A Synthetic Oligonucleotide Model for Evaluating the Oxidation and Crosslinking Propensities of Natural Furan‐Modified DNA

We have previously developed a crosslinking methodology for oligonucleotides based on the incorporation of furan moieties, which can be selectively oxidised to reactive intermediates that will quickly react with the opposite bases in DNA, forming toxic interstrand crosslinks (ICLs). Furan moieties also occur in natural DNA, as a result of oxidative stress. Moreover, the furan‐containing degradation product of this modified DNA—kinetin—has been found to display beneficial anti‐ageing effects. To investigate the apparent discrepancy between the effects of the synthetic and the natural furan modifications in DNA, a quick and easy postsynthetic method providing access to the natural modification in short synthetic oligonucleotides was developed. On checking for potential crosslinking propensity, we found that the furan moiety does indeed undergo oxidation, in this way functioning as an important scavenger for oxidative stress. The reactive intermediate, however, was shown to degrade without producing toxic crosslinked products.     

Assembly of designed oligonucleotides as an efficient method for gene recombination: a new tool in directed evolution

A new and practical method for gene recombination with formation of libraries of mutant genes is presented. The method is based on the assembly of appropriately prepared oligonucleotides whose design is guided by sequence information. High recombination frequency with formation of full-length products is achieved by controlled overlapping of the designed oligomers. This process (ADO) minimizes self-hybridization of parental genes, which constitutes a significant advantage over conventional family shuffling as used in the directed evolution of functional enzymes. ADO was applied to the recombination of two lipase family genes from Bacillus subtilis (LipA and LipB). In a library of 3000 lipase variants created by this method, several were found that display increased enantioselectivity in a model reaction involving the hydrolysis of a meso-diacetate.     

大肠杆菌GBP的定点突变与制备方法研究

在mglB基因上进行了E149C、 A213S、 L238S三个定点突变,并利用大肠杆菌BL21(DE3)/pET28c系统,构建了表达突变型GBP的基因工程菌BL21(DE3)/pLE3.工程菌经诱导培养后收获细胞,利用渗透休克法提取周质空间蛋白,经Ni-NTA柱纯化,从1 L培养液中可得到约3.5 mg SDS-PAGE纯度的GBP.     

环境工程专业点源与农业面源结合型教学体系的构建

农业面源污染已成为我国污染的重要来源,但我国环境工程专业教育的教学课程、手段和理念均以工程技术教育为核心,缺乏对面源污染基本理念的教育,致使我国面源污染治理技术人员极度缺乏。本文阐明了环境工程专业教学面临的困境及存在的问题,提出了围绕教学课程、手段、理论及实习基地相结合而转变的理念:增加面源污染课程,及时更新教学手段,丰富农业面源污染控制实习基地,让学生及时地掌握农业和生态的新知识、新理论和新技能。意在为我国环境工程毕业生完善知识结构、拓宽就业面提供重要思路。     

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.     

Beating Bias in the Directed Evolution of Proteins: Combining High‐Fidelity on‐Chip Solid‐Phase Gene Synthesis with Efficient Gene Assembly for Combinatorial Library Construction

Saturation mutagenesis (SM) constitutes a widely used technique in the directed evolution of selective enzymes as catalysts in organic chemistry and in the manipulation of metabolic paths and genomes, but the quality of the libraries is far from optimal due to the inherent amino acid bias. Herein, it is shown how this fundamental problem can be solved by applying high‐fidelity solid‐phase chemical gene synthesis on silicon chips followed by efficient gene assembly. Limonene epoxide hydrolase was chosen as the catalyst in the model desymmetrization of cyclohexene oxide with the stereoselective formation of (R,R)‐ and (S,S)‐cyclohexane‐1,2‐diol. A traditional combinatorial PCR‐based SM library, produced by simultaneous randomization at several residues by using a reduced amino acid alphabet, and the respective synthetic library were constructed and compared. Statistical analysis at the DNA level with massive sequencing demonstrates that, in the synthetic approach, 97 % of the theoretically possible DNA mutants are formed, whereas the traditional SM library contained only about 50 %. Screening at the protein level also showed the superiority of the synthetic library; many highly (R,R)‐ and (S,S)‐selective variants being discovered are not found in the traditional SM library. With the prices of synthetic genes decreasing, this approach may point the way to future directed evolution.     

Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.     

