An outcome model for human bladder cancer: A comprehensive study based on weighted gene co‐expression network analysis

The precision evaluation of prognosis is crucial for clinical treatment decision of bladder cancer (BCa). Therefore, establishing an effective prognostic model for BCa has significant clinical implications. We performed WGCNA and DEG screening to initially identify the candidate genes. The candidate genes were applied to construct a LASSO Cox regression analysis model. The effectiveness and accuracy of the prognostic model were tested by internal/external validation and pan‐cancer validation and time‐dependent ROC. Additionally, a nomogram based on the parameter selected from univariate and multivariate cox regression analysis was constructed. Eight genes were eventually screened out as progression‐related differentially expressed candidates in BCa. LASSO Cox regression analysis identified 3 genes to build up the outcome model in E‐MTAB‐4321 and the outcome model had good performance in predicting patient progress free survival of BCa patients in discovery and test set. Subsequently, another three datasets also have a good predictive value for BCa patients' OS and DFS. Time‐dependent ROC indicated an ideal predictive accuracy of the outcome model. Meanwhile, the nomogram showed a good performance and clinical utility. In addition, the prognostic model also exhibits good performance in pan‐cancer patients. Our outcome model was the first prognosis model for human bladder cancer progression prediction via integrative bioinformatics analysis, which may aid in clinical decision‐making.     

宫颈癌差异表达基因筛选及功能分析

目的:筛选参与宫颈癌发生、发展的关键基因,为临床诊疗提供新的靶点。方法:在NCBI-GEO数据库中筛选多组宫颈癌基因表达检测数据集,利用GEO2R分析工具筛选各组数据集的差异表达基因;应用R分析筛选不同数据集之间共有的差异表达基因;利用DAVID在线分析对差异表达基因进行功能聚类和通路分析;利用STRING分析差异表达基因编码蛋白之间的相互作用关系。结果:共选择6组表达数据集,筛选得到59个差异表达基因(宫颈癌组织vs正常组织),表达差异至少达2倍,其中包含50个表达上调基因及9个表达下调基因。这些差异表达基因参与细胞周期、DNA复制、细胞分裂等生物进程。蛋白互作分析表明,这些差异表达基因多数存在相互作用。结论:利用生物信息学方法对不同来源的基因检测数据进行整合分析,有助于更准确的筛选对宫颈癌发生、发展过程具有重要作用的关键基因,本文筛选的宫颈癌差异基因为进一步研究宫颈癌发生、发展的分子机制及临床诊疗提供思路。     

PPARγ与c/EBPα基因在苏太猪不同组织中的表达水平探究

为探究PPARγ与c/EBPα基因在苏太猪不同组织中的表达与脂肪沉积的关系,本实验以10月龄苏太猪为研究对象,运用实时荧光定量PCR (q RT-PCR)技术检测PPARγ与c/EBPα基因mRNA在苏太猪心、肝、脾、肺、肾、胃、背最长肌和皮下脂肪8个组织中的表达水平。结果表明,PPARγ与c/EBPα基因在苏太猪的8个组织中均有不同程度的表达,其中,PPARγ基因在苏太猪脾脏组织中的表达量最高,皮下脂肪中的表达水平仅次于脾;以背最长肌中PPARγ基因的相对表达量作对比,背最长肌与脾、肺和皮下脂肪的相对表达差异极显著(p<0.01),其余为差异不显著(p>0.05),表达量高低顺序为脾>皮下脂肪>肺>心>胃>肾>肝>背最长肌;c/EBPα基因在苏太猪的皮下脂肪的表达量最高,以背最长肌中c/EBPα基因的相对表达量作对比,在肝、脾、皮下脂肪组织中表达差异极显著(p<0.01),肺的相对表达差异显著(p<0.05),其余组织中差异不显著(p>0.05),表达量的高低顺序为皮下脂肪>肝>脾>肺>肾>心>胃>背最长肌。两基因在各组织中表达趋势趋于一致。试验结果表明PPARγ和c/EBPα基因可能对猪脂肪沉积有重要影响。     

A high‐throughput BAC end analysis protocol (BAC‐anchor) for profiling genome assembly and physical mapping

