👤 Longbo Hu

Circulating trans fatty acids (TFAs), which cannot be synthesized by humans, are linked to adverse health outcomes. Although TFAs are obtained from diet, little is known about subsequent influences (e.g., relating to incorporation, metabolism, or intercompetition with other fatty acids) that could alter circulating concentrations and possibly modulate or mediate impacts on health. The objective was to elucidate novel biologic pathways that may influence circulating TFAs by evaluating associations between common genetic variation and TFA biomarkers. We performed meta-analyses using 7 cohorts of European-ancestry participants (n = 8013) having measured genome-wide variation in single-nucleotide polymorphisms (SNPs) and circulating TFA biomarkers (erythrocyte or plasma phospholipids), including trans-16:1n-7, total trans-18:1, trans/cis-18:2, cis/trans-18:2, and trans/trans-18:2. We further evaluated SNPs with genome-wide significant associations among African Americans (n = 1082), Chinese Americans (n = 669), and Hispanic Americans (n = 657) from 2 of these cohorts. Among European-ancestry participants, 31 SNPs in or near the fatty acid desaturase (FADS) 1 and 2 cluster were associated with cis/trans-18:2; a top hit was rs174548 (β = 0.0035, P = 4.90 × 10(-15)), an SNP previously associated with circulating n-3 and n-6 polyunsaturated fatty acid concentrations. No significant association was identified for other TFAs. rs174548 in FADS1/2 was also associated with cis/trans-18:2 in Hispanic Americans (β = 0.0053, P = 1.05 × 10(-6)) and Chinese Americans (β = 0.0028, P = 0.002) but not African Americans (β = 0.0009, P = 0.34); however, in African Americans, fine mapping identified a top hit in FADS2 associated with cis/trans-18:2 (rs174579: β = 0.0118, P = 4.05 × 10(-5)). The association between rs174548 and cis/trans-18:2 remained significant after further adjustment for individual circulating n-3 and n-6 fatty acids, except arachidonic acid. After adjustment for arachidonic acid concentrations, the association between rs174548 and cis/trans-18:2 was nearly eliminated in European-ancestry participants (β-coefficient reduced by 86%), with similar reductions in Hispanic Americans and Chinese Americans. Our findings provide novel evidence for genetic regulation of cis/trans-18:2 by the FADS1/2 cluster and suggest that this regulation may be influenced/mediated by concentrations of arachidonic acid, an n-6 polyunsaturated fat. Show less

Tissue concentrations of omega-3 fatty acids may reduce cardiovascular disease risk, and genetic variants are associated with circulating fatty acids concentrations. Whether dietary fatty acids interact with genetic variants to modify circulating omega-3 fatty acids is unclear. We evaluated interactions between genetic variants and fatty acid intakes for circulating alpha-linoleic acid, eicosapentaenoic acid, docosahexaenoic acid, and docosapentaenoic acid. We conducted meta-analyses (N = 11 668) evaluating interactions between dietary fatty acids and genetic variants (rs174538 and rs174548 in FADS1 (fatty acid desaturase 1), rs7435 in AGPAT3 (1-acyl-sn-glycerol-3-phosphate), rs4985167 in PDXDC1 (pyridoxal-dependent decarboxylase domain-containing 1), rs780094 in GCKR (glucokinase regulatory protein), and rs3734398 in ELOVL2 (fatty acid elongase 2)). Stratification by measurement compartment (plasma versus erthyrocyte) revealed compartment-specific interactions between FADS1 rs174538 and rs174548 and dietary alpha-linolenic acid and linoleic acid for docosahexaenoic acid and docosapentaenoic acid. Our findings reinforce earlier reports that genetically based differences in circulating fatty acids may be partially due to differences in the conversion of fatty acid precursors. Further, fatty acids measurement compartment may modify gene-diet relationships, and considering compartment may improve the detection of gene-fatty acids interactions for circulating fatty acid outcomes. Show less

Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells. Show less

Schizophrenia is a heritable, heterogeneous common psychiatric disorder. In this study, we evaluated the hypothesis that de novo variants (DNVs) contribute to the pathogenesis of schizophrenia. We performed exome sequencing in Chinese patients (N = 45) with schizophrenia and their unaffected parents (N = 90). Forty genes were found to contain DNVs. These genes had enriched transcriptional co-expression profile in prenatal frontal cortex (Bonferroni corrected p < 9.1 × 10(-3)), and in prenatal temporal and parietal regions (Bonferroni corrected p < 0.03). Also, four prenatal anatomical subregions (VCF, MFC, OFC and ITC) have shown significant enrichment of connectedness in co-expression networks. Moreover, four genes (LRP1, MACF1, DICER1 and ABCA2) harboring the damaging de novo mutations are strongly prioritized as susceptibility genes by multiple evidences. Our findings in Chinese schizophrenic patients indicate the pathogenic role of DNVs, supporting the hypothesis that schizophrenia is a neurodevelopmental disease. Show less

Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. Show less

Coffee, a major dietary source of caffeine, is among the most widely consumed beverages in the world and has received considerable attention regarding health risks and benefits. We conducted a genome-wide (GW) meta-analysis of predominately regular-type coffee consumption (cups per day) among up to 91,462 coffee consumers of European ancestry with top single-nucleotide polymorphisms (SNPs) followed-up in ~30 062 and 7964 coffee consumers of European and African-American ancestry, respectively. Studies from both stages were combined in a trans-ethnic meta-analysis. Confirmed loci were examined for putative functional and biological relevance. Eight loci, including six novel loci, met GW significance (log10Bayes factor (BF)>5.64) with per-allele effect sizes of 0.03-0.14 cups per day. Six are located in or near genes potentially involved in pharmacokinetics (ABCG2, AHR, POR and CYP1A2) and pharmacodynamics (BDNF and SLC6A4) of caffeine. Two map to GCKR and MLXIPL genes related to metabolic traits but lacking known roles in coffee consumption. Enhancer and promoter histone marks populate the regions of many confirmed loci and several potential regulatory SNPs are highly correlated with the lead SNP of each. SNP alleles near GCKR, MLXIPL, BDNF and CYP1A2 that were associated with higher coffee consumption have previously been associated with smoking initiation, higher adiposity and fasting insulin and glucose but lower blood pressure and favorable lipid, inflammatory and liver enzyme profiles (P<5 × 10(-8)).Our genetic findings among European and African-American adults reinforce the role of caffeine in mediating habitual coffee consumption and may point to molecular mechanisms underlying inter-individual variability in pharmacological and health effects of coffee. Show less

Adenosine triphosphate-binding cassette transporter A1 (ABCA1) is a crucial cholesterol transporter and plays a central role in the high density lipoproteins (HDL) cholesterol metabolism and lipid clearance from the foam cell. Lipoxin A4 (LXA4) is an endogenous lipid mediator that requires cell-cell interaction or cell-platelet interaction for its synthesis. The roles of LXA4 on inflammatory responses are well described, while its effects on mediating ABCA1 and underlying mechanisms remain unclear. In this study, we showed that LXA4 significantly increases expression of ABCA1 and LXRα in a dose-dependent manner in THP-1 macrophage-derived foam cells. Cellular cholesterol content was decreased while cholesterol efflux was increased by LXA4 treatment. However, after short interfering RNA of LXRα, the effects of LXA4 on ABCA1 expression and cholesterol metabolism were significantly abolished. These results provide evidence that LXA4 increases ABCA1 expression and promotes cholesterol efflux through LXRα pathway in THP-1 macrophage-derived foam cells. Show less

Curcumin, a traditional Chinese derivative from the rhizomes of Curcuma longa, is beneficial to health by modulating lipid metabolism and suppressing atherogenesis. A key part of atherosclerosis is the failure of macrophages to restore their cellular cholesterol homeostasis and the formation of foam cells. In this study, results showed that curcumin dramatically increased the expression of ATP-binding cassette transporter 1 (ABCA1), promoted cholesterol efflux from THP-1 macrophage-derived foam cells, and reduced cellular cholesterol levels. Curcumin activated AMP-activated protein kinase (AMPK) and SIRT1, and then activated LXRα in THP-1 macrophage-derived foam cells. Inhibiting AMPK/SIRT1 activity by its specific inhibitor or by small interfering RNA could inhibit LXRα activation and abolish curcumin-induced ABCA1 expression and cholesterol efflux. Thus, curcumin enhanced cholesterol efflux by upregulating ABCA1 expression through activating AMPK-SIRT1-LXRα signaling in THP-1 macrophage-derived foam cells. This study describes a possible mechanism for understanding the antiatherogenic effects of curcumin on attenuating the progression of atherosclerosis. Show less

Macrophage foam cell formation is the most prominent characteristic of the early stages of atherosclerosis. Ubiquitin Fold Modifier 1 (UFM1) is a new member of the ubiquitin-like protein family, and its underlying mechanism of action in macrophage foam cell formation is poorly understood. Our current study focuses on UFM1 and investigates its role in macrophage foam cell formation. Using real-time quantitative PCR (qRT-PCR) and western blot analysis, we first analyzed the UFM1 expression in mouse peritoneal macrophages (MPMs) from ApoE－/－ mice in vivo and in human macrophages treated with oxLDL in vitro. Subsequently, the effects of UFM1 on macrophages foam cell formation were determined by Nile Red staining and direct lipid analysis. We then examined whether UFM1 affects the process of lipid metabolism in macrophages. Lastly, with the method of small interfering RNA (siRNA), we delineated the mechanism of UFM1 to attenuate lipid accumulation in THP-1 macrophages. UFM1 is dramatically upregulated under atherosclerosis conditions both in vivo and in vitro. Moreover, UFM1 markedly decreased macrophage foam cell formation. Mechanistic studies revealed that UFM1 increased the macrophage cholesterol efflux, which was due to the increased expression of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1). Furthermore, the upregulation of ABCA1 and ABCG1 by UFM1 resulted from liver X receptor α (LXRα) activation, which was confirmed by the observation that LXRα siRNA prevented the expression of ABCA1 and ABCG1. Consistent with this, the UFM1-mediated attenuation of lipid accumulation was abolished by such inhibition. Taken together, our results showed that UFM1 could suppress foam cell formation via the LXRα-dependent pathway. Show less

