Expression and Characterization of Monomeric Recombinant Isocitrate Dehydrogenases from Corynebacterium glutamicum and Azotobacter vinelandii for NADPH Regeneration

The enzymatic transformation of various chemicals, especially using NADPH-dependent hydroxylase, into more soluble and/or high value-added products has steadily garnered increasing attention. However, the industrial application of these NADPH-dependent hydroxylases has been limited due to the high cost of the cofactor NADPH. As an alternative, enzymatic NADPH-regeneration systems have been developed and are frequently used in various fields. Here, we expressed and compared two recombinant isocitrate dehydrogenases (IDHs) from Corynebacterium glutamicum and Azotobacter vinelandii in Escherichia coli. Both enzymes were hyper-expressed in the soluble fraction of E. coli and were single-step purified to apparent homogeneity with yields of more than 850 mg/L. These enzymes also functioned well when paired with NADPH consumption systems. Specifically, NADPH was regenerated from NADP⁺ when an NADPH-consuming cytochrome P450 BM3 from Bacillus megaterium was incorporated. Therefore, both enzymes could be used as alternatives to the commonly used regeneration system for NADPH. These enzymes also have promising potential as genetic fusion partners with NADPH-dependent enzymes due to the monomeric nature of their quaternary structure, thereby resulting in self-sufficient biocatalysts via NADPH regeneration in a single polypeptide with NADPH-dependent activity.     

A Novel Antibody-Drug Conjugate Targeting Nectin-2 Suppresses Ovarian Cancer Progression in Mouse Xenograft Models

Ovarian cancer is the fifth leading cause of cancer, followed by front line is mostly platinum agents and PARP inhibitors, and very limited option in later lines. Therefore, there is a need for alternative therapeutic options. Nectin-2, which is overexpressed in ovarian cancer, is a known immune checkpoint that deregulates immune cell function. In this study, we generated a novel anti-nectin-2 antibody (chimeric 12G1, c12G1), and further characterized it using epitope mapping, enzyme-linked immunosorbent assay, surface plasmon resonance, fluorescence-activated cell sorting, and internalization assays. The c12G1 antibody specifically bound to the C2 domain of human nectin-2 with high affinity (K_D = 2.90 × 10⁻¹⁰ M), but not to mouse nectin-2. We then generated an antibody-drug conjugate comprising the c12G1 antibody conjugated to DM1 and investigated its cytotoxic effects against cancer cells in vitro and in vivo. c12G1-DM1 induced cell cycle arrest at the mitotic phase in nectin-2-positive ovarian cancer cells, but not in nectin-2-negative cancer cells. c12G1-DM1 induced ~100-fold cytotoxicity in ovarian cancer cells, with an IC₅₀ in the range of 0.1 nM~7.4 nM, compared to normal IgG-DM1. In addition, c12G1-DM1 showed ~91% tumor growth inhibition in mouse xenograft models transplanted with OV-90 cells. These results suggest that c12G1-DM1 could be used as a potential therapeutic agent against nectin-2-positive ovarian cancers.     

Hypoxia Promotes Angiogenic Effect in Extracranial Arteriovenous Malformation Endothelial Cells

Arteriovenous malformation (AVM) is characterized by high-flow blood vessels connecting arteries and veins without capillaries. This disease shows increased angiogenesis and a pathophysiological hypoxic environment in proximal tissues. Here, we analyzed the effects of hypoxia on angiogenesis in the endothelial cells (ECs) of AVM and normal tissues. ECs from human normal and AVM tissues were evaluated using immunocytochemistry with CD31. In vitro tube formation under hypoxia was tested in both ECs using Matrigel. The relative expression of angiogenesis-related genes was measured using real-time PCR. Under normoxia, CD31 was significantly higher in AVM ECs (79.23 ± 0.65%) than in normal ECs (74.15 ± 0.70%). Similar results were observed under hypoxia in AVM ECs (63.85 ± 1.84%) and normal ECs (60.52 ± 0.51%). In the tube formation test under normoxic and hypoxic conditions, the junction count and total vessel length were significantly greater in AVM ECs than normal ECs. Under both normoxia and hypoxia, the angiogenesis-related gene FSTL1 showed a significantly higher expression in AVM ECs than in normal ECs. Under hypoxia, CSPG4 expression was significantly lower in AVM ECs than in normal ECs. Accordingly, the angiogenic effect was increased in AVM ECs compared with that in normal ECs. These results provide a basic knowledge for an AVM treatment strategy.     

