Coordinate regulation by transcription factors and DNA methylation in the core promoter region of SIRT6 in bovine adipocytes.

Sirtuin6 (SIRT6) is an ADP-ribosyltransferase and NAD+-dependent deacylase of acetyl groups and long-chain fatty acyl groups, and has been shown as a regulator of insulin secretion, glucose metabolism, lipid metabolism, and cancer. In this study, we determined that the bovine SIRT6 showed higher levels of mRNA expression in the testis, longissimus thoracis, and subcutaneous fat tissue. To elucidate the molecular regulation mechanism of bovine SIRT6 expression, we obtained a 2-kb fragment containing the 5'-regulatory region, and the functional proximal minimal promoter of bovine SIRT6 was identified in the -472/-73 bp region. The CCAAT enhancer binding protein beta (CEBPß), paired box 6 (PAX6), Kruppel-like factor 2 (KLF2), myb proto-oncogene protein (CMYB), nuclear respiratory factor 1 (NRF1), and E2F transcription factor 1 (E2F1) binding sites, as transcriptional activators or repressors in the core promoter region of SIRT6, were determined by electrophoretic mobility shift assay (EMSA) experiments and luciferase reporter assays. In addition, the results from methylation assay and luciferase report assay showed that the bovine SIRT6 promoter activity was coordinately regulated by methylation and NRF1 or E2F1 during bovine adipocyte differentiation. Taken together, this study illuminated the underlying mechanism of methylation and transcription regulation of SIRT6 expression in bovine adipocytes.

Myocyte enhancer factor 2A promotes proliferation and its inhibition attenuates myogenic differentiation via myozenin 2 in bovine skeletal muscle myoblast.

Myocyte enhancer factor 2A (MEF2A) is widely distributed in various tissues or organs and plays crucial roles in multiple biological processes. To examine the potential effects of MEF2A on skeletal muscle myoblast, the functional role of MFE2A in myoblast proliferation and differentiation was investigated. In this study, we found that the mRNA expression level of Mef2a was dramatically increased during the myogenesis of bovine skeletal muscle primary myoblast. Overexpression of MEF2A significantly promoted myoblast proliferation, while knockdown of MEF2A inhibited the proliferation and differentiation of myoblast. RT-PCR and western blot analysis revealed that this positive effect of MEF2A on the proliferation of myoblast was carried out by triggering cell cycle progression by activating CDK2 protein expression. Besides, MEF2A was found to be an important transcription factor that bound to the myozenin 2 (MyoZ2) proximal promoter and performed upstream of MyoZ2 during myoblast differentiation. This study provides the first experimental evidence that MEF2A is a positive regulator in skeletal muscle myoblast proliferation and suggests that MEF2A regulates myoblast differentiation via regulating MyoZ2.

Transcriptome analysis of mRNA and microRNAs in intramuscular fat tissues of castrated and intact male Chinese Qinchuan cattle.

Intramuscular fat (IMF) is known to enhance beef palatability and can be markedly increased by castration. However, there is little understanding of the molecular mechanism underlying the IMF deposition after castration of beef cattle. We hypothesize that genetic regulators function differently in IMF from bulls and steers. Therefore, after detecting serum testosterone and lipid parameter, as well as the contents of IMF at 6, 12, 18 and 24 months, we have investigated differentially expressed (DE) microRNAs (miRNAs) and mRNAs in IMF of bulls and steers at 24 months of age in Qinchuan cattle using next-generation sequencing, and then explored the possible biopathways regulating IMF deposition. Serum testosterone levels were significantly decreased in steers, whereas IMF content, serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TGs) were markedly increased in steers. Comparing the results of steers and bulls, 580 upregulated genes and 1,120 downregulated genes in IMF tissues were identified as DE genes correlated with IMF deposition. The upregulated genes were mainly associated with lipid metabolism, lipogenesis and fatty acid transportation signalling pathways, and the downregulated genes were correlated with immune response and intracellular signal transduction. Concurrently, the DE miRNAs-important players in adipose tissue accumulation induced by castration-were also examined in IMF tissues; 52 DE miRNAs were identified. The expression profiles of selected genes and miRNAs were also confirmed by quantitative real-time PCR (qRT-PCR) assays. Using integrated analysis, we constructed the microRNA-target regulatory network which was supported by target validation using the dual luciferase reporter system. Moreover, Ingenuity Pathway Analysis (IPA) software was used to construct a molecular interaction network that could be involved in regulating IMF after castration. The detected molecular network is closely associated with lipid metabolism and adipocyte differentiation, which is supported by functional identification results of bta-let-7i on bovine preadipocytes. These results provided valuable insights into the molecular mechanisms of the IMF phenotype differences between steers and bulls.

