Impact of aeration strategy on CHO cell performance during antibody production

Stirred tank bioreactors using suspension adapted mammalian cells are typically used for the production of complex therapeutic proteins. The hydrodynamic conditions experienced by cells within this environment have been shown to directly impact growth, productivity, and product quality and therefore an improved understanding of the cellular response is critical. Here we investigate the sub‐lethal effects of different aeration strategies on Chinese hamster ovary cells during monoclonal antibody production. Two gas delivery systems were employed to study the presence and absence of the air–liquid interface: bubbled direct gas sparging and a non‐bubbled diffusive silicone membrane system. Additionally, the effect of higher gas flow rate in the sparged bioreactor was examined. Both aeration systems were run using chemically defined media with and without the shear protectant Pluronic F‐68 (PF‐68). Cells were unable to grow with direct gas sparging without PF‐68; however, when a silicone membrane aeration system was implemented growth was comparable to the sparged bioreactor with PF‐68, indicating the necessity of shear protectants in the presence of bubbles. The cultures exposed to increased hydrodynamic stress were shown by flow cytometry to have decreased F‐actin intensity within the cytoskeleton and enter apoptosis earlier. This indicates that these conditions elicit a sub‐lethal physiological change in cells that would not be detected by the at‐line assays which are normally implemented during cell culture. These physiological changes only result in a difference in continuous centrifugation performance under high flow rate conditions. Product quality was more strongly affected by culture age than the hydrodynamic conditions tested. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.     

Novel nanoimaging approach: Antibodious polymeric nanolabel for intracellular alpha‐fetoprotein targeted monitoring

This study describes preparation and use of novel labeled and antibodious polymeric nanolabels (anti‐alpha fetoprotein cross‐linked nanolabels) as an immunogenic and semisynthetic nanolabel with potential prognostic and therapeutic roles for hepatoma cancer. Specificity, uptake, and binding efficiencies of the nanolabel have been examined in a human hepatosarcoma cell line HepG2, a human colorectal cell line DLD‐1, and a mouse myoblast cell line C2. Labeling of the cells has been performed by treating live and fixed cells with varying concentrations of the nanolabels and then, the cells have been examined under a fluorescence microscope. In addition, all cell lines have also been labeled using FITC‐conjugated nanotrastuzumab to compare the results obtained with those of the binding of the FITC‐nanoanti‐alpha fetoprotein nanolabels. Results show that FITC‐conjugated anti‐alpha fetoprotein cross‐linked nanolabels have been taken up by both live and fixed cells and have efficiently and specifically labeled HepG2 cells at a quite low concentration. Taken all together, the results indicate that the novel targeted nanoimaging tools and technique demonstrated their ability to detect the distribution of the nanolabels as probes in hepatoma cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 472–479, 2013     

DNA data and morphology reveal the existence of Kentrochrysalis streckeri Staudinger, 1880 (Lepidoptera: Sphingidae) instead of K. consimilis in South Korea

In the present study, we determined that specimens of Kentrochrysalis consimilis collected from South Korea were K. streckeri, rather than K. consimilis, based on morphology, DNA barcodes and nuclear elongation factor 1 alpha (EF‐1α) sequences. The major morphological differences between K. streckeri and K. consimilis include the shape of forewing and hind‐wing pattern elements and male and female genitalia. The DNA barcode analysis of the South Korean specimens and the Russia‐originated K. streckeri showed a maximum sequence divergence of only 0.659% (4 bp), whereas that of the South Korean specimens and Japan‐originated K. consimilis showed a minimum sequence divergence of 2.965% (18 bp), indicating that the Korean specimens are, in fact, K. streckeri and not K. consimilis. Phylogenetic analyses both by Bayesian inference and maximum likelihood methods strongly clustered the South Korean specimens and Russian K. streckeri into one group, excluding K. consimilis. The EF‐1α‐based sequence and phylogenetic analyses of the two species also supported data from the DNA barcode, indicating the distribution of K. streckeri in South Korea, instead of K. consimilis.     

Notch-Hif-1-alpha信号通路参与调控大鼠肝再生过程的研究

目的：探讨使用酌分泌酶抑制剂Fli-06 特异性阻断Notch 信号通路后,大鼠肝部分切除术后肝再生的情况并初步阐明 Notch-Hif-1-alpha信号通路调控肝再生的可能机制。方法：取SD 大鼠分为对照（生理盐水注射组,n=24）和抑制剂组（酌分泌酶抑制剂 注射组,n=24）。给予药物处理后,两组分别施行大鼠肝部分切除术,术后0 d,1 d,3 d,5 d,每个时间点分别留取对照组（n=6）及抑 制剂组（n=6）再生的肝组织,并检测相应肝重体重比,免疫组化法检测再生肝PCNA（增殖细胞核抗原,Proliferation Cell Nuclear Antigen）表达,RT-PCR 检测再生肝组织中的Notch1、Hes1、VEGF mRNA的变化,Western-Blot 法检测NICD（Notch 胞内段,Notch Intracellular Domain）、Hif-1-alpha（低氧诱导因子-1-alpha,Hypoxia Inducible Factor-1琢)蛋白在肝再生过程的变化情况。结果：1、肝部分切除 术后3 d和5 d,抑制剂组肝重体重比明显低于对照组差异有统计学意义（P<0.05）;2、免疫组织化学染色结果提示：抑制剂组再生 肝PCNA阳性细胞率在术后1 d,3 d,5 d 均明显低于对照组（P<0.05）;3、Western blot 结果表明：NICD 和Hif-1-alpha蛋白水平明显低 于对照组（P<0.05）;同时RT-PCR 结果提示：抑制剂组Hes1 的mRNA表达量术后1 d,3 d明显低于对照组,差异具有统计学意义 （P<0.05）。同时,抑制剂组VEGF mRNA 水平在术后3 d,5 d明显低于对照组,差异具有统计学意义（P<0.05）。结论：在大鼠肝部分 切除术后肝再生过程中,使用酌分泌酶抑制剂Fli-06 抑制Notch 信号通路后,大鼠的肝再生能力明显降低,Notch-Hif-1alpha信号通 路可能参与调控了大鼠肝部分切除术后肝再生过程。     