Menin, encoded by the MEN1 gene, was initially identified as a tumor suppressor for endocrine neoplasia. Our previous report showed that Menin enhances PPARα transactivity preventing triglyceride accumulation in the liver. Here, we further explore the role of Menin in liver steatosis. Transient transfection assays demonstrate that Menin inhibits the transcriptional activity of nuclear receptor liver X receptor α (LXRα). Accordingly, Menin overexpression results in reduced expression of LXRα target genes, such as lipogenic enzymes including SREBP-1c, FASN and SCD-1. Co-immunoprecipitation assays revealed physical interaction between Menin and LXRα. Collectively, our data suggest that Menin acts as a novel corepressor of LXRα and functions as a negative regulator of hepatic lipogenesis. Show less

ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor-B1 (SR-B1) promote cholesterol efflux from cells to lipid-poor apolipoprotein A-I and play an important role in the development of atherosclerosis. Liver X receptor (LXRα) and peroxisome proliferator-activated receptor-gamma (PPARγ) operate as cholesterol sensors, which may protect from cholesterol overload by stimulating cholesterol efflux from cells to high-density lipoprotein through ABCA1, ABCG1, and SR-B1. Propofol administration is associated with cardiovascular protective effects, including anti-inflammatory and antioxidant properties. Here, we examined the effect of propofol on ABCA1, ABCG1, and SR-B1 expression and explored whether PPARγ and LXRα were involved in the regulation of propofol-induced ABCA1, ABCG1, and SR-B1 expression in THP-1 macrophage-derived foam cells. Propofol significantly increased expression levels of ABCA1, ABCG1, and SR-B1 in a time-dependent manner. Cellular cholesterol content was decreased while cholesterol efflux was increased by propofol treatment. Moreover, PPARγ and LXRα were up-regulated by propofol treatment. In addition, the up-regulated expression of ABCA1, ABCG1, and SR-B1 by propofol was significantly abolished by both PPARγ siRNA and LXRα siRNA in THP-1 macrophage-derived foam cells. These results provide evidence that propofol up-regulates expression of ABCA1, ABCG1, and SR-B1 through the PPARγ/LXRα pathway in THP-1 macrophage-derived foam cells. Show less

A high level of RGS17 expression is observed in diverse human cancers and correlates with tumor progression. Herein, we aim to investigate its expression and function in breast cancer. The expression of RGS17 was detected by immunohistochemical analysis and western blot analysis. The level of miR-32 expression was investigated by qRT-PCR. Western blot analysis was used to determine the relationship between RGS17 and miR-32. A series of loss or gain of function assays was performed to measure the effects of RGS17 or miR-32 on tumor migration, invasion, and proliferation. Compared to that in normal breast specimen, the expression of RGS17 had a significantly higher expression level in breast cancer tissues and cell lines. Although the potential relationship of RGS17 expression with clinicopathological features was not observed, there was a significant correlation of RGS17 expression with p63 expression. In cells, inhibition of RGS17 expression impaired cell migration, invasion, and proliferation. Further, RGS17 was identified as a direct and functional target of miR-32. Overexpression of miR-32 in cells could decrease the expression of RGS17 and inhibit cell migration, invasion, and proliferation. In contrast, ectopic expression of RGS17 could attenuate phenotypes caused by miR-32 overexpression. The expression of RGS17 was upregulated in breast cancer, which could enhance cell migration, invasion, and proliferation. Moreover, the RGS17 was identified as a target of miR-32. Our results suggest that RGS17 might play an important role in breast cancer progression and could be a potential target for human breast cancer treatment. Show less

