Generation of Five Human Lactoferrin Transgenic Cloned Goats Using Fibroblast Cells and Their Methylation Status of Putative Differential Methylation Regions of IGF2R and H19 Imprinted Genes

Background

Somatic cell nuclear transfer (SCNT) is a promising technique to produce transgenic cloned mammalian, including transgenic goats which may produce Human Lactoferrin (hLF). However, success percentage of SCNT is low, because of gestational and neonatal failure of transgenic embryos. According to the studies on cattle and mice, DNA methylation of some imprinted genes, which plays a vital role in the reprogramming of embryo in NT maybe an underlying mechanism.

Methodology/Principal Findings

Fibroblast cells were derived from the ear of a two-month-old goat. The vector expressing hLF was constructed and transfected into fibroblasts. G418 selection, EGFP expression, PCR, and cell cycle distribution were applied sequentially to select transgenic cells clones. After NT and embryo transfer, five transgenic cloned goats were obtained from 240 cloned transgenic embryos. These transgenic goats were identified by 8 microsatellites genotyping and southern blot. Of the five transgenic goats, 3 were lived after birth, while 2 were dead during gestation. We compared differential methylation regions (DMR) pattern of two paternally imprinted genes (H19 and IGF2R) of the ear tissues from the lived transgenic goats, dead transgenic goats, and control goats from natural reproduction. Hyper-methylation pattern appeared in cloned aborted goats, while methylation status was relatively normal in cloned lived goats compared with normal goats.

Conclusions/Significance

In this study, we generated five hLF transgenic cloned goats by SCNT. This is the first time the DNA methylation of lived and dead transgenic cloned goats was compared. The results demonstrated that the methylation status of DMRs of H19 and IGF2R were different in lived and dead transgenic goats and therefore this may be potentially used to assess the reprogramming status of transgenic cloned goats. Understanding the pattern of gene imprinting may be useful to improve cloning techniques in future.     

Effects of Different Grazing Intensities on Grassland Production in China: A Meta-Analysis

Background

Grazing is one of the main grassland disturbances in China, and it is essential to quantitatively evaluate the effects of different grazing intensities on grassland production for grassland carbon budget and sustainable use.

Methods

A meta-analysis was conducted to reveal general response patterns of grassland production to grazing in China. We used weighted log response ratio to assess the effect size, and 95% confidence intervals to give a sense of the precision of the estimate. Grazing effects were estimated as a percentage change relative to control (%).

Results

A total of 48 studies, including 251 data sets, were included in the meta-analysis. Grazing significantly decreased total biomass by 58.34% (95% CI: −72.04%∼−37.94%, CI: Confidence Interval), increased root/shoot ratio by 30.58% and decreased litter by 51.41% (95% CI: −63.31%∼−35.64%). Aboveground biomass and belowground biomass decreased significantly by 42.77% (95% CI: −48.88%∼−35.93%) and 23.13% (95% CI: −39.61%∼−2.17%), respectively. However, biomass responses were dependent on grazing intensity and environmental conditions. Percentage changes in aboveground biomass to grazing showed a quadratic relationship with precipitation in light grazing intensity treatment and a linear relationship in moderate and heavy grazing intensity treatment, but did not change with temperature. Grazing effects on belowground biomass did not change with precipitation or temperature. Compared to the global average value, grazing had greater negative effects on grassland production in China.

Conclusions

Grazing has negative effects on grassland biomass and the grazing effects change with environmental conditions and grazing intensity, therefore flexible rangeland management tactics that suit local circumstances are necessary to take into consideration for balancing the demand of grassland utilization and conservation.     

Using Oxidized Low-Density Lipoprotein Autoantibodies to Predict Restenosis after Balloon Angioplasty in Patients with Acute Myocardial Infarction

Objectives

Oxidized low-density lipoproteins (oxLDL) and oxidized low-density lipoprotein autoantibodies (OLAB) have been detected in human plasma and atherosclerotic lesions. OLAB appear to play a role in the clearance of oxLDL from circulation. Higher levels of OLAB appear to be associated with a reduced risk of a wide range of cardiovascular diseases. We investigated the prognostic value of plasma oxLDL and OLAB in patients undergoing primary coronary balloon angioplasty for acute ST-elevation myocardial infarction (STEMI).

