👤 Nan Sun

Mouse embryonic fibroblasts (MEFs) differentiate into fully functional chondrocytes in response to bone morphogenetic protein-2 (BMP-2). However, the comprehensive proteomic aspect of BMP-2-induced chondrogenesis remains unknown. We took advantage of quantitative proteomic analysis based on isobaric tag for relative and absolute quantitation (iTRAQ) and on-line 2D nano-liquid chromatography/tandem mass spectrometry (LC/MS/MS) to identify proteins differentially expressed during BMP-2-induced chondrogenic differentiation of MEFs. We found 85 downregulated proteins, and ingenuity pathways analysis (IPA) revealed a protein-protein network with chromodomain-helicase-DNA-binding protein 4 (Chd4) in the center. Chromatin immunoprecipitation (ChIP) and nuclease hypersensitivity assays showed that Chd4, interacting with Hdac1/2, cooperates with its related proteins Kap1 and Cbx1 to bind at -207/-148 of the Sox9 promoter. We also provided evidence that let-7a targets the 3'UTR of Chd4 to promote chondrogenesis of MEFs. Together, our findings indicate that BMP-2 induced the upregulation of let-7a, targeting Chd4 and positively controlling the chondrogenic differentiation of MEFs. These findings illustrate epigenetic regulation of the chondrogenic differentiation process and also expand the understanding of the involved intracellular mechanisms. Show less

Zinc ions highly concentrate in hippocampus and play a key role in modulating spatial learning and memory. At a time when dietary fortification and supplementation of zinc have increased the zinc consuming level especially in the youth, the toxicity of zinc overdose on brain function was underestimated. In the present study, weaning ICR mice were given water supplemented with 15 ppm Zn (low dose), 60 ppm Zn (high dose) or normal lab water for 3 months, the behavior and brain zinc homeostasis were tested. Mice fed high dose of zinc showed hippocampus-dependent memory impairment. Unexpectedly, zinc deficiency, but not zinc overload was observed in hippocampus, especially in the mossy fiber-CA3 pyramid synapse. The expression levels of learning and memory related receptors and synaptic proteins such as NMDA-NR2A, NR2B, AMPA-GluR1, PSD-93 and PSD-95 were significantly decreased in hippocampus, with significant loss of dendritic spines. In keeping with these findings, high dose intake of zinc resulted in decreased hippocampal BDNF level and TrkB neurotrophic signaling. At last, increasing the brain zinc level directly by brain zinc injection induced BDNF expression, which was reversed by zinc chelating in vivo. These results indicate that zinc plays an important role in hippocampus-dependent learning and memory and BDNF expression, high dose supplementation of zinc induces specific zinc deficiency in hippocampus, which further impair learning and memory due to decreased availability of synaptic zinc and BDNF deficit. Show less

Plasma lipid levels are important risk factors for cardiovascular disease and are influenced by genetic and environmental factors. Recent genome wide association studies (GWAS) have identified several lipid-associated loci, but these loci have been identified primarily in European populations. In order to identify genetic markers for lipid levels in a Chinese population and analyze the heterogeneity between Europeans and Asians, especially Chinese, we performed a meta-analysis of two genome wide association studies on four common lipid traits including total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL) and high-density lipoprotein cholesterol (HDL) in a Han Chinese population totaling 3,451 healthy subjects. Replication was performed in an additional 8,830 subjects of Han Chinese ethnicity. We replicated eight loci associated with lipid levels previously reported in a European population. The loci genome wide significantly associated with TC were near DOCK7, HMGCR and ABO; those genome wide significantly associated with TG were near APOA1/C3/A4/A5 and LPL; those genome wide significantly associated with LDL were near HMGCR, ABO and TOMM40; and those genome wide significantly associated with HDL were near LPL, LIPC and CETP. In addition, an additive genotype score of eight SNPs representing the eight loci that were found to be associated with lipid levels was associated with higher TC, TG and LDL levels (P = 5.52 × 10(-16), 1.38 × 10(-6) and 5.59 × 10(-9), respectively). These findings suggest the cumulative effects of multiple genetic loci on plasma lipid levels. Comparisons with previous GWAS of lipids highlight heterogeneity in allele frequency and in effect size for some loci between Chinese and European populations. The results from our GWAS provided comprehensive and convincing evidence of the genetic determinants of plasma lipid levels in a Chinese population. Show less

