口虾蛄性腺组织蛋白质双向电泳体系的建立及优化

旨在通过口虾蛄雄性、雌性性腺组织蛋白质双向电泳技术体系的优化,获得雄性、雌性口虾蛄性腺蛋白质的表达图谱。结果表明,不同的蛋白提取方法,上样前处理方法,上样量,聚焦时间及雌、雄性口虾蛄性腺蛋白表达图谱存在一定的差异。雌性、雄性口虾蛄用Tris-HCl提取后用丙酮沉淀方法提取蛋白质、不经上样缓冲液处理、上样量为10μg时,得到较好图谱。对图谱分析发现在pH4-6.5范围内,雌性口虾蛄性腺可溶性蛋白质种类多于雄性。雄性口虾蛄蛋白质点相对于雌性较少,且蛋白质大部分分布于酸性端。经过蛋白质双向电泳体系的优化,能显著提高双向电泳图谱的分辨率,为进一步研究口虾蛄性别差异表达蛋白的筛选,后续的口虾蛄蛋白质组学研究提供技术保障。     

天冬氨酸家族主要氨基酸高产菌株的选育策略

L-苏氨酸与L-赖氨酸是L-天冬氨酸家族氨基酸(AFAAs)中的重要成员,近年来由于其在食品、化妆品、动物饲料添加剂等方面的广泛应用而备受关注,市场需求逐年上升。运用代谢工程手段构建高产菌,可有效地提高L-苏氨酸和L-赖氨酸的生产水平。本文详述了L-苏氨酸与L-赖氨酸的合成途径、调控机制以及两种氨基酸高产菌株的构建策略。     

一种构建马链球菌兽疫亚种血红素受体基因缺失突变株的方法

【目的】探讨一种构建马链球菌兽疫亚种基因缺失突变株的方法。【方法】PCR扩增目的基因,用pJR700温度敏感载体系统,构建目的基因载体;反向PCR扩增目的基因缺失载体片段,连接,产生目的基因缺失载体;电转化缺失重组质粒导入感受态细胞,先在37℃卡拉霉素(kan)培养基中连续培养,然后在30℃不含kan液体培养基中传代,挑取抗生素敏感菌落,PCR扩增检测抗生素敏感菌染色体上目的基因片段和链黑霉素抗性实验确认血红素受体基因缺失。【结果】获得不含抗生素基因的马链球菌兽疫亚种血红素受体基因缺失突变株。【结论】用pJR700温度敏感载体系统,构建马链球菌兽疫亚种基因缺失突变株是可行的。     

Association between the Polymorphism rs3217927 of CCND2 and the Risk of Childhood Acute Lymphoblastic Leukemia in a Chinese Population

CyclinD proteins, the ultimate recipients of mitogenic and oncogenic signals, play a crucial role in cell-cycle regulation. CyclinD2, one of the cyclinD family, is overexpressed in T-acute lymphoblastic leukemia (ALL) and B-cell chronic lymphocytic leukemia and involved in the pathogenesis of leukemias. Recent reports indicated that CCND2 polymorphisms are associated with human cancer risk, thusly we hypothesized that CCND2 gene polymorphisms may contribute to childhood ALL susceptibility. We selected the polymorphism rs3217927 located in the 3′UTR region of CCND2 to assess its associations with childhood ALL risk in a case-control study. A significant difference was found in the genotype distributions of rs3217927 polymorphism between cases and controls (P = 0.019) and homozygous GG genotype may be an increased risk factor for childhood ALL (adjusted OR  =  1.84, 95% CI  =  1.14 —2.99). Furthermore, this increased risk was more pronounced with GG genotype among high-risk ALL (adjusted OR  = 1.95, 95% CI  =  1.04–3.67), low-risk ALL (adjusted OR  =  2.09, 95% CI  =  1.13–3.87), B-phenotype ALL patients (adjusted OR  =  1.78, 95% CI  =  1.08–2.95) and T-phenotype ALL patients (adjusted OR  =  2.87, 95% CI  =  1.16–7.13). Our results provide evidence that CCND2 polymorphism rs3217927 may be involved in the etiology of childhood ALL, and the GG genotype of rs3217927 may modulate the genetic susceptibility to childhood ALL in the Chinese population. Further functional studies and investigations in larger populations should be conducted to validate our findings.     

