Detecting epistasis within chromatin regulatory circuitry reveals CAND2 as a novel susceptibility gene for obesity.

BACKGROUND: Genome-wide association studies have identified many susceptibility loci for obesity. However, missing heritability problem is still challenging and ignorance of genetic interactions is believed to be an important cause. Current methods for detecting interactions usually do not consider regulatory elements in non-coding regions. Interaction analyses within chromatin regulatory circuitry may identify new susceptibility loci. METHODS: We developed a pipeline named interaction analyses within chromatin regulatory circuitry (IACRC), to identify genetic interactions impacting body mass index (BMI). Potential interacting SNP pairs were obtained based on Hi-C datasets, PreSTIGE (Predicting Specific Tissue Interactions of Genes and Enhancers) algorithm, and super enhancer regions. SNP × SNP analyses were next performed in three GWAS datasets, including 2286 unrelated Caucasians from Kansas City, 3062 healthy Caucasians from the Gene Environment Association Studies initiative, and 3164 Hispanic subjects from the Women's Health Initiative. RESULTS: A total of 16,643,227 SNP × SNP analyses were performed. Meta-analyses showed that two SNP pairs, rs6808450-rs9813534 (combined P = 2.39 × 10-9) and rs6808450-rs3773306 (combined P = 2.89 × 10-9) were associated with BMI after multiple testing corrections. Single-SNP analyses did not detect significant association signals for these three SNPs. In obesity relevant cells, rs6808450 is located in intergenic enhancers, while rs9813534 and rs3773306 are located in the region of strong transcription regions of CAND2 and RPL32, respectively. The expression of CAND2 was significantly downregulated after the differentiation of human Simpson-Golabi-Behmel syndrome (SGBS) preadipocyte cells (P = 0.0241). Functional validation in the International Mouse Phenotyping Consortium database showed that CAND2 was associated with increased lean body mass and decreased total body fat amount. CONCLUSIONS: Detecting epistasis within chromatin regulatory circuitry identified CAND2 as a novel obesity susceptibility gene. We hope IACRC could facilitate the interaction analyses for complex diseases and offer new insights into solving the missing heritability problem.

Plasma Pharmacokinetic Determination of Canagliflozin and Its Metabolites in a Type 2 Diabetic Rat Model by UPLC-MS/MS.

Canagliflozin is a novel, orally selective inhibitor of sodium-dependent glucose co-transporter-2 (SGLT2) for the treatment of patients with type 2 diabetes mellitus. In this study, a sensitive and efficient UPLC-MS/MS method for the quantification of canagliflozin and its metabolites in rat plasma was established and applied to pharmacokinetics in a type 2 diabetic rat model. We firstly investigated the pharmacokinetic changes of canagliflozin and its metabolites in type 2 diabetic rats in order to use canagliflozin more safely, reasonably and effectively. We identified three types of O-glucuronide metabolites (M5, M7 and M17), two kinds of oxidation metabolites (M8 and M9) and one oxidation and glucuronide metabolite (M16) using API 5600 triple-TOF-MS/MS. Following liquid⁻liquid extraction by tert-butyl methyl ether, chromatographic separation of canagliflozin and its metabolites were performed on a Waters XBridge BEH C18 column (100 × 2.1 mm, 2.5 µm) using 0.1% acetonitrile⁻formic acid (75:15, v/v) as the mobile phase at a flow rate of 0.7 mL/min. Selected ion monitoring transitions of m/z 462.00→191.10, 451.20→153.10, 638.10→191.10 and 478.00→267.00 were chosen to quantify canagliflozin, empagliflozin (IS), O-glucuronide metabolites (M5, M7 and M17), and oxidation metabolites (M9) using an API 5500-triple-MS/MS in the positive electrospray ionization mode. The validation of the method was found to be of sufficient specificity, accuracy and precision. The pathological condition of diabetes could result in altered pharmacokinetic behaviors of canagliflozin and its metabolites. The pharmacokinetic parameters (AUC0⁻t, AUC0⁻∞, CLz/F, and Vz/F) of canagliflozin were significantly different between the CTRL and DM group rats (p < 0.05 or p < 0.01), which may subsequently cause different therapeutic effects.

[Research progress on the asymmetric division in mammalian oocytes].

