. [ABSTRACT FROM AUTHOR]

Congenital heart disease (CHD) is a complex illness with high rates of morbidity and mortality. In embryonic development, the heart is the first formed organ, which is strictly controlled by gene regulatory networks, including transcription factors, signaling pathways, epigenetic factors and microRNAs. Bone morphogenetic protein (BMP)-2 and - 4 are essential in cardiogenesis as they can induce the expression of transcription factors, NKX2-5 and GATA binding protein 4, which are important in the development of the heart. The inhibition of BMP-2 and -4 inhibits the late expression of NKX2-5 and affects cardiac differentiation. The aim of the present study was to investigate whether BMP-2 and -4 variations may be associated with CHD in Chinese Han populations. The rs1049007, rs235768 and rs17563 single nucleotide polymorphisms (SNPs), which are genetic variations located within the translated region of the BMP-2 and -4, were evaluated in 230 patients with CHD from the Chinese Han population and 160 non-CHD control individuals. Statistical analyses were performed using the χ² test, implemented using SPSS software (version 13.0). The Hardy-Weinberg equilibrium test was performed on the population using online Online Encyclopedia for Genetic Epidemiology studies software, and multiple-sequence alignments of the BMP proteins were performed using Vector NTI software. No statistically significant associations were identified between these genetic variations and the risk of CHD (rs1049007, P-value=0.560; rs235768, P-value=0.972; rs17563, P-value=0.787). In addition, no correlation was found between the patients with CHD and the non-CHD control individuals. Therefore, the rs1049007, rs235768 and rs17563 genetic variations of BMP-2 were not associated with CHD in the Chinese Han population. [ABSTRACT FROM AUTHOR]

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement and exhibits putative anti-aging properties. However, the molecular basis of the actions of DHEA, particularly on the biological characteristics of target cells, remain unclear. The aim of the current study was to investigate the effects of DHEA on cell viability, cell proliferation, cell cycle and mitochondrial function in primary rat Leydig cells. Adult Leydig cells were purified by Percoll gradient centrifugation, and cell proliferation was detected using a Click-iT® EdU Assay kit and cell cycle assessment performed using flow cytometry. Mitochondrial membrane potential was detected using JC-1 staining assay. The results of the current study demonstrate that DHEA decreased cell proliferation in a dose-dependent manner, whereas it improved cell viability in a time-dependent and dose-dependent manner. Flow cytometry analysis demonstrated that DHEA treatment increased the S phase cell population and decreased the G2/M cell population. Cyclin A and CDK2 mRNA levels were decreased in primary rat Leydig cells following DHEA treatment. DHEA treatment decreased the transmembrane electrical gradient in primary Leydig cells, whereas treatment significantly increased succinate dehydrogenase activity. These results indicated that DHEA inhibits primary rat Leydig cell proliferation by decreasing cyclin mRNA level, whereas it improves cells viability by modulating the permeability of the mitochondrial membrane and succinate dehydrogenase activity. These findings may demonstrate an important molecular mechanism by which DHEA activity is mediated. [ABSTRACT FROM AUTHOR]

Oxidative damage to mitochondrial DNA (mtDNA) and cell apoptosis are heavily implicated in aging. Our previous study established a mimetic rat model of aging in the cochleae using D.galactose (D.gal), and revealed that chronic injection of D.gal can increase oxidative stress and mtDNA common deletions (CD). The aim of the present study was to investigate the sources of reactive oxygen species and the occurrence of apoptosis in the cochleae of rats following 8 weeks of D.gal exposure. The results of the present study indicated that an elevated accumulation of the mtDNA CD and mitochondrial ultrastructural damage occurred in the cochleae of rats injected with D.gal for 8 weeks. In addition, the levels of 8.hydroxy.2.deoxyguanosine, NADPH oxidase (NOX) 3, P22phox and cleaved caspase 3, and the number of terminal deoxynucleotidyl transferase.mediated deoxyuridine triphosphate nick.end.labelling.positive cells were increased in the cochleae of D.gal.treated rats, compared with the controls. These findings suggested that nitric oxide synthase NOX3.associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase 3.dependent apoptotic signalling pathway in the cochleae during aging. The present study also provided novel insights into the development of age.associated hearing loss, also termed presbycusis. [ABSTRACT FROM AUTHOR]

