High-level expression of the <Emphasis Type="Italic">Penicillium notatum</Emphasis> glucose oxidase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using codon optimization

The glucose oxidase (GOD) gene from Penicillium notatum was expressed in Pichia pastoris. The 1,815 bp gene, god-w, encodes 604 amino acids. Recombinant GOD-w had optimal activity at 35–40°C and pH 6.2 and was stable, from pH 3 to 7 maintaining >75% maximum activity after incubation at 50°C for 1 h. GOD-w worked as well as commercial GODs to improve bread making. To achieve high-level expression of recombinant GOD in P. pastoris, 272 nucleotides involving 228 residues were mutated, consistent with the codon bias of P. pastoris. The optimized recombinant GOD-m yielded 615 U ml⁻¹ (2.5 g protein l⁻¹) in a 3 l fermentor—410% higher than GOD-w (148 U ml⁻¹), and thus is a low-cost alternative for the bread baking industry.     

Identification of differentially expressed genes in primary Sjögren's syndrome

Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease that affects exocrine glands. To study the molecular mechanism and identify crucial genes/pathways in pSS pathogenesis, the microarray-based whole-genome gene expression profiles from salivary glands of patients with pSS and non-sicca controls were retrieved. After normalization and subsequent batch effect adjustment, significance analysis of microarrays method was applied to five available datasets, and 379 differentially expressed genes (DEGs) were identified. The 300 upregulated DEGs were enriched in Gene Ontology terms of immune and inflammatory responses, including antigen processing and presentation, interferon-mediated signaling pathway, and chemotaxis. Previously reported pSS-associated genes, including HLA-DRA, TAP2, PRDM1, and IFI16, were found to be significantly upregulated. The downregulated DEGs were enriched in pathways of salivary secretion, carbohydrate digestion and absorption, and starch and sucrose metabolism, implying dysfunction of salivary glands during pathogenesis. Next, a protein-protein interaction network was constructed, and B2M, an upregulated DEG, was shown to be a hub, suggesting its potential involvement in pSS development. In summary, we found the activation of pSS-associated genes in pathogenesis, and provide clues for salivary glands dysfunction. Experimental investigation on the identified DEGs in this study will deepen our understanding on pSS.     

云南48种木兰科野生植物资源遗传多样性研究

为了解云南省木兰科（Magnoliaceae）野生植物资源的遗传多样性,利用ISSR分子标记技术对48种木兰科野生植物资源进行研究。结果表明,10对引物共扩增出151条带,均为多态性条带,多态性条带百分率为100%。总的观测等位基因数（N_a）为2.000 0,有效等位基因数（N_e）为1.564 5,Nei’s基因多样性指数（H）0.337 9,Shannon’s信息指数（I）为0.510 1。总的基因多样性指数（H_t）为0.368 0,属间基因多样性指数（D_st）为0.251 9,占68.4%,基因分化系数（G_st）为0.684 0,基因流（N_m）为0.231 0。UPGMA聚类分析将48种木兰科植物划分为7个类群,各类群并非按照属聚在一起,而是不同属植物相间分布,长喙厚朴（Magnolia rostrata）、素黄含笑（Michelia flaviflora）和球花含笑（M.sphaerantha）可能为云南省木兰科植物中的原始种。48种木兰科野生植物总体具有较高的遗传多样性,但属间遗传变异较高,基因流较小,存在遗传漂变的风险,聚类结果与刘玉壶的分类系统存在分歧,这从分子水平为木兰科植物间的起源、进化与分类提供了重要依据。     

HaCaT细胞中FUT4表达与其启动子区甲基化的相关性分析

岩藻糖基转移酶Ⅳ(Fucosyltransferase Ⅳ,FUT4)在正常细胞中表达量很低,但其低表达的调控机制以及是否受其启动子甲基化调控并不十分清楚。文章采用Western blot、免疫荧光和Real-time PCR的方法检测正常人永生化表皮细胞系HaCaT细胞FUT4的表达,观察DNA甲基转移酶抑制剂5-aza-dC处理对FUT4表达的影响。应用甲基化特异性PCR方法分析HaCaT细胞中FUT4启动子甲基化状态。结果表明,HaCaT细胞中FUT4的表达水平明显低于人表皮鳞癌细胞A431和SCC12。5 μmol/L的5-aza-dC处理72 h的HaCaT细胞,其FUT4 mRNA水平明显升高,并且与未经5-aza-dC处理的对照组相比,U引物扩增检测到的产物量增加,M 引物扩增检测到的产物量明显减少。这些结果表明,HaCaT细胞中FUT4的低表达可能与其启动子区CpG岛甲基化有关。     

微生物降解3,5,6-三氯-2-吡啶醇的研究进展

随着高毒性有机磷杀虫剂的限制和禁止使用,近年来以毒死蜱为代表的低毒性有机磷杀虫剂的市场份额有所增加。然而,毒死蜱的使用也导致了环境中3,5,6-三氯-2-吡啶醇(TCP)的产生,因为TCP是毒死蜱和甲基毒死蜱在环境中降解的主要中间代谢产物。它具有较高的水溶性和迁移性,容易进入深层土壤及水体环境,从而引起广泛的污染。释放到环境中的TCP不仅可以抑制TCP及其母体化合物毒死蜱和甲基毒死蜱的生物降解,而且也能抑制其他有机污染物的生物降解,从而进一步加重环境中TCP以及其他有机污染物的累积残留,影响环境系统的自我修复功能。本文概述了TCP及其母体化合物的结构、TCP的生态毒性、TCP降解菌的多样性及其微生物降解的最新研究进展,为毒死蜱和TCP污染地区进行经济可行的生物修复提供参考。     

