Dual-trait genomic analysis in highly stratified Arabidopsis thaliana populations using genome-wide association summary statistics.

Genome-wide association study (GWAS) is a powerful tool to identify genomic loci underlying complex traits. However, the application in natural populations comes with challenges, especially power loss due to population stratification. Here, we introduce a bivariate analysis approach to a GWAS dataset of Arabidopsis thaliana. We demonstrate the efficiency of dual-phenotype analysis to uncover hidden genetic loci masked by population structure via a series of simulations. In real data analysis, a common allele, strongly confounded with population structure, is discovered to be associated with late flowering and slow maturation of the plant. The discovered genetic effect on flowering time is further replicated in independent datasets. Using Mendelian randomization analysis based on summary statistics from our GWAS and expression QTL scans, we predicted and replicated a candidate gene AT1G11560 that potentially causes this association. Further analysis indicates that this locus is co-selected with flowering-time-related genes. The discovered pleiotropic genotype-phenotype map provides new insights into understanding the genetic correlation of complex traits.

Isolation, culture, and characterisation of bovine ovarian fetal fibroblasts and gonadal ridge epithelial-like cells and comparison to their adult counterparts.

During ovarian development, gonadal ridge epithelial-like (GREL) cells arise from the epithelial cells of the ventral surface of the mesonephros. They ultimately develop into follicular granulosa cells or into ovarian surface epithelial cells. Stromal fibroblasts arise from the mesonephros and penetrate the ovary. We developed methods for isolating and culturing fetal ovarian GREL cells and ovarian fibroblasts by expansion of colonies without passage. In culture, these two cell types were morphologically different. We examined the expression profile of 34 genes by qRT-PCR, of which 24 genes had previously been studied in whole fetal ovaries. Expression of nine of the 10 newly-examined genes in fetal ovaries correlated with gestational age (MUC1, PKP2, CCNE1 and CCNE2 negatively; STAR, COL4A1, GJA1, LAMB2 and HSD17B1 positively). Comparison between GREL cells and fetal fibroblasts revealed higher expression of KRT19, PKP2, OCLN, MUC1, ESR1 and LGR5 and lower expression of GJA1, FOXL2, NR2F2, FBN1, COL1A1, NR5A1, CCND2, CCNE1 and ALDH1A1. Expression of CCND2, CCNE1, CCNE2, ESR2 and TGFBR1 was higher in the fetal fibroblasts than in adult fibroblasts; FBN1 was lower. Expression of OCLN, MUC1, LAMB2, NR5A1, ESR1, ESR2, and TGFBR3 was lower in GREL cells than ovarian surface epithelial cells. Expression of KRT19, DSG2, PKP2, OCLN, MUC1, FBN1, COL1A1, COL3A1, STAR and TGFBR2 was higher and GJA1, CTNNB1, LAMB2, NR5A1, CYP11A1, HSD3B1, CYP19A1, HSD17B1, FOXL2, ESR1, ESR2, TGFBR3 and CCND2 was lower in GREL cells compared to granulosa cells. TGFß1 altered the expression of COL1A1, COL3A1 and FBN1 in fetal fibroblasts and epidermal growth factor altered the expression of FBN1 and COL1A1. In summary, the two major somatic cell types of the developing ovary have distinct gene expression profiles. They, especially GREL cells, also differ from the cells they ultimately differentiate in to. The regulation of cell fate determination, particularly of the bi-potential GREL cells, remains to be elucidated.

Placental Transcription Profiling in 6-23 Weeks' Gestation Reveals Differential Transcript Usage in Early Development.

The human placenta is a rapidly developing transient organ that is key to pregnancy success. Early development of the conceptus occurs in a low oxygen environment before oxygenated maternal blood begins to flow into the placenta at ~10-12 weeks' gestation. This process is likely to substantially affect overall placental gene expression. Transcript variability underlying gene expression has yet to be profiled. In this study, accurate transcript expression profiles were identified for 84 human placental chorionic villus tissue samples collected across 6-23 weeks' gestation. Differential gene expression (DGE), differential transcript expression (DTE) and differential transcript usage (DTU) between 6-10 weeks' and 11-23 weeks' gestation groups were assessed. In total, 229 genes had significant DTE yet no significant DGE. Integration of DGE and DTE analyses found that differential expression patterns of individual transcripts were commonly masked upon aggregation to the gene-level. Of the 611 genes that exhibited DTU, 534 had no significant DGE or DTE. The four most significant DTU genes ADAM10, VMP1, GPR126, and ASAH1, were associated with hypoxia-responsive pathways. Transcript usage is a likely regulatory mechanism in early placentation. Identification of functional roles will facilitate new insight in understanding the origins of pregnancy complications.

Peptidomic Analysis on Mouse Lung Tissue Reveals AGDP as a Potential Bioactive Peptide against Pseudorabies Virus Infection.