Recent findings have reemphasized the importance of epistasis, or gene-gene interactions, as a contributing factor to the unexplained heritability of obesity. Network-based methods such as statistical epistasis networks (SEN), present an intuitive framework to address the computational challenge of studying pairwise interactions between thousands of genetic variants. In this study, we aimed to analyze pairwise interactions that are associated with Body Mass Index (BMI) between SNPs from twelve genes robustly associated with obesity (BDNF, ETV5, FAIM2, FTO, GNPDA2, KCTD15, MC4R, MTCH2, NEGR1, SEC16B, SH2B1, and TMEM18). We used information gain measures to identify all SNP-SNP interactions among and between these genes that were related to obesity (BMI > 30 kg/m(2)) within the Framingham Heart Study Cohort; interactions exceeding a certain threshold were used to build an SEN. We also quantified whether interactions tend to occur more between SNPs from the same gene (dyadicity) or between SNPs from different genes (heterophilicity). We identified a highly connected SEN of 709 SNPs and 1241 SNP-SNP interactions. Combining the SEN framework with dyadicity and heterophilicity analyses, we found 1 dyadic gene (TMEM18, P-value = 0.047) and 3 heterophilic genes (KCTD15, P-value = 0.045; SH2B1, P-value = 0.003; and TMEM18, P-value = 0.001). We also identified a lncRNA SNP (rs4358154) as a key node within the SEN using multiple network measures. This study presents an analytical framework to characterize the global landscape of genetic interactions from genome-wide arrays and also to discover nodes of potential biological significance within the identified network. Show less

MicroRNA-452 (miRNA-452) was overexpressed in docetaxel-resistant human breast cancer MCF-7 cells (MCF-7/DOC). However, its role in modulating the sensitivity of breast cancer cells to docetaxel (DOC) remains unclear. The aim of this study is to investigate the role of miRNA-452 in the sensitivity of breast cancer cells to DOC.Real-time quantitative PCR (RT-qPCR) were used to identify the differential expression of miRNA-452 between MCF-7/DOC and MCF-7 cells. MiRNA-452 mimic was transfected into MCF-7 cells and miRNA-452 inhibitor was transfected into MCF-7/DOC cells. The role of miRNA-452 in these transfected cells was evaluated using RT-qPCR, MTT assay, and flow cytometry assay. The relationship of miRNA-452 and its predictive target gene "anaphase-promoting complex 4" (APC4) was analyzed by RT-qPCR and Western blot.MiRNA-452 showed significantly higher expression (78.9-folds) in MCF-7/DOC cells compared to parental MCF-7 cells. The expression of miRNA-452 in the mimic transfected MCF-7 cells was upregulated 212.2-folds (P < 0.05) compared to its negative control (NC), and the half maximal inhibitory concentration (IC50) value of DOC (1.98 ± 0.15 μM) was significantly higher than that in its NC (0.85 ± 0.08 μM, P < 0.05) or blank control (1.01 ± 0.19 μM, P < 0.05). Furthermore, its apoptotic rate (6.3 ± 1.3 %) was distinctly decreased compared with that in its NC (23.8 ± 6.6 %, P < 0.05) or blank control (18.6 ± 4.7 %, P < 0.05). In contrast, the expression of miRNA-452 in the inhibitor-transfected MCF-7/DOC cells was downregulated 0.58-fold (P < 0.05) compared to its NC, the IC50 value of DOC (44.5 ± 3.2 μM) was significantly lower than that in its NC (107.3 ± 6.63 μM, P < 0.05) or blank control (102.22 ± 11.34 μM, P < 0.05), and the apoptotic rate (45.5 ± 10.8 %) was distinctly increased compared with its NC (9.9 ± 2.2 %, P < 0.05) and blank control (9.4 ± 2.5 %, P < 0.05). Further, there was an inverse association between miRNA-452 and APC4 expression in breast cancer cells in vitro.Dysregulation of miRNA-452 involved in the DOC resistance formation of breast cancer cells may be, in part, via targeting APC4. Show less

Single nucleotide polymorphisms (SNPs) in apolipoprotein A5 (APOA5) gene are associated with triglyceride (TG) levels. However, the minor allele frequencies and linkage disequilibriums (LDs) of the SNPs in addition to their effects on TG levels vary greatly between Caucasians and East Asians. The distributions of the SNPs/haplotypes and their associations with TG levels in Uyghur population, an admixture population of Caucasians and East Asians, have not been reported to date. Here, we performed a cross-sectional study to address these. Genotyping of four SNPs in APOA5 (rs662799, rs3135506, rs2075291, and rs2266788) was performed in 1174 unrelated Uyghur subjects. SNP/haplotype and TG association analyses were conducted. The frequencies of the SNPs in Uyghurs were in between those in Caucasians and East Asians. The LD between rs662799 and rs2266788 in Uyghurs was stronger than that in East Asians but weaker than that in Caucasians, and the four SNPs resulted in four haplotypes (TGGT, CGGC, TCGT, and CGTT arranged in the order of rs662799, rs3135506, rs2075291, and rs2266788) representing 99.2% of the population. All the four SNPs were significantly associated with TG levels. Compared with non-carriers, carriers of rs662799-C, rs3135506-C, rs2075291-T, and rs2266788-C alleles had 16.0%, 15.1%, 17.1%, and 12.4% higher TG levels, respectively. When haplotype TGGT was defined as the reference, the haplotypes CGGC, TCGT, and CGTT resulted in 16.1%, 19.0%, and 19.8% higher TG levels, respectively. The proportions of variance in TG explained by APOA5 locus were 2.5%, 0.3%, 0.4%, and 1.9% for single SNP rs662799, rs3135506, rs2075291, and rs2266788, respectively, and 3.0% for the haplotypes constructed by them. The association profiles between the SNPs and haplotypes at APOA5 locus and TG levels in this admixture population differed from those in Caucasians and East Asians. The functions of these SNPs and haplotypes need to be elucidated comprehensively. Show less