The dried succulent stem of Cistanche tubulosa (Schenk) R. Wight is one component of traditional Chinese medicine prescriptions for diabetes. However, there have been no modern scientific reports to confirm this traditional claim for the Cistanche species until now. Thus, we investigated the effects of Cistanche tubulosa on glucose homeostasis and serum lipids in male BKS.Cg-Dock7(m) +/+ Lepr(db)/J (db/db) mice, a model of type 2 diabetes. The verbascoside and echinacoside contents of Cistanche tubulosa powder were evaluated using HPLC. The total phenolic content, polysaccharide content and antioxidant activity of Cistanche tubulosa powder were also evaluated. Then, different doses of Cistanche tubulosa (equivalent to 120.9, 72.6 or 24.2mg verbascoside/kg) were administered orally once daily for 45 days to male db/db mice. Age matched db/+ mice were used as normal controls. Body weight, fasting blood glucose, postprandial blood glucose and insulin tolerance test were measured during the experiment. At the time of sacrifice, blood was collected for measurement of insulin level, the homeostatic model assessment of insulin resistance (HOMA-IR), and total cholesterol, triglyceride, HDL-c, LDL-c and VLDL-c levels; liver and muscle were harvested for measurement of glycogen levels. Cistanche tubulosa significantly suppressed the elevated fasting blood glucose and postprandial blood glucose levels, improved insulin resistance and dyslipidemia, and suppressed body weight loss in db/db mice. However, Cistanche tubulosa did not significantly affect serum insulin levels or hepatic and muscle glycogen levels. This study provides scientific evidence for the traditional use of Cistanche tubulosa to treat diabetes, suggesting that Cistanche tubulosa has the potential for development into a functional food ingredient or drug to prevent hyperglycemia and treat hyperlipidemia. Show less

Little is known about the associations of FADS1 genetic variants with circulating levels of PUFA and lipids in Asian populations who have a different dietary pattern and dyslipidemia prevalence compared with Western populations. In a population-based sample of 3,210 unrelated Han Chinese living in Beijing and Shanghai, we examined a FADS1 genetic variant, rs174550, in relation to blood PUFA and lipid levels. C-allele of rs174550 was significantly associated with levels of erythrocyte PUFAs in upstream and downstream pathways of delta-5 desaturase (D5D) (P ≤ 0.003). Moreover, rs174550 C-allele was associated with a lower HDL cholesterol level (P = 0.02) in total population and a higher triglyceride level (P = 0.0002) in Beijing residents. Interestingly, erythrocyte levels of 18:2n-6 and 18:3n-3 modified the effect of rs174550 on HDL cholesterol level: stronger associations between rs174550 C-allele and lower HDL cholesterol levels were exhibited when erythrocyte 18:2n-6 or 18:3n-3 level was low (P for interaction = 0.02 and 0.03, respectively). These data suggested that FADS1 genetic variant was associated with circulating PUFA and lipid levels and that its effect on HDL cholesterol might depend on PUFA status in the Han Chinese population. Show less

We aimed to investigate the effects of LXRα, ChREBP and Elovl6 in the development of insulin resistance-induced by medium- and long-chain fatty acids. Sprague Dawley rats were fed a standard chow diet (Control group) or a high-fat, high sucrose diet with different fat sources (coconut oil, lard, sunflower and fish oil) for 8 weeks. These oils were rich in medium-chain saturated fatty acids (MCFA group), long-chain saturated fatty acids (LCFA group), n-6 and n-3 long-chain polyunsaturated fatty acids (n-6 PUFA and n-3 PUFA groups), respectively, which had different chain lengths and degrees of unsaturation. Hyperinsulinemic-euglycemic clamp with [6-(3)H] glucose infusion was performed in conscious rats to assess hepatic insulin sensitivity. LCFA and n-6 PUFA groups induced hepatic insulin resistance and increased liver X receptor α (LXRα), carbohydrate response element binding protein (ChREBP) and long-chain fatty acid elongase 6 (Elovl6) expression in liver and white adipose tissue (WAT). Furthermore, LCFA and n-6 PUFA groups suppressed Akt serine 473 phosphorylation in liver and WAT. By contrast, in liver and WAT, MCFA and n-3 PUFA groups decreased LXRα, ChREBP and Elovl6 expression and improved insulin signaling and insulin resistance, but Akt serine 473 phosphorylation was not restored by MCFA group in WAT. This study demonstrated that the mechanism of the different effects of medium- and long-chain fatty acids on hepatic insulin resistance involves LXRα, ChREBP and Elovl6 alternations in liver and WAT. It points to a new strategy for ameliorating insulin resistance and diabetes through intervention on Elovl6 or its control genes. Show less

Genotype-phenotype correlation of hypertrophic cardiomyopathy (HCM) has been challenging because of the genetic and clinical heterogeneity. To determine the mutation profile of Chinese patients with HCM and to correlate genotypes with phenotypes, we performed a systematic mutation screening of the eight most commonly mutated genes encoding sarcomere proteins in 200 unrelated Chinese adult patients using direct DNA sequencing. A total of 98 mutations were identified in 102 mutation carriers. The frequency of mutations in MYH7, MYBPC3, TNNT2 and TNNI3 was 26.0, 18.0, 4.0 and 3.5 % respectively. Among the 200 genotyped HCM patients, 83 harbored a single mutation, and 19 (9.5 %) harbored multiple mutations. The number of mutations was positively correlated with the maximum wall thickness. We found that neither particular gene nor specific mutation was correlated to clinical phenotype. In summary, the frequency of multiple mutations was greater in Chinese HCM patients than in the Caucasian population. Multiple mutations in sarcomere protein may be a risk factor for left ventricular wall thickness. Show less

