利用功能分类基因芯片研究不同品种猪脂肪中特定基因的发育性变化

利用Oligo功能分类基因芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在1、2、3、4和5月龄间背部皮下脂肪中脂肪沉积代谢和细胞生长调控相关基因的动态表达变化。差异表达分析结果显示1~5月龄的品种间分别有10、6、11、8和19个基因的表达差异倍数大于2倍, 且长白猪有25个基因在不同月龄间的表达差异达显著水平(P<0.05)。其中血管生成素样蛋白4 (ANGPTL4)、组织蛋白酶K (CTSK) 、异柠檬酸脱氢酶2(NADP+) (IDH2)、脂蛋白脂酶 (LPL)、苹果酸酶1 (ME1)、 硬酯酰辅酶A去饱和酶 (SCD)和解藕联蛋白2 (UCP2)这7个基因不仅在同月龄的品种间和品种内的不同月龄间差异表达, 主成分分析结果也显示其表达模式明显偏离其他基因, 提示受到了特殊的调控。聚类分析结果显示1~5月龄间长白猪中正调控脂肪酸代谢基因的表达量逐渐上调, 太湖猪中参与细胞生长调控基因的表达量平缓波动且变化幅度相对较小。另外, 5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势。结果成功筛选出了对猪胴体和肉质性状可能具有重要影响并值得深入研究的一些候选基因, 初步揭示了相关基因的表达变化规律, 为了解生长发育过程中脂肪酸合成与水解的动态平衡过程提供了基础数据。     

利用功能分类基因芯片研究不同品种猪脂肪中特定基因的发育性变化

利用Oligo功能分类基因芯片检测了瘦肉型的长白猪和脂肪型的太湖猪在1、2、3、4和5月龄间背部皮下脂肪中脂肪沉积代谢和细胞生长调控相关基因的动态表达变化。差异表达分析结果显示1~5月龄的品种间分别有10、6、11、8和19个基因的表达差异倍数大于2倍, 且长白猪有25个基因在不同月龄间的表达差异达显著水平(P<0.05)。其中血管生成素样蛋白4 (ANGPTL4)、组织蛋白酶K (CTSK) 、异柠檬酸脱氢酶2(NADP+) (IDH2)、脂蛋白脂酶 (LPL)、苹果酸酶1 (ME1)、 硬酯酰辅酶A去饱和酶 (SCD)和解藕联蛋白2 (UCP2)这7个基因不仅在同月龄的品种间和品种内的不同月龄间差异表达, 主成分分析结果也显示其表达模式明显偏离其他基因, 提示受到了特殊的调控。聚类分析结果显示1~5月龄间长白猪中正调控脂肪酸代谢基因的表达量逐渐上调, 太湖猪中参与细胞生长调控基因的表达量平缓波动且变化幅度相对较小。另外, 5个差异表达基因的荧光定量RT-PCR验证结果均与芯片结果呈正相关趋势。结果成功筛选出了对猪胴体和肉质性状可能具有重要影响并值得深入研究的一些候选基因, 初步揭示了相关基因的表达变化规律, 为了解生长发育过程中脂肪酸合成与水解的动态平衡过程提供了基础数据。     

猪背最长肌中胰岛素样生长因子(IGFs)系统基因的发育表达模式

采用荧光定量PCR技术检测了长白猪和梅山猪的背最长肌组织中胰岛素样生长因子1和2(IGF-1和-2)、胰岛素样生长因子1受体和2受体(IGF-1R和-2R)、胰岛素样生长因子结合蛋白3和5(IGFBP-3和-5)基因mRNA丰度在初生(0月龄)、1、2、3、4和5月龄间的表达变化并分析品种间和不同月龄间基因表达的差异及其对肌肉生长发育的影响。结果表明: 两猪种出生后IGF-1 mRNA表达量均表现为逐渐上调, 而IGF-2则恰好相反, 表现为逐渐下调。这与IGF-2主要在胚胎期发挥作用, 而IGF-1则主要在动物个体出生后才发挥促进细胞增殖和个体发育功能的特点相符。IGFRs mRNA与IGFs mRNA 表达的发育性变化模式并不相似, 提示背最长肌组织中IGFRs mRNA 的表达可能没有受到组织局部产生的IGFs调节。长白猪的IGF-1R、IGF-2R 和IGFBP-3 mRNA表达量均在2月龄时达到最高峰, 提示2月龄可能是长白猪IGFs系统发挥作用最为明显的生长发育阶段。以上结果初步揭示猪生长发育过程中胰岛素样生长因子系统基因表达的发育性变化模式和品种差异, 为深入研究胰岛素样生长因子系统基因的相互调控机制提供了基础数据。     