[Concentration and Particle Size Distribution of Microbiological Aerosol During Haze Days in Beijing].

In this study, we evaluated the bacterial, fungal aerosol concentration, and particle size distribution using microbiological aerosol sampler, and analyzed the particles count concentration of PM1.0, PM2.5, PM5.0 and PM10.0 using aerodynamic particle sizer during clear and haze days in Beijing during Jan 8th, 2013 to Feb 4th, 2013. The concentration of bacterial, fungal aerosol, air particulate matter and aerosol distribution were compared between haze days and clear days. Our results indicated that the proportion of fungal particles smaller than 5 micron, which could deposit in lungs or deeper regions, was much higher than bacterial particles. The biological concentration of bacteria and fungi were higher in clear days than in haze days, and there was no statistic difference of the microbiological aerosol distribution. The concentration of air particulate matter were higher in haze days than in clear days, PM10 was the main particulate matters both in clear days and haze days.

TNFRSF11B polymorphisms are associated with metabolic traits in Uyghur and Han ethnic groups.

UNLABELLED: Abstract Introduction: To test whether genetic variants of osteoprotegerin gene (TNFRSF11B) affect metabolic traits (body mass index [BMI], glucose, triglyceride, total cholesterol) and bone mass traits. METHODS: We conducted a population based association study to investigate associations of eight tagging single nucleotide polymorphisms (tSNPs) of the TNFRSF11B gene with the aforementioned traits in a Chinese Han population and an ethnic group admixed with Caucasians and Asians - Uyghur. The associations between the tSNPs and bone mass density (BMD) were also tested in Han population. RESULTS: We found that SNP rs3102727, located in the first intron of the TNFRSF11B gene, was significantly associated with triglyceride levels in Uyghur population and Han population simultaneously. T allele of the rs3102727 variant was associated with a 0.10 mmol/L and 0.09 mmol/L lower level of triglyceride than C allele in Uyghur (p = 0.019) and Han subjects (p = 0.037), respectively. In addition, the T allele is also associated with a lower level of hip BMD (p = 0.025) and total BMD (p = 0.048). Further, we found significant associations between SNP rs11573869 and BMI in Uyghur subjects and SNP rs3134062 with hip BMD in Han sbujects. Rs11573869-T allele was associated with a 0.81 kg/m(2) lower level of BMI than C allele (p = 0.002) and the hip BMD decreases with the copy of rs3134062-T allele increases (p = 0.002). CONCLUSION: We detected novel associations between TNFRSF11B polymorphisms and metabolic traits in Uyghur and Han populations. In addition, we found associations between TNFRSF11B polymorphisms and bone mass traits in Han population.

[Value of adrenal venous sampling in the subtype diagnosis of primary aldosteronism].