教学课程与思政教育知识构建的探析——以制药工程专业有机化学课程为例

课程思政是专业教育与德育的有机融合。如何将德育潜移默化地滲透、贯穿于教学过程中,助力学生的全面发展,培养学生的爱国主义情怀,引导学生树立报效祖国的理想,是专业课教师必须思考的问题。文章以制药工程专业有机化学课程为例,探讨了在专业课程中开展思政教育的具体措施,并通过教学实践结果分析确认了课程思政对于学生的积极影响。     

化工原理案例教学的构建与实施--以“流体流动”为例

以"流体流动"一章为例,分析确定该章的知识框架和知识要点,设计并建立开篇案例、问题案例和综合案例三层次的案例内容,教学中通过不同案例的组合,实现了由点到面、由浅入深的工程知识系统化学习及运用,取得了良好的效果。     

Aspen plus在化工过程分析与合成课程教学中的应用

化工过程分析与合成是化工类实践性非常强的专业基础课,精馏塔的实践教学是教学的重点和难点。文章采用Aspen Plus软件,以甲醇制二甲醚的实际工艺为案例,对二甲醚精馏塔进行模拟、分析,同时使学生熟悉Aspen Plus软件的使用,为后续化工设计打下基础。     

地方高校医化类《专业英语》课程建设研究

根据地方性高校的实际情况,结合多年来专业英语教学的经验,将我校医药化工类《专业英语》作为平台课进行课程建设的研究和实践。分别从教学内容、教学方式和能力培养等方面深化教学改革,取得了良好的教学效果。     

Experimental Construction of BMP2 and VEGF Gene Modified Tissue Engineering Bone in Vitro

The purpose of this study was to investigate the feasibility and advantages of constructing a novel tissue engineering bone, using β-tricalcium phosphate (β-TCP) and rat bone marrow mesenchymal stem cells (MSCs), modified with human bone morphogenetic protein 2 gene (hBMP2) and human vascular endothelial growth factor 165 gene (hVEGF165), through lentiviral transfection. Both genes were successfully co-expressed in the co-transfection group for up to eight weeks confirmed by enzyme-linked immunosorbent assay (ELISA). After seeding MSCs onto the scaffolds, scanning electron microscopy (SEM) observation showed that MSCs grew and proliferated well in co-transfection group at 7 and 14 days. There was no significant difference among all the groups in hoechst DNA assay for cell proliferation for 14 days after cell seeding (P > 0.05), but the highest alkaline phosphatase (ALP) activity was observed in the co-transfection group at 14 days after cell seeding (p < 0.01). These results demonstrated that it was advantageous to construct tissue engineering bone using β-TCP combined with MSCs lentivirally co-transfected with BMP2 and VEGF165, providing an innovative way for treating bone defects.     

Unravelling the Contribution of the rs7041 and rs4588 Polymorphisms of the GC Gene and Serum VDBP Levels for Developing Metabolic Syndrome in the Mexican Population

Metabolic syndrome (MetS) is a multifactorial disorder integrated by a constellation of cardiovascular risk factors. The genetic and environmental determinants of MetS are not fully elucidated. This study investigated the association of two common single nucleotide polymorphisms (SNPs) on GC, rs7041 and rs4588, derived haplotypes, and serum vitamin D binding protein (VDBP) levels with the susceptibility to suffer MetS in Mexican adults. We included 1924 individuals; clinical and biochemical data were obtained through standard methods. Genotyping was performed through predesigned TaqMan assays. Logistic regression models were used to assess the associations of interest. Prevalence of MetS was 52.9% in the whole population, being more frequent in women. We observed that some association results differed between sexes. The GG genotype of the rs7041 was associated with increased odds of MetS in women. For the rs4588, the CA genotype had a protective effect against MetS in women. The haplotype GC2 was associated with reduced odds for MetS and some of its components in women. Our data suggest that VDBP serum levels were influenced by genotypes/haplotypes and this interplay seems to influence the risk of MetS. Our data provide reliable evidence regarding the association of GC polymorphisms with MetS risk in Mexican women.     

新工科背景下化妆品技术与工程专业创新创业教育现状及启示——以美国、欧洲、亚洲部分大学为例

化妆品技术与工程专业是新工科背景下由轻化工程专业衍生而来的新的工科专业.我国化妆品市场规模位居全球第二,然而目前全国仅有12所高校开设此专业,现有的教育体系不能满足化妆品产业的复合型创新创业人才需求.因此,科学合理地建构化妆品技术与工程专业创新创业教育体系,对于产业的发展具有战略性意义.美国、欧洲、亚洲部分大学化妆品工...     