Several studies have investigated whether the polymorphism in the apolipoprotein A5 (APOA5) is associated with type 2 diabetes mellitus (T2DM) risk. However, those studies have produced inconsistent results. The purpose of this study was to investigate whether the APOA5 -1131T/C polymorphism (rs662799) confers significant susceptibility to T2DM using a meta-analysis. PubMed, Embase, Web of Science, Cochrane database, CBMdisc, CNKI and Google Scholar were searched to get the genetic association studies. All statistical analyses were done with Stata 11.0. A total of 19 studies included 4,767 T2DM cases and 10,370 controls (four studies involving 555 T2DM cases and 2958 controls were performed among Europeans and 15 studies involving 4212 T2DM cases and 7412 controls were performed among Asians) were combined showing significant association between the APOA5 -1131T/C polymorphism and T2DM risk (for C allele vs. T allele: OR = 1.28, 95% CI = 1.17-1.40, p<0.00001; for C/C vs. T/T: OR = 1.57, 95% CI = 1.35-1.83, p<0.00001; for C/C vs. T/C+T/T: OR = 1.36, 95% CI = 1.18-1.57, p<0.0001; for C/C+T/C vs. T/T: OR = 1.32, 95% CI = 1.16-1.51, p<0.0001). In the subgroup analysis by ethnicity, significant association was also found among Asians (for C allele vs. T allele: OR = 1.31, 95% CI = 1.22-1.40, p<0.00001; for C/C vs. T/T: OR = 1.61, 95% CI = 1.38-1.88, p<0.00001; for C/C vs. T/C+T/T: OR = 1.39, 95% CI = 1.20-1.61, p<0.0001; for C/C+T/C vs. T/T: OR = 1.42, 95% CI = 1.25-1.62, p<0.00001). However, no significant association was found between the APOA5 -1131T/C polymorphism and T2DM risk among Europeans. The present meta-analysis suggests that the APOA5 -1131T/C polymorphism is associated with an increased T2DM risk in Asian population. Show less

The genetic background of ischemic vascular disease is actively being explored. Several studies have shown that inhibition of APOC3 significantly reduces plasma levels of apolipoprotein C3 and triglycerides. Recently, the TG and HDL Working Group and Jørgensen et al. reported that loss-of-function mutations in APOC3 are associated with decreased triglyceride levels and a reduced risk of ischemic vascular disease in European and African individuals. We performed a replication study in 4470 Chinese participants. The coding regions of APOC3 were amplified and re-sequenced. However, only synonymous and intronic variants with no functional consequences were identified. None of the loss-of-function mutations reported in European and African individuals were observed. Therefore, APOC3 may not be an ideal predictor for risk of ischemic vascular disease in the Chinese population. Show less

Plasma triglyceride levels are heritable and are correlated with the risk of coronary heart disease. Sequencing of the protein-coding regions of the human genome (the exome) has the potential to identify rare mutations that have a large effect on phenotype. We sequenced the protein-coding regions of 18,666 genes in each of 3734 participants of European or African ancestry in the Exome Sequencing Project. We conducted tests to determine whether rare mutations in coding sequence, individually or in aggregate within a gene, were associated with plasma triglyceride levels. For mutations associated with triglyceride levels, we subsequently evaluated their association with the risk of coronary heart disease in 110,970 persons. An aggregate of rare mutations in the gene encoding apolipoprotein C3 (APOC3) was associated with lower plasma triglyceride levels. Among the four mutations that drove this result, three were loss-of-function mutations: a nonsense mutation (R19X) and two splice-site mutations (IVS2+1G→A and IVS3+1G→T). The fourth was a missense mutation (A43T). Approximately 1 in 150 persons in the study was a heterozygous carrier of at least one of these four mutations. Triglyceride levels in the carriers were 39% lower than levels in noncarriers (P<1×10(-20)), and circulating levels of APOC3 in carriers were 46% lower than levels in noncarriers (P=8×10(-10)). The risk of coronary heart disease among 498 carriers of any rare APOC3 mutation was 40% lower than the risk among 110,472 noncarriers (odds ratio, 0.60; 95% confidence interval, 0.47 to 0.75; P=4×10(-6)). Rare mutations that disrupt APOC3 function were associated with lower levels of plasma triglycerides and APOC3. Carriers of these mutations were found to have a reduced risk of coronary heart disease. (Funded by the National Heart, Lung, and Blood Institute and others.). Show less