OBJECTIVE: To assess the diagnostic value of adrenal venous sampling (AVS) in the subtype diagnosis of primary aldosteronism (PA). METHODS: The diagnosis of PA was made in 36 patients based on an elevated ratio of plasma aldosterone (ALD) to plasma rennin activity (PRA) (ARR) and confirmed tests (saline infusion or captopril challenge) in recent 3 years. All PA patients underwent adrenal computed tomographic scan (CT) and AVS. The diagnostic accuracy of CT and AVS in the subtype differentiation of PA were evaluated by comparing the differences of CT findings, AVS results and clinical outcomes. RESULTS: Fifteen of 36 patients (42%) had a final diagnosis of aldosterone-producing adenoma (APA) and another 21 patients (58%) with bilateral adrenal hyperplasia (BAH). The level of ALD was significantly higher in APA group than that in BAH group (298.9 ± 91.0 vs 226.3 ± 59 ng/L, P < 0.05). PRA (ng×ml(-1)×h(-1)) in APA patients were markedly lower than that in BAH counterparts (0.18 ± 0.14 vs 0.28 ± 0.29 ng×ml(-1)×h(-1), P < 0.01). Consequently, ARR in APA group was evidently higher than that in BAH group (2444.7 ± 1405.2 vs 1550.0 ± 1059.8, P < 0.05). Plasma potassium in APA patients was lower than that in those with BAH (2.71 ± 0.57 vs 3.17 ± 0.40 mmol/L). But there was no statistic significance (P > 0.05). The CT findings were discordant with the AVS results in 27.8% of patients (10/36). The accuracy of adrenal CT scan was only 72.2% in the subtype diagnosis of PA, provided AVS was the gold standard for distinguishing between APA and BAH. Reliance on CT findings could lead to inappropriate management in 25% of PA patients. Conversely, the AVS results were concordant with the clinical outcomes in 94.4% of all patients. CONCLUSION: CT scan is not a reliable method of differentiating primary aldosteronism. Compared with CT, AVS is more accurate in establishing a correct diagnosis of primary aldosteronism. AVS should be performed routinely before operation in PA patients opting for adrenalectomy.

Effect of rosuvastatin on ROCK activity, endothelial function, and inflammation in Asian patients with atherosclerosis.

BACKGROUND: HMG-CoA reductase inhibitors (statins) inhibit cholesterol biosynthesis, and also decrease the formation of isoprenoid intermediates required for the activation of Rho kinase (ROCK) pathway. ROCK pathway plays pivotal roles in cardiovascular diseases including arteriosclerosis. It has been implicated that inhibition of ROCK can reverse vascular dysfunction in humans with atherosclerosis. However, it is not clear whether statins, at doses used to lower cholesterol levels, inhibit ROCK activity in humans with atherosclerosis. METHODS: We treated 40 subjects with stable atherosclerosis with rosuvastatin 10 mg/day, or rosuvastatin 40 mg/day for 28 days in a randomized, double-blinded study. We assessed the change in the lipid levels, C-reactive protein (CRP), ROCK activity, and flow-mediated dilation (FMD) of the brachial artery before and after statins therapy. RESULTS: Treatment with rosuvastatin 10 mg and 40 mg significantly reduced LDL cholesterol by 43.2% to 55.9% and increased FMD by 29.3% to 42.5% (p<0.05 for both compared with baselines). Both doses inhibited ROCK activity (p<0.05), and the extent of inhibition was greater with rosuvastatin 40 mg compared with 10 mg (p<0.05). Only rosuvastatin 40 mg significantly reduced hsCRP (p<0.05).There was no correlation between changes in ROCK activity and changes in low-density lipoprotein cholesterol (r=0.37, p>0.05 vs. r=0.41, p>0.05) among patients randomized to rosuvastatin 10 mg group or 40 mg group. There was a correlation between ROCK inhibition and change in FMD among patients with rosuvastatin 10 mg therapy (r=0.43, p<0.05), and 40 mg therapy (r=0.54, p<0.05). Correlation was found between changes in ROCK inhibition and changes in CRP in rosuvastatin 40 mg/day group (r=0.47, p<0.05). CONCLUSION: These results demonstrate that high dose rosuvastatin exerts greater effects on LDL-C, ROCK activity, and CRP than low dose rosuvastatin. These findings provide clinical evidence that statins are effective in improving endothelium dysfunction by a cholesterol-independent mechanism in patients with atherosclerosis.

[Effects of heart rate variability and smoothness index on the reversal of hypertensive left ventricular hypertrophy].