Traditional approaches for sequencing insertion ends of bacterial artificial chromosome (BAC) libraries are laborious and expensive, which are currently some of the bottlenecks limiting a better understanding of the genomic features of auto‐ or allopolyploid species. Here, we developed a highly efficient and low‐cost BAC end analysis protocol, named BAC‐anchor, to identify paired‐end reads containing large internal gaps. Our approach mainly focused on the identification of high‐throughput sequencing reads carrying restriction enzyme cutting sites and searching for large internal gaps based on the mapping locations of both ends of the reads. We sequenced and analysed eight libraries containing over 3 200 000 BAC end clones derived from the BAC library of the tetraploid potato cultivar C88 digested with two restriction enzymes, Cla I and Mlu I. About 25% of the BAC end reads carrying cutting sites generated a 60–100 kb internal gap in the potato DM reference genome, which was consistent with the mapping results of Sanger sequencing of the BAC end clones and indicated large differences between autotetraploid and haploid genotypes in potato. A total of 5341 Cla I‐ and 165 Mlu I‐derived unique reads were distributed on different chromosomes of the DM reference genome and could be used to establish a physical map of target regions and assemble the C88 genome. The reads that matched different chromosomes are especially significant for the further assembly of complex polyploid genomes. Our study provides an example of analysing high‐coverage BAC end libraries with low sequencing cost and is a resource for further genome sequencing studies.     

Integration of high-throughput analytics and cell imaging enables direct early productivity and product quality assessment during Chinese Hamster ovary cell line development for a complex multi-subunit vaccine antigen

Mammalian cell line generation typically includes stable pool generation, single cell cloning and several rounds of clone selection based on cell growth, productivity and product quality criteria. Individual clone expansion and phenotype-based ranking is performed initially for hundreds or thousands of mini-scale cultures, representing the major operational challenge during cell line development. Automated cell culture and analytics systems have been developed to enable high complexity clone selection workflows; while ensuring traceability, safety, and quality of cell lines intended for biopharmaceutical applications. Here we show that comprehensive and quantitative assessment of cell growth, productivity, and product quality attributes are feasible at the 200–1,200 cell colony stage, within 14 days of the single cell cloning in static 96-well plate culture. The early cell line characterization performed prior to the clone expansion in suspension culture can be used for a single-step, direct selection of high quality clones. Such clones were comparable, both in terms of productivity and critical quality attributes (CQAs), to the top-ranked clones identified using an established iterative clone screening approach. Using a complex, multi-subunit antigen as a model protein, we observed stable CQA profiles independently of the cell culture format during the clonal expansion as well as in the batch and fed-batch processes. In conclusion, we propose an accelerated clone selection approach that can be readily incorporated into various cell line development workstreams, leading to significant reduction of the project timelines and resource requirements.     

草地贪夜蛾海藻糖合成酶基因的克隆、时空表达谱及对温度胁迫的响应

【目的】本研究旨在通过克隆草地贪夜蛾Spodoptera frugiperda的海藻糖合成酶(trehalose-6-phosphate synthase)基因SfTPS,分析其在草地贪夜蛾不同发育阶段、不同组织中的表达水平及不同温度胁迫时5龄幼虫中的相对表达量,为进一步探究TPS在草地贪夜蛾生长发育及抗逆应激反应中的功能奠定基础。【方法】运用RT-PCR技术克隆草地贪夜蛾SfTPS的全长编码区,并进行生物信息学分析。运用RT-qPCR技术检测SfTPS在草地贪夜蛾不同发育阶段(卵、1-6龄幼虫、蛹和成虫)、5龄幼虫不同组织(体壁、中肠和脂肪体)中和经短期(2, 4和8 h)高(35℃)低温(10℃)胁迫后5龄幼虫中的表达变化。【结果】克隆获得2 571 bp的草地贪夜蛾TPS cDNA序列,命名为SfTPS(GenBank登录号： MT920672),全长开放阅读框(ORF)长2 481 bp,编码的826个氨基酸具有TPS和TPP两个保守结构域。同源比对和系统进化分析表明,昆虫TPS蛋白具有较高的保守性,SfTPS与斜纹夜蛾S. litura的TPS亲缘关系最近,序列一致性达到99.15%。SfTPS中α-螺旋、β-折叠和无规则卷曲占比分别为38.14%, 12.23%和48.55%;SfTPS的三级结构为同源二聚体。RT-qPCR结果表明,SfTPS在草地贪夜蛾卵期和1-5龄幼虫期低表达,在6龄幼虫期、蛹期和成虫期高表达,且草地贪夜蛾变态前后SfTPS的表达量变化较大。组织分布结果显示,SfTPS在5龄幼虫脂肪体中表达量最高。草地贪夜蛾5龄幼虫经2~8 h低温(10℃)和高温(35℃)胁迫后,SfTPS的相对表达量显著高于对照(25℃),分别为对照的4.43~9.34和2.50~6.03倍。【结论】SfTPS基因在草地贪夜蛾生长发育过程及抵御高低温度胁迫中可能具有重要作用。