A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO‐DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO‐DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA‐based CHO‐DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self‐organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up‐regulating NCK1 and down‐regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial‐ and endoplasmic reticulum‐mediated cell death pathways by up‐regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1026–1038, 2015     

Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015     

Predicting fine‐scale distributions of peripheral aquatic species in headwater streams

Headwater species and peripheral populations that occupy habitat at the edge of a species range may hold an increased conservation value to managers due to their potential to maximize intraspecies diversity and species' adaptive capabilities in the context of rapid environmental change. The southern Appalachian Mountains are the southern extent of the geographic range of native Salvelinus fontinalis and naturalized Oncorhynchus mykiss and Salmo trutta in eastern North America. We predicted distributions of these peripheral, headwater wild trout populations at a fine scale to serve as a planning and management tool for resource managers to maximize resistance and resilience of these populations in the face of anthropogenic stressors. We developed correlative logistic regression models to predict occurrence of brook trout, rainbow trout, and brown trout for every interconfluence stream reach in the study area. A stream network was generated to capture a more consistent representation of headwater streams. Each of the final models had four significant metrics in common: stream order, fragmentation, precipitation, and land cover. Strahler stream order was found to be the most influential variable in two of the three final models and the second most influential variable in the other model. Greater than 70% presence accuracy was achieved for all three models. The underrepresentation of headwater streams in commonly used hydrography datasets is an important consideration that warrants close examination when forecasting headwater species distributions and range estimates. Additionally, it appears that a relative watershed position metric (e.g., stream order) is an important surrogate variable (even when elevation is included) for biotic interactions across the landscape in areas where headwater species distributions are influenced by topographical gradients.     

Adaptive Evolution of p53 Thermodynamic Stability

The thermodynamic stability of a protein plays an important role during evolution and adaptation in order to maintain a folded and active conformation. p53 is a tumour suppressor involved in the regulation of numerous genes. Human p53 has an unusually low thermodynamic stability and is frequently inactivated by oncogenic missense mutations. Here, we examined the thermodynamic and kinetic stability of p53 DNA binding domains from selected invertebrate and vertebrate species by differential scanning calorimetry and equilibrium urea denaturation. There is a correlation in the apparent melting temperature of p53 with the body temperature of homeotherm vertebrates. We found that p53 from these organisms has a half-life for spontaneous unfolding at organismal body temperature of 10-20 min. We also found that p53 from invertebrates has higher stability, bearing more resemblance towards p63 and p73 from humans. Using structure-guided mutagenesis on the human p53 scaffold, we demonstrated that the amino acid changes on the protein surface and in the protein interior lead to the elevated stability of p53 orthologs. We propose a model in which the p53 DNA binding domain has been shaped by the complex interplay of different selective pressures and underwent adaptive evolution leading to pronounced effects on its stability. p53 from vertebrates has evolved to have a low thermodynamic stability and similarly short spontaneous half-life at organismal body temperature, which is related to function.     

厚朴营养成分分析

利用高效液相色谱法及重量法等对野生植物厚朴的营养成分进行了分析,结果表明,厚朴中含有矿质元素、蛋白质、维生素等多种营养成分,旨在为开发利用厚朴的植物资源提供科学依据.     

传统中草药对肠道益生微生物的作用研究进展

探讨传统中草药对肠道益生微生物的调整作用,重点概述了不同种类与剂量中草药对益生微生物生长的影响及相应的研究方法。在上述基础上对中草药与肠道益生微生物的作用机制、研究前景和发展趋势进行了展望。     

对治疗糖尿病的传统中药中生命元素含量测定初步研究

利用原子吸收分光光度法对几味治疗糖尿病的常用中药进行锌、镁、铁、铜、铬、硒元素的含量测定。结果表明,实验选取的中药中与糖尿病关系密切的生命元素含量较丰富,且所测元素含量与糖尿病患者体内其含量呈负相关性。为探讨中药的作用机理、中药配制工艺提供一定的信息和理论依据,对于糖尿病患者治疗具有参考价值。     

秸秆浸提液漂浮栽培对不结球白菜产量及相关品质特性的影响

以1/4园试营养液为对照,以腐熟发酵的农业秸秆(苋菜和番茄植株残体)浸提液模拟富含N、P的水体进行不结球白菜(Brassica campestris ssp.chinensis Makino)的漂浮栽培,对茎叶的单株鲜质量和干质量及叶片的VC、可溶性蛋白质、蔗糖、果糖、葡萄糖含量及可溶性糖总含量进行了比较分析。结果表明:在24 d的栽培周期内,随生长时间的延长,处理组和对照组茎叶的单株鲜质量和干质量均呈增加趋势,但从第10天开始,处理组茎叶的单株鲜质量和干质量均显著低于对照组。栽培初期叶片VC含量均最高且处理组叶片VC含量显著低于对照组;从第10天开始,叶片VC含量降低,但处理组叶片VC含量与对照组无显著差异。处理组与对照组叶片可溶性蛋白质含量均呈波动的变化趋势,但总体差异不显著。处理组叶片蔗糖含量总体上与对照组差异不显著,仅在第24天显著低于对照组。处理组叶片的果糖含量均高于对照组,特别是在第24天为对照组的1.81倍,差异显著。叶片葡萄糖含量均呈先升高后降低的趋势,可溶性糖总含量则呈波动的变化趋势,对照组叶片葡萄糖含量和可溶性糖总含量总体上高于处理组,但差异不显著。研究结果显示:用秸秆浸提液漂浮栽培,虽然不结球白菜的生长量(即产量)有所减少,但相关的品质指标变化不大,对果糖合成和积累还有一定的促进作用,因而,此种秸秆浸提液可用于栽培商品不结球白菜。