DNA Methylation Signature of Post-injury Neointimal Cells During Vascular Remodeling in the Rat Balloon Injury Model.

Vascular smooth muscle cell (VSMC) accumulation in the neointimal is a common feature in vascular diseases such as atherosclerosis, transplant arteriosclerosis and restenosis. In this study, we isolated the neointimal cells and uninjured residential vascular smooth muscle cells by laser micro dissection and carried out single-cell whole-genome methylation sequencing. We also sequenced the bisulfite converted genome of circulating bone-marrow-derived cells such as peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC). We found totally 2,360 differential methylation sites (DMS) annotated to 1,127 gene regions. The majority of differentially methylated regions (DMRs) were located in intergenic regions, outside those CpG islands and island shores. Interestingly, exons have less DMRs than promotors and introns, and CpG islands contain more DMRs than islands shores. Pearson correlation analysis showed a clear clustering of neointimal cells with PBMC/BMMC. Gene set enrichment analysis of differentially methylated CpG sites revealed that many genes were important for regulation of VSMC differentiation and stem cell maintenance. In conclusion, our results showed that neointimal cells are more similar to the progenitor cells in methylation profile than the residential VSMCs at the 30th day after the vascular injury.

High-accuracy haplotype imputation using unphased genotype data as the references.

Enormously growing genomic datasets present a new challenge on missing data imputation, a notoriously resource-demanding task. Haplotype imputation requires ethnicity-matched references. However, to date, haplotype references are not available for the majority of populations in the world. We explored to use existing unphased genotype datasets as references; if it succeeds, it will cover almost all of the populations in the world. The results showed that our HiFi software successfully yields 99.43% accuracy with unphased genotype references. Our method provides a cost-effective solution to breakthrough the bottleneck of limited reference availability for haplotype imputation in the big data era.

Functional pseudogenes inhibit the superoxide production.

We recently discovered a dynamic copy number variation on the NCF1 (neutrophil cytosolic factor 1) pseudogenes in human populations. In this study, we investigated whether these pseudogenes are functional or junk as described before. We sequenced the RNAs transcribed from the genome of this locus, and discovered over 10 splicing isoforms from the NCF1 pseudogenes. We cloned 4 splicing isoforms into expression vectors and introduced them into human vascular endothelial cells by transient transfection. We then used two chemical approaches to measure the superoxide production in the cells with and without these pseudogene overexpression. Our data showed that three pseudogene splicing products remarkably reduced the superoxide production after the GFP (Green fluorescent protein) normalization. We used an anti-HA (Hemagglutinin A) tag antibody to stain the cells and confirmed that the proteins transcribed from the NCF1 pseudogene were exclusively localized in the cytoplasm in the perinuclear area in the transient transfection assays. We further examined the tissue distribution of these splicing isoforms of NCF1 pseudogenes in human tissues by quantitative real-time PCR analysis. Our data showed that although these splicing variants are ubiquitously expressed in non-immune tissues in human, they seem to be under a tight control of transcription regulation and show a non-random distribution pattern across tissues. This study challenges the concept that pseudogenes in human genome are only junks without biological functions. Moreover, it suggests that those pseudogenes in human genome may serve as a natural resource for novel drug discovery.

A preliminary exploration on DNA methylation of transgene across generations in transgenic rats.

Epigenetic heritability is an important issue in the field of genetics and also in the development of many human diseases. In this study, we created a transgenic rat model and investigated the transgenerational methylation patterns in these animals. The transgene DNA fragment was unmethylated before it was injected into the pronucleus, so it is a good model to study the inheritance of DNA methylation patterns. We performed bisulfite sequencing on 23 CpG dinucleotides on the transgene across three generations in two tissues. We observed that the transgene was heavily methylated in the liver (87.53%) from the founder generation, whereas its methylation rate was much lower in the kidney (70.47%). Spearman correlation analysis showed that there was a strong correlation on the methylation status between different generations in the same tissue, which was observed in both liver and kidney, and among all individuals in this pedigree. This study provided some evidence that DNA methylation patterns acquired in the founder animal can be passed to the offspring.

The mirror RNA expression pattern in human tissues.

