拟南芥PKS5点突变体对盐碱胁迫的响应特性

拟南芥蛋白激酶PKS5参与植物对外界的盐碱胁迫信号响应过程.为探求PKS5不同结构域对外界盐碱胁迫下的响应功能,以PKS5点突变体pks5-2、pks5-4、pks5-5、pks5-6、pks5-7、pks5-8和pks5-9为材料,分析PKS5不同点突变体在外界盐碱胁迫下的特性.结果显示:(1)pks5-2、pks5-6、pks5-7和pks5-8对外界盐碱胁迫的主根生长表型与野生型存在差异,pks5-4、pks5-5和pk5-9则无差异,其中的pks5-2和pks5-8主根生长对盐碱胁迫表现出抗性表型,而pks5-6和pks5-7表现出敏感表型.(2)当PKS5发生点突变后其突变基因的表达发生改变,与野生型基因相比,PKS5-2、PKS5-6与PKS5-7的表达均有所降低.(3) PKS5点突变蛋白的亚细胞定位与野生型相比不存在差异,在细胞核、细胞质及细胞膜中均有分布.(4)pks5各点突变体内的Na+含量在野生型与点突变体间存在显著差异,pks5-2体内的Na+含量较野生型降低,而pks5-6和pks5-7体内的Na+则升高.研究表明,PKS5不同位置点突变导致植物对外界盐碱胁迫有着不同的响应过程,预示PKS5不同的结构域在其功能上存在差异.     

高寒草地狼毒与阴山扁蓿豆种群的空间格局

不同的格局类型和空间关联性可以反映出干扰状态下天然草地植物种群的资源环境适应对策.采用草地群落学调查与点格局分析方法,分析了祁连山北坡高寒草地优势种群更替过程中狼毒和阴山扁蓿豆种群的空间格局及其种间关联关系.结果表明:随着草地退化程度加剧,狼毒种群地上生物量、密度、植株高度持续增加,空间分布类型由聚集分布转变为非聚集分布,阴山扁蓿豆种群高度逐渐降低,种群密度和地上生物量先增大后减小,空间分布类型由均匀或聚集分布转为随机分布;狼毒和阴山扁蓿豆的空间关联性由显著正关联区间不断增大转为关联性不显著.草地退化过程中群落上层禾草西北针茅种群的衰退以及狼毒和阴山扁蓿豆的植株高度差异,导致了植物对光资源的非对称性竞争,使二者的资源分配策略发生了调整,并影响了空间分布格局和种间关联性.     

Triple mutated antibody scFv2F3 with high GPx activity: insights from MD, docking, MDFE, and MM-PBSA simulation

By combining computational design and site-directed mutagenesis, we have engineered a new catalytic ability into the antibody scFv2F3 by installing a catalytic triad (Trp²⁹–Sec⁵²–Gln⁷²). The resulting abzyme, Se-scFv2F3, exhibits a high glutathione peroxidase (GPx) activity, approaching the native enzyme activity. Activity assays and a systematic computational study were performed to investigate the effect of successive replacement of residues at positions 29, 52, and 72. The results revealed that an active site Ser⁵²/Sec substitution is critical for the GPx activity of Se-scFv2F3. In addition, Phe²⁹/Trp–Val⁷²/Gln mutations enhance the reaction rate via functional cooperation with Sec⁵². Molecular dynamics simulations showed that the designed catalytic triad is very stable and the conformational flexibility caused by Tyr¹⁰¹ occurs mainly in the loop of complementarity determining region 3. The docking studies illustrated the importance of this loop that favors the conformational shift of Tyr⁵⁴, Asn⁵⁵, and Gly⁵⁶ to stabilize substrate binding. Molecular dynamics free energy and molecular mechanics-Poisson Boltzmann surface area calculations estimated the pK _a shifts of the catalytic residue and the binding free energies of docked complexes, suggesting that dipole–dipole interactions among Trp²⁹–Sec⁵²–Gln⁷² lead to the change of free energy that promotes the residual catalytic activity and the substrate-binding capacity. The calculated results agree well with the experimental data, which should help to clarify why Se-scFv2F3 exhibits high catalytic efficiency.     