Pseudorabies virus (PRV) infection could cause severe histopathological damage via releasing multiple factors, including cytokines, peptides, etc. Here, peptidomic results showed that 129 peptides were identified in PRV-infected mouse lungs and were highly involved in the process of PRV infection. The role of one down-regulated biological peptide (designated as AGDP) during PRV infection was investigated. To verify the expression profiles of AGDP in response to PRV infection, the expression level of the precursor protein of AGDP mRNA was significantly decreased in PRV-infected mouse lungs and cells. The synthesized AGDP-treating cells were less susceptible to PRV challenges than the controls, as demonstrated by the decreased virus production and gE expression. AGDP not only inhibited the expression of TNF-α and IL-8 but also appeared to suppress the extracellular release of high-mobility group box 1 (HMGB1) by inhibiting the output of nuclear HMGB1 in cells. AGDP could also inhibit the degradation of IκBα and the phosphorylation levels of P65 after PRV infection. In total, our results revealed many meaningful peptides involved in PRV infection, thereby enhancing the current understanding of the host response to PRV infection, and how AGDP may serve as a promising candidate for developing novel anti-PRV drugs.

Neuropeptide S and its receptor NPSR enhance the susceptibility of hosts to pseudorabies virus infection.

The neuropeptide S (NPS) and its receptor (NPSR) represent a signaling system in the brain. Increased levels of NPS and NPSR have been observed in PK15 cells and murine brains in response to pseudorabies virus (PRV) infection, but it remains unclear whether elevated levels of NPS and NPSR are involved in the pathogenic process of PRV infection. In this study, the activities of both NPS and NPSR during PRV pathogenesis were explored in vitro and in vivo by reverse transcription polymerase chain reaction (RT-PCR), PCR, real-time quantitative RT-PCR (qRT-PCR), qPCR, TCID50, and Western blotting methods. NPSR-deficient cells were less susceptible to PRV infection, as evidenced by decreased viral production and PRV-glycoprotein E (gE) expression. In vitro studies showed that exogenous NPS promoted the expression of interleukin 6 (IL-6) mRNA but inhibited interferon ß (IFN-ß) mRNA expression in PK15 cells after PRV infection. In vivo studies showed that NPS-treated mice were highly susceptible to PRV infection, with decreased survival rates and body weights. In addition, NPS-treated mice showed elevated levels of IL-6 mRNA and STAT3 phosphorylation. However, the expression of IFN-ß mRNA was greatly decreased after virus challenge. Contrasting results were obtained from the NPSR-ir-treated groups, which further highlighted the effects of NPS. This study revealed that NPS-treated hosts are more susceptible to PRV infection than controls. Moreover, excessive IL-6/STAT3 and defective IFN-ß responses in NPS-treated mice may contribute to the pathogenesis of PRV.

Large-scale transcriptome-wide profiling of microRNAs in human placenta and maternal plasma at early to mid gestation.

MicroRNAs (miRNAs) are increasingly seen as important regulators of placental development and opportunistic biomarker targets. Given the difficulty in obtaining samples from early gestation and subsequent paucity of the same, investigation of the role of miRNAs in early gestation human placenta has been limited. To address this, we generated miRNA profiles using 96 placentas from presumed normal pregnancies, across early gestation, in combination with matched profiles from maternal plasma. Placenta samples range from 6 to 23 weeks' gestation, a time period that includes placenta from the early, relatively low but physiological (6-10 weeks' gestation) oxygen environment, and later, physiologically normal oxygen environment (11-23 weeks' gestation).We identified 637 miRNAs with expression in 86 samples (after removing poor quality samples), showing a clear gestational age gradient from 6 to 23 weeks' gestation. We identified 374 differentially expressed (DE) miRNAs between placentas from 6-10 weeks' versus 11-23 weeks' gestation. We see a clear gestational age group bias in miRNA clusters C19MC, C14MC, miR-17 ~ 92 and paralogs, regions that also include many DE miRNAs. Proportional change in expression of placenta-specific miRNA clusters was reflected in maternal plasma.The presumed introduction of oxygenated maternal blood into the placenta (between ~10 and 12 weeks' gestation) changes the miRNA profile of the chorionic villus, particularly in placenta-specific miRNA clusters. Data presented here comprise a clinically important reference set for studying early placenta development and may underpin the generation of minimally invasive methods for monitoring placental health.

Quality control measures for placental sample purity in DNA methylation array analyses.

The purity of tissue samples can affect the accuracy and utility of DNA methylation array analyses. This is particularly important for the placenta which is globally hypomethylated compared to other tissues. Placental villous tissue from early pregnancy terminations can be difficult to separate from non-villous tissue, resulting in potentially inaccurate results. We used several methods to identify mixed placenta samples using DNA methylation array datasets from our laboratory and those contained in the NCBI GEO database, highlighting the importance of determining sample purity during quality control processes.

