An Integrative Transcriptomic Analysis of Systemic Juvenile Idiopathic Arthritis for Identifying Potential Genetic Markers and Drug Candidates

Systemic juvenile idiopathic arthritis (sJIA) is a rare subtype of juvenile idiopathic arthritis, whose clinical features are systemic fever and rash accompanied by painful joints and inflammation. Even though sJIA has been reported to be an autoinflammatory disorder, its exact pathogenesis remains unclear. In this study, we integrated a meta-analysis with a weighted gene co-expression network analysis (WGCNA) using 5 microarray datasets and an RNA sequencing dataset to understand the interconnection of susceptibility genes for sJIA. Using the integrative analysis, we identified a robust sJIA signature that consisted of 2 co-expressed gene sets comprising 103 up-regulated genes and 25 down-regulated genes in sJIA patients compared with healthy controls. Among the 128 sJIA signature genes, we identified an up-regulated cluster of 11 genes and a down-regulated cluster of 4 genes, which may play key roles in the pathogenesis of sJIA. We then detected 10 bioactive molecules targeting the significant gene clusters as potential novel drug candidates for sJIA using an in silico drug repositioning analysis. These findings suggest that the gene clusters may be potential genetic markers of sJIA and 10 drug candidates can contribute to the development of new therapeutic options for sJIA.     

Co-Expression of Neighboring Genes in the Zebrafish (Danio rerio) Genome

Neighboring genes in the eukaryotic genome have a tendency to express concurrently, and the proximity of two adjacent genes is often considered a possible explanation for their co-expression behavior. However, the actual contribution of the physical distance between two genes to their co-expression behavior has yet to be defined. To further investigate this issue, we studied the co-expression of neighboring genes in zebrafish, which has a compact genome and has experienced a whole genome duplication event. Our analysis shows that the proportion of highly co-expressed neighboring pairs (Pearson’s correlation coefficient R>0.7) is low (0.24% ~ 0.67%); however, it is still significantly higher than that of random pairs. In particular, the statistical result implies that the co-expression tendency of neighboring pairs is negatively correlated with their physical distance. Our findings therefore suggest that physical distance may play an important role in the co-expression of neighboring genes. Possible mechanisms related to the neighboring genes’ co-expression are also discussed.     

基于离散时序基因表达数据的双聚类算法

目前应用于基因表达数据上的双聚类算法大多是基于真实数据提出的, 因此易受噪声干扰, 且这些算法很少考虑样本间的时序性。提出了一种有效的时间点连续的双聚类挖掘算法DTCB, 从离散的时序基因表达数据中挖掘出时间点连续的最大共表达双聚类。该算法使用了一种新的数据离散化方法, 同时提出了三种在离散数据集下基因间的共表达关系; 为了提高挖掘效率, DTCB使用了有效的剪枝和输出策略, 可以在不产生候选集的情况下一次性挖掘出所有的最大共表达双聚类。通过实验分析, 证明DTCB具有高效的性能和良好的鲁棒性, 且结果具有较好的统计和生物意义。     

共表达谷氨酰-tRNA还原酶增强染料脱色过氧化物酶在大肠杆菌中的表达活性

染料脱色过氧化物酶(dye-decoloorizing peroxidase,DyP)属于以血红素为辅基的新型过氧化物酶类,常因缺乏辅因子而导致催化活性低。将来源于褐色嗜热裂孢菌(Thermobifida fusca)的染料脱色过氧化物酶基因(TfuDyP)与大肠杆菌谷氨酰-tRNA还原酶基因(hemA),构建重组质粒phemA-DyP,转化至E.coli BL21中进行共表达。分别以2,2-联氮-二(3-乙基苯并噻唑-6-磺酸)二铵盐(ABTS)和顽固性染料活性蓝19(RB19)、溴酚蓝、溴甲酚绿为底物检测TfuDyP的催化活力以及染料脱色效率。结果表明,诱导后的共表达菌株pAD胞内血红素含量为9. 8μmol/L,而单独过表达基因TfuDyP的菌株pD仅为3. 4μmol/L。TfuDyP纯酶的全波长扫描分析表明,在菌株pAD中DyP酶与血红素的结合度相比pD有较大幅度的提升。pAD菌株表达的DyP酶活力较pD菌株提高了110%,酶活力的提高使其在染料脱色应用方面也得到增强。在pAD菌株培养基中分别添加谷氨酸(Glu)、FeCl2使得胞内血红素含量、DyP酶活力和染料脱色效率比未添加时进一步提高。以上结果为TfuDyP的功能开发奠定了基础,同时也为其他血红素依赖性过氧化物酶的研发提供借鉴。     