OBJECTIVE: To investigate the relationship of reversal of hypertensive left ventricular hypertrophy (LVH) with heart rate variability (HRV) and smoothness index (SI). METHODS: A total of 127 patients with untreated essential hypertension associated with LVH were enrolled to receive a 20-week treatment. The drugs included losartan potassium & hydrochlorothiazide (1 tablet/day) and metoprolol (12.5 mg - 50 mg twice daily). The sitting systolic and diastolic blood pressures (SBP & DBP), M-mode and pulsed Doppler echocardiography, 24-hour ambulatory blood pressure monitoring (ABPM) and 24-hour ambulatory ECG (Holter) were performed at pre- and post-treatment. The changes in various parameters such as echocardiography left ventricular end-systolic dimension (LVDs), left ventricular end-diastolic dimension (LVDd) and the thickness of interventricular septum (IVST) and posterior wall (PWT) were measured. And left ventricular mass index (LVMI) and smoothness index (SI) were also examined. The evaluated parameters of ABPM were average 24-hour, daytime and nighttime SBP & DBP. As to 24-hour ambulatory ECG (Holter), the parameters were standard deviation of normal to normal intervals (SDNN), rate mean square of the differences of successive RR intervals (RMSSD), percentage of RR intervals differing > 50 ms (PNN50), high frequency (HF) and low frequency (LF). RESULTS: After a 20-week treatment, the levels of sitting blood pressure (SBP 158.72 ± 12.11 mm Hg vs 132.21 ± 14.03 mm Hg; DBP 97.20 ± 7.71 vs 86.36 ± 6.48 mm Hg, P < 0.001), parameters of 24-hour ABPM (24-hour mean SBP 146.20 ± 10.11 mm Hg vs 129.68 ± 6.12 mm Hg, P < 0.001; 24-hour mean DBP 93.45 ± 5.46 mm Hg vs 81.77 ± 6.71 mm Hg, P < 0.01; daytime mean SBP 149.53 ± 8.67 mm Hg vs 133.60 ± 6.27 mm Hg, P < 0.001; daytime mean DBP 94.68 ± 4.96 mm Hg vs 83.55 ± 7.03 mm Hg, P < 0.001; nighttime mean SBP 137.21 ± 8.73 mm Hg vs 122.74 ± 7.58 mm Hg, P < 0.001; nighttime mean DBP 86.75 ± 6.22 mm Hg vs 72.81 ± 5.47 mm Hg, P < 0.001) and LVMI significantly decreased (128.90 ± 32.35 g/m(2) vs 118.39 ± 31.10 g/m(2), P < 0.01) while the indicators of HRV changes such as SDNN, RMSSD, PNN50 and HF significantly increased (SDNN 97.28 ± 16.67 ms vs 152.27 ± 34.23 ms, P < 0.01; RMSSD 21.32 ± 8.70 ms vs 41.91 ± 10.38 ms, P < 0.001; PNN50 3.17 ± 1.23 vs 5.89 ± 2.18, P < 0.01; HF 239.82 ± 98.26 ms(2)/Hz vs 367.32 ± 188.37 ms(2)/Hz, P < 0.01)accompanied by the decreases in LF and LF/HF (LF 485.22 ± 217.34 ms(2)/Hz vs 287.94 ± 128.61 ms(2)/Hz, P < 0.01; LF/HF 2.03 ± 0.56 vs 0.79 ± 0.38, P < 0.001). The post-treatment SIs of SBP and DBP were 1.35 and 1.2 respectively. CONCLUSION: The combination treatment of angiotensin II receptor blocker (ARB), diuretics and ß1-receptor blockers can lower the blood pressures stably, improve heart rate variability and lead to a reversal of hypertensive LVH.

Three novel SNPs in the coding region of PPARγ gene and their associations with meat quality traits in cattle.

The peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear hormone receptor that regulates adipogenesis and many other biological processes. In the present study, we carried out PCR-SSCP and DNA sequencing analyses to examine SNPs in coding region of the PPARγ gene. A total of 660 individuals from five Chinese cattle breeds were genotyped. We identified three SNPs and their associations with meat quality traits were analyzed in 108 Qinchuan cattle. Two missense mutations and one synonymous mutation were found: 200 A>G (genotypes AA, AB and BB) resulting in D7G change, the silent substitution 42895 C>T (genotypes JJ and JI) and 72472 G>T (genotypes CC, DC and DD) producing Q448H change, respectively. The frequencies of PPARγ-A allele were 0.86, 0.83, 0.80, 0.72 and 0.87 for Qinchuan, Nanyang, Jiaxian, Luxi and Xianan populations, respectively. The frequencies of PPARγ-J allele varied from 0.87 to 0.96 in the five populations. In the 72472 G>T locus, the frequencies of PPARγ-C allele were higher than PPARγ-D allele in the five populations, and ranged from 0.58 to 0.82. Least squares analysis revealed that in 42895 C>T locus, there was a significant effect on tenderness in 18-20 months Qinchuan cattle (P<0.01), and in the 72472 G>T locus, animals with the genotype DC had lower mean values than these with genotype CC (P<0.01) for back fat thickness in 18-20 months, and animals with the genotype DD had lower mean values than these with genotypes CC and DC (P<0.01) for water holding capacity in 21-24 months (P<0.01). The SNPs we have identified may contribute to establishing a more efficient selection program for improving of genetic characteristics in indigenous Chinese cattle.

[Genome array on differentially expressed genes of muscle tissue in intact male and castrated Qinchuan cattle].

Bovine genome array was used to construct gene expression profile to screen differentially expressed genes of the Longuissimus dorsi muscle (LDM) tissue between intact male and castrated Qinchuan cattle and investigate the molecular mechanism related to meat quality differences between the two groups. Significance Analysis of Microarray (SAM) methods was used to identify the differentially expressed genes. Go (gene ontology) and the pathway analyses were conducted on differentially expressed genes using a free web-based Molecular Annotation System 2.0 (MAS 2.0). About 11,000 probe sets represented about 10,000 genes were detected in LDM of 36 months old Qinchuan cattle. A total of 143 genes were identified to be differentially expressed genes. They were mainly involved in collagen fibril organization and synthesis, regulation of cell growth and development, ubiquitin-dependent protein catabolism, and striated muscle contraction etc. The enriched pathways mainly included ECM-receptor interaction, cell communication, and focal adhesion etc. Subsequently, real-time PCR was performed to validate eight differentially expressed genes screened out by the microarray approach and sufficient consistency was observed between the two methods. According to our study and published papers, the regulated pathways including ECM-receptor interaction, cell communication, focal adhesion, tight junction and genes including COL3A1, COL1A1, COL1A2, SPP1, FBN1, MMP2, ECM1, MYH3, MYH8, S100A4, ASPN, CFD etc were considered as the most important pathways and genes involved in meat quality differences between males and castrated Qinchuan cattle. Moreover, some genes had no annotation in GenBank were screened out, which were presumed to be unknown new genes. The roles that they may play in muscle metabolism need to be clarified in future.

Profiling of chicken adipose tissue gene expression by genome array.

