Synthesis of positive plasmas with known chromosomal abnormalities for validation of non-invasive prenatal screening

Non-invasive prenatal screening (NIPS) is a DNA sequencing-based screening test for fetal aneuploidies and possibly other pathogenic genomic abnormalities, such as large deletions and duplications. Validation and quality assurance (QA) of this clinical test using plasmas with and without targeted chromosomal abnormalities from pregnant women as negative and positive controls are required. However, the positive plasma controls may not be available for many laboratories that are planning to establish NIPS. Limited synthetic positive plasmas are commercially available, but the types of abnormalities and the number/quantity of synthetic plasmas for each abnormality are insufficient to meet the minimal requirements for the initial validation. We report here a method of making synthetic positive plasmas by adding cell-free DNA (cfDNA) isolated from culture media of prenatal cells with chromosomal abnormalities to the plasmas from non-pregnant women. Thirty-eight positive plasmas with various chromosomal abnormalities, including autosomal and sex chromosomal aneuploidies, large deletions and duplications, were synthesized. The synthetic plasmas were characterized side-by-side with real positive plasmas from pregnant women and commercially available synthetic positive plasmas using the Illumina VeriSeq NIPT v2 system. All chromosomal abnormalities in the synthetic plasmas were correctly identified with the same testing sensitivity and specificity as in the real and commercial synthetic plasmas. The findings demonstrate that the synthetic positive plasmas are excellent alternatives of real positive plasmas for validation and QA of NIPS. The method described here is simple and straightforward, and can be readily used in clinical genetics laboratories with accessibility to prenatal cultures

Neuroprotective Effects of Pre-Treament with l-Carnitine and Acetyl-l-Carnitine on Ischemic Injury In Vivo and In Vitro

The therapeutic effect of stroke is hampered by the lack of neuroprotective drugs against ischemic insults beyond the acute phase. Carnitine plays important roles in mitochondrial metabolism and in modulating the ratio of coenzyme A (CoA)/acyl-CoA. Here, we investigate the neuroprotective effects of l-carnitine (LC) and Acetyl-l-carnitine (ALC) pre-treatment on ischemic insults under the same experimental conditions. We used a transient middle cerebral artery occlusion (MCAO) model to evaluate the protective roles of LC and ALC in acute focal cerebral ischemia in vivo and to understand the possible mechanisms using model of PC12 cell cultures in vitro. Results showed that ALC, but not LC, decreased infarction size in SD rats after MCAO in vivo. However, both LC and ALC pretreatment reduced oxygen-glucose deprivation (OGD)-induced cell injury and decreased OGD-induced cell apoptosis and death in vitro; at the same time, both of them increased the activities of super oxide dismutase (SOD) and ATPase, and decreased the concentration of malondialdehyde (MDA) in vitro. Thus, our findings suggested that LC and ALC pre-treatment are highly effective in the prevention of neuronal cell against ischemic injury in vitro, however, only ALC has the protective effect on neuronal cell injury after ischemia in vivo

Dynamical alterations of brain function and gut microbiome in weight loss

ObjectiveIntermittent energy restriction (IER) is an effective weight loss strategy. However, little is known about the dynamic effects of IER on the brain-gut-microbiome axis.MethodsIn this study, a total of 25 obese individuals successfully lost weight after a 2-month IER intervention. FMRI was used to determine the activity of brain regions. Metagenomic sequencing was performed to identify differentially abundant gut microbes and pathways in from fecal samples.ResultsOur results showed that IER longitudinally reduced the activity of obese-related brain regions at different timepoints, including the inferior frontal orbital gyrus in the cognitive control circuit, the putamen in the emotion and learning circuit, and the anterior cingulate cortex in the sensory circuit. IER longitudinally reduced E. coli abundance across multiple timepoints while elevating the abundance of obesity-related Faecalibacterium prausnitzii, Parabacteroides distasonis, and Bacterokles uniformis. Correlation analysis revealed longitudinally correlations between gut bacteria abundance alterations and brain activity changes.ConclusionsThere was dynamical alteration of BGM axis (the communication of E. coli with specific brain regions) during the weight loss under the IER

Upregulation of Cyclin B1 by miRNA and its implications in cancer

It is largely recognized that microRNAs (miRNAs) function to silence gene expression by targeting 3′UTR regions. However, miRNAs have also been implicated to positively-regulate gene expression by targeting promoter elements, a phenomenon known as RNA activation (RNAa). In the present study, we show that expression of mouse Cyclin B1 (Ccnb1) is dependent on key factors involved in miRNA biogenesis and function (i.e. Dicer, Drosha, Ago1 and Ago2). In silico analysis identifies highly-complementary sites for 21 miRNAs in the Ccnb1 promoter. Experimental validation identified three miRNAs (miR-744, miR-1186 and miR-466d-3p) that induce Ccnb1 expression in mouse cell lines. Conversely, knockdown of endogenous miR-744 led to decreased Ccnb1 levels. Chromatin immunoprecipitation (ChIP) analysis revealed that Ago1 was selectively associated with the Ccnb1 promoter and miR-744 increased enrichment of RNA polymerase II (RNAP II) and trimethylation of histone 3 at lysine 4 (H3K4me3) at the Ccnb1 transcription start site. Functionally, short-term overexpression of miR-744 and miR-1186 resulted in enhanced cell proliferation, while prolonged expression caused chromosomal instability and in vivo tumor suppression. Such phenotypes were recapitulated by overexpression of Ccnb1. Our findings reveal an endogenous system by which miRNA functions to activate Ccnb1 expression in mouse cells and manipulate in vivo tumor development/growth

