A comprehensive study of construction and analysis of competitive endogenous RNA networks in lung adenocarcinoma

BackgroundLong noncoding RNAs (lncRNAs) have gain increasing attention in lung adenocarcinoma. In this study, we aimed at constructing and analyzing the lncRNAs and the related proteins based competitive endogenous RNA (ceRNA) network.MethodsRNA expression data of lung adenocarcinoma were extracted from the TCGA database. Differentially expressed (DE) lncRNAs, messenger RNAs (mRNAs) and microRNAs (miRNAs) were identified and then a DElncRNA-DEmiRNA-DEmRNA ceRNA network was constructed for lung adenocarcinoma. We also analyzed the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of the DEgenes. Kaplan-Meier survival curves were also been further utilized for exploring the prognostic factors.ResultsAfter compared and calculated lncRNA, mRNA and miRNA expression profiles between lung adenocarcinoma and normal samples, 1709 differential expressed lncRNAs, 2554 differential expressed mRNAs and 116 differential expressed miRNAs were finally identified. Afterwards, a lncRNA mediated ceRNA network was constructed, according to the interactions among 544 pairs of DElncRNA-DEmiRNA relationships and 47 pairs of DEmiRNA-DEmRNA relationships. As for the survival analyses, we found 10 DElncRNAs, 25 DEmRNAs and 7 miRNAs have statistically prognostic significance for overall survival, respectively.ConclusionsThis study provides meaningful information for deeper understanding the underlying molecular mechanism of lung adenocarcinoma and for evaluating prognosis, which could monitor recurrence, guide clinical treatment drugs and subsequent related researches.     

CRISPR/Cas9介导的USP30基因敲除细胞系的建立及其对塞内卡病毒增殖的影响

【目的】利用规律成簇的间隔短回文重复序列/Cas9核酸酶（clustered regularly interspaced short palindromic repeats/Cas9 nuclease,CRISPR/Cas9）技术建立USP30基因敲除的人胚胎肾细胞（human embryonic kidney 293T cells,HEK-293T）细胞系,为开展宿主泛素特异性蛋白酶30（ubiquitin-specific protease 30,USP30）蛋白的功能研究建立了细胞模型;同时,初步探究USP30蛋白在病毒感染过程中的作用。【方法】根据Ensemble数据库查询USP30基因序列,定位USP30在基因组中不同转录本重叠区的第一个外显子段,设计并合成2对引导RNA （single guide RNAs,sgRNA）,分别构建在pX459载体中;将pX459-USP30-sgRNA质粒转染HEK-293T细胞,并用嘌呤霉素处理,筛选出转染阳性的细胞,然后通过有限稀释法筛选单克隆细胞,通过Western blotting及测序检测USP30基因的敲除。通过Western blotting及实时荧光定量PCR分析比较塞内卡病毒（Senecavirus A,SVA）在野生型和USP30基因敲除HEK-293T细胞中的复制差异。【结果】Western blotting及测序证实USP30基因敲除单克隆细胞系构建成功。进一步实验发现,SVA在USP30基因敲除细胞中的复制水平显著低于野生型细胞。【结论】成功构建USP30基因敲除的HEK-293T细胞系,首次证明USP30对SVA的复制具有促进作用,为进一步揭示USP30相关免疫反应和SVA感染过程的作用机制提供了良好的细胞模型,也为开展宿主USP30蛋白调控病毒复制的机制研究提供了关键工具和一定的理论依据。     

表达GFP/mCherry白色念珠菌的构建及其在与巨噬细胞互作中的应用

白色念珠菌(Candida albicans)被巨噬细胞吞噬的效率与被吞噬后的形态观察是研究白色念珠菌与巨噬细胞互作的重要内容。【目的】以野生型菌株SC5314为母本,构建能够表达绿色荧光蛋白(green fluorescent protein, GFP)/mCherry的白色念珠菌,应用于巨噬细胞与白色念珠菌互作的研究。【方法】通过生长与形态观察、细胞活性检测及小鼠系统性感染模型确定荧光蛋白的表达对菌株生长、形态与毒力的影响;在共培养条件下,通过流式细胞术及荧光显微镜检测巨噬细胞的吞噬率及白色念珠菌的形态变化。【结果】构建的菌株在表型上与野生型菌株一致,并可用于在共培养下测定巨噬细胞吞噬率的流式细胞术以及观察白色念珠菌的形态变化。【结论】表达荧光蛋白的菌株为研究巨噬细胞与白色念珠菌的互作提供了新方法。     