The mammalian oocyte maturation process consists of two consecutive asymmetric divisions, and produces three daughter cells of vastly different sizes: one larger egg cell and two smaller polar bodies. Asymmetric division is a typical feature of mammalian oocyte meiosis that results in a highly polar egg cell. The mitosis of the cell after fertilization exhibits restored symmetric division, but the polarity characteristics formed during meiosis of oocytes are preserved and affect the polarity of early embryos. In this review, we summarize the research progress on asymmetric division of mammalian oocytes in recent years, and mainly focus on the asymmetric division of cytoplasmic and the asymmetric division of nucleus, including the functions of chromosome and cytoskeleton in asymmetric division of mammalian oocytes, the redistribution of cytoplasmic organelles occurring in oocyte maturation, and chromosome nonrandom separation. We aim to demonstrate the main mechanism of asymmetry division in mammalian oocytes from both cellular and molecular levels.

Effect of cisplatin-based neoadjuvant chemotherapy on survival in patients with bladder cancer: a meta-analysis.

PURPOSE: Cisplatin-based neoadjuvant chemotherapy (NAC) has been shown to improve survival in patients with muscle-invasive bladder cancer (MIBC) who underwent radical cystectomy as compared with patients who underwent surgery alone. It has also been suggested as current standard of care in surgically-fit patients with MIBC. This meta-analysis assessed the effect of cisplatin-based NAC on survival in patients with bladder cancer. SOURCE: PubMed, CENTRAL, and Embase were searched until November 22, 2016. Two-arm randomized controlled trials that compared cisplatin-based neoadjuvant chemotherapy plus local treatment versus the same local treatment without neoadjuvant chemotherapy were selected. Patients with histologically-confirmed bladder cancer (adenocarcinoma, transitional, or squamous-cell carcinoma) were included. The primary outcome was overall survival (OS). PRINCIPAL FINDINGS: Of the 292 articles initially identified, 14 were included in the final analysis. Patients in the NAC group had similar OS as the local treatment (i.e., radiation therapy or cystectomy) group (pooled hazard ratio [HR] = 0.92, 95% confidence interval [CI]: 0.84 to 1.00, P=0.056). No difference in progress-free survival between two groups was observed (P=0.725). Subgroup analysis showed that OS was similar in patients treated with NAC plus radiotherapy or cystectomy compared with patients who received local treatment alone. CONCLUSIONS: Platinum-based NAC was associated with similar survival benefit as patients undergoing cystectomy and/or radiotherapy. No conclusion can be drawn about the optimal platinum-based combination to be used in the neoadjuvant setting.

MiR-429 prohibited NF-κB signalling to alleviate contrast-induced acute kidney injury via targeting PDCD4.

MiR-429 was reported to be downregulated in contrast-induced acute kidney injury (CI-AKI). However, whether miR-429 is functionally relevant with CI-AKI needs further investigation. Human renal tubular epithelial cell (HK-2) cells were stimulated with contrast media iodixanol to establish in vitro CI-AKI model. Cell Counting Kit-8 (CCK-8) was applied to access cell viability. Flow cytometry was performed to determine apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to evaluate level of programmed cell death 4 (PDCD4) mRNA and miR-429 while western blot was applied to evaluate level of proteins including PDCD4, B-cell leukaemia/lymphoma 2 (Bcl-2), BCL2-associated X protein (Bax), cleaved caspase 3, cleaved caspase 9, p65, phosphorylated p65. Dual luciferase assay was used to validate miR-429 targeting PDCD4. MiR-429 was downregulated whereas PDCD4 was upregulated in contrast media iodixanol-stimulated HK-2 cells. MiR-429 overexpression elevated cell viability and attenuated cell apoptosis. Moreover, the activation of nuclear factor kappa-B (NF-κB) signalling was suppressed after miR-429 overexpression, while PDCD4 overexpression reversed these effects. MiR-429 directly targeted PDCD4 and negatively regulated its expression. CI-AKI induced NF-κB signalling activation and PDCD4 overexpression further promoted NF-κB signalling activation. However, the treatment of BAY11-7082 reversed above results. Overexpression of miR-429 attenuated apoptosis and elevated cell viability in a CI-AKI cell model via targeting PDCD4 and thus restraining NF-κB signalling.