Cardiotoxicity is one of the leading causes of drug attrition. Current in vitro models insufficiently predict cardiotoxicity, and there is a need for alternative physiologically relevant models. Here we describe the gene expression profile of human-induced pluripotent stem cell-derived cardiocytes (iCC) postthaw over a period of 42 days in culture and compare this profile to human fetal and adult as well as adult cynomolgus nonhuman primate (NHP, Macaca fascicularis) heart tissue. Our results indicate that iCC express relevant cardiac markers such as ion channels (SCN5A, KCNJ2, CACNA1C, KCNQ1, and KCNH2), tissue-specific structural markers (MYH6, MYLPF, MYBPC3, DES, TNNT2, and TNNI3), and transcription factors (NKX2.5, GATA4, and GATA6) and lack the expression of stem cell markers (FOXD3, GBX2, NANOG, POU5F1, SOX2, and ZFP42). Furthermore, we performed a functional evaluation of contractility of the iCC and showed functional and pharmacological correlations with myocytes isolated from adult NHP hearts. These results suggest that stem cell-derived cardiocytes may represent a novel in vitro model to study human cardiac toxicity with potential ex vivo and in vivo translation. Show less

Both L-4F, one apolipoprotein A-1 mimetic peptide, and statins can reduce progression of atherosclerosis by different mechanisms. The combination of the two drugs can cause lesion regression by rendering HDL anti-inflammatory. We postulated that combination of L-4F and simvastatin may stimulate cholesterol efflux and related proteins expressions to alleviate atherosclerosis. Thirty male wild-type (W-T) C57 BL/6 mice and apo E(-/-) mice were divided into five groups: W-T group, atherosclerosis (AS) group, simvastatin group, L-4F group and the combination of simvastatin and L-4F group. After 16 weeks, serum lipids, atherosclerotic lesion areas, cholesterol efflux and the expressions of related proteins including ABCA1, SR-BI, ABCG1, LXRα and PPARγ were evaluated. The aortic atherosclerotic lesion areas were reduced more significantly by combination of both drugs than single agent, and cholesterol efflux was promoted more in combination group than simvastatin and L-4F group. Besides, the combination group promoted expressions of cholesterol efflux related proteins. The combination of L-4F and simvastatin reduced atherosclerotic lesions, which stimulates cholesterol efflux by promoting the expressions of related proteins. In addition, these results help us further understand that the regression of the atherosclerosis would be assessed by reduction in LDL-C with increase of cholesterol efflux. Show less

Cholesterol efflux from macrophages is a critical mechanism to prevent the development of atherosclerosis. Here, we sought to investigate the effects of arctigenin, a bioactive component of Arctium lappa, on the cholesterol efflux in oxidized low-density lipoprotein (oxLDL)-loaded THP-1 macrophages. Our data showed that arctigenin significantly accelerated apolipoprotein A-I- and high-density lipoprotein-induced cholesterol efflux in both dose- and time-dependent manners. Moreover, arctigenin treatment enhanced the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, and apoE, all of which are key molecules in the initial step of cholesterol efflux, at both mRNA and protein levels. Arctigenin also caused a concentration-dependent elevation in the expression of peroxisome proliferator-activated receptor-gamma (PPAR-γ) and liver X receptor-alpha (LXR-α). The arctigenin-mediated induction of ABCA1, ABCG1, and apoE was abolished by specific inhibition of PPAR-γ or LXR-α using small interfering RNA technology. Our results collectively indicate that arctigenin promotes cholesterol efflux in oxLDL-loaded THP-1 macrophages through upregulation of ABCA1, ABCG1 and apoE, which is dependent on the enhanced expression of PPAR-γ and LXR-α. Show less

To explore the function of PPAR γ in the goat mammary gland, we cloned the whole cDNA of the PPAR γ gene. Homology alignments revealed that the goat PPAR γ gene is conserved among goat, bovine, mouse, and human. Luciferase assays revealed that rosiglitazone enhanced the activity of the PPAR γ response element (PPRE) in goat mammary epithelial cells (GMECs). After rosiglitazone (ROSI) treatment of GMECs, there was a significant (P < 0.05) increase in the expression of genes related to triacylglycerol synthesis and secretion: LPL, FASN, ACACA, PLIN3, FABP3, PLIN2, PNPLA2, NR1H3, SREBF1, and SCD. The decreases in expression observed after knockdown of PPAR γ relative to the control group (Ad-NC) averaged 65%, 52%, 67%, 55%, 65%, 58%, 85%, 43%, 50%, and 24% for SCD, DGAT1, AGPAT6, SREBF1, ACACA, FASN, FABP3, SCAP, ATGL, and PLIN3, respectively. These results provide direct evidence that PPAR γ plays a crucial role in regulating the triacylglycerol synthesis and secretion in goat mammary cells and underscore the functional importance of PPAR γ in mammary gland tissue during lactation. Show less