No evidence for a major role of polymorphisms during bupropion treatment

This study evaluated the ability of polymorphisms in five candidate genes to predict weight gain among patients taking bupropion or placebo in a smoking cessation trial. Five hundred fifty-three smokers were enrolled into a randomized double-blind, placebo-controlled trial and followed for 12 months. Five candidate genes [DRD2 Taq1 (rs1800497), DRD2-141 (rs1799732), C957T (rs6277), COMT (rs4818), and SLC6A3] were genotyped. Weights at baseline, at end of treatment, and after 6 and 12 months of follow-up were self-reported. Smoking abstinence at each endpoint was self-reported and confirmed biochemically. A self-reported average weight gain after 12 months of 1.1 +/- 6.0 kg (mean +/- standard deviation) in the bupropion group and 1.8 +/- 4.8 kg in the placebo group was noted. For subjects with biochemically confirmed abstinence from smoking, the HL genotype (alleles coding Val at codon 108 are denoted as H, and those coding Met are denoted as L) at the COMT locus and A1A1 genotype at the DRD2 Taq1 locus were associated with less weight gain at the end of treatment. The TC genotype at the C957T locus was associated with increased weight gain at 6 months of follow-up. However, no polymorphisms or their interactions with bupropion consistently and significantly predicted baseline BMI or weight change during treatment for all study subjects. Overall, our results do not support a major role for these five candidate genes in weight gain after smoking cessation.     

Analyzing time-series microarray data reveals key genes in spinal cord injury

Although many scholars have utilized high-throughput microarrays to delineate gene expression patterns after spinal cord injury (SCI), no study has evaluated gene changes in raphe magnus (RM) and somatomotor cortex (SMTC), two areas in brain primarily affected by SCI. In present study, we aimed to analyze the differentially expressed genes (DEGs) of RM and SMTC between SCI model and sham injured control at 4, 24 h, 7, 14, 28 days, and 3 months using microarray dataset GSE2270 downloaded from gene expression omnibus and unpaired significance analysis of microarray method. Protein–protein interaction (PPI) network was constructed for DEGs at crucial time points and significant biological functions were enriched using DAVID. The results indicated that more DEGs were identified at 14 days in RM and at 4 h/3 months in SMTC after SCI. In the PPI network for DEGs at 14 days in RM, interleukin 6, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), FBJ murine osteosarcoma viral oncogene homolog (FOS), tumor necrosis factor, and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) were the top 5 hub genes; In the PPI network for DEGs at 3 months in SMTC, the top 5 hub genes were ubiquitin B, Ras‐related C3 botulinum toxin substrate 1 (rho family, small GTP binding protein Rac1), FOS, Janus kinase 2 and vascular endothelial growth factor A. Hedgehog and Wnt signaling pathways were the top 2 significant pathways in RM. These hub DEGs and pathways may be underlying therapeutic targets for SCI.     

Hepatic xenobiotic metabolizing enzyme and transporter gene expression through the life stages of the mouse

Background

Differences in responses to environmental chemicals and drugs between life stages are likely due in part to differences in the expression of xenobiotic metabolizing enzymes and transporters (XMETs). No comprehensive analysis of the mRNA expression of XMETs has been carried out through life stages in any species.