Phorbol myristate acetate (PMA), as a potent tumor promoter, may induce microglial senescence. The present study investigated the effect of PMA infection on microglial senescence. From 58 male Sprague-Dawley rats, 10 were randomly selected and divided into a PMA injection group, containing five rats (0.5 μg/μl PMA) and a control group, containing five rats (commensurable 0.9% saline). Immunofluorescent staining of Iba-1 and enzyme-linked immunosorbent assay analyses of the expression levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1 β were performed in these two groups. The remaining 48 rats were randomly divided into the following three groups, each containing 16 rats: Repeated injection control group (commensurable normal saline, once a week for 4 weeks), single PMA injection group (0.5 μg/μl PMA, once in the first week) and repeated injection PMA group (0.5 μg/μl PMA, once a week for 4 weeks). The expression levels of p21, detected using double immunofluorescence staining with Iba-1, and β-galactosidase, via double immunohistochemical staining of Iba-1, were examined in these three groups. The results indicated that a single injection of PMA did not change the microglial morphology and had no significant effects on the expression levels of TNF-α and IL-1β, compared with the control group (P>0.05). Following four repeated injections of PMA, the microglia in the substantia nigra presented with features of senescence, characterized by increased expression levels of β-galactosidase (P<0.001) and p21 (P<0.001), compared with the repeated injection control group. In conclusion, repeated intra-nigrostriatal treatment with PMA induced microglial senescence with increased expression levels of β-galactosidase and p21 in the substantia nigra of the rats. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells (MSCs) with the ability to differentiate into insulin-producing cells (IPCs) have become the most promising means of therapy for diabetes mellitus. Adipose-derived stromal cells (AdSCs), having similar characteristics to those of derived MSCs, are known to exhibit extensive proliferation potential and are able to undergo multi-lineage differentiation. Whether AdSCs can differentiate into insulin-producing cells (IPCs), however, has not been sufficiently elucidated. Therefore, the present study sought to investigate the in vitro differentiation of rabbit (r)AdSCs into IPCs, which may provide an abundant source of cells to treat diabetes. rADSCs were obtained from liposuction aspirates and then induced with glucagon-like peptide-1 and nicotinamide to differentiate into insulin-secreting cells. Differentiation was evaluated by the analysis of morphology, dithizone (DTZ) staining, reverse transcription polymerase chain reaction (RT-PCR), western blot analysis and a glucose challenge assay with detection of insulin secretion by ELISA. Morphological phase-contrast microscopic observation revealed typical islet-like cell clusters following 21 days of differentiation. DTZ staining also showed that differentiated cells were positive and undifferentiated cells were negative for insulin production. Furthermore, RT-PCR analysis confirmed the mRNA expression of insulin, PDX1 and GLUT2 in differentiated cells. Western blot analysis showed that insulin was expressed by the differentiated cells. The glucose challenge assay showed that insulin secretion of the IPCs was in a glucose dependent manner. These findings implied that AdSCs are able to differentiate into IPC in vitro, and are therefore promising candidates for the treatment of diabetes. [ABSTRACT FROM AUTHOR]

Serum exosomal microRNAs (miRNAs) have received considerable attention as potential biomarkers for tumor diagnosis. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is commonly used to detect miRNA expression levels in various types of cancer. One prerequisite for valid RT-qPCR data is the correct normalization of miRNAs to stably expressed endogenous reference genes (RGs). The study of liver carcinoma resection requires the use of reliable RGs in order to assess the expression levels of serum exosomal target miRNAs. However, the assessment of RG suitability for optimum serum exosomal miRNA expression analysis has yet to be investigated. The present study investigated the expression stability of 10 candidate RGs. The candidate genes included eight miRNAs (miR-16, miR-103, miR-191, let-7a, miR-26a, miR-221, miR-181a, and miR-451) and two small RNAs (5S and U6). The stability values of the candidate genes were calculated using the following algorithms: geNorm, NormFinder, BestKeeper, and the comparative Ct method. The overall ranking obtained from these analyses revealed that miR-221, let-7a, and miR-26a were appropriate internal RGs for analysis of serum miRNAs in patients with hepatocellular carcinoma. In addition, normalization with miR-221 and let-7a combined, as recommended by geNorm, or with miR-26a, as recommended by NormFinder, increased the accuracy of interpretation of the target miRNA expression levels in hepatopathy studies. [ABSTRACT FROM AUTHOR]

Chronic administration of D-galactose (D-gal) is a useful method for establishing a model of natural aging in the auditory system. Previous studies have demonstrated that NADPH oxidases (NOXs) may be an important source of reactive oxygen species (ROS) in the peripheral auditory system (PAS) and cause an increase in mitochondrial DNA (mtDNA) common deletion (CD) levels in the PAS and central auditory system (CAS) of rats with D-gal-induced aging. However, the source of the ROS in the CAS and the mechanisms of age-related hearing loss (ARHL) have yet to be elucidated. In the present study, male Sprague Dawley rats were administered a daily injection of D-gal (150, 300 and 500 mg/kg, respectively) for eight weeks. All three doses of D-gal caused a significant increase in the expression of NOX2, 8-hydroxy-2-deoxyguanosine, a biomarker of DNA oxidative damage, and uncoupling protein 2, together with a decrease in the mitochondrial total antioxidant capabilities in the auditory cortex, as compared with the control rats (injected daily with the same volume of 0.9% saline for eight weeks). The levels of the mtDNA CD were also increased in the auditory cortex of the D-gal-induced aging rats. These findings suggest that both NOX- and mitochondria-associated ROS generation may contribute to mtDNA oxidative damage in the auditory cortex of the CAS of D-gal-induced aging rats. This study may provide novel insight into the development of ARHL. [ABSTRACT FROM AUTHOR]