Apoptosis constitutes a system for the removal of aged, or damaged cells, which is regulated by the interplay of pro-apoptotic and antiapoptotic proteins. Previous study has shown that Juvenile Batten disease protein, CLN3, is antiapoptotic gene in NT2 neuronal precursor cells and a few types of cancers. However, in colorectal cancer, whether CLN3 also play its antiapoptotic role and the effect of targeted controlling CLN3 on the biological behavior of human colorectal cancer cell is unknown. We employed the sequence-specific siRNA silencing the CLN3 gene and investigated its effects on growth and apoptosis of colorectal cancer HCT116 cells, which has highest elevation of CLN3 expression among four colorectal cancer cell lines. After CLN3 specific siRNA transfection, mRNA and protein expression levels of CLN3 in HCT116 cells were noticeably decreased. Moreover, CLN3-siRNA inhibited the proliferation of colorectal cancer cells, promoted their apoptosis and induced G0/G1 cell cycle arrest. Our current study demonstrated that CLN3 was expressed in colorectal cancer cells at a high frequency. Moreover, CLN3 down-regulation with RNA interference can inhibit proliferation, apoptosis, and cell cycle progression of colorectal cancer cells. Our study represented a potential new approach to understanding the role of CLN3 in cancer and provides a potential novel strategy colorectal cancer therapy. Show less

Carbamoyl phosphate synthetase 1 (CPS1) deficiency due to CPS1 mutations is a rare autosomal-recessive urea cycle disorder causing hyperammonemia that can lead to death or severe neurological impairment. CPS1 catalyzes carbamoyl phosphate formation from ammonia, bicarbonate and two molecules of ATP, and requires the allosteric activator N-acetyl-L-glutamate. Clinical mutations occur in the entire CPS1 coding region, but mainly in single families, with little recurrence. We characterized here the only currently known recurrent CPS1 mutation, p.Val1013del, found in eleven unrelated patients of Turkish descent using recombinant His-tagged wild type or mutant CPS1 expressed in baculovirus/insect cell system. The global CPS1 reaction and the ATPase and ATP synthesis partial reactions that reflect, respectively, the bicarbonate and the carbamate phosphorylation steps, were assayed. We found that CPS1 wild type and V1013del mutant showed comparable expression levels and purity but the mutant CPS1 exhibited no significant residual activities. In the CPS1 structural model, V1013 belongs to a highly hydrophobic β-strand at the middle of the central β-sheet of the A subdomain of the carbamate phosphorylation domain and is close to the predicted carbamate tunnel that links both phosphorylation sites. Haplotype studies suggested that p.Val1013del is a founder mutation. In conclusion, the mutation p.V1013del inactivates CPS1 but does not render the enzyme grossly unstable or insoluble. Recurrence of this particular mutation in Turkish patients is likely due to a founder effect, which is consistent with the frequent consanguinity observed in the affected population. Show less

Carbamoyl phosphate synthetase 1 deficiency (CPS1D) is an inborn error of the urea cycle that is due to mutations in the CPS1 gene. In the first large repertory of mutations found in CPS1D, a small CPS1 domain of unknown function (called the UFSD) was found to host missense changes with high frequency, despite the fact that this domain does not host substrate-binding or catalytic machinery. We investigate here by in vitro expression studies using baculovirus/insect cells the reasons for the prominence of the UFSD in CPS1D, as well as the disease-causing roles and pathogenic mechanisms of the mutations affecting this domain. All but three of the 18 missense changes found thus far mapping in this domain in CPS1D patients drastically decreased the yield of pure CPS1, mainly because of decreased enzyme solubility, strongly suggesting misfolding as a major determinant of the mutations negative effects. In addition, the majority of the mutations also decreased from modestly to very drastically the specific activity of the fraction of the enzyme that remained soluble and that could be purified, apparently because they decreased V(max). Substantial although not dramatic increases in K(m) values for the substrates or for N-acetyl-L-glutamate were observed for only five mutations. Similarly, important thermal stability decreases were observed for three mutations. The results indicate a disease-causing role for all the mutations, due in most cases to the combined effects of the low enzyme level and the decreased activity. Our data strongly support the value of the present expression system for ascertaining the disease-causing potential of CPS1 mutations, provided that the CPS1 yield is monitored. The observed effects of the mutations have been rationalized on the basis of an existing structural model of CPS1. This model shows that the UFSD, which is in the middle of the 1462-residue multidomain CPS1 protein, plays a key integrating role for creating the CPS1 multidomain architecture leading us to propose here a denomination of "Integrating Domain" for this CPS1 region. The majority of these 18 mutations distort the interaction of this domain with other CPS1 domains, in many cases by causing improper folding of structural elements of the Integrating Domain that play key roles in these interactions. Show less