Lack of Association of Two Common Polymorphisms rs2910164 and rs11614913 with Susceptibility to Hepatocellular Carcinoma: A Meta-Analysis

BACKGROUND: Single nucleotide polymorphisms (SNPs) in microRNA-coding genes may participate in the process of carcinogenesis by altering the expression of tumor-related microRNAs. It has been suggested that two common SNPs rs2910164 in miR-146a and rs11614913 in miR-196a2 are associated with susceptibility to hepatocellular carcinoma (HCC). However, published results are inconsistent and inconclusive. In the present study, we performed a meta-analysis to systematically summarize the possible association between the two SNPs and the risk for HCC. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a search of case-control studies on the associations of SNPs rs2910164 and/or rs11614913 with susceptibility to HCC in PubMed, EMBASE, ISI Web of Science, Cochrane Central Register of Controlled Trials, ScienceDirect, Wiley Online Library and Chinese National Knowledge Infrastructure databases. Data from eligible studies were extracted for meta-analysis. HCC risk associated with the two polymorphisms was estimated by pooled odds ratios (ORs) and 95% confidence intervals (95% CIs). 5 studies on rs2910164 and 4 studies on rs11614913 were included in our meta-analysis. Our results showed that neither allele frequency nor genotype distribution of the two polymorphisms was associated with risk for HCC in all genetic models. Similarly, subgroup analysis in Chinese population showed no association between the two SNPs and the susceptibility to HCC. CONCLUSIONS/SIGNIFICANCE: This meta-analysis suggests that two common SNPs rs2910164 and rs11614913 are not associated with the risk of HCC. Well-designed studies with larger sample size and more ethnic groups are required to further validate the results

Transgenic Expression of Entire Hepatitis B Virus in Mice Induces Hepatocarcinogenesis Independent of Chronic Liver Injury

Hepatocellular carcinoma (HCC), the third leading cause of cancer deaths worldwide, is most commonly caused by chronic hepatitis B virus (HBV) infection. However, whether HBV plays any direct role in carcinogenesis, other than indirectly causing chronic liver injury by inciting the host immune response, remains unclear. We have established two independent transgenic mouse lines expressing the complete genome of a mutant HBV (“preS2 mutant”) that is found at much higher frequencies in people with HCC than those without. The transgenic mice show evidence of stress in the endoplasmic reticulum (ER) and overexpression of cyclin D1 in hepatocytes. These mice do not show any evidence of chronic liver injury, but by 2 years of age a majority of the male mice develop hepatocellular neoplasms, including HCC. Unexpectedly, we also found a significant increase in hepatocarcinogenesis independent of necroinflammation in a transgenic line expressing the entire wildtype HBV. As in the mutant HBV mice, HCC was found only in aged—2-year-old—mice of the wildtype HBV line. The karyotype in all the three transgenic lines appears normal and none of the integration sites of the HBV transgene in the mice is near an oncogene or tumor suppressor gene. The significant increase of HCC incidence in all the three transgenic lines—expressing either mutant or wildtype HBV—therefore argues strongly that in absence of chronic necroinflammation, HBV can contribute directly to the development of HCC

Haploinsufficiency and triploinsensitivity of the same 6p25.1p24.3 region in a family

BACKGROUND: Chromosome 6pter-p24 deletion syndrome (OMIM #612582) is a recognized chromosomal disorder. Most of the individuals with this syndrome carry a terminal deletion of the short arm of chromosome 6 (6p) with a breakpoint within the 6p25.3p23 region. An approximately 2.1 Mb terminal region has been reported to be responsible for some major features of the syndrome. The phenotypic contributions of other deleted regions are unknown. Interstitial deletions of the region are uncommon, and reciprocal interstitial duplication in this region is extremely rare. CASE PRESENTATION: We present a family carrying an interstitial deletion and its reciprocal duplication within the 6p25.1p24.3 region. The deletion is 5.6 Mb in size and was detected by array comparative genomic hybridization (aCGH) in a 26-month-old female proband who presented speech delay and mild growth delay, bilateral conductive hearing loss and dysmorphic features. Array CGH studies of her family members detected an apparently mosaic deletion of the same region in the proband’s mildly affected mother, but a reciprocal interstitial duplication in her phenotypically normal brother. Further chromosomal and fluorescence in situ hybridization (FISH) analyses revealed that instead of a simple mosaic deletion of 6p25.1p24.3, the mother actually carries three cell populations in her peripheral blood, including a deletion (~70 %), a duplication (~8 %) and a normal (~22 %) populations. Therefore, both the deletion and duplication seen in the siblings were apparently inherited from the mother. CONCLUSIONS: Interstitial deletion within the 6p25.1p24.3 region and its reciprocal duplication may co-exist in the same individual and/or family due to mitotic unequal sister chromatid exchange. While the deletion causes phenotypes reportedly associated with the chromosome 6pter-p24 deletion syndrome, the reciprocal duplication may have no or minimal phenotypic effect, suggesting possible triploinsensitivity of the same region. In addition, the cells with the duplication may compensate the phenotypic effect of the cells with the deletion in the same individual as implied by the maternal karyotype and her mild phenotype. Chromosomal and FISH analyses are essential to verify abnormal cytogenomic array findings