IGSF1: A novel oncogene regulates the thyroid cancer progression

Thyroid cancer has been continuously increasing and extraordinarily prevalent worldwide. The genetic diagnosis has been widely used in fine needle aspiration. IGSF1, an immunoglobulin superfamily member 1, has been shown to be associated with the regulation of thyroid hormone. But the function of IGSF1 in thyroid cancer has not been explored yet. In this article, we will illuminate the correlation between IGSF1 expression and thyroid cancer. We analysed the level of IGSF1 expression in 55 pairs of tissue samples by real‐time polymerase chain reaction (PCR) and The Cancer Genome Atlas (TCGA) data portal. After that, we transfected small interfering RNA to silence IGSF1 in thyroid cancer cell lines (KTC‐1 and BCPAP) and confirmed the function of IGSF1 by performed colony formation, migration, invasion, cell counting kit‐8, and apoptosis assays. IGSF1 was upregulated in thyroid cancer tissues compared with the adjacent normal tissues (t = 5.783, df = 54; P < .0001) and TCGA (T: N = 65.91 ± 3.998, n = 501: 2.824 ± 0.273, n = 58; P < .0001). In thyroid cell lines, experiments showed that downregulated IGSF1 inhibited proliferation, metastasis, and promoted cell apoptosis. Meanwhile, inhibited IGSF1 expression could downregulate N‐cadherin, vimentin, and EZH2, which is associated with metastasis. Thyroid cancer cells IGSF1 expression levels are a correlation with its ability to growth, metastasis, and apoptosis.     

Design,synthesis and insulin-sensitizing activity of indomethacin and diclofenac derivatives

A series of aromatic acetic acid compounds were designed and synthesized on the basis of Non-steroidal anti-inflammatory drugs indomethacin and diclofenac. Compounds 5a, 7a, 5h, 7h and 17 could strongly promote insulin-regulated differentiation of 3T3-L1 cells in vitro. They acted as full or partial PPARγ agonist, or improved insulin resistance through non-PPARγ pathway.     

Effect of High-Density Lipoprotein Metabolic Pathway Gene Variations and Risk Factors on Neovascular Age-Related Macular Degeneration and Polypoidal Choroidal Vasculopathy in China

Purpose

To investigate the effect of genetic variants in the high-density lipoprotein (HDL) metabolic pathway and risk factors on neovascular age-related macular degeneration (nAMD) and polypoidal choroidal vasculopathy (PCV) in China.

Methods

A total of 742 Chinese subjects, including 221 controls, 230 cases with nAMD, and 291 cases with PCV, were included in the present study. Five single nucleotide polymorphisms (SNPs) from three genes in the HDL metabolic pathway (HDLMP) including cholesteryl ester transfer protein (CETP), hepatic lipase (LIPC) and lipoprotein lipase (LPL) were genotyped in all study subjects with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Risk factors including gender, hypertension, hyperlipidemia, diabetes mellitus, and coronary artery disease were identified. Chi-square tests or Fisher’s exact tests were applied to discover associations between SNPs and risk factors for PCV and nAMD. Gene-gene interactions and gene-environment interactions were evaluated by the multifactor-dimensionality reduction (MDR) method.

Results

CETP rs3764261 were significantly associated with an increased risk for PCV (odds ratio (OR) = 1.444, P = 0.0247). LIPC rs1532085 conferred an increased risk for PCV (OR = 1.393, P = 0.0094). We found no association between PCV and LPL rs12678919, LIPC rs10468017 or CETP rs173539. No association was found between five SNPs with nAMD. Regarding risk factors, females were found to have significantly decreased risks for both PCV and nAMD (P = 0.006 and 0.001, respectively). Coronary artery disease (CAD) was a risk factor in PCV patients but played a protective role in nAMD patients. Hyperlipidemia was associated with PCV but not with nAMD. Neither hypertension nor diabetes mellitus was associated with PCV or nAMD. The MDR analysis revealed that a three-locus model with rs12678919, rs1532085, and gender was the best model for nAMD, while a five-locus model consisting of rs10468017, rs3764261, rs1532085, gender, and hyperlipidemia was best for PCV.

Conclusion

Our large-sample study suggested that CETP rs3764261 conferred an increased risk for PCV. We also first found the association between rs1532085 and PCV. The result of present study also showed that gender and CAD are associated with PCV and nAMD. Significant association was found between hyperlipidemia and PCV but not nAMD.     