Endocrine disrupting chemicals (EDCs) have emerged as a major public health issue because of their potentially disruptive effects on physiological hormonal actions. SXR (steroid xenobiotic receptor), also known as NR1I2, regulates CYP3A expression in response to exogenous chemicals, such as EDCs, after binding to SXRE (SXR response element). In our study, luciferase assay showed that 14 out of 55 EDCs could enhance SXR-mediated rat or human CYP3A gene transcription nearly evenly, and could also activate rat CYP7A1 gene transcription by cross-interaction of SXR and LXRE (LXRα response element). SXR diffused in the nucleus without ligand, whereas intranuclear foci of liganded SXR were produced. Furthermore, endogenous mRNA expression of CYP3A4 gene was enhanced by the 14 positive EDCs. Our results suggested a probable mechanism of EDCs disrupting the steroid or xenobiotic metabolism homeostasis via SXR. Show less

Forkhead box Q1 (FoxQ1) is a member of the forkhead transcription factor family, and it has recently been found to participate in cancer development. However, whether FoxQ1 expression contributes to glioma development and progression is not known. We investigate FoxQ1 expression in gliomas and the role of FoxQ1 during tumorgenesis. Reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot were used to determine the FoxQ1 and Neurexins 3 (NRXN3) expression in gliomas. Chromatin immunoprecipitation (ChIP) and luciferase assays were used to determine the regulation between FoxQ1 and NRXN3. We established depleted FoxQ1 stable clones in U-87MG cells and overexpressed FoxQ1 stable clones in SW1088 cells. MTT and transwell were used to evaluate the ability of proliferation and migration, respectively. FoxQ1 mRNA and protein were up-regulated in gliomas and negatively related to the NRXN3 expression (r = -0.373, P = 0.042). FoxQ1 directly binds to NRXN3 promoter region and suppresses the promoter activity. Furthermore, the ability of proliferation and migration is reduced in depleted FoxQ1 cells. FoxQ1 promotes glioma cell proliferation and migration by down-regulation of NRXN3 expression. Show less

To investigate the relationship between SNPs reported in previous studies and the blood lipid level in the Tibetan population. Random cluster sampling was employed in 5 areas (Lhasa, Shigatse, Shannan, Nagqu, and Nyingchi). The levels of cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) from blood samples were determined and DNA was extracted for genotyping and statistical analyses. Among 1 318 subjects aged >18 years enrolled in this study, 367 had dyslipidemia with a prevalence of 27.8%, of whom dyslipidemia males accounted for 33.1% and dyslipidemia females -24.5%. Results of the correlation analysis between all SNPs and TG showed that the SNPs of rs714052 and rs964184 were related to the serum TG level. Subjects with rs714052 CC genotype had the lowest TG level, and the highest TG level was found in those with rs714052 TT genotype. The serum TG level in individuals with TC genotype lied in between the above two population groups. Subjects with rs964184 CC genotype had the lowest TG level, and the highest serum TG level was noted in those with rs964184 GG genotype. Several SNPs were found to be related to the serum TG level in the Tibetan population. The APOA5 gene and MLXIPL gene may be closely associated with the serum TG level in this ethnic population group. Show less

Triglyceride (TG) is a complex phenotype influenced by both genetic and environmental factors. Recent genome-wide association studies (GWAS) have identified genes or loci affecting lipid levels; however, such studies in Chinese populations are limited. A two-stage GWAS were conducted to identify genetic variants that were associated with TG in a Chinese population of 3495 men. Gene-environment interactions on serum TG levels were further investigated for the seven single nucleotide polymorphisms (SNPs) that were studied in both stages. Two previously reported SNPs (rs651821 in APOA5, rs328 in LPL) were replicated in the second stage, and the combined P-values were 9.19 × 10(-26) and 1.41 × 10(-9) for rs651821 and rs328, respectively. More importantly, a significant interaction between aldehyde dehydrogenase 2 (ALDH2) rs671 and alcohol consumption on serum TG levels were observed (P = 3.34 × 10(-5)). Rs671 was significantly associated with serum TG levels in drinkers (P = 1.90 × 10(-10)), while no association was observed in non-drinkers (P > 0.05). For drinkers, men carrying the AA/AG genotype have significantly lower serum TG levels, compared with men carrying the GG genotype. For men with the GG genotype, the serum TG levels increased with the quantity of alcohol intake (P = 1.28 × 10(-8) for trend test). We identified a novel, significant interaction effect between alcohol consumption and the ALDH2 rs671 polymorphism on TG levels, which suggests that the effect of alcohol intake on TG occurs in a two-faceted manner. Just one drink can increase TG level in susceptible individuals who carry the GG genotype, while individuals carrying AA/AG genotypes may actually benefit from moderate drinking. Show less

Axin1 and its homolog Axin2 are scaffold proteins essential for regulating Wnt signaling. Axin-dependent regulation of Wnt is important for various developmental processes and human diseases. However, the involvement of Axin1 and Axin2 in host defense and inflammation remains to be determined. Here, we report that Axin1, but not Axin2, plays an essential role in host-pathogen interaction mediated by the Wnt pathway. Pathogenic Salmonella colonization greatly reduces the level of Axin1 in intestinal epithelial cells. This reduction is regulated at the posttranslational level in early onset of the bacterial infection. Further analysis reveals that the DIX domain and Ser614 of Axin1 are necessary for the Salmonella-mediated modulation through ubiquitination and SUMOylation. Axin1 apparently has a preventive effect on bacterial invasiveness and inflammatory response during the early stages of infection. The results suggest a distinct biological function of Axin1 and Axin2 in infectious disease and intestinal inflammation while they are functionally equivalent in developmental settings. Show less