We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks. Show less

Vascular endothelial growth factor-C (VEGF-C), which contributes to lymphatic metastasis (LM) in malignant disease, is one of the most important factors involved in physical and pathological lymphangiogenesis. Some VEGF-C related factors such as sine oculis homeobox homolog (SIX) 1, contactin (CNTN) 1 and dual specificity phosphatase (DUSP) 6 have been extensively studied in malignancies, but their expression levels and associations have still to be elucidated in stomach cancer. We detected their expression levels in 30 paired stomach cancer tissues using quantitative real-time reverse transcription-PCR (qRT-PCR). The expression and clinical significance of each factor was analyzed using Wilcoxon signed rank sum test. The correlation among all the factors was performed by Spearman rank correlation analysis. The results suggest that VEGF-C and CNTN1 are significantly correlated with tumor size, SIX1 with the age and CNTN1 also with the cTNM stage. There are significant correlations of expression levels among VEGF-C, SIX1, CNTN1 and DUSP6. There exists an important regulatory crosstalk involving SIX1, VEGF-C, CNTN1 and DUSP6 in stomach cancer. Show less

Cysteine proteases play important roles in pathobiology. Here we reveal that cathepsin K (CatK) has a role in ischaemia-induced neovascularization. Femoral artery ligation-induced ischaemia in mice increases CatK expression and activity, and CatK-deficient mice show impaired functional recovery following hindlimb ischaemia. CatK deficiency reduces the levels of cleaved Notch1 (c-Notch1), Hes1 Hey1, Hey2, vascular endothelial growth factor, Flt-1 and phospho-Akt proteins of the ischaemic muscles. In endothelial cells, silencing of CatK mimicked, whereas CatK overexpression enhanced, the levels of c-Notch1 and the expression of Notch downstream signalling molecules, suggesting CatK contributes to Notch1 processing and activates downstream signalling. Moreover, CatK knockdown leads to defective endothelial cell invasion, proliferation and tube formation, and CatK deficiency is associated with decreased circulating endothelial progenitor cells-like CD31(+)/c-Kit(+) cells in mice following hindlimb ischaemia. Transplantation of bone marrow-derived mononuclear cells from CatK(+/+) mice restores the impairment of neovascularization in CatK(-/-) mice. We conclude that CatK may be a potential therapeutic target for ischaemic disease. Show less

The bone morphogenetic protein antagonist Gremlin 2 (Grem2) is required for atrial differentiation and establishment of cardiac rhythm during embryonic development. A human Grem2 variant has been associated with familial atrial fibrillation, suggesting that abnormal Grem2 activity causes arrhythmias. However, it is not known how Grem2 integrates into signaling pathways to direct atrial cardiomyocyte differentiation. Here, we demonstrate that Grem2 expression is induced concurrently with the emergence of cardiovascular progenitor cells during differentiation of mouse embryonic stem cells (ESCs). Grem2 exposure enhances the cardiogenic potential of ESCs by 20-120-fold, preferentially inducing genes expressed in atrial myocytes such as Myl7, Nppa, and Sarcolipin. We show that Grem2 acts upstream to upregulate proatrial transcription factors CoupTFII and Hey1 and downregulate atrial fate repressors Irx4 and Hey2. The molecular phenotype of Grem2-induced atrial cardiomyocytes was further supported by induction of ion channels encoded by Kcnj3, Kcnj5, and Cacna1d genes and establishment of atrial-like action potentials shown by electrophysiological recordings. We show that promotion of atrial-like cardiomyocytes is specific to the Gremlin subfamily of BMP antagonists. Grem2 proatrial differentiation activity is conveyed by noncanonical BMP signaling through phosphorylation of JNK and can be reversed by specific JNK inhibitors, but not by dorsomorphin, an inhibitor of canonical BMP signaling. Taken together, our data provide novel mechanistic insights into atrial cardiomyocyte differentiation from pluripotent stem cells and will assist the development of future approaches to study and treat arrhythmias. Show less

Phenotypic switching of vascular smooth muscle cells (VSMCs) plays an initial role in neointimal hyperplasia, the main cause of many occlusive vascular diseases. The aim of this study was to measure the effects of resveratrol (RSV) on the phenotypic transformation of VSMCs and to investigate its mechanism of action. Cultured VSMCs isolated from rat thoracic aorta were prepared with serum starvation for 72 hours followed by RSV treatment (50-200 μmol/L) and 10% serum stimulation. Male Sprague-Dawley rats, subjected to carotid arteries injury from a balloon catheter, were exposed to intraperitoneal injection of RSV (1 mg/kg) or saline and were killed after 7 or 28 days. Compared with cells in the serum-induced group, VSMCs in the RSV or N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment group exhibited significant decreases of proliferation and migration. The total and cytoplasmic Notch-1 levels were declined by RSV, accompanied by a significant increase in smooth muscle α-actin and smooth muscle myosin heavy chain protein. The expression of Notch-1, Jagged-1, Hey-1, and Hey-2 mRNA in balloon-injured arteries at 7 days was decreased by RSV treatment. Arteries from RSV-treated rats showed less neointimal hyperplasia, lower collagen content, and a lower rate of cells positive for proliferating cell nuclear antigen 28 days after injury, compared with saline controls. The results indicate that RSV can attenuate phenotypic switching of VSMCs after arterial injury through inhibition of the Notch pathway. Show less