It has been realized in recent years that non-coding RNAs are playing important roles in genome functions and human diseases. Here we developed a new technology and observed a new pattern of gene expression. We observed that over 72% of RNAs in human genome are expressed in forward-reverse pairs, just like mirror images of each other between forward expression and reverse expression; the overview showed that it cannot be simply described as transcript overlapping, so we designated it as mirror expression. Furthermore, we found that the mirror expression is gene-specific and tissue-specific, and less common in the proximal promoter regions. The size of the shadows varies between different genes, different tissues and different classes. The shadow expression is most significant in the Alu element, it was also observed among L1, Simple Repeats and LTR elements, but rare in other repeats such as low-complexity, LINE/L2, DNA and MIRs. Although there is no evidence yet about the relationship of this mirror pattern and double-strand RNA (dsRNA), this new striking pattern provides a new clue and a new direction to unveil the role of RNAs in the genome functions and diseases.

A method to evaluate genome-wide methylation in archival formalin-fixed, paraffin-embedded ovarian epithelial cells.

BACKGROUND: The use of DNA from archival formalin and paraffin embedded (FFPE) tissue for genetic and epigenetic analyses may be problematic, since the DNA is often degraded and only limited amounts may be available. Thus, it is currently not known whether genome-wide methylation can be reliably assessed in DNA from archival FFPE tissue. METHODOLOGY/PRINCIPAL FINDINGS: Ovarian tissues, which were obtained and formalin-fixed and paraffin-embedded in either 1999 or 2011, were sectioned and stained with hematoxylin-eosin (H&E).Epithelial cells were captured by laser micro dissection, and their DNA subjected to whole genomic bisulfite conversion, whole genomic polymerase chain reaction (PCR) amplification, and purification. Sequencing and software analyses were performed to identify the extent of genomic methylation. We observed that 31.7% of sequence reads from the DNA in the 1999 archival FFPE tissue, and 70.6% of the reads from the 2011 sample, could be matched with the genome. Methylation rates of CpG on the Watson and Crick strands were 32.2% and 45.5%, respectively, in the 1999 sample, and 65.1% and 42.7% in the 2011 sample. CONCLUSIONS/SIGNIFICANCE: We have developed an efficient method that allows DNA methylation to be assessed in archival FFPE tissue samples.

A promoter that drives gene expression preferentially in male transgenic rats.

Gender-preferential gene expression is a widespread phenomenon in humans. It is important to study how gender differences influence the pathogenesis of various diseases and response to specific drugs. The aim of this study is to determine if the mouse albumin enhancer/promoter may serve as the promoter to introduce gender-preferential gene expression in transgenic animals. We created four independent transgenic rat lines in which the human C-reactive protein transgene was under the control of mouse albumin enhancer/promoter. Quantitative real time RT-PCR analysis showed that transgene expression in the liver of male rats was significantly higher than transgene expression in the female rats (P < 0.05).There was a 5.3-fold (male/female) difference in line-519, and a 12.2-fold (male/female) difference in line-488. Enzyme-linked immunosorbent assay showed that the serum of male transgenic rats had a 13- to 679-fold difference at the protein level on transgene production compared with female transgenic rats. The male-to-female difference in gene expression was 10- to 17-fold in the liver of transgenic rats. Orchiectomy dramatically reduced protein production from the transgene in the liver. Testosterone administration into female rats did not increase the transgene expression, but estrogen administration into the male rats reduced transgene expression. This study provides a valuable tool for investigating the pathological roles of genes that are expressed in a gender-preferential manner in human disease.

Multimeric stability of human C-reactive protein in archived specimens.

BACKGROUND: C-reactive protein (CRP) is a marker of inflammation and a risk predictor of cardiovascular disease. Current CRP assays are focused on the quantification of the CRP levels as pentamers. However, CRP can be present as other multimeric forms. There will be a market need to measure the CRP multimeric structure in addition to the levels in human populations. To meet this need, we investigated whether the long-term archived samples could be used instead of freshly collected samples. METHODOLOGY/PRINCIPAL FINDINGS: The specimens of serum, plasma and tissues were collected from transgenic rats expressing the human CRP. These samples were stored at 4°C, -20°C and -80°C for different periods. Non-denaturing Western blot analysis was used to observe the influence of storage conditions to multimeric structures of human CRP. Our results showed that there was no difference on multimeric structures of human CRP between samples stored at 4°C, -20°C and -80°C, between samples stored at -80°C for twenty-four hours and three months, and between plasma and serum. CONCLUSIONS/SIGNIFICANCE: This study implicated that archived samples stored at these conditions in those large longitudinal studies could be used for investigating the multimeric structures of CRP. Our report may speed up these researches and save labors and budget by enabling them to use currently available archived samples rather than freshly collected samples.