There is increasing evidence to show that 2-cell stage mouse blastomeres have differing developmental properties. Additionally, it has been suggested that such a difference might be due to their distribution of mRNA and/or protein asymmetry. However, to date, the exact genes that are involved in the orientation and order of blastomere division are not known. In this study, some differentially expressed transcripts were identified. Axin1, cell division cycle 25 homolog C (Cdc25c) and cyclin-dependent inhibitor 2D (Cdkn2d) were selected for validation by real-time polymerase chain reaction (PCR) based on published data. Our real-time PCR results demonstrated that Axin1, Cdc25c and Cdkn2d genes had different levels of expression among blastomeres of the mouse 2-cell embryo i.e. the level of Axin1 mRNA was significantly higher in one blastomere when compared with the other blastomeres of the 2-cell embryo (p < 0.05). The variation in Cdc25c (p < 0.05) and Cdkn2d (p < 0.01) mRNA expression followed a similar trend to that of Axin1. In addition, the highest levels of expression of these three genes were detected in the same blastomere in the 2-cell embryo. We confirmed that there was an asymmetrical distribution pattern for Axin1, Cdc25c and Cdkn2d transcripts in 2-cell embryos. In conclusion, this study demonstrated clearly that there is embryonic asymmetry at the 2-cell stage and that these differentially expressed genes may result in differentiation in expression in embryo development. Show less

Cln3(Δex7/8) mice harbor the most common genetic defect causing juvenile neuronal ceroid lipofuscinosis (JNCL), an autosomal recessive disease involving seizures, visual, motor and cognitive decline, and premature death. Here, to more thoroughly investigate the manifestations of the common JNCL mutation, we performed a broad phenotyping study of Cln3(Δex7/8) mice. Homozygous Cln3(Δex7/8) mice, congenic on a C57BL/6N background, displayed subtle deficits in sensory and motor tasks at 10-14 weeks of age. Homozygous Cln3(Δex7/8) mice also displayed electroretinographic changes reflecting cone function deficits past 5 months of age and a progressive decline of retinal post-receptoral function. Metabolic analysis revealed increases in rectal body temperature and minimum oxygen consumption in 12-13 week old homozygous Cln3(Δex7/8) mice, which were also seen to a lesser extent in heterozygous Cln3(Δex7/8) mice. Heart weight was slightly increased at 20 weeks of age, but no significant differences were observed in cardiac function in young adults. In a comprehensive blood analysis at 15-16 weeks of age, serum ferritin concentrations, mean corpuscular volume of red blood cells (MCV), and reticulocyte counts were reproducibly increased in homozygous Cln3(Δ) (ex7/8) mice, and male homozygotes had a relative T-cell deficiency, suggesting alterations in hematopoiesis. Finally, consistent with findings in JNCL patients, vacuolated peripheral blood lymphocytes were observed in homozygous Cln3(Δ) (ex7/8) neonates, and to a greater extent in older animals. Early onset, severe vacuolation in clear cells of the epididymis of male homozygous Cln3(Δ) (ex7/8) mice was also observed. These data highlight additional organ systems in which to study CLN3 function, and early phenotypes have been established in homozygous Cln3(Δ) (ex7/8) mice that merit further study for JNCL biomarker development. Show less

Multiple osteochondroma (MO) is an autosomal dominant disease characterized by abnormal skeleton development: one or more exostoses localized mainly at the end of long bones. Three pathogenic gene loci have been identified and cloned: EXT1, 2, and 3. Only EXT1 and 2 mutations were reported to cause MO. Here, we report on a large Chinese family with MO and a disease-causing mutation in EXT. We extracted DNA from peripheral blood samples of 25 family members, 9 with MO. Polymerase chain reaction and direct DNA sequencing of the entire coding regions of EXT1 and 2 for the nine patients revealed a novel pathogenic mutation, insertion of a T in exon 2 (c.72-73 insT) of EXT2. Our results extend the mutational spectrum of MO and can help with genetic counseling and prenatal diagnosis for this family. Show less

Circulating androgen levels are often used as indicators of physiological or pathological conditions. More than half of the variance for circulating androgen levels is thought to be genetically influenced. A genome-wide association study (GWAS) has identified two loci, SHBG at 17p13 and FAM9B at Xp22, for serum testosterone (T) levels; however, these explain only a small fraction of inter-individual variability. To identify additional genetic determinants of androgen levels, a GWAS of baseline serum T and dihydrotestosterone (DHT) levels was conducted in 3225 men of European ancestry from the REduction by DUtasteride of Prostate Cancer Events (REDUCE) study. Cross-validation was used to confirm the observed associations between the drug (n = 1581) and placebo (n = 1644) groups of REDUCE. In addition to confirming the associations of two known loci with serum T levels (rs727428 in SHBG: P = 1.26 × 10(-12); rs5934505 in FAM9B: P = 1.61 × 10(-8)), we identified a new locus, JMJD1C at 10q21 that was associated with serum T levels at a genome-wide significance level (rs10822184: P = 1.12 × 10(-8)). We also observed that the SHBG locus was associated with serum DHT levels (rs727428: P = 1.47 × 10(-11)). Moreover, two additional variants in SHBG [rs72829446, in strong linkage equilibrium with the missense variant D356N (rs6259), and rs1799941] were also independently associated with circulating androgen levels in a statistical scale. These three loci (JMJD1C, SHBG and FAM9B) were estimated to account for ~5.3 and 4.1% of the variance of serum T and DHT levels. Our findings may provide new insights into the regulation of circulating androgens and potential targets for androgen-based therapy. Show less