基于Golden Gate技术的齐整小核菌转录单元高效组装系统

【目的】为齐整小核菌代谢工程研究建立高效的转录单元组装系统。【方法】通过应用Golden Gate技术,以mobius assembly为基础,分别设计并构建DNA元件标准化接口改造、单转录单元组装、应用质粒(多转录单元)组装等功能的载体,从而形成一套完整的多转录单元组装系统。【结果】构建了2个用于DNA元件标准化接口改造的Level 0载体,4个用于单转录单元组装的Level 1载体,4个用于应用质粒组装的Level 2载体和13个应用质粒组装的辅助质粒。然后应用此系统为齐整小核菌组装了若干经过标准化接口改造的DNA元件质粒、单转录单元质粒和硬葡聚糖相关基因的功能分析质粒。所构建的最终应用质粒可以同时适用于齐整小核菌的根癌农杆菌介导转化法、电穿孔转化法和原生质体转化法。【结论】此质粒系统具有强大的DNA设计、组装和容纳能力,为未来齐整小核菌代谢工程和功能基因组学研究提供了高效的质粒构建技术平台。     

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...     

A high-quality model for predicting the prognosis of breast neuroendocrine carcinoma to help clinicians decide on appropriate treatment methods: A population-based analysis

BackgroundBreast neuroendocrine carcinoma (NEC) is a rare malignancy with unclear treatment options and prognoses. This study aimed to construct a high-quality model to predict overall survival (OS) and breast cancer-specific survival (BCSS) and help clinicians choose appropriate breast NEC treatments.Patients and methodsA total of 378 patients with breast NEC and 349,736 patients with breast invasive ductal carcinoma (IDC) were enrolled in the Surveillance, Epidemiology, and End Results (SEER) database between 2010 and 2018. Propensity score matching (PSM) was performed to balance the clinical baseline. Prognostic factors determined by multivariate Cox analysis were included in the nomogram. C-index and calibration curves were used to verify the performance of the nomogram.ResultsNomograms were constructed for the breast NEC and breast IDC groups after PSM. The C–index of the nomograms ranged from 0.834 to 0.880 in the internal validation and 0.818–0.876 in the external validation, indicating that the nomogram had good discrimination. The risk stratification system showed that patients with breast NEC had worse prognoses than those with breast IDC in the low-risk and intermediate-risk groups but had a similar prognosis that those in the high-risk group. Moreover, patients with breast NEC may have a better prognosis when undergoing surgery plus chemotherapy than when undergoing surgery alone or chemotherapy alone.ConclusionsWe established nomograms with a risk stratification system to predict OS and BCSS in patients with breast NEC. This model could help clinicians evaluate prognosis and provide individualized treatment recommendations for patients with breast NEC.     

新型酵母蛋白表位标记和基因敲除质粒系统的构建及可行性验证

【目的】构建一套用于酿酒酵母基因功能研究的质粒。该套质粒结合pUG系列和pFA6a系列的优点,同时采用同尾酶实现蛋白表位标签的串联插入。【方法】利用PCR技术分别克隆pUG系列质粒的lox P位点、pFA6a质粒多酶切位点和ADH1终止子模块;通过重组连接各片段,构建pCLHN-TRP和pCLHN-URA质粒。在此基础上利用同尾酶实现多种蛋白表位标签的单个或串联重复插入,获得一系列蛋白表位标记质粒。最后,以ATG1、COX4和NHX1为例验证本质粒系列的性能。【结果】在本项工作中,我们共构建2种基因敲除用质粒和17种表位标记用质粒(涵盖1-8 FLAG、1-12 V5、3-9 HA、2-8MYC、GFP和m Cherry)。在几个靶基因上的应用证实了本套质粒的实用性。尤其值得指出的是,通过组合采用不同重复度的串联表位标签,在同一张膜上同时检测表达差异极大的不同蛋白而不使高表达蛋白信号饱和成为可能。【结论】本文所构建的pCLHN质粒系列是对现有酵母质粒工具的有益补充。     