Elevated levels of IL-10 and G-CSF associated with asymptomatic malaria in pregnant women.

In sub-Saharan Africa, approximately 30 million pregnant women are at risk of contracting malaria annually. Nearly 36% of healthy pregnant women receiving routine antenatal care tested positive for Plasmodium falciparum HRP-II antigen in Ghana. We tested the hypothesis that asymptomatic HRP II positive pregnant women expressed a unique Th1 and Th2 phenotype that differs from healthy controls. Plasma from healthy (n = 15) and asymptomatic (n = 25) pregnant women were evaluated for 27 biomarkers (IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL- 17, Eotaxin, bFGF-2, G-CSF, GM-CSF, IFN-gamma, IP-10, MCP-1, MIP-1alpha, MIP-1beta, PDGF-bb, RANTES, TNF, and VEGF) associated with Th1 and Th2 cytokine homeostasis. IL-10 and G-CSF levels were elevated in the asymptomatic group when compared with the healthy group (P = .031 and .041, resp.). The median ratios of IL-1beta:5, IL-1beta:10, IL-1beta:G-CSF, IL-1beta:Eotaxin, IL-12:G-CSF, IL-15:10, IL-17:G-CSF, IL-17:Eotaxin, TNF:IL-4, TNF:IL-5, and TNF:G-CSF were significantly different among the two groups. Thus, asymptomatic malaria carriage may be linked to circulating levels of IL-10 and G-CSF.

Stability of Trichomonas vaginalis DNA in urine specimens.

Trichomonas vaginalis is an important pathogen in both men and women. Culture is considered the diagnostic gold standard, although studies have shown that PCR is more sensitive than either culture or wet mount for the diagnosis of T. vaginalis infections. We sought to identify a simple method for stabilizing T. vaginalis DNA in urine samples that could be easily applied to molecular testing. The stability of T. vaginalis DNA in 40 urine samples was assessed by storage for various times at either 4 degrees C or room temperature with or without the Becton Dickinson urine preservative transport (UPT) kit. Overall, there was better stability of T. vaginalis DNA when specimens were stored at 4 degrees C than when they were stored at 20 to 22 degrees C and when the UPT system was used. T. vaginalis DNA was stable in specimens stored without using the UPT at 4 degrees C for about 3 days and at room temperature for only 1 day. For specimens placed in the UPT within 24 h (times of 1, 6, and 24 h) of collection, the DNA was stable for up to 30 days when stored at 4 degrees C. For specimens stored at room temperature, the urine should be added to the UPT ideally within 1 hour of collection, and in this case the DNA remained stable for up to 30 days. When storing specimens at room temperature, a delay of 24 h prior to adding to UPT led to an unacceptably high loss of assay sensitivity.

Evaluation of real-time PCR laboratory-developed tests using analyte-specific reagents for cytomegalovirus quantification.

Viral load testing for cytomegalovirus (CMV) has become the standard for the diagnosis of infection and monitoring of therapy at many transplant centers. However, no viral load test has been approved by the FDA. Therefore, many laboratories rely on laboratory-developed assays. This study evaluated the performance characteristics of two real-time PCR tests developed using the artus CMV analyte-specific reagents (ASRs). One version is distributed by Abbott Molecular and the other by QIAGEN. For plasma specimens, the Abbott test had a limit of detection of 2.3 log10 copies/ml and a linear range up to at least 6.0 log10 copies/ml. Comparison of plasma viral loads using the Abbott test and the Roche Amplicor Monitor test showed a mean difference of -0.012 log10 copies/ml. In addition, the Abbott test viral loads correlated with the Digene Hybrid Capture assay ratios. Viral loads obtained from plasma specimens tested by the Abbott and QIAGEN tests were in very close agreement (mean difference, 0.144 log10 copies/ml). When the QIAGEN test was evaluated with the QIAGEN, MagNA Pure, and easyMAG extraction methods, the viral loads for all three methods were within 0.370 log10 copies/ml. Thus, there is good agreement between viral loads obtained by the different tests using the same extraction method or by the same test using different extraction methods. The availability of real-time PCR ASRs provides additional reagents that can be used for CMV viral load testing.