The liver X receptor α (LXRα) is a nuclear receptor of the transcription factor and is known to play a crucial role in lipid metabolism processes such as bile acid and fatty acid synthesis in humans and rodents. However, very little information is available on the role of LXRα in the regulation of fatty acid synthesis in the goat mammary gland. In this investigation, a cDNA was isolated from the mammary gland of Xinong Saanen dairy goats and designated as goat LXRα. RT-PCR and RACE gave rise to the full-length cDNA of LXRα, which was comprised of 1654 bp and characterized by an ORF of 1344 bp and 5'- and 3'-UTR regions of 150 and 160 bp, respectively. The deduced amino acid sequence encodes 477 amino acids with a predicted molecular weight (MW) of 50.4kDa and a theoretical isoelectric point (pI) of 6.3. Additionally, homology search and sequence multi-alignment indicated that the putative goat LXRα amino acid sequence is very similar to those of cattle, mice, rats, swine, and humans. Bioinformatic predictions demonstrated that the LXRα protein is located in the nucleus, containing characteristic signatures of a nuclear receptor with DNA-binding domain (DBD) and ligand-binding domain (LBD). Real-time quantitative PCR suggested that LXRα was predominantly expressed in the small intestine, liver, spleen and mammary gland. Treatment of goat mammary gland epithelial cells (GMEC) with different concentrations (i.e., 0.01, 0.1, 1 μM) of T0901317, a synthetic agonist of LXRα, resulted in elevated sterol regulatory element binding protein 1 (SREBP1) and fatty acid synthase (FASN) mRNA levels in response to LXRα activation. The association between different T0901317 concentrations and fatty acid composition in GMEC also was examined using gas chromatography (GC). The results showed that activation of LXRα significantly increased GMEC C18:1 and C18:2 contents, but did not affect levels of saturated fatty acids (SFA). These discoveries are consistent with the notion that LXRα plays a key role in controlling lipogenesis and regulating synthesis of unsaturated fatty acids (UFA) in the mammary gland of goats, which may prove useful in regulation of milk fat production. Show less

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Diabetes is known to alter the amount of retinal expression of the water-selective channels aquaporin 4 (AQP4). However, the function and impact of AQP4 in diabetic retinopathy is not well understood. In the present work, diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley rats. Two weeks later, AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were delivered by intravitreal injection to the eyes. Gene delivery was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting analysis. Eight weeks later, BRB breakdown was measured using Evans blue dye. Images of retinal sections were obtained and the thicknesses of the retinas were determined. Retinal leukostasis measurement was performed using acridine orange leukocyte fluorography. The mRNA levels of IL-1β, IL-6, intercellular adhesion molecule 1 (ICAM-1), glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) were determined using qRT-PCR method. AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were transfected into rMC-1 cells to investigate its effect on inflammation induced by high glucose. Incubation with IL-1β or IL-6 was performed to test their effect on AQP4 expression in rMC-1 cells. In the current work, it was found that AQP4 expression was enhanced in the retina of diabetic rats. AQP4 knockdown led to exacerbation of retinopathy including enhancing retinal vascular permeability, retinal thickness, pro-inflammatory factors expression, and VEGF and GFAP expression in retinas of diabetic rats. AQP4 knockdown enhanced the expression of pro-inflammatory cytokines induced by high glucose in rMC-1 cells. In addition, AQP4 knockdown enhanced the release of IL-6 and VEGF from rMC-1 cells into the medium. Moreover, it was found that incubation with IL-1β or IL-6 suppressed AQP4 expression in rMC-1 cells. These results suggested that streptozotocin injection induced diabetes resulted in compensatory increases of AQP4 expression, and downregulation of AQP4 exacerbated diabetic retinopathy through aggravating inflammatory response, at last in part. Therefore, regulation of retinal function by AQP4 may attenuate diabetic retinopathy, offering a promising therapeutic strategy for diabetic retinopathy. Show less

