Cholesteryl Ester Transfer Protein (CETP) Deficiency and CETP Inhibitors

Epidemiologic studies have shown that low-density lipoprotein cholesterol (LDL-C) is a strong risk factor, whilst high-density lipoprotein cholesterol (HDL-C) reduces the risk of coronary heart disease (CHD). Therefore, strategies to manage dyslipidemia in an effort to prevent or treat CHD have primarily attempted at decreasing LDL-C and raising HDL-C levels. Cholesteryl ester transfer protein (CETP) mediates the exchange of cholesteryl ester for triglycerides between HDL and VLDL and LDL. We have published the first report indicating that a group of Japanese patients who were lacking CETP had extremely high HDL-C levels, low LDL-C levels and a low incidence of CHD. Animal studies, as well as clinical and epidemiologic evidences, have suggested that inhibition of CETP provides an effective strategy to raise HDL-C and reduce LDL-C levels. Four CETP inhibitors have substantially increased HDL-C levels in dyslipidemic patients. This review will discuss the current status and future prospects of CETP inhibitors in the treatment of CHD. At present anacetrapib by Merck and evacetrapib by Eli Lilly are under development. By 100mg of anacetrapib HDL-C increased by 138%, and LDL-C decreased by 40%. Evacetrapib 500 mg also showed dramatic 132% increase of HDL-C, while LDL-C decreased by 40%. If larger, long-term, randomized, clinical end point trials could corroborate other findings in reducing atherosclerosis, CETP inhibitors could have a significant impact in the management of dyslipidemic CHD patients. Inhibition of CETP synthesis by antisense oligonucleotide or small molecules will produce more similar conditions to human CETP deficiency and may be effective in reducing atherosclerosis and cardiovascular events. We are expecting the final data of prospective clinical trials by CETP inhibitors in 2015.     

Evaluation of Th17 related cytokines associated with clinical and laboratorial parameters in sickle cell anemia patients with leg ulcers

Leg ulcers (LUs) represent one of the main causes of morbidity in sickle cell anemia (SCA). This manifestation has been related to hemolysis, infections predisposition and inflammation that leads cytokines secretion. In this context, our study aimed to evaluate Th17 related cytokines (IL-6, IL-17A, IL-22 and IL-23) in serum and peripheral mononuclear cells culture supernatants with and without lymphoproliferative stimulation (anti-human CD3 and anti-human CD28). The cytokines levels were also correlated to clinical, hematological and biochemical parameters in SCA patients with and without LUs history (SCALU and SCAWH) as well as in healthy controls. In SCALU patients, high levels of IL-17A were associated with absence of acute chest syndrome (ACS, p = 0.0328). The other clinical parameters analyzed (osteonecrosis, stroke, priapism, splenectomy and blood transfusions history) were not significantly related with other cytokine levels. In SCALU patients was also observed that IL-17A increased levels were associated with high levels of LDH (p = 0.0130), the same association pattern was found for IL-6 (0.0160) and IL-22 (p = 0.0165) in the SCALU group. Interestingly, we did not find statistical correlations with these parameters in SCAWH group. The other hematological parameters (hemoglobin, leucocyte and reticulocyte count) and indirect bilirrubin did not show any correlation with analyzed cytokines in both groups. So, for the first time, we show that IL-17A present in SCALU patients may exert a preventive role in the ACS development. Furthermore, IL-6, IL-17A and IL-22 accompanied the LDH levels only in SCALU patients suggesting to serve as additional markers of hemolysis or to be related with immunity response against extracellular pathogens.     

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population^1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide³. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA^4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells^5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks^5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the “pilus”⁵. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface^9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.     

Improvement of small seed for big nutritional feed

Exploding global population, rapid urbanization, salinization of soils, decreasing arable land availability, groundwater resources, and dynamic climatic conditions pose impending damage to our food security by reducing the grain quality and quantity. This issue is further compounded in arid and semi-arid regions due to the shortage of irrigation water and erratic rainfalls. Millets are gluten (a family of proteins)-free and cultivated all over the globe for human consumption, fuel, feed, and fodder. They provide nutritional security for the under- and malnourished. With the deployment of strategies like foliar spray, traditional/marker-assisted breeding, identification of candidate genes for the translocation of important minerals, and genome-editing technologies, it is now tenable to biofortify important millets. Since the bioavailability of iron and zinc has been proven in human trials, the challenge is to make such grains accessible. This review encompasses nutritional benefits, progress made, challenges being encountered, and prospects of enriching millet crops with essential minerals.     