Expression and Iron Storage Properties of Recombinant Human Ferritins in Escherichia Coli: Relevance for Functional Ferritins in Food Systems

Ferritin is an iron storage protein found in most living organisms. Human heavy chain (H-) and light chain (L-) ferritin were amplified from human heart cDNA library. Each ferritin gene was inserted down stream of the T7 promoter of bacterial expression, and finally four types of H-, L-, and co-expression vectors were constructed. Recombinant human ferritins were overexpressed and identified with SDS-PAGE and western blotting. The expression levels of recombinant ferritin proteins ranged about 29–36% of whole cell total protein. From atomic absorption spectrometry (AAS) analysis, the rate of iron uptake for H-ferritin was significantly faster than that for the HL-, LH-, and L‐ferritin, respectively. From AAS analysis, the levels of iron content in cells progressively increased in transformants with 0–30 mM ferric citrate in the media. Among these ferritin transformants, the highest amount of cellular iron was observed with H‐ferritin transformant. The total amounts of iron content in E. coli could be sequentially ranked as H-ferritin > HL-ferritin > LH-ferritin > L-ferritin > negative transformants. Expressed recombinant human ferritins indicated that their assembled subunits were able to store iron in the cells. The results of this study further enhances our understanding of iron uptake properties in vivo and suggest similar strategies for using food-grade ferritins for functional foods or iron-fortified food ingredients.     

敲除ptsG基因及共表达透明颤菌血红蛋白提高大肠杆菌SHMT产量

利用Red同源重组技术敲除大肠杆菌BL21(DE3)的ptsG基因,得到ptsG基因缺失菌株BL21(DE3)ΔptsG。构建重组质粒,得到表达大肠杆菌丝氨酸羟甲基转移酶(serine hydroxymethyltransferase,SHMT)工程菌株BL21(DE3)/pET-glyA、BL21(DE3)ΔptsG/pET-glyA,及共表达SHMT和透明颤菌血红蛋白(Vitreoscilla hemoglobin,VHb)工程菌株BL21(DE3)ΔptsG/pET-SV。在LB培养基中,BL21(DE3)ΔptsG/pET-glyA菌株与BL21(DE3)/pET-glyA菌株生长情况没有明显差异,BL21(DE3)ΔptsG/pET-SV菌株稳定期的OD 600 nm值比BL21(DE3)ΔptsG/pET-glyA菌株提高了21.3%,在LBG培养基中,稳定期BL21(DE3)ΔptsG/pET-glyA菌株OD600 nm值比BL21(DE3)/pET-glyA菌株提高了19.4%,BL21(DE3)ΔptsG/pET-SV菌株OD600 nm值比BL21(DE3)ΔptsG/pET-glyA菌株提高了21.5%。在LBG培养基中,经过异丙基-β-D-硫代半乳糖苷诱导,与对照菌株BL21(DE3)/pET-28a相比较,两种单表达工程菌株产SHMT活力分别是其6.4倍和7.7倍,共表达工程菌株产SHMT活力是其9.6倍。实验结果表明,敲除ptsG基因能够增加大肠杆菌在含葡萄糖培养基中的生长量及SHMT表达量,共表达VHb能进一步提高菌株生长量和SHMT产量。     

串联表达NAD激酶和谷氨酸脱氢酶基因对L-谷氨酸产量的影响

由ppnk和gdh编码的NAD激酶和谷氨酸脱氢酶是L-谷氨酸合成途径中的两个重要酶。以谷氨酸生产菌CN1021基因组为模板PCR扩增ppnk和gdh基因并转化至该菌中,检测上述两个基因单独表达和串联表达对L-谷氨酸产量的影响。结果表明,当两个基因单独过表达时,其酶活性分别提高2.4和2.1倍,L-谷氨酸产量分别提高7.9%和1.4%。当将两个基因串联表达时,其酶活性分别提高2.0和1.5倍,L-谷氨酸的产量却提高了13.2%。说明过表达ppnk和gdh能够有效提高L-谷氨酸产量,且具有协同作用。