4019例妇科门诊不同症状患者阴道微生态状况分析

目的探讨妇科门诊不同症状患者的阴道微生态状况。方法回顾性分析2011年11月至2012年9月西安交通大学医学院第一附属医院妇科门诊因不同症状就诊患者的阴道微生态评价（菌群的密集度、多样性、优势菌、病原菌、AV评分、Nugent评分及五项菌群功能及炎症反应指标）检测结果。结果4019例被检查者平均年龄为33．56岁。其中阴道微生态正常占n70％（28／4019）;阴道微生态失调者占99．30％（3991／4019）;后者包括未明确感染的阴道微生态失调患者占66．36％（2667／4019）;明确感染的阴道微生态失调患者占32．94％（1324／4019）;其中外阴阴道假丝酵母菌病（vulvovaginal candidiasis,VVC）患者占24．33％（978／4019）;需氧菌陛阴道炎（aerobic vaginitis,AV）患者占3．11％（125／4019）;细菌性阴道病（bacterial vaginosis,BV）患者占3．14％（126／4019）;滴虫阴道炎（trichomonas vaginitis,TV）患者占5．15％（207／4019）;混合感染患者占8．31％（110／1324）。优势菌群异常患者占54．32％（2183／4019）。结论妇科门诊中未明确感染的阴道微生态失调患者构成比最高;在明确感染的患者中外阴阴道假丝酵母菌病构成比最高;因此阴道微生态评价成为阴道感染性疾病诊断首选检测方法,在临床诊疗实践中具有重要意义。     

Waist Circumference,Not Body Mass Index,Is Associated with Renal Function Decline in Korean Population: Hallym Aging Study

Background

Prospective investigation of obesity and renal function decline in Asia is sparse. We examined the associations of body mass index (BMI) and waist circumference (WC) with renal function decline in a prospective study of Korean population.

Methods

A total of 454 participants who had baseline estimated glomerular filtration rate (eGFR) levels of more than 60 mL/min/1.73 m² in Hallym Aging Study (HAS) were included and followed for 6 years. Renal function decline was defined as follows: (1) an eGFR decline ≥3 mL/min/1.73 m²/year (n = 82 cases); (2) an eGFR decrease of 20% or greater (n = 87 cases) at follow-up; (3) an eGFR decrease of 20% greater at follow-up or eGFR decline ≥3 mL/min/1.73 m²/year (n = 91 cases); and (4) an eGFR <60 mL/min/1.73 m² at follow-up (n = 54 cases). eGFR was determined based on the Modification of Diet in Renal Disease (MDRD) Study equation. Multivariate logistic regression model was used to determine the association between obesity and renal function decline.

Results

We found that central obesity was associated with faster renal function decline. Comparing WC of >95 cm in men or >90 cm in women with ≤90 cm in men or ≤85 cm in women, ORs (95% CIs) ranged from 2.31 (1.14–4.69) to 2.78 (1.19–6.50) for the 4 definitions of renal function decline (all p-values for trend <0.05). Waist-to-hip ratio (WHR) and waist-to-height ratio (WHtR) also was associated with renal function decline. There was no significant association of BMI with renal function decline.

Conclusions

Central obesity, but not BMI, is associated with faster renal function decline in Korean population. Our results provide important evidence that simple measurement of central fat deposition rather than BMI could predict decline in renal function in Korean population.     

Protective Effect of the Silkworm Protein 30Kc6 on Human Vascular Endothelial Cells Damaged by Oxidized Low Density Lipoprotein (Ox-LDL)

Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases.     

Promoter Hypermethylation of ARID1A Gene Is Responsible for Its Low mRNA Expression in Many Invasive Breast Cancers

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.     

Enhancing the evaluation of PI3K inhibitors through 3D melanoma models

Targeted therapies for mutant BRAF metastatic melanoma are effective but not curative due to acquisition of resistance. PI3K signaling is a common mediator of therapy resistance in melanoma; thus, the need for effective PI3K inhibitors is critical. However, testing PI3K inhibitors in adherent cultures is not always reflective of their potential in vivo. To emphasize this, we compared PI3K inhibitors of different specificity in two‐ and three‐dimensional (2D, 3D) melanoma models and show that drug response predictions gain from evaluation using 3D models. Our results in 3D demonstrate the anti‐invasive potential of PI3K inhibitors and that drugs such as PX‐866 have beneficial activity in physiological models alone and when combined with BRAF inhibition. These assays finally help highlight pathway effectors that could be involved in drug response in different environments (e.g. p4E‐BP1). Our findings show the advantages of 3D melanoma models to enhance our understanding of PI3K inhibitors.