原核生物全基因组中16S rRNA基因的识别

【目的】识别原核生物全基因组中的16S rRNA基因。【方法】本文依据基因序列的GC碱基含量、碱基3-周期性和马尔可夫链3个方面的特性,构建了识别原核生物全基因组中16S rRNA基因的三层过滤模型。【结果】经检验,模型的特异性、敏感性和马修斯相关系数分别为99.58%、91.60%和91.49%。【结论】结果表明,本文所提出的方法可以高效、准确地识别出16S rRNA基因。     

枯草芽胞杆菌异戊二烯酶ComQ的生物信息学分析及对生物膜形态的影响

[目的]枯草芽胞杆菌ComQ是一种类异戊二烯生物合成酶.利用生物信息学预测分析了ComQ的生物学特性,对comQ基因进行过表达和敲除,构建突变菌,孔板发酵培养验证生物膜形态变化.[方法]运用NCBI (National Center for Biotechnology Information)网站里的Protein数据...     

Identification of a Degradation Intermediate of the Momilactone A Rice Phytoalexin by the Rice Blast Fungus

We have already shown that major rice diterpene phytoalexin, momilactone A, was detoxified by Magnaporthe oryzae. We report here the identification by NMR, MS, and chemical synthesis of 3,6-dioxo-19-nor-9β-pimara-7,15-diene (1) as the degradation intermediate. Compound 1 exhibited similar antifungal activity to that of momilactone A, indicating 1 to be a precursor of possible detoxified compounds.     

人源脂肪酸合酶在酿酒酵母中的表达纯化和结构的初步分析

[背景] 聚酮类化合物在医药领域有重要的应用,相关药物研发依赖聚酮合酶多变的结构认知,人源脂肪酸合酶的组成结构和催化机制与聚酮合酶相近,研究人源脂肪酸合酶结构可为聚酮合酶的研究奠定基础。[目的] 在酿酒酵母中表达纯化人源脂肪酸合酶蛋白,确定合适的体外纯化条件。[方法] 以酿酒酵母BJ5464为表达载体,构建带有His和Strep双亲和层析标签的重组质粒,诱导表达蛋白后用亲和层析方法获取目标蛋白,并结合凝胶电泳和快速蛋白质液相层析技术,确定合适的蛋白纯化条件。[结果] 成功构建重组表达质粒pxw55-hfas-cSHII, 并在体外纯化得到合适浓度和纯度的人源脂肪酸合酶蛋白,筛选不同缓冲液条件并结合电子显微镜观察结果反馈,确定合适的蛋白体外纯化体系。[结论] 蛋白电镜结构分析需要有高纯度、合适浓度并且形成正确构象的蛋白样品,而人源脂肪酸合酶蛋白纯化体系的建立和纯化条件的确定为其电镜结构分析提供了良好的样品,为人源脂肪酸合酶的结构解析及结构相似但更为复杂的聚酮合酶蛋白解析奠定了良好基础。     

The Effect of Perfusion on Soft Tissue Mechanical Properties: A Computational Model

Some simple finite element models were constructed to investigate the magnitude and character of changes in mechanical properties of very soft tissues due to the loss of perfusion. Changes in the apparent stress-strain curve were used to characterise the effect of simulated blood perfusion pressure on the engineering stress-strain curve. The results indicated that the blood to tissue volume ratio and the perfusion pressure have the strongest effect on the effective stress-strain response of a representative tissue cell. Tissue viscoelasticity increased the sensitivity of the system to perfusion pressure changes.     

多酶组合催化制备L-高苯丙氨酸

【目的】L-高苯丙氨酸(L-HPA)是许多医药化学品的重要中间体,化学合成法生产L-HPA反应复杂、环境污染严重,本研究旨在开发高效环保的L-HPA酶法合成路线。【方法】采用模块化组装的方法,构建了一条以甘氨酸和苯乙醛为底物高产L-HPA的新途径。【结果】首先,根据文献挖掘设计了一条由苏氨酸醛缩酶(TA)、苏氨酸脱氨酶(TD)、苯丙氨酸脱氢酶(PheDH)和甲酸脱氢酶(FDH)组成的多酶组合催化途径,用于L-HPA的合成。其次,根据氨基基团的引入和重构,将L-HPA多酶组合催化途径分为基础单元和扩增单元,基础单元包括TA和TD,扩增单元包括PheDH和FDH。然后,利用不同表达水平的质粒,对基础单元和扩增单元进行蛋白表达的组合调节,获得最优工程菌BL21-C-M1-R-M2,使L-HPA产量达到208.6mg/L。最后,我们对全细胞转化体系进行优化,使L-HPA产量进一步提高到1226.6 mg/L,苯乙醛摩尔转化率为34.2%。【结论】该工艺路线绿色高效,为未来大规模生产L-HPA奠定基础。     