利用单链构象多态性检测牛白细胞黏附缺陷症方法的建立

牛白细胞黏附缺陷症(BLAD)是由常染色体上CD18基因单碱基突变(A→G)引起的隐性遗传疾病,隐性基因纯合时导致白细胞表面的β2整合素表达明显减少或缺乏而引起临床发病,患病牛的主要特征是机体免疫力降低、易患病从而影响其生产性能的表现,严重影响了奶牛场的经济效益。该工作利用自行设计的特异性PCR引物,通过对PCR、PCR-SSCP条件的筛选和优化,并结合DNA序列测定,建立了PCR-SSCP检测BLAD 有害基因的新方法,该方法简便快捷、准确率高,使用样品宽泛,适合大样本筛选,为奶牛BLAD有害基因的分型和筛选提供了新的方法和思路,为我国奶牛的分子选育提供了可靠的理论依据。     

马传染性贫血病毒基因非编码区LTR嵌合克隆的构建

在已有全长感染性克隆pLGFD3-8和pD70344的基础上,根据马传贫弱毒疫苗致弱过程中不同代次毒株LTR序列的分析,在LTR U3区选取特定的酶切位点对EIAV非编码区LTR基因进行了部分替换.将替换的全基因克隆转染驴胎皮肤细胞(FDD)并以驴白细胞(DL)传代,用逆转录酶活性检测、RT-PCR方法及Real-time RT-PCR验证其感染性.结果发现,其衍生病毒感染上述两种细胞均出现明显的细胞病变;细胞培养上清可检测到RT酶活性和RT-PCR阳性.电镜下可见大量典型的EIAV颗粒.pLGFD9-12嵌合克隆衍生病毒与其父本克隆衍生病毒pLGFD3-8具有相似的复制水平,pLGFD9-12嵌合克隆衍生病毒在DL细胞上复制水平略高于FDD细胞.此结果为进一步深入研究LTR对马传染性贫血病毒复制水平和毒力的影响奠定了基础.     

鸡β-肌动蛋白第一内含子对EIAV转移载体外源蛋白表达能力的影响

以马传染性贫血病毒（equine infectious anemia virus, EIAV）基因转移载体pcPPTWPRE为基础,用含不同长度片段的鸡β-肌动蛋白启动子替换原有的人巨细胞病毒立即早期启动子（CMVIEp）启动外源基因的表达,分别构建了2个基因转移载体,含部分第一内含子的pWCAGP0.8和含全长内含子的pWCAGP1.6。连同在其它研究中构建的不含内含子的质粒pcPPTWCAG（另文发表）,采用磷酸钙法分别转染HEK293细胞和DF-1细胞, 利用荧光显微镜观察报告基因eGFP蛋白的表达。并利用流式细胞仪定量。统计学分析结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8和 pWCAGP1.6表达阳性率分别为47.9%、46.1%和23.8%,转染后48h,收获细胞,利用流式细胞仪比较转染细胞中EGFP表达阳性细胞的百分率。结果表明,在HEK293细胞中,pcPPTWCAG、pWCAGP0.8 和pWCAGP1.6的阳性细胞分别占计数细胞的47.9%、46.1%和23.8%;在DF-1细胞中,依次分别为12.4%、9.5%和4.2%,pcPPTWCAG与pWCAGP1.6差异显著。本研究表明不含内含子的EIAV载体质粒表达EGFP的能力高于含部分和全长内含子的载体。     

Synergistic effect ofrib80 andhit Mutations on riboflavin biosynthesis and iron transport in the yeastPichia guilliermondii

Mutant strains of the yeastPichia guilliermondii, carrying bothrib80 andhit mutations in a haploid genome, were derived from previously obtained strains with defectiverib80 orhit genes, exerting negative control of the riboflavin biosynthesis and iron transport inPichia guilliermondii. The double mutant rib80hit strains exhibited an increased level of riboflavin biosynthesis and higher activities of GTP cyclohydrolase and riboflavin synthetase. Iron deficiency caused an additional increase in riboflavin overproduction. These results suggest the synergistic interaction of therib80 andhit mutations. A combination of both mutations in a single genome did not affect iron assimilation by the cells: ferrireductase activity, the rate of⁵⁵Fe uptake, and the iron content in cells of the double mutants remained at the level characteristic of the parent strains.     

Biochemical properties of canine serum ferritin: iron content and nonbinding to concanavalin A

A sandwich enzyme-linked immunosorbent assay using H-subunit-rich canine heart ferritin as a standard has been developed for measuring canine serum ferritin which is H-subunit-rich. Serum ferritin concentrations in 51 normal dogs ranged from 143 to 1766 ng ml^–1, with a mean value of 479±286 (SD) ng ml^–1. Serum ferritin iron concentrations as determined by an immunoprecipitation technique ranged from 30.4 to 115.9 ng ml^–1 in 15 normal dogs with serum ferritin protein levels of 298 to 959 ng ml^–1. There was a significant linear correlation between the serum ferritin iron and protein levels (r=0.9441, P<0.001), and the mean iron/protein ratio of serum ferritin was 0.112±0.017. When canine sera were incubated with concanavalin A-Sepharose 4B, we observed the apparent binding of serum ferritin to concanavalin A. However, ferritin obtained by heat-treating the sera at pH 4.8 to remove the ferritin-binding proteins did not bind to the lectin. These results suggest that canine serum ferritin contains a considerable amount of iron but no concanavalin A-binding G subunit present in human serum ferritin.