Results

Using full-genome arrays, the mRNA expression of all XMETs and their regulatory proteins was examined during fetal (gestation day (GD) 19), neonatal (postnatal day (PND) 7), prepubescent (PND32), middle age (12 months), and old age (18 and 24 months) in the C57BL/6J (C57) mouse liver and compared to adults. Fetal and neonatal life stages exhibited dramatic differences in XMET mRNA expression compared to the relatively minor effects of old age. The total number of XMET probe sets that differed from adults was 636, 500, 84, 5, 43, and 102 for GD19, PND7, PND32, 12 months, 18 months and 24 months, respectively. At all life stages except PND32, under-expressed genes outnumbered over-expressed genes. The altered XMETs included those in all of the major metabolic and transport phases including introduction of reactive or polar groups (Phase I), conjugation (Phase II) and excretion (Phase III). In the fetus and neonate, parallel increases in expression were noted in the dioxin receptor, Nrf2 components and their regulated genes while nuclear receptors and regulated genes were generally down-regulated. Suppression of male-specific XMETs was observed at early (GD19, PND7) and to a lesser extent, later life stages (18 and 24 months). A number of female-specific XMETs exhibited a spike in expression centered at PND7.

Conclusions

The analysis revealed dramatic differences in the expression of the XMETs, especially in the fetus and neonate that are partially dependent on gender-dependent factors. XMET expression can be used to predict life stage-specific responses to environmental chemicals and drugs.     

Tau/APP/PS1三转基因小鼠模型的建立及生物学特征

目的:建立Tau/APP/PS1三转基因小鼠模型,从分子生物学、行为学及病理学角度研究其生物学特征。方法:将自行建立的Tau转基因小鼠与Jackson实验室引种的APP/PS1双转基因小鼠杂交、传代;PCR鉴定小鼠基因型;RT-PCR检测外源基因的转录;Western blot测定外源基因的蛋白表达;Bielschowsky氏染色法和ABC免疫组化法观察大脑神经纤维缠结和老年斑等病理改变;Morris水迷宫观测学习记忆的改变。结果:Tau/APP/PS1三转基因小鼠的大脑可转录和表达Tau、APP和PS1三种外源基因,6~8月龄时大脑皮层和海马可见神经元纤维缠结和老年斑,其学习记忆获得能力在6月龄开始受损。结论:建立的Tau/APP/PS1三转基因小鼠具有Tau和Aβ两种病理改变和学习记忆障碍,为深入探究Tau与Aβ的关系、阐明AD的发病机制以及研发靶点治疗药物提供实验工具。     

apoE/LDLR双基因缺失幼龄小鼠主动脉中动脉粥样硬化相关基因的表达

利用RT-PCR以及实时定量RT-PCR检测11个动脉粥样硬化(atherosclerosis,AS)相关基因在1、2和3月龄的载脂蛋白E(apolipoproteinE,aopE)/低密度脂蛋白受体(low-density lipoprotein receptor,LDLR)双基因缺失(apoE-/-/LDLR-/-)小鼠主动脉中的表达变化,同时应用血生化指标和病理形态学观察AS早期病变特点,探讨apoE和LDLR基因联合缺失引发的血脂代谢紊乱和血管炎症损伤的关系以及AS的炎症反应机制.结果显示,apoE-/-/LDLR-/-小鼠IL-18、TLR2、MCP-1、ICAM-1、VCAM-1、GM-CSF、CD36和ET-1表达在1月龄时较同龄野生型(wild type,WT)小鼠显著上调(P<0.05,P<0.01),PDGF-α和TNF-α表达在2月龄时较同龄WT小鼠显著上调,除ET-1表达在2月龄时以及了LR2、VCAM-1和ICAM-1表达在3月龄时降至WT小鼠水平以外,其余各基因表达随年龄增长继续升高(P<0.05,P<0.01),其中MCP-1表达在2月龄时达到峰值.NF-kB在各年龄段apoE-/-/LDLR-/-小鼠中的表达与同龄WT小鼠相比均无显著差异.各年龄段apoE-/-/LDLR-/-/小鼠血清了C、TG、LDL、HDL、TNF-α、IL-1β和ox-LDL含量均显著高于同龄WT小鼠(P<0.05,P<0.01),并随年龄增长逐渐升高.apoE-/-/LDLR-/-小鼠1月龄时主动脉内膜出现少量的散在的脂质沉积,随着年龄增长病变区域增多,脂质沉积增厚.上述结果提示:apoE和LDLR双基因缺失形成的高脂血症可能通过刺激主动脉中炎症基因时序表达,起始并扩大病变部位的炎症反应,共同促进AS的发生发展.     

Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater

Aims: In this study, the main objective was to verify the hypothesis of induction of ‘viable but non‐culturable’ (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat‐labile (LT) or heat‐stable (ST) enterotoxins was observed using GM1‐ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real‐time RT‐PCR on cDNA derived from the bacteria. Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long‐term incubation in water.     

Identification and characterization of genes related to the development of skeletal muscle in the Hainan black goat

In total, 185 unigenes were identified from 380 clones of postnatal skeletal muscle of Hainan Black goats by suppression subtractive hybridization (SSH) technology. Most of the differentially expressed genes involved energy metabolism and muscle contraction. The expression of 19 genes was analyzed in the longissimus dorsi muscles of 2-, 6-, 12-, 24-month olds, and four gene expression patterns were found by hierarchical cluster analysis. Most genes in first expression pattern belonged to myofibrillar proteins and had higher expression levels at 2 months old; genes of the secondary expression pattern had higher expression levels at 12 months old; tropomyoain 1 (alpha) (TPM1) was classified into the third expression pattern, and its expression level showed decreases tendency as age increased. Tropomyoain 2 (beta) (TPM2) was classified into the third expression pattern, which had the opposite expression pattern against TPM1.     

Detecting ALK,ROS1 and RET Fusion Genes in Cell Block Samples

Whether Cell block (CB) samples are applicable to detect anaplastic lymphoma kinase (ALK), c-ros oncogene 1 (ROS1) and ret proto-oncogene (RET) fusion genes in lung adenocarcinoma is still unknown. In this study, 108 cytological samples that contained lung adenocarcinoma cells were collected, and made into CB. The CB samples all contained at least 30% lung adenocarcinoma cells. In these patients, 48 harbored EGFR mutation. Among the 50 EGFR wild type patients who detected fusion genes, 14 carried EML4-ALK fusion (28%), 2 had TPM3-ROS1 fusion (4%), and 3 harbored KIF5B-RET fusion (6%). No double fusions were found in one sample. Patients with fusion genes were younger than those without fusion genes (p = 0.032), but no significant difference was found in sex and smoking status (p > 0.05). In the thirty-five patients who received first-line chemotherapy, patients with fusion gene positive had disease control rate (DCR) (72.7% VS 50%, p > 0.05) and objective response rate (ORR) (9.1% VS 4.2%, p > 0.05) compared with those having fusion gene negative. The median progression free survival (mPFS) were 4.0 and 2.7 months in patients harbored fusion mutations and wild type, respectively (p > 0.05). We conclude that CB samples could be used to detect ALK, ROS1 and RET fusions in NSCLC. The frequency distribution of three fusion genes is higher in lung adenocarcinoma with wild-type EGFR, compared with unselected NSCLC patient population. Patients with fusion genes positive are younger than those with fusion gene negative, but they had no significantly different PFS in first-line chemotherapy.     

Both sulfate-reducing bacteria and Enterobacteriaceae take part in marine biocorrosion of carbon steel

AIMS: In order to evaluate the part played in biocorrosion by microbial groups other than sulfate-reducing bacteria (SRB), we characterized the phylogenetic diversity of a corrosive marine biofilm attached to a harbour pile structure as well as to carbon steel surfaces (coupons) immersed in seawater for increasing time periods (1 and 8 months). We thus experimentally checked corroding abilities of defined species mixtures. METHODS AND RESULTS: Microbial community analysis was performed using both traditional cultivation techniques and polymerase chain reaction cloning-sequencing of 16S rRNA genes. Community structure of biofilms developing with time on immersed coupons tended to reach after 8 months, a steady state similar to the one observed on a harbour pile structure. Phylogenetic affiliations of isolates and cloned 16S rRNA genes (rrs) indicated that native biofilms (developing after 1-month immersion) were mainly colonized by gamma-proteobacteria. Among these, Vibrio species were detected in majority with molecular methods while cultivation techniques revealed dominance of Enterobacteriaceae such as Citrobacter, Klebsiella and Proteus species. Conversely, in mature biofilms (8-month immersion and pile structure), SRB, and to a lesser extent, spirochaetes were dominant. CONCLUSIONS: Corroding activity detection assays confirmed that Enterobacteriaceae (members of the gamma-proteobacteria) were involved in biocorrosion of metallic material in marine conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: In marine biofilms, metal corrosion may be initiated by Enterobacteriaceae.     