萌芽黑豆中蛋白质组分分子量分布及其抗氧化活性研究

本文目的是研究萌芽黑豆中不同蛋白质组分的分子量分布,以及各组分的抗氧化活性。采用碱提酸沉法获得萌芽黑豆中的蛋白质,通过硫酸铵盐析法逐级分离各蛋白质组分,SDS-PAGE法测定各蛋白质组分分子量分布,并从清除自由基和抗人皮肤成纤维细胞氧化损伤两个方面研究了各蛋白质组分的抗氧化活性。萌芽黑豆中蛋白质组分可以集中分离为三个部分,其分子量分布为140.0¹66.0、45.0⁷2.0、17.0²3.0ku;清除自由基和抗人皮肤成纤维细胞氧化损伤的实验结果均表明分子量在17.0²3.0ku的蛋白质组分具有最优的抗氧化活性,说明低分子量的萌芽黑豆蛋白质具有更强的抗氧化活性。      

混合菌种发酵酸马乳的降血脂功能研究

为研究混合菌种发酵酸马乳的降血脂作用,采用混合菌种发酵酸马乳对小鼠进行实验,研究其对高脂模型小鼠体重、血脂水平、脏器指数和动脉硬化（AI）指数的影响。结果表明,混合菌种发酵酸马乳中、高剂量能够显著降低高血脂症小鼠TC、TG、LDL-C、AI水平（p<0.05）,显著升高HLD-C水平（p<0.05）,并且能降低小鼠体重和肝脏指数,说明混合菌种发酵酸马乳对实验性高脂血症小鼠具有降低血脂的作用,对高脂血症及动脉粥样硬化具有一定的预防作用。      

单增李斯特菌非可培养状态的诱导与PMA-qPCR检测

在9%（w/v）NaCl条件下,通过温度、pH两种因素诱导一株单增李斯特菌进入\     

基于PLS-LDA和拉曼光谱快速定性识别食用植物油

以6种食用油共计23个样本为分析对象,采用偏最小二乘线性判别分析法（PLS-LDA）和拉曼光谱进行单一种类（橄榄油、花生油和玉米油）食用油快速定性检测,通过自适应迭代惩罚最小二乘法（airPLS）对拉曼信号进行背景扣除,以及蒙特卡洛无信息变量消除法筛选波长变量,不但有效减少了波长点数,降低了建模运算量,而且提高了单一种类食用油的识别率,使得总体识别率均高于90%,并在此基础上进一步提出了采用PLS-LDA进行多种类食用油识别的检测流程。实验结果表明PLS-LDA在食用油定性识别检测中具有较好的应用前景和可行性,该方法也可为定性检测食品及农产品品质提供借鉴。      

花椒精非挥发性成分对大鼠肠道健康的影响

目的:研究花椒精中非挥发性组成成分对大鼠肠道健康的影响。方法:4组实验鼠分别被灌胃相同量的大豆油、花椒精去除挥发油成分(RE)、花椒麻味物质(NTC)和花椒精除去以上两部分后的剩余物(REN)的大豆油溶液,4周后处死取样,测定各相关指标。结果:灌胃NTC组盲肠壁面积减小,游离氨浓度显著升高,短链脂肪酸含量无显著变化,盲肠内容物中乳酸菌等有益菌数量减少,大肠杆菌等有害菌数量显著上升;RE组各指标变化趋势与NTC组相同,但没有NTC作用效果显著;灌胃REN组短链脂肪酸浓度显著增大,游离氨浓度显著升高,pH呈下降趋势,盲肠壁面积增大,盲肠内容物中部分有益菌和有害菌数量均有上升趋势。结论:RE和NTC均会抑制大鼠肠道发酵,对肠道健康有害,且NTC的作用强于RE组分。      

三层链式客户/服务模型及其分析

为了改进传统的2层客户/服务结构的缺陷,提出了3层链式客户/服务计算模型,研究了其中间层任务的分布.     

基于Revit二次开发实现三维装饰算量

本文针对Revit无法满足按国内清单计价规范计算装饰工程量的不足,采用插件开发的方法,基于Revit二次开发技术对装饰算量进行了研究与实践,通过研发装饰建模算量一体化平台,实现三维装饰算量,提高装饰算量效率和精度。