Interfacial electron transfer of glucose oxidase on poly(glutamic acid)-modified glassy carbon electrode and glucose sensing

The interfacial electron transfer of glucose oxidase (GOx) on a poly(glutamic acid)-modified glassy carbon electrode (PGA/GCE) was investigated. The redox peaks measured for GOx and flavin adenine dinucleotide (FAD) are similar, and the anodic peak of GOx does not increase in the presence of glucose in a mediator-free solution. These indicate that the electroactivity of GOx is not the direct electron transfer (DET) between GOx and PGA/GCE and that the observed electroactivity of GOx is ascribed to free FAD that is released from GOx. However, efficient electron transfer occurred if an appropriate mediator was placed in solution, suggesting that GOx is active. The PGA/GCE-based biosensor showed wide linear response in the range of 0.5–5.5 mM with a low detection limit of 0.12 mM and high sensitivity and selectivity for measuring glucose.     

Coexpression of EGFR and CXCR4 Predicts Poor Prognosis in Resected Pancreatic Ductal Adenocarcinoma

BackgroundEpidermal growth factor receptor (EGFR) is highly expressed in pancreatic ductal adenocarcinoma (PDAC) and is involved in tumorigenesis and development. However, EGFR expression alone has limited clinical and prognostic significance. Recently, the cross-talk between EGFR and G-protein-coupled chemokine receptor CXCR4 has become increasingly recognized.MethodsIn the present study, immunohistochemical staining of EGFR and CXCR4 was performed on paraffin-embedded specimens from 131 patients with surgically resected PDAC. Subsequently, the associations between EGFR expression, CXCR4 expression, EGFR/CXCR4 coexpression and clinicopathologic factors were assessed, and survival analyses were performed.ResultsIn total, 64 (48.9%) patients expressed EGFR, 68 (51.9%) expressed CXCR4, and 33 (25.2%) coexpressed EGFR and CXCR4. No significant association between EGFR and CXCR4 expression was observed (P = 0.938). EGFR expression significantly correlated with tumor differentiation (P = 0.031), whereas CXCR4 expression significantly correlated with lymph node metastasis (P = 0.001). EGFR/CXCR4 coexpression was significantly associated with lymph node metastasis (P = 0.026), TNM stage (P = 0.048), and poor tumor differentiation (P = 0.004). By univariate survival analysis, both CXCR4 expression and EGFR/CXCR4 coexpression were significant prognostic factors for poor disease-free survival (DFS) and overall survival (OS). Moreover, EGFR/CXCR4 coexpression significantly increased the hazard ratio for both recurrence and death compared with EGFR or CXCR4 protein expression alone. Multivariate survival analysis demonstrated that EGFR/CXCR4 coexpression was an independent prognostic factor for DFS (HR = 2.33, P<0.001) and OS (HR = 2.48, P = 0.001).ConclusionsIn conclusion, our data indicate that although EGFR expression alone has limited clinical and prognostic significance, EGFR/CXCR4 coexpression identified a subset of PDAC patients with more aggressive tumor characteristics and a significantly worse prognosis. Our results suggest a potentially important "cross-talk" between CXCR4 and EGFR intracellular pathways and indicate that the simultaneous inhibition of these pathways might be an attractive therapeutic strategy for PDAC.     

Demographic History,Population Structure,and Local Adaptation in Alpine Populations of Cardamine impatiens and Cardamine resedifolia

Species evolution depends on numerous and distinct forces, including demography and natural selection. For example, local adaptation and population structure affect the evolutionary history of species living along environmental clines. This is particularly relevant in plants, which are often characterized by limited dispersal ability and the need to respond to abiotic and biotic stress factors specific to the local environment. Here we study the demographic history and the possible existence of local adaptation in two related species of Brassicaceae, Cardamine impatiens and Cardamine resedifolia, which occupy separate habitats along the elevation gradient. Previous genome-wide analyses revealed the occurrence of distinct selective pressures in the two species, with genes involved in cold response evolving particularly fast in C. resedifolia. In this study we surveyed patterns of molecular evolution and genetic variability in a set of 19 genes, including neutral and candidate genes involved in cold response, across 10 populations each of C. resedifolia and C. impatiens from the Italian Alps (Trentino). We inferred the population structure and demographic history of the two species, and tested the occurrence of signatures of local adaptation in these genes. The results indicate that, despite a slightly higher population differentiation in C. resedifolia than in C. impatiens, both species are only weakly structured and that populations sampled at high altitude experience less gene flow than low-altitude ones. None of the genes showed signatures of positive selection, suggesting that they do not seem to play relevant roles in the current evolutionary processes of adaptation to alpine environments of these species.