BACKGROUND: Excessive accumulation of lipids in the adipose tissue is a major problem in the present-day broiler industry. However, few studies have analyzed the expression of adipose tissue genes that are involved in pathways and mechanisms leading to adiposity in chickens. Gene expression profiling of chicken adipose tissue could provide key information about the ontogenesis of fatness and clarify the molecular mechanisms underlying obesity. In this study, Chicken Genome Arrays were used to construct an adipose tissue gene expression profile of 7-week-old broilers, and to screen adipose tissue genes that are differentially expressed in lean and fat lines divergently selected over eight generations for high and low abdominal fat weight. RESULTS: The gene expression profiles detected 13,234-16,858 probe sets in chicken adipose tissue at 7 weeks, and genes involved in lipid metabolism and immunity such as fatty acid binding protein (FABP), thyroid hormone-responsive protein (Spot14), lipoprotein lipase(LPL), insulin-like growth factor binding protein 7(IGFBP7) and major histocompatibility complex (MHC), were highly expressed. In contrast, some genes related to lipogenesis, such as leptin receptor, sterol regulatory element binding proteins1 (SREBP1), apolipoprotein B(ApoB) and insulin-like growth factor 2(IGF2), were not detected. Moreover, 230 genes that were differentially expressed between the two lines were screened out; these were mainly involved in lipid metabolism, signal transduction, energy metabolism, tumorigenesis and immunity. Subsequently, real-time RT-PCR was performed to validate fifteen differentially expressed genes screened out by the microarray approach and high consistency was observed between the two methods. CONCLUSION: Our results establish the groundwork for further studies of the basic genetic control of growth and development of chicken adipose tissue, and will be beneficial in clarifying the molecular mechanism of obesity in chickens.

[Beta1-adrenergic receptor (Arg389Gly) polymorphism and response to bisoprolol in patients with chronic heart failure].

OBJECTIVE: The purpose of this study was to investigate the relation between the Arg389Gly polymorphism of the beta(1)-AR gene and chronic heart failure (CHF) and to evaluate the effect of this polymorphism on clinical response to beta-adrenoceptor blockade (bisoprolol) in patients with CHF. METHODS: One hundred and ten patients with stable CHF receiving basic therapy for heart failure were included. Before initiation and 3 months after the maximal tolerated dose of bisoprolol was reached, all indices (including BP, HR, LAD, LVEDD, LVESD, LVEF, BNP level, 6 min walk distance) were measured and compared with the Arg389Gly genotypes, which identified by PCR-restriction fragment length polymorphism analysis. We also determined the Arg389Gly genotypes in 100 healthy control subjects, and compared the distribution of Arg389Gly genotypes with that in CHF. RESULTS: No difference was observed between the two groups in any of the three genotypes (CC, CG and GG). The prevalences of the three genotypes in normal subjects and patients with CHF were Arg389Arg 0.53 vs. 0.51, Arg389Gly 0.40 vs. 0.40, Gly38Gly 0.07 vs. 0.09, respectively. After 3 months of bisoprolol usage, a significant improvement in LVEF was observed in CC group, which increased from (36.7 +/- 8.63)% to (44.1 +/- 9.53)%, CG group, from (35.76 +/- 8.39)% to (42.90 +/- 9.41)%, but not GG group, from (36.00 +/- 5.66)% to (37.33 +/- 5.64)%. The improvement in BNP was also observed in CC [from (502.93 +/- 160.80) ng/L to (325.26 +/- 135.63) ng/L], CG [from (525.76 +/- 157.66) ng/L to (331.79 +/- 133.97) ng/L], but not GG [from (505.33 +/- 125.07) ng/L to (429.67 +/- 182.39) ng/L]. Arg389-homozygous patients showed a substantially greater improvement in LVEF and BNP, compared with Gly389-homozygous patients (all P < 0.01). CONCLUSIONS: There was no difference in the prevalence of the three genotypes between healthy and CHF subjects. The Gly389 polymorphism of the beta(1)-AR gene was not associated with an increased risk of CHF. The Arg389 variant of the beta(1)-AR gene was associated with a greater response to bisoprolol than that of the Gly389 variant in patients with CHF.

[CDNA microarray on differentially expressed genes of adipose tissue in two breeds chicken].

cDNA microarray containing 9024 cDNAs was used to construct gene expression profile in order to screen differentially expressed genes of adipose tissue between broiler and Bai' er and investigate the molecular mechanism related with body fatness traits between the two breeds. Sixty seven differentially expressed genes, being involved in fat metabolism, energy metabolism, cytoskeleton, transcription and splicing factor, protein synthesis and degradation, were screened out. Furthermore, some genes that had no annotation in GenBank were screened out, they were presumed to be unknown new genes. The roles that they may play in chicken fat metabolism need clarify later.