Bivariate genomic analysis identifies a hidden locus associated with bacteria hypersensitive response in Arabidopsis thaliana.

Multi-phenotype analysis has drawn increasing attention to high-throughput genomic studies, whereas only a few applications have justified the use of multivariate techniques. We applied a recently developed multi-trait analysis method on a small set of bacteria hypersensitive response phenotypes and identified a single novel locus missed by conventional single-trait genome-wide association studies. The detected locus harbors a minor allele that elevates the risk of leaf collapse response to the injection of avrRpm1-modified Pseudomonas syringae (P = 1.66e-08). Candidate gene AT3G32930 with in the detected region and its co-expressed genes showed significantly reduced expression after P. syringae interference. Our results again emphasize that multi-trait analysis should not be neglected in association studies, as the power of specific multi-trait genotype-phenotype maps might only be tractable when jointly considering multiple phenotypes.

Identification and Analysis of Regulatory Elements in Porcine Bone Morphogenetic Protein 15 Gene Promoter.

Bone morphogenetic protein 15 (BMP15) is secreted by the mammalian oocytes and is indispensable for ovarian follicular development, ovulation, and fertility. To determine the regulation mechanism of BMP15 gene, the regulatory sequence of porcine BMP15 was investigated in this study. The cloned BMP15 promoter retains the cell-type specificity, and is activated in cells derived from ovarian tissue. The luciferase assays in combination with a series of deletion of BMP15 promoter sequence show that the -427 to -376 bp region of BMP15 promoter is the primary regulatory element, in which there are a number of transcription factor binding sites, including LIM homeobox 8 (LHX8), newborn ovary homeobox gene (NOBOX), and paired-like homeodomain transcription factor 1 (PITX1). Determination of tissue-specific expression reveals that LHX8, but not PITX1 and NOBOX, is exclusively expressed in pig ovary tissue and is translocated into the cell nuclei. Overexpression of LHX8 in Chinese hamster ovary (CHO) cells could significantly promote BMP15 promoter activation. This study confirms a key regulatory element that is located in the proximal region of BMP15 promoter and is regulated by the LHX8 factor.

[Generation of human oocyte-like cell differentiation in vivo].

Oocyte-like cells (OLC) can be generated by stem cells after the induction and differentiation in vitro, and maturated when transplanted in vivo to improve the development potential. Human amniotic fluid stem cells (hAFSC) were cultured for 10 days in porcine follicle fluid (pFF) that was extracted from the medium follicle with high levels of hormones and Bmp 15 protein. After the induction, the cell aggregates showed the germ cell-like cells and produced the germ cell marker oct4, and triggered epigenetic changes with high expression of methylation transferase gene dnmt3b. The cell aggregates were packaged into porcine theca folliculi to form grafts, which were then transplanted into mouse renal capsule. After one month of transplantation, the morphology of OLC from a graft was not only similar to oocytes, but also expressed the germ cells markers (oct4, nanog, stella, ifitm3, dazl, nanos3, bmp15, and gd9). The results demonstrate that the in vivo differentiation model was useful for OLC development.

Human amniotic fluid stem cells possess the potential to differentiate into primordial follicle oocytes in vitro.

Previous reports have demonstrated that embryonic stem cells were capable of differentiating into primordial germ cells through the formation of embryoid bodies that subsequently generated oocyte-like cells (OLCs). Such a process could facilitate studies of primordial follicle oocyte development in vitro and regenerative medicine. To investigate the pluripotency of human amniotic fluid stem cells (hAFSCs) and their ability to differentiate into germ cells, we isolated a CD117(+)/CD44(+) hAFSC line that showed fibroblastoid morphology and intrinsically expressed both stem cell markers (OCT4, NANOG, SOX2) and germ cell markers (DAZL, STELLA). To encourage differentiation into OLCs, the hAFSCs were first cultured in a medium supplemented with 5% porcine follicular fluid for 10 days. During the induction period, cell aggregates formed and syntheses of steroid hormones were detected; some OLCs and granulosa cell-like cells could be loosened from the surface of the culture dish. Cell aggregates were collected and replated in oocyte culture medium for an additional 7-10 days. OLCs ranging from 50 to 120 µm presenting zona pellucida were observed in cumulus-oocyte complexes; some OLCs developed spontaneously into multicell structures similar to preimplantation embryos. Approximately 2% of the hAFSCs differentiated to meiotic germ cells that expressed folliculogenesis- and oogenesis-associated markers. Although the in vitro maturation and fertilization potentials are as yet unproven, short-term (<25 days) and high-efficiency (>2%) derivation of OLCs from hAFSCs might provide a new approach to the study of human germ cell development in vitro.