Neointimal formation after vessel injury is a complex process involving multiple cellular and molecular processes. Inhibition of intimal hyperplasia plays an important role in preventing proliferative vascular diseases, such as restenosis. In this study, we intended to identify whether sodium ferulate could inhibit neointimal formation and further explore potential mechanisms involved. Cultured vascular smooth muscle cells (VSMCs) isolated from rat thoracic aorta were pre-treated with 200 µmol/L sodium ferulate for 1 hour and then stimulated with 1 µmol/L angiotensin II (Ang II) for 1 hour or 10% serum for 48 hours. Male Sprague-Dawley rats subjected to balloon catheter insertion were administrated with 200 mg/kg sodium ferulate (or saline) for 7 days before sacrificed. In presence of sodium ferulate, VSMCs exhibited decreased proliferation and migration, suppressed intracellular reactive oxidative species production and NADPH oxidase activity, increased SOD activation and down-regulated p38 phosphorylation compared to Ang II-stimulated alone. Meanwhile, VSMCs treated with sodium ferulate showed significantly increased protein expression of smooth muscle α-actin and smooth muscle myosin heavy chain protein. The components of Notch pathway, including nuclear Notch-1 protein, Jagged-1, Hey-1 and Hey-2 mRNA, as well as total β-catenin protein and Cyclin D1 mRNA of Wnt signaling, were all significantly decreased by sodium ferulate in cells under serum stimulation. The levels of serum 8-iso-PGF2α and arterial collagen formation in vessel wall were decreased, while the expression of contractile markers was increased in sodium ferulate treated rats. A decline of neointimal area, as well as lower ratio of intimal to medial area was observed in sodium ferulate group. Sodium ferulate attenuated neointimal hyperplasia through suppressing oxidative stress and phenotypic switching of VSMCs. Show less

Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the central nervous system (CNS), resulting in myelin sheath damage and axonal loss. Leucine-rich repeat and immunoglobulin domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1 (LINGO-1) have been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1 inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. In this study, we cloned the zebrafish homolog of the human LINGO-1 and found that lingo1b regulated myelination and oligodendrocyte differentiation. The expression of lingo1b started 1 (mRNA) and 2 (protein) days post-fertilization (dpf) in the CNS. Morpholino oligonucleotide knockdown of lingo1b resulted in developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord. The lack of lingo1b enhanced myelination and oligodendrocyte differentiation during embryogenesis. Furthermore, immunohistochemistry and movement analysis showed that lingo1b was involved in the axon development of primary motor neurons. These results suggested that Lingo1b protein functions as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development. Show less

The nuclear receptor liver X receptor (LXR) plays an important role in the metabolism and homeostasis of cholesterol, lipids, bile acids, and steroid hormones. In this study, we uncovered a function of LXRα (NR1H3) in regulating the human hydroxysteroid sulfotransferase SULT2A1, a phase II conjugating enzyme known to sulfonate bile acids, hydroxysteroid dehydroepiandrosterone, and related androgens. We showed that activation of LXR induced the expression of SULT2A1 at mRNA, protein, and enzymatic levels. A combination of promoter reporter gene and chromatin immunoprecipitation assays showed that LXRα transactivated the SULT2A1 gene promoter through its specific binding to the -500- to -258-base pair region of the SULT2A1 gene promoter. LXR small interfering RNA knockdown experiments suggested that LXRα, but not LXRβ, played a dominant role in regulating SULT2A1. In primary human hepatocytes, we found a positive correlation between the expression of SULT2A1 and LXRα, which further supported the regulation of SULT2A1 by LXRα. In summary, our results established human SULT2A1 as a novel LXRα target gene. The expression of LXRα is a potential predictor for the expression of SULT2A1 in human liver. Show less

To investigate associations of single nucleotide polymorphisms (SNPs) rs2228314 of sterol regulatory element-binding protein-2 (SREBP-2) or rs11039155 of liver X receptor α (LXRα) with susceptibility to polycystic ovary syndrome (PCOS) in a Chinese Han population. SREBP-2 rs2228314 and LXRα rs11039155 polymorphisms were genotyped in patients with PCOS and age- and sex-matched PCOS-free controls from a Chinese Han population. A total of 605 patients with PCOS and 615 controls were recruited in this study. We found that GC and CC genotypes of rs2228314, and variant C, were associated with a significantly increased risk of PCOS. In addition, GA and AA genotypes of rs11039155, as well as variant A, were also associated with a significantly increased risk of PCOS. Our results showed that SREBP-2 rs2228314 G to C change and variant C genotype as well as LXRα rs11039155 G to A change and variant A may contribute to PCOS in Chinese Han population. Show less