Hepatocellular carcinoma (HCC) is believed to arise from tumor-initiating cells (T-ICs), although little is known about their stem cell-like properties. We quantified levels of p28(GANK) (Gankyrin), OV6, and Oct4 in 130 human HCC samples using immunohistochemistry. Magnetic-activated cell sorting was used to isolate OV6+ HCC cells. T-IC properties were evaluated by quantitative reverse-transcription polymerase chain reaction, flow cytometry, and spheroid formation. We used a coimmunoprecipitation assay to study interactions among p28(GANK), Oct4, and WWP2. Tumorigenicity and pulmonary metastasis were examined in nonobese diabetic and severe combined immunodeficient mice. In HCC samples, high levels of p28(GANK) correlated with expansion of OV6+ tumor cells; the combination of high levels of p28(GANK) and OV6 was associated with progression of HCC. p28(GANK) was predominantly expressed in liver T-ICs, isolated by magnetic sorting, and undifferentiated primary HCC spheroids. Increased levels of p28(GANK) in T-ICs increased their percentages in HCC samples, expression of stem cell genes, self-renewal potential, chemoresistance in vitro, and tumorigenicity and ability to develop into pulmonary metastases in mice. Conversely, knockdown of p28(GANK) reduced their T-IC properties. p28(GANK) likely activates liver T-ICs by impeding ubiquitination and degradation of the transcription factor Oct4 by WWP2. In support of this concept, levels of p28(GANK) correlated with those of Oct4 in HCC samples. p28(GANK) activates and maintains liver T-ICs in HCCs by preventing degradation of Oct4. Inhibitors of p28(GANK) might therefore be developed to inactivate T-ICs and slow tumor progression. Show less

OBJECTIVE The metabolic syndrome (MetS) is defined as concomitant disorders of lipid and glucose metabolism, central obesity, and high blood pressure, with an increased risk of type 2 diabetes and cardiovascular disease. This study tests whether common genetic variants with pleiotropic effects account for some of the correlated architecture among five metabolic phenotypes that define MetS. RESEARCH DESIGN AND METHODS Seven studies of the STAMPEED consortium, comprising 22,161 participants of European ancestry, underwent genome-wide association analyses of metabolic traits using a panel of ∼2.5 million imputed single nucleotide polymorphisms (SNPs). Phenotypes were defined by the National Cholesterol Education Program (NCEP) criteria for MetS in pairwise combinations. Individuals exceeding the NCEP thresholds for both traits of a pair were considered affected. RESULTS Twenty-nine common variants were associated with MetS or a pair of traits. Variants in the genes LPL, CETP, APOA5 (and its cluster), GCKR (and its cluster), LIPC, TRIB1, LOC100128354/MTNR1B, ABCB11, and LOC100129150 were further tested for their association with individual qualitative and quantitative traits. None of the 16 top SNPs (one per gene) associated simultaneously with more than two individual traits. Of them 11 variants showed nominal associations with MetS per se. The effects of 16 top SNPs on the quantitative traits were relatively small, together explaining from ∼9% of the variance in triglycerides, 5.8% of high-density lipoprotein cholesterol, 3.6% of fasting glucose, and 1.4% of systolic blood pressure. CONCLUSIONS Qualitative and quantitative pleiotropic tests on pairs of traits indicate that a small portion of the covariation in these traits can be explained by the reported common genetic variants. Show less

To investigate the association between the polymorphisms of fatty acid desaturase 1 (FADS1), fatty acid desaturase 2 (FADS2) and elongation of very long chain fatty acids like 2 (ELOVL2) gene and coronary artery disease (CAD) in a Chinese Han population. Three single nucleotide polymorphisms (SNPs) from these genes were genotyped using PCR-based restriction fragment length polymorphism analysis in 199 CAD cases and 192 controls of Han Chinese origin. rs174556 in the FADS1 gene showed allelic (P=0.002) and genotypic (P=0.030) association with the disease, while there was no disease association for the other two SNPs. The frequency of rs174556 minor allele (T) was significantly higher in the case group than the control group. The trans phase gene-gene interaction analysis showed that the combined genotype of rs174556 (T/T) and rs3756963 (T/T) was weakly associated with the disease (P=0.043). rs174556 in the FADS1 gene is very likely to be associated with CAD in the Chinese Han population. Show less

To investigate an association between gene polymorphisms of FADS 1 (fatty acid desaturase 1) , FADS2 (fatty acid desaturase 2) and ELOVL2 (elongation of very long chain fatty acids (FEN1/ Elo2, SUR4/Elo3, yeast) -like 2) and paranoid schizophrenia of the Han ethnicity in Jilin province of China. We genotyped 3 single nucleotide polymorphisms from 3 candidate genes in 100 paranoid schizophrenia cases and 95 healthy controls using polymerase chain reaction-restriction fragment length polymorphism method. All samples are from Han ethnicity in Jilin province of China. The genotype distributions of rs174556 in FADS1 gene and rs174617 in FADS2 gene showed no significant difference between case and control groups (P > 0.05). The genotype distribution of rs3756963 in ELOVL2 gene showed significant difference between case group and control group (P < 0.05). The distribution proportion of allele T carriers in case group was higher than that in control group. Trans-phase gene interaction analysis showed that the distribution proportion of combined genotypes included rs3756963 (T/T) was higher in case group than that in control group (P < 0.05). rs174556 in FADS1 gene and rs174617 in FADS2 gene may not be associated with paranoid schizophrenia. rs3756963 in ELOVL2 gene may be associated with paranoid schizophrenia. Show less