Tibial loading increases osteogenic gene expression and cortical bone volume in mature and middle-aged mice

There are conflicting data on whether age reduces the response of the skeleton to mechanical stimuli. We examined this question in female BALB/c mice of different ages, ranging from young to middle-aged (2, 4, 7, 12 months). We first assessed markers of bone turnover in control (non-loaded) mice. Serum osteocalcin and CTX declined significantly from 2 to 4 months (p<0.001). There were similar age-related declines in tibial mRNA expression of osteoblast- and osteoclast-related genes, most notably in late osteoblast/matrix genes. For example, Col1a1 expression declined 90% from 2 to 7 months (p<0.001). We then assessed tibial responses to mechanical loading using age-specific forces to produce similar peak strains (-1300 με endocortical; -2350 με periosteal). Axial tibial compression was applied to the right leg for 60 cycles/day on alternate days for 1 or 6 weeks. qPCR after 1 week revealed no effect of loading in young (2-month) mice, but significant increases in osteoblast/matrix genes in older mice. For example, in 12-month old mice Col1a1 was increased 6-fold in loaded tibias vs. controls (p = 0.001). In vivo microCT after 6 weeks revealed that loaded tibias in each age group had greater cortical bone volume (BV) than contralateral control tibias (p<0.05), due to relative periosteal expansion. The loading-induced increase in cortical BV was greatest in 4-month old mice (+13%; p<0.05 vs. other ages). In summary, non-loaded female BALB/c mice exhibit an age-related decline in measures related to bone formation. Yet when subjected to tibial compression, mice from 2-12 months have an increase in cortical bone volume. Older mice respond with an upregulation of osteoblast/matrix genes, which increase to levels comparable to young mice. We conclude that mechanical loading of the tibia is anabolic for cortical bone in young and middle-aged female BALB/c mice.     

Comparative Survey of Rumen Microbial Communities and Metabolites across One Caprine and Three Bovine Groups, Using Bar-Coded Pyrosequencing and 1H Nuclear Magnetic Resonance Spectroscopy

Pyrosequencing of 16S rRNA genes (targeting Bacteria and Archaea) and (1)H nuclear magnetic resonance were applied to investigate the rumen microbiota and metabolites of Hanwoo steers in the growth stage (HGS), Hanwoo steers in the late fattening stage (HFS), Holstein-Friesian dairy cattle (HDC), and Korean native goats (KNG) in the late fattening stage. This was a two-part investigation. We began by comparing metabolites and microbiota of Hanwoo steers at two stages of husbandry. Statistical comparisons of metabolites and microbial communities showed no significant differences between HFS and HGS (differing by a dietary shift at 24 months and age [67 months versus 12 months]). We then augmented the study by extending the investigation to HDC and KNG. Overall, pyrosequencing of 16S rRNA genes showed that the rumens had highly diverse microbial communities containing many previously undescribed microorganisms. Bioinformatic analysis revealed that the bacterial sequences were predominantly affiliated with four phyla-Bacteroidetes, Firmicutes, Fibrobacteres, and Proteobacteria-in all ruminants. However, interestingly, the bacterial reads belonging to Fibrobacteres were present at a very low abundance (<0.1%) in KNG. Archaeal community analysis showed that almost all of these reads fell into a clade related to, but distinct from, known cultivated methanogens. Statistical analyses showed that the microbial communities and metabolites of KNG were clearly distinct from those of other ruminants. In addition, bacterial communities and metabolite profiles of HGS and HDC, fed similar diets, were distinctive. Our data indicate that bovine host breeds override diet as the key factor that determines bacterial community and metabolite profiles in the rumen.