Methods

Plasma oxLDL and OLAB concentrations were measured in 56 patients with acute STEMI before primary angioplasty, and then 3 days, 7 days and 1 month after the acute event. Follow-up angiography was repeated 6 months later to detect the presence of restensosis (defined as >50% luminal diameter stenosis). The thrombolysis in myocardial infarction (TIMI) risk score was calculated to determine the relationship between OLAB/oxLDL ratio and TIMI risk scores.

Results

Of the 56 patients, 18 (31%) had angiographic evidence of restenosis. Plasma OLAB concentrations were significantly lower in the restenosis group before angioplasty (181±114 vs. 335±257 U/L, p = 0.003), and at day 3 (155±92 vs. 277±185 U/L, p<0.001) and day 7 (177±110 vs. 352±279 U/L, p<0.001) after the acute event. There was no difference in oxLDL concentration between the two groups. The ratio of OLAB/oxLDL positively correlated with TIMI risk scores before angioplasty (p for trend analysis, p = 0.004), at day 3 (p = 0.008) and day 7 (p<0.001) after STEMI.

Significance

A relative deficit of OLAB, and hence likely impaired clearance of oxLDL, is associated with the risk of arterial restenosis after primary angioplasty for acute STEMI.     

Evaluation of Five Candidate Genes from GWAS for Association with Oligozoospermia in a Han Chinese Population

Background

Oligozoospermia is one of the severe forms of idiopathic male infertility. However, its pathology is largely unknown, and few genetic factors have been defined. Our previous genome-wide association study (GWAS) has identified four risk loci for non-obstructive azoospermia (NOA).

Objective

To investigate the potentially functional genetic variants (including not only common variants, but also less-common and rare variants) of these loci on spermatogenic impairment, especially oligozoospermia.

Design, Setting, and Participants

A total of 784 individuals with oligozoospermia and 592 healthy controls were recruited to this study from March 2004 and January 2011.

Measurements

We conducted a two-stage study to explore the association between oligozoospermia and new makers near NOA risk loci. In the first stage, we used next generation sequencing (NGS) in 96 oligozoospermia cases and 96 healthy controls to screen oligozoospermia-susceptible genetic variants. Next, we validated these variants in a large cohort containing 688 cases and 496 controls by SNPscan for high-throughput Single Nucleotide Polymorphism (SNP) genotyping.

Results and Limitations

Totally, we observed seven oligozoospermia associated variants (rs3791185 and rs2232015 in PRMT6, rs146039840 and rs11046992 in Sox5, rs1129332 in PEX10, rs3197744 in SIRPA, rs1048055 in SIRPG) in the first stage. In the validation stage, rs3197744 in SIRPA and rs11046992 in Sox5 were associated with increased risk of oligozoospermia with an odds ratio (OR) of 4.62 (P  =  0.005, 95%CI 1.58-13.4) and 1.82 (P  =  0.005, 95%CI 1.01-1.64), respectively. Further investigation in larger populations and functional characterizations are needed to validate our findings.

Conclusions

Our study provides evidence of independent oligozoospermia risk alleles driven by variants in the potentially functional regions of genes discovered by GWAS. Our findings suggest that integrating sequence data with large-scale genotyping will serve as an effective strategy for discovering risk alleles in the future.     

Measles Contributes to Rheumatoid Arthritis: Evidence from Pathway and Network Analyses of Genome-Wide Association Studies

Growing evidence from epidemiological studies indicates the association between rheumatoid arthritis (RA) and measles. However, the exact mechanism for this association is still unclear now. We consider that the strong association between both diseases may be caused by shared genetic pathways. We performed a pathway analysis of large-scale RA genome-wide association studies (GWAS) dataset with 5,539 cases and 20,169 controls of European descent. Meanwhile, we evaluated our findings using previously identified RA loci, protein-protein interaction network and previous results from pathway analysis of RA and other autoimmune diseases GWAS. We confirmed four pathways including Cytokine-cytokine receptor interaction, Jak-STAT signaling, T cell receptor signaling and Cell adhesion molecules. Meanwhile, we highlighted for the first time the involvement of Measles and Intestinal immune network for IgA production pathways in RA. Our results may explain the strong association between RA and measles, which may be caused by the shared genetic pathway. We believe that our results will be helpful for future genetic studies in RA pathogenesis and may significantly assist in the development of therapeutic strategies.     