Transcription Factor Enrichment Analysis in Enhancers Identifies EZH2 as a Susceptibility Gene for Osteoporosis.

PURPOSE: Genome-wide association studies (GWASs) have identified hundreds of single nucleotide polymorphisms (SNPs) associated with osteoporosis. Most of these SNPs are noncoding variants and could be mapped to enhancers. Transcription factors (TFs) play important roles in gene regulation via enhancers harboring these SNPs; thus, we aimed to identify common regulatory TFs binding to enhancers associated with osteoporosis. METHODS: We first annotated all the osteoporosis-related SNPs identified by GWASs to enhancers and conducted TF enrichment analyses to identify common TFs binding to osteoporosis-associated enhancers. We further conducted genetic association analyses between the identified TFs and bone mineral density (BMD) in a Han Chinese population. RESULTS: After functional annotation, a total of 5081 osteoporosis-related SNPs were mapped to enhancers. TF enrichment analyses identified 2 significant TFs after multiple testing adjustments, which are EZH2 (Padj = .028) and NRSF (Padj = .038). We also found 1 SNP, rs111851041, in EZH2 was significantly associated with BMD both at the hip and spine after multiple testing adjustments (hip BMD: P = 4.32 × 10-4; spine BMD: P = 2.72 × 10-3). The expression of EZH2 decreased significantly from 12 to 48 hours of osteogenic differentiation. And functional validation showed that EZH2 was associated with osteoporosis-related phenotypes in knockout mice. CONCLUSIONS: By conducting TF enrichment analyses, we identified EZH2 as a common TF binding to osteoporosis-associated enhancers, and EZH2 was also associated with BMD in a Chinese population. EZH2 is functionally related to bone phenotypes. The identified gene could provide new insight into osteoporosis pathophysiology and highlight opportunities for future clinical and pharmacological research on osteoporosis.

Alteration of UDP-glucuronosyltransferase 1a1, 1a7 and P-glycoprotein expression in hepatic fibrosis rats and the impact on pharmacokinetics of puerarin.

BACKGROUND: Puerarin, derived from a traditional Chinese herb Pueraria lobata (Willd.) Ohwi which was distributed globally and planted in most parts of China, has been extensively applied in patients with cardiovascular diseases in China. Yet a considerable proportion of the patients were accompanied with liver illnesses simultaneously because of all sorts of reasons. HYPOTHESIS/PURPOSE: It had been implied by some previous research that the absorption and the metabolism of puerarin were susceptible to liver issues due to changed P-gp and Ugt1a level, but pharmacokinetics of puerarin under such conditions were few concerned. Our study aimed to make sure whether and how much the behavior of puerarin in vivo was affected by hepatic diseases, and to explore the potential mechanisms. METHODS: A CCl4 induced rat model of hepatic fibrosis (HF) was prepared and verified. Single low/high doses of oral and intravenous administration of puerarin to HF and normal rats were performed. Pharmacokinetics of puerarin were determined by a validated HPLC method. The expression of P-gp, Ugt1a1, and Ugt1a7 in both liver and intestines were determined by quantitative RT-PCR and Western blot analysis respectively. RESULTS: The systemic exposure of puerarin in HF rats of experimental groups were found decreased remarkably except for that of the high dose intravenous group. Moreover, the expression of P-gp, Ugt1a1, and Ugt1a7 in liver and intestines of HF rats were figured out increased. CONCLUSION: The results indicated that the HF originated overexpression of Ugt1a1, Ugt1a7, and P-gp level played important roles in pharmacokinetics of puerarin, suggested the clinical regimen of puerarin based on normal populations might be inappropriate for patients with chronic liver diseases. It was implied drugs whose absorption or elimination were related to P-gp, Ugt1a1, or Ugt1a7 might also be affected by hepatic illnesses.

Characterization of porcine extraembryonic endoderm cells.