A maximum-likelihood approach for building cell-type trees by lifting

Background

In cell differentiation, a less specialized cell differentiates into a more specialized one, even though all cells in one organism have (almost) the same genome. Epigenetic factors such as histone modifications are known to play a significant role in cell differentiation. We previously introduce cell-type trees to represent the differentiation of cells into more specialized types, a representation that partakes of both ontogeny and phylogeny.

Results

We propose a maximum-likelihood (ML) approach to build cell-type trees and show that this ML approach outperforms our earlier distance-based and parsimony-based approaches. We then study the reconstruction of ancestral cell types; since both ancestral and derived cell types can coexist in adult organisms, we propose a lifting algorithm to infer internal nodes. We present results on our lifting algorithm obtained both through simulations and on real datasets.

Conclusions

We show that our ML-based approach outperforms previously proposed techniques such as distance-based and parsimony-based methods. We show our lifting-based approach works well on both simulated and real data.

     

A tumor-associated macrophages related model for predicting biochemical recurrence and tumor immune environment in prostate cancer

ObjectiveTo identify tumor-associated macrophages (TAMs) related molecular subtypes and develop a TAMs related prognostic model for prostate cancer (PCa).MethodsConsensus clustering analysis was used to identify TAMs related molecular clusters. A TAMs related prognostic model was developed using univariate and multivariate Cox analysis.ResultsThree TAMs related molecular clusters were identified and were confirmed to be associated with prognosis, clinicopathological characteristics, PD-L1 expression levels and tumor microenvironment. A TAMs related prognostic model was constructed. Patients in low-risk group all showed a more appreciable biochemical recurrence-free survival (BCRFS) than patients in high-risk group in train cohort, test cohort, entire TCGA cohort and validation cohort. SLC26A3 attenuated progression of PCa and prevented macrophage polarizing to TAMs phenotype, which was initially verified.ConclusionsWe successfully identified molecular clusters related to TAMs. Additionally, we developed a prognostic model involving TAMs that exhibits excellent predictive performance for biochemical recurrence-free survival in PCa.     

利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株

【目的】利用CRISPR/Cas9系统构建稳定敲除anxa6基因的Caco-2细胞株,为研究大肠杆菌O157:H7效应蛋白Esp F与宿主膜联蛋白A6 (ANXA6)相互作用及其致病机制奠定基础。【方法】根据CRISPR/Cas9靶向原理设计并合成3个特异性识别anxa6基因的向导RNA (single guide RNA,sgRNA),基于Lenti CRISPRv2载体构建Lenti CRISPRv2-sg RNA重组质粒,转入293T细胞中,制备sgRNA-Cas9慢病毒,将慢病毒感染Caco-2细胞,经嘌呤霉素筛选阳性细胞,有限稀释法分离培养单克隆细胞,提取单克隆细胞基因组DNA,并对敲除位点附近的DNA片段进行PCR扩增,测序并进行脱靶效应评估;免疫印记法检测ANXA6蛋白表达情况,细胞计数试剂盒8 (cell counting kit 8,CCK8)试剂盒检测细胞增殖能力,免疫荧光法检测细胞紧密连接分布情况。【结果】Western blotting及序列测序表明anxa6基因敲除单克隆细胞构建成功;脱靶效应评估结果显示预测的10个脱靶位点均无脱靶现象;基因敲除对细胞增殖能力...     