SPA患者循环EPCs及SDF-1-alpha与冠状动脉侧支循环的相关性分析

目的：研究冠状动脉严重狭窄稳定型心绞痛(Stable angina pectoris, SPA)患者循环内皮祖细胞（endothelial progenitor cells, EPCs）及基质细胞衍生因子（SDF）-1-alpha与冠状动脉侧支循环（CCC）形成的相关性,以期为治疗冠心病提供新的思路。方法：选择 2012 年8 月到2014 年12月在我院就诊的88 例冠状动脉严重狭窄的稳定型心绞痛患者（CCC 良好40 例、不良48 例）,均采集 外周血测定EPC 数量、体外生成血管能力,并用ELISA 法检测其血浆SDF-1alpha 水平,采用直线相关和Pearson 检验分析CCC良好 与不良者各指标间及与CCC 分级的相关性;将所有入选病例随机分为6 组,并分离外周血单个核细胞并分别加入不同的培养 液,培养7 天后体外测定EPCs 数量以及生成血管的能力,并通过ELISA 法检测培养液上清中VEGF 的蛋白水平。结果：CCC 不 良组EPCs 数量、体外生成血管能力及SDF-1-alpha水平均明显低于CCC 良好组（P<0.05）。体外生成血管能力、循环EPCs 数量以及 SDF-1alpha水平均与CCC分级呈现显著的正相关性（r =0.72、0.67、0.79,均P<0.05）;循环EPCs 数量、SDF-1alpha水平以及体外生成血 管能力亦均呈现显著正相关性（r =0.78、0.62,均P<0.05）。与PBS、SDF-1alpha+ AMD3100 及SDF-1alpha+ KI8751 干预物质比较,SDF-1琢 能够呈剂量依赖性的明显提高EPCs 数量、增强其体外生成血管的能力及VEGF水平（P<0.05）。结论：冠状动脉严重狭窄稳定型 心绞痛患者循环EPCs及SDF-1琢与CCC 形成有关,VEGF可能参与该过程。     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

PTPRN2 and PLCβ1 promote metastatic breast cancer cell migration through PI(4,5)P2‐dependent actin remodeling

Altered abundance of phosphatidyl inositides (PIs) is a feature of cancer. Various PIs mark the identity of diverse membranes in normal and malignant cells. Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) resides predominantly in the plasma membrane, where it regulates cellular processes by recruiting, activating, or inhibiting proteins at the plasma membrane. We find that PTPRN2 and PLCβ1 enzymatically reduce plasma membrane PI(4,5)P₂ levels in metastatic breast cancer cells through two independent mechanisms. These genes are upregulated in highly metastatic breast cancer cells, and their increased expression associates with human metastatic relapse. Reduction in plasma membrane PI(4,5)P₂ abundance by these enzymes releases the PI(4,5)P₂‐binding protein cofilin from its inactive membrane‐associated state into the cytoplasm where it mediates actin turnover dynamics, thereby enhancing cellular migration and metastatic capacity. Our findings reveal an enzymatic network that regulates metastatic cell migration through lipid‐dependent sequestration of an actin‐remodeling factor.     

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Molecular-assisted alpha taxonomy of the genus Rhodymenia (Rhodymeniaceae,Rhodymeniales) from Australia reveals overlooked species diversity

A previously published DNA barcode survey of red macroalgae in Australia revealed significant cryptic and overlooked diversity for the genus Rhodymenia with recognition of R. novahollandica, R. prolificans, R. stenoglossa, R. wilsonis and an additional four uncharacterized genetic species groups. Since that study, increased sampling effort in Australia has warranted reassessment and reinvestigation of the number of genetic species groups attributed to Rhodymenia and their respective taxonomic affiliations. Using molecular-assisted alpha taxonomy employing the DNA barcode (COI-5P), the present study resolved 188 Australian specimens in 12 genetic species groups assignable to the genus Rhodymenia. Four of these groups were attributed to the previously recognized species (above), whereas some collections from Lord Howe Island were attributed to the New Zealand species R. novazelandica, expanding its biogeographic range. The following seven genetic groups were inconsistent with existing species of Rhodymenia and established as novel taxa: R. compressa sp. nov., R. contortuplicata sp. nov., R. gladiata sp. nov., R. insularis sp. nov., R. lociperonica sp. nov., R. norfolkensis sp. nov. and R. womersleyi sp. nov. Although morphological and biogeographic features were adequate for distinguishing some species of Rhodymenia from Australia, DNA sequencing in combination with morphology and biogeography provided the most reliable means of identification.