OBJECTIVES: To date, many efforts have been made to establish porcine embryonic stem (pES) cells without success. Extraembryonic endoderm (XEN) cells can self-renew and differentiate into the visceral endoderm and parietal endoderm. XEN cells are derived from the primitive endoderm of the inner cell mass of blastocysts and may be an intermediate state in cell reprogramming. MATERIALS AND METHODS: Porcine XEN cells (pXENCs) were generated from porcine pluripotent stem cells (pPSCs) and were characterized by RNA sequencing and immunofluorescence analyses. The developmental potential of pXENCs was investigated in chimeric mouse embryos. RESULTS: Porcine XEN cells derived from porcine pPSCs were successfully expanded in N2B27 medium supplemented with bFGF for least 30 passages. RNA sequencing and immunofluorescence analyses showed that pXENCs expressed the murine and canine XEN markers Gata6, Gata4, Sox17 and Pdgfra but not the pluripotent markers Oct4, Sox2 and TE marker Cdx2. Moreover, these cells contributed to the XEN when injected into four-cell stage mouse embryos. Supplementation with Chir99021 and SB431542 promoted the pluripotency of the pXENCs. CONCLUSIONS: We successfully derived pXENCs and showed that supplementation with Chir99021 and SB431542 confer them with pluripotency. Our results provide a new resource for investigating the reprogramming mechanism of porcine-induced pluripotent stem cells.

Comprehensive functional annotation of susceptibility SNPs prioritized 10 genes for schizophrenia.

Nearly 95% of susceptibility SNPs identified by genome-wide association studies (GWASs) are located in non-coding regions, which causes a lot of difficulty in deciphering their biological functions on disease pathogenesis. Here, we aimed to conduct a comprehensive functional annotation for all the schizophrenia susceptibility loci obtained from GWASs. Considering varieties of epigenomic regulatory elements, we annotated all 22,688 acquired susceptibility SNPs according to their genomic positions to obtain functional SNPs. The comprehensive annotation indicated that these functional SNPs are broadly involved in diverse biological processes. Histone modification enrichment showed that H3K27ac, H3K36me3, H3K4me1, and H3K4me3 were related to the development of schizophrenia. Transcription factors (TFs) prediction, methylation quantitative trait loci (meQTL) analyses, expression quantitative trait loci (eQTL) analyses, and proteomic quantitative trait loci analyses (pQTL) identified 447 target protein-coding genes. Subsequently, differential expression analyses between schizophrenia cases and controls, nervous system phenotypes from mouse models, and protein-protein interaction with known schizophrenia-related pathways and genes were carried out with our target genes. We finaly prioritized 10 target genes for schizophrenia (CACNA1C, CLU, CSNK2B, GABBR1, GRIN2A, MAPK3, NOTCH4, SRR, TNF, and SYNGAP1). Our results may serve as an encyclopedia of schizophrenia susceptibility SNPs and offer holistic guides for post-GWAS functional experiments.

Comparison of Clinical and Ultrasonographic Features of Poorly Differentiated Thyroid Carcinoma and Papillary Thyroid Carcinoma.

BACKGROUND: The clinical behavior and management of poorly differentiated thyroid carcinoma (PDTC) are very different from papillary thyroid carcinoma (PTC). By comparing the clinical and ultrasonographic features between the two tumors, we proposed to provide more possibilities for recognizing PDTC before treatment. METHODS: The data of 13 PDTCs and 39 age- and gender-matched PTCs in Peking Union Medical College Hospital between December 2003 and September 2013 were retrospectively reviewed. The clinical and ultrasonic features between the two groups were compared. RESULTS: The frequencies of family history of carcinoma, complication with other thyroid lesions, lymph node metastases, recurrent laryngeal nerve injuries, and distant metastases were higher in PDTCs (30.8%, 61.6%, 69.2%, 23.1%, and 46.2%, respectively) than those in PTCs (2.6%, 23.1%, 25.6%, 2.6%, and 2.6%, respectively) (P < 0.05). The mortality rate of PDTCs was greatly higher than PTCs (P < 0.01). Conventional ultrasound showed that the size of PDTCs was larger than that of PTCs (3.1 ± 1.9 cm vs. 1.7 ± 1.0 cm). Clear margins and rich and/or irregular blood flow were found in 92.3% of PDTCs, which differed substantially from PTCs (51.7% and 53.8%, respectively) (P < 0.05). CONCLUSIONS: PDTC is more aggressive and its mortality rate is higher than PTCs. Accordingly, more attention should be given to suspicious thyroid cancer nodules that show large size, regular shape, and rich blood flow signals on ultrasound to exclude the possibility of PDTCs.