Utilization of serum procalcitonin as a biomarker in the diagnosis and treatment of children with bacterial hospital-acquired pneumonia

Hospital-acquired pneumonia (HAP) is one of the common infections in hospitalized patients. Early and prompt diagnosis of HAP is important because it aids in the appropriate selection of antibiotics and decreases the mortality and morbidity of patients. The investigation on serum procalcitonin (PCT) levels in pediatric patients is limited. Herein we aimed to evaluate the role of PCT in the early diagnosis of children with bacterial HAP. The study enrolled 264 children (<?14 years old) who were radiographically detected by pulmonary condensation chest X-rays. The HAP patients were stratified by patterns of microbiological detection of pathogens. Baseline white blood cell (WBC) count, neutrophil proportion, PCT, and C-reactive protein (CRP) were measured on admission. The laboratory findings and microbiological findings were analyzed and compared among groups. The median PCT concentration of patients with typical bacterial pathogens (3.95?±?3.75 ng/mL) was significantly higher than the one of the patients with other pathogen types (median lower than 1.20 ng/mL). Correlation analysis indicated a significant correlation between PCT concentrations and the main inflammation makers including WBC count, neutrophil proportion, and CRP. PCT level was significantly decreased to 0.86?±?1.46 ng/mL in post-treatment patients (p?<?0.001). This cohort study with 264 pediatric HAP patients demonstrated the reliability of PCT level as a biomarker in patients with typical bacterial pathogens. Specifically, PCT cutoffs of 2 ng/mL accurately identified HAP children with typical bacterial pathogens. This finding suggested that PCT may serve as a reliable biomarker for the early diagnosis and treatment indicator of children with HAP.

     

大黄酸抗耐甲氧西林金黄色葡萄球菌的抗菌机制

【背景】耐甲氧西林金黄色葡萄球菌(Methicillin-Resistant Staphylococcus aureus,MRSA)是医院及社区常见的机会性致病菌,具有多重耐药性、高发病率和高死亡率的特点。MRSA感染已成为全球医学界的普遍难题之一。【目的】研究大黄酸对MRSA的抗菌机制。【方法】以二倍稀释法测定大黄酸对MRSA的最小抑菌浓度(Minimum Inhibitory Concentration,MIC);通过MIC测定大黄酸对MRSA动态抑菌作用;建立生物膜模型,测定大黄酸在生物膜屏障下对生物膜内存活菌的影响,并通过扫描电镜观察不同浓度大黄酸作用后的MRSA菌体形态变化;用免疫荧光染色法、荧光分析法测定大黄酸对MRSA细胞质膜去极化和细胞膜通透性的影响。【结果】大黄酸对MRSA的MIC为8μg/mL;大黄酸能对生物膜内存活菌有明显的抑制作用,而且在大黄酸作用下菌体的形态发生明显皱缩和破损,破损严重程度与浓度呈正相关;随着大黄酸浓度和作用时间的增加,荧光强度出现明显变化,细胞膜的通透性发生改变。【结论】大黄酸主要通过损伤细菌的细胞膜,从而抑制细菌的生长和繁殖。     

山黧豆β 腈基丙氨酸合成酶基因LsCAS的原核表达及蛋白聚合状态分析

该研究从山黧豆种子萌发6 d后的幼苗根中扩增到β 腈基丙氨酸合成酶基因(LsCAS)的CDS序列,并构建pGEX2T LsCAS表达载体;经诱导表达后,通过GST亲和层析进行LsCAS蛋白纯化,并利用GST标签抗体和大豆半胱氨酸合成酶(Cysteine synthase,CS)抗体对纯化蛋白进行Western blot验证;纯化后的LsCAS蛋白经凝血酶切除GST标签抗体后,利用凝胶过滤预装柱Superdex 200 Increase 10/300 GL分析判断分子量。结果显示：(1)山黧豆LsCAS基因的CDS序列为1 035 bp,编码344个氨基酸;其编码的蛋白质具有典型的CBS like 蛋白功能结构域胱硫醚β 合酶(CBS)和半胱氨酸合成酶(CS)。(2)成功构建LsCAS基因的原核表达载体pGEX2T LsCAS并进行蛋白纯化;SDS PAGE检测表明,所获融合蛋白条带单一,大小在64 kD左右;Western blot分析发现,诱导后的菌体蛋白和纯化后的重组蛋白中均能检测到特征条带,说明所获融合蛋白为山黧豆LsCAS蛋白。(3)纯化的山黧豆LsCAS蛋白在412 nm的特征吸收峰显示,LsCAS隶属于磷酸吡哆醛(PLP)依赖的半胱氨酸合成酶家族;分子排阻试验证实山黧豆LsCAS为PLP依赖性蛋白酶,可能以四聚体方式发挥作用。研究结果为进一步探讨山黧豆LsCAS的调控及其功能奠定了基础。