Ipr1和GFP基因真核共表达穿梭质粒的构建及其在人肺癌细胞中的表达

目的构建胞内病原体抗性基因1(Ipr1)和绿色荧光蛋白(GFP)基因真核共表达穿梭质粒,并在人肺腺癌细胞A549中表达。方法采用PCR方法,分别从质粒pEGFP-C1-Ipr1和pEGFP-C1中扩增Ipr1和GFP基因,将GFP基因、分枝杆菌复制子OriM和Ipr1基因同时克隆入多启动子真核共表达载体pBudCE4.1中,构建pBud-GFP-OriM-Ipr1穿梭质粒,脂质体法转染A549细胞,荧光显微镜观察GFP的表达,免疫组化方法检测Ipr1蛋白的表达。结果酶切和测序分析表明,pBud-GFP-OriM-Ipr1真核共表达穿梭质粒构建正确。转染A549细胞后,荧光显微镜下可观察到转染细胞中有GFP表达,免疫组化法可检测到Ipr1蛋白的表达,且定位于细胞核内。结论已成功构建Ipr1和GFP基因真核共表达穿梭质粒,为进一步研究Ipr1抗结核的功能奠定了基础。     

颗粒溶素活性肽与增强型绿色荧光蛋白融合基因真核表达质粒的构建及其在小鼠黑色素瘤细胞中的表达

目的构建人颗粒溶素(GLS)活性肽(9ku-GLS)与增强型绿色荧光蛋白(EGFP)融合基因的真核表达载体,并检测其在小鼠黑色素瘤细胞B16中的表达。方法经PCR扩增9ku-GLS基因,插入质粒pBudCE4.1中,经酶切及测序鉴定正确后,亚克隆至质粒pEGFP-C1中,将融合基因真核表达质粒pEGFP-C1/S9K转染B16细胞,采用荧光显微镜及RT-PCR法检测融合蛋白的表达。结果融合基因真核表达质粒pEGFP-C1/S9K经双酶切鉴定证明构建正确,转染B16细胞24h后,荧光显微镜下可观察到绿色荧光,且经RT-PCR可扩增出267bp的目的基因片段。结论已成功构建9ku-GLS与EGFP融合基因的真核表达质粒,且在B16细胞中表达了融合蛋白。     

Construction and Validation of a Prognostic Gene-Based Model for Overall Survival Prediction in Hepatocellular Carcinoma Using an Integrated Statistical and Bioinformatic Approach

Hepatocellular carcinoma (HCC) is one of the most common lethal cancers worldwide and is often related to late diagnosis and poor survival outcome. More evidence is demonstrating that gene-based prognostic models can be used to predict high-risk HCC patients. Therefore, our study aimed to construct a novel prognostic model for predicting the prognosis of HCC patients. We used multivariate Cox regression model with three hybrid penalties approach including least absolute shrinkage and selection operator (Lasso), adaptive lasso and elastic net algorithms for informative prognostic-related genes selection. Then, the best subset regression was used to identify the best prognostic gene signature. The prognostic gene-based risk score was constructed using the Cox coefficient of the prognostic gene signature. The model was evaluated by Kaplan–Meier (KM) and receiver operating characteristic curve (ROC) analyses. A novel four-gene signature associated with prognosis was identified and the risk score was constructed based on the four-gene signature. The risk score efficiently distinguished the patients into a high-risk group with poor prognosis. The time-dependent ROC analysis revealed that the risk model had a good performance with an area under the curve (AUC) of 0.780, 0.732, 0.733 in 1-, 2- and 3-year prognosis prediction in The Cancer Genome Atlas (TCGA) dataset. Moreover, the risk score revealed a high diagnostic performance to classify HCC from normal samples. The prognosis and diagnosis prediction performances of risk scores were verified in external validation datasets. Functional enrichment analysis of the four-gene signature and its co-expressed genes involved in the metabolic and cell cycle pathways was constructed. Overall, we developed a novel-gene-based prognostic model to predict high-risk HCC patients and we hope that our findings can provide promising insight to explore the role of the four-gene signature in HCC patients and aid risk classification.     

新升格本科院校专科生物理化学实验教学探究——以钦州学院化学化工学院为例

结合新升格本科院校里专科生的特点,参考本科教学大纲,从实验内容、实验成员分工、实验教学顺序、实验辅助教学、实验理念、实验秩序等方面阐述本科院校中专科生的物理化学实验教学改革思路。