A Noninvasive Technique for Determining Sex of Live Adult North American Sturgeons

We have developed a noninvasive technique to determine the sex of adult North American sturgeon by examination of the external morphology of live individuals. We analyzed four sturgeon species taken by commercial and scientific harvest: white Acipenser transmontanus, green A. medirostris, Atlantic A. oxyrinchus and shortnose A. brevirostrum. Males have a urogenital opening in the shape of a letter Y, while the opening of females is in the shape of the letter O. We accurately sexed 26 of 36 sturgeon using this criterion. Accuracy was highest in live fish (82%), and significantly lower in dead fish (29%). Dead sturgeon usually have the rectum prolapsed so that the urogenital opening is protruded, and thus making the sexes indistinguishable.     

Typical tumor immune microenvironment status determine prognosis in lung adenocarcinoma

BackgroundImmune cells, vital components of tumor microenvironment, regulate tumor survival and progression. Lung adenocarcinoma (LUAD), the tumor with the highest mortality rate worldwide, reconstitutes tumor immune microenvironment (TIME) to avoid immune destruction. Data have shown that TIME influences LUAD prognosis and predicts immunotherapeutic efficacy. The related information about the role of TIME's characteristics in LUAD is limited.MethodsWe performed unsupervised consensus clustering via machine-learning techniques to identify TIME clusters among 1906 patients and gathered survival data. The characteristics of TIME clusters of LUAD were visualized by multi-omics analysis, pseudo-time dynamic analysis, and enrichment analysis. TIME score model was constructed by principal component analysis. Comprehensive analysis and validation were conducted to test the prognostic efficacy and immunotherapeutic response of TIME score.ResultsTIME clusters (A, B and C) were constructed and exhibited different immune infiltration states. Multi-omics analyses included significant mutated genes (SMG), copy number variation (CNV) and cancer stemness that were significantly different among the three clusters. TIME cluster A had a lower SMG, lower CNV, and lower stemness but a higher immune infiltration level compared to TIME clusters B and C. TIME score showed that patients in low TIME score group had higher overall survival rates, higher immune infiltration level and high expression of immune checkpoints. In validation cohorts, low TIME score subgroup had better drug sensitivity and favorable immunotherapeutic response.ConclusionWe constructed a stable model of LUAD immune microenvironment characteristics that may improve the prognostic accuracy of patients, provide improved explanations of LUAD responses to immunotherapy, and provide new strategies for LUAD treatment.     

Computed tomography-based radiomic model at node level for the prediction of normal-sized lymph node metastasis in cervical cancer

PurposeRadiomic models have been demonstrated to have acceptable discrimination capability for detecting lymph node metastasis (LNM). We aimed to develop a computed tomography–based radiomic model and validate its usefulness in the prediction of normal-sized LNM at node level in cervical cancer.MethodsA total of 273 LNs of 219 patients from 10 centers were evaluated in this study. We randomly divided the LNs from the 2 centers with the largest number of LNs into the training and internal validation cohorts, and the rest as the external validation cohort. Radiomic features were extracted from the arterial and venous phase images. We trained an artificial neural network (ANN) to develop two single-phase models. A radiomic model reflecting the features of two-phase images was also built for directly predicting LNM in cervical cancer. Moreover, four state-of-the-art methods were used for comparison. The performance of all models was assessed using the area under the receiver operating characteristic curve (AUC).ResultsAmong the models we built, the models combining the features of two phases surpassed the single-phase models, and the models generated by ANN had better performance than the others. We found that the radiomic model achieved the highest AUCs of 0.912 and 0.859 in the training and internal validation cohorts, respectively. In the external validation cohort, the AUC of the radiomic model was 0.800.ConclusionWe constructed a radiomic model that exhibited great ability in the prediction of LNM. The application of the model could optimize clinical staging and decision-making.     

托光柄菇属一中国新记录种

[背景] 托光柄菇属（Volvopluteus）隶属于光柄菇科（Pluteaceae）,目前世界上仅有4个种。[目的] 调查我国东北地区大型真菌资源。[方法] 采集大型真菌标本,对其形态进行详细的观察和描述,提取DNA,测定rDNA ITS序列,基于最大似然法和贝叶斯法构建系统发育树。[结果] 2019–2020年在吉林省延边朝鲜族自治州敦化市采集的标本中,有6份标本为密执安托光柄菇V.michiganensis,该种真菌此前未在我国发现。在系统发育分析中,采自我国的密执安托光柄菇V.michiganensis与该种的模式标本聚为一个分支。[结论] 密执安托光柄菇V.michiganensis为中国新记录种。