Involvement of and Interaction between WNT10A and EDA Mutations in Tooth Agenesis Cases in the Chinese Population

Background

Dental agenesis is the most common, often heritable, developmental anomaly in humans. Although WNT10A gene mutations are known to cause rare syndromes associated with tooth agenesis, including onycho-odontodermal dysplasia (OODD), Schöpf-Schulz-Passarge syndrome (SSPS), hypohidrotic ectodermal dysplasia (HED), and more than half of the cases of isolated oligodontia recently, the genotype-phenotype correlations and the mode of inheritance of WNT10A mutations remain unclear. The phenotypic expression with WNT10A mutations shows a high degree of variability, suggesting that other genes might function with WNT10A in regulating ectodermal organ development. Moreover, the involvement of mutations in other genes, such as EDA, which is also associated with HED and isolated tooth agenesis, is not clear. Therefore, we hypothesized that EDA mutations interact with WNT10A mutations to play a role in tooth agenesis. Additionally, EDA, EDAR, and EDARADD encode signaling molecules in the Eda/Edar/NF-κB signaling pathways, we also checked EDAR and EDARADD in this study.

Methods

WNT10A, EDA, EDAR and EDARADD were sequenced in 88 patients with isolated oligodontia and 26 patients with syndromic tooth agenesis. The structure of two mutated WNT10A and two mutated EDA proteins was analyzed.

Results

Digenic mutations of both WNT10A and EDA were identified in 2 of 88 (2.27%) isolated oligodontia cases and 4 of 26 (15.38%) syndromic tooth agenesis cases. No mutation in EDAR or EDARADD gene was found.

Conclusions

WNT10A and EDA digenic mutations could result in oligodontia and syndromic tooth agenesis in the Chinese population. Moreover, our results will greatly expand the genotypic spectrum of tooth agenesis.     

Serum Uric Acid Levels and the Risk of Impaired Fasting Glucose: A Prospective Study in Adults of North China

Objective

To prospectively investigate the association between serum uric acid (SUA) level and incidence of impaired fasting glucose (IFG) in adult Chinese.

Methods

We evaluated 13,328 women and 41,350 men without diabetes and IFG. The participants were classified into quintile according to baseline level of SUA. Data were analyzed to examine the association between SUA levels and the incidence of IFG. We used Cox regression models to estimate the relative risk of IFG after adjusting for known risk factors.

Results

For men, the second quintile of SUA has the lowest cumulative incidence of IFG (29.9%); the fifth quintile of SUA has the highest cumulative incidence of IFG (35.6%). After corrected with Cox regression, the first quartile and the fourth quartile have higher cumulative incidence of IFG than the second quintile, with the HR of 1.11(1.05-1.17) and 1.07(1.01-1.13), respectively. For women, the first quartile of SUA has the lowest cumulative incidence of IFG (20.7%), while the fifth quintile of SUA has the highest cumulative incidence of IFG (30.0%). However, there is no significant difference in IFG between different quintile after adjusted with Cox regression.

Conclusions

The results of this prospective study suggest that there is a higher risk of developing IFG in association with low or high SUA concentrations for men. These relationships were independent of other known risk factors. There is no significant correlation in the risk of developing IFG in association with SUA concentrations for women. Analyses excluding participants with hypertension or with hyperlipidemia and analyses with participants stratified by age reached similar conclusion.     

Effects of Vascular Endothelial Growth Factor 165 on Bone Tissue Engineering

To study the relationship between vascular endothelial growth factor (VEGF) and formation and repair of engineering bone, second-generation bone marrow stromal cells (BMSCs) of New Zealand white rabbits that were separated in vitro were transfected with VEGF 165 gene vectors by adenovirus to detect gene expressions. Transfected BMSCs and β-tricalcium phosphate material were complexed and implanted at the femoral injury sites of the study group (n = 12), and the control group (n = 12) were implanted with engineering bones that were not transfected with VEGF. Femoral recoveries of the two groups were observed on the 15th, 30th, 45th and 60th days, and their vascularization and ossification statuses were observed by immunohistochemical methods. The BMSCs transfected with VEGF highly expressed VEGF genes and excreted VEGF. The two groups both experienced increased vascularization and bone volume after implantation (t = 7.92, P<0.05), and the increases of the study group were significantly higher than those of the control group (t = 6.92, P<0.05). VEGF is clinically applicable because it can accelerate the formation and repair of engineering bone by promoting vascularization and ossification.