Our previous work suggested that there was no significant association between plasma steroid hormone levels and prostate cancer tumor grade at diagnosis. In this study, we systematically tested the hypothesis that inherited variations in the androgen and estrogen metabolic pathways may be associated with plasma levels of steroid hormones, or prostate cancer aggressiveness at diagnosis. Plasma hormone levels including total testosterone, total estradiol, and sex hormone-binding globulin were measured in a cohort of 508 patients identified with localized prostate cancer. D'Amico risk classification at diagnosis was also determined. A total of 143 single-nucleotide polymorphisms (SNPs) from 30 genes that are involved in androgen and estrogen metabolism were selected for analysis. The global association of genotypes with plasma hormone levels and prostate cancer aggressiveness (D'Amico risk classification) was statistically analyzed. Q values were estimated to account for multiple testing. We observed significant associations between plasma testosterone level and SNPs in HSD17B2 (rs1424151), HSD17B3 (rs9409407), and HSD17B1 (rs12602084), with P values of 0.002, 0.006, and 0.006, respectively. We also observed borderline significant associations between prostate aggressiveness at diagnosis and SNPs in AKR1C1 (rs11252845; P = 0.005), UGT2B15 (rs2045100; P = 0.007), and HSD17B12 (rs7932905; P = 0.008). No individual SNP was associated with both clinical variables. Genetic variants of genes in hormone metabolic pathways may influence plasma androgen levels or prostate cancer aggressiveness. However, it seems that the inherited variations affecting plasma hormone levels differ from those affecting disease aggressiveness. Show less

The use of LINGO-1 antagonists to promote repair of damaged myelin is an emerging therapeutic opportunity for treatment of CNS diseases caused by demyelination such as multiple sclerosis. The Li33 anti-LINGO-1 antibody is a potent inducer of myelination in vitro and in vivo, but aggregation issues prevented the engineering of an optimal development candidate. PEGylated Li33 Fab' is one of several versions of the Li33 antibody that is being investigated in an attempt to identify the most favorable anti-LINGO-1 antibody design. For targeted PEGylation, a Li33 Fab' construct was engineered with a single unpaired cysteine in the heavy-chain hinge sequence. The Fab' was expressed in CHO cells, purified, and PEGylated with 20 kDa methoxy-poly(ethylene glycol) maleimide using a reaction strategy optimized to improve the yield of the PEG-Fab'. Biochemical analysis of the Li33 PEG-Fab' verified the selectivity of the PEGylation reaction. The in vitro and in vivo attributes of the PEG-Fab' were benchmarked against a Li33 full antibody. Both the Li33 PEG-Fab' and intact antibody bound LINGO-1 with nanomolar affinity, promoted myelination in an in vitro signaling assay, and promoted the repair of damaged myelin in the rat lysolecithin model. These studies extend our understanding of the biological activity of the Li33 mAb and validate the use of an anti-LINGO-1 PEG-Fab' for treatment of CNS diseases caused by demyelination. Show less

Fms-like tyrosine kinase-3 (FLT3) is a growth factor receptor normally expressed on hematopoietic progenitor cells. Approximately one third of all patients with AML carry an activating mutation in FLT3 that drives proliferation and survival of the leukemic cells. The most common activating mutation is the so-called internal tandem duplication (ITD), which involves an in-frame duplication of a segment of varying length in the region of the FLT3 gene that encodes the juxtamembrane domain. The pathways downstream of FLT3-ITD are partially known but further knowledge regarding the downstream signal transduction molecules is important in order to develop alternative strategies for pharmacological intervention. In this paper we have studied the role of MEK/ERK5 in FLT3-ITD mediated transformation. We have found that both wild-type FLT3 and FLT3-ITD activate MEK5 leading to the activation of ERK5. By use of the selective inhibitor of MEK5, BIX02188, we have shown that activation of AKT downstream of FLT3 is partially dependent on ERK5. Furthermore, inhibition of MEK5/ERK5 induces apoptosis of both FLT3-ITD transfected Ba/F3 cells as well as the FLT3-ITD carrying leukemic cell lines MV4-11 and MOLM-13. These results suggest that MEK5/ERK5 is important for FLT3-ITD induced hematopoietic transformation and may thus represent an alternative therapeutic target in the treatment of FLT3-ITD positive leukemia. Show less

Liver X receptor α (LXRα) and sterol regulatory element binding protein-1c (SREBP-1c) were studied in rats with non-alcoholic steatohepatitis (NASH) induced by a high-fat diet. Forty 5-week-old rats were fed either a high-fat diet (n = 30) or a normal diet (n = 10) for 9, 13 or 17 weeks. The mRNA and protein levels for LXRα and SREBP-1c were measured at each time point, as was fatty acid synthase (FAS) activity and the serum levels of free fatty acid (FFA) and triglyceride (TG). The mRNA and protein levels for LXRα and SREBP-1c, FAS activity and serum levels of FFA and TG all significantly increased from week 9 in the high-fat diet rats versus controls. In conclusion, a high-fat diet upregulates LXRα which, in turn, upregulates SREBP-1c, increasing the activity of FAS and FFA and accumulation of TG in hepatocytes. Thus, LXRα and SREBP-1c contribute to the development of NASH. Show less