Characterization of an aerated submerged hollow fiber ultrafiltration device for efficient microalgae harvesting

The present work characterizes a submerged aerated hollow fiber polyvinylidene fluorid (PVDF) membrane (0.03 μm) device (Harvester) designed for the ultrafiltration (UF) of microalgae suspensions. Commercial baker''s yeast served as model suspension to investigate the influence of the aeration rate of the hollow fibers on the critical flux (CF, J _c) for different cell concentrations. An optimal aeration rate of 1.25 vvm was determined. Moreover, the CF was evaluated using two different Chlorella cultures (axenic and non‐axenic) of various biomass densities (0.8–17.5 g DW/L). Comparably high CFs of 15.57 and 10.08 L/m/²/h were measured for microalgae concentrations of 4.8 and 10.0 g DW/L, respectively, applying very strict CF criteria. Furthermore, the J _c‐values correlated (negative) linearly with the biomass concentration (0.8–10.0 g DW/L). Concentration factors between 2.8 and 12.4 and volumetric reduction factors varying from 3.5 to 11.5 could be achieved in short‐term filtration, whereat a stable filtration handling biomass concentrations up to 40.0 g DW/L was feasible. Measures for fouling control (aeration of membrane fibers, periodic backflushing) have thus been proven to be successful. Estimations on energy consumption revealed very low energy demand of 17.97 kJ/m³ treated microalgae feed suspension (4.99 × 10⁻³ kWh/m³) and 37.83 kJ/kg treated biomass (1.05 × 10⁻² kWh/kg), respectively, for an up‐concentration from 2 to 40 g DW/L of a microalgae suspension.     

Development of an activated carbon filtration step and high throughput screening method to remove host cell proteins from a recombinant enzyme process

An increasing number of non-mAb recombinant proteins are being developed today. These biotherapeutics provide greater purification challenges where multiple polishing steps may be required to meet final purity specifications or the process steps may require extensive optimization. Recent studies have shown that activated carbon can be employed in downstream purification processes to selectively separate host cell proteins (HCPs) from monoclonal antibodies (mAb). However, the use of activated carbon as a unit operation in a cGMP purification process is relatively new. As such, the goal of this work is to provide guidance on development approaches, insight into operating parameters and solution conditions that can impact HCP removal, as well as further investigate the mechanism of removal by using mass spectrometry. In this work, activated carbon was evaluated to remove HCPs in the downstream purification process of a recombinant enzyme. Impact of process placement, flux (or residence time), and mass loading on HCP removal was investigated. Feasibility of high throughput screening (HTS) using loose activated carbon was assessed to reduce the amount of therapeutic protein needed and enable testing of a larger number of solution conditions. Finally, mass spectrometry was used to determine the population of HCPs removed by activated carbon. Our work demonstrates that activated carbon can be used effectively in downstream processes of biopharmaceuticals to remove HCPs (up to a 3 log₁₀ reduction) and that an HTS format can be implemented to reduce material demands by up to 23x and allow for process optimization of this adsorbent for purification purposes.     

Telomerase Activity in the Various Regions of Mouse Brain: Non-Radioactive Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase, a ribonucleoprotein, is responsible for maintaining the telomere length and therefore promoting genomic integrity, proliferation, and lifespan. In addition, telomerase protects the mitochondria from oxidative stress and confers resistance to apoptosis, suggesting its possible importance for the surviving of non-mitotic, highly active cells such as neurons. We previously demonstrated the ability of novel telomerase activators to increase telomerase activity and expression in the various mouse brain regions and to protect motor neurons cells from oxidative stress. These results strengthen the notion that telomerase is involved in the protection of neurons from various lesions. To underline the role of telomerase in the brain, we here compare the activity of telomerase in male and female mouse brain and its dependence on age. TRAP assay is a standard method for detecting telomerase activity in various tissues or cell lines. Here we demonstrate the analysis of telomerase activity in three regions of the mouse brain by non-denaturing protein extraction using CHAPS lysis buffer followed by modification of the standard TRAP assay.In this 2-step assay, endogenous telomerase elongates a specific telomerase substrate (TS primer) by adding TTAGGG 6 bp repeats (telomerase reaction). The telomerase reaction products are amplified by PCR reaction creating a DNA ladder of 6 bp increments. The analysis of the DNA ladder is made by 4.5% high resolution agarose gel electrophoresis followed by staining with highly sensitive nucleic acid stain.Compared to the traditional TRAP assay that utilize ³²P labeled radioactive dCTP''s for DNA detection and polyacrylamide gel electrophoresis for resolving the DNA ladder, this protocol offers a non-toxic time saving TRAP assay for evaluating telomerase activity in the mouse brain, demonstrating the ability to detect differences in telomerase activity in the various female and male mouse brain region.     

Calpain II induced insolubilization of lens β-crystallin polypeptides may induce cataract

Addition of calpain II (EC 3.4.22.17) to soluble proteins from 10-day-old rat lens caused an increase in turbidity and production of water-insoluble protein. The insolubilization increased with higher concentrations of both lens protein and calpain II, it could be prevented by the cysteine protease inhibitor E-64; it required at least 0.5 mM Ca²⁺, it was limited to 6% of the soluble protein present and resulted from precipitation β-crystallin polypeptides. When compared by two-dimensional electrophoresis, the insoluble β-crystallin polypeptides produced by calpain II were similar to insoluble β-crystallin polypeptides found incataractous lenses. Trypsin also caused insolubilization of β-crystallin polypeptides, but these polypeptides were unlike polypeptides produced during cataract formation. These data suggested that the loss of solubility was due to a specific removal of N/or C-terminal extensions from β-crystallin polypeptides by calpain II, and that a similar process may occur in vivo during cataract formation. It is hypothesized that the insoluble protein produced by calpain II causes cataract by increasing light scatter in the lens.     

应用微核试验和单细胞凝胶电泳技术来检测农药对青蛙蝌蚪及成体的遗传毒性

应用青蛙红细胞微核试验和单细胞凝胶电泳试验研究了两种新型杀虫剂 -吡虫啉和抑食肼对青蛙蝌蚪和成体的遗传毒性 ,结果表明 :当吡虫啉为 2mg/L时 ,蝌蚪红细胞微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;浓度升高到 8mg/L时 ,微核率与对照组相比 ,有显著性差异 (p <0 .0 5) ;当浓度为 3 2mg/L时 ,微核率与对照组相比 ,有极显著性差异 (p <0 .0 1) ;并有明显的剂量 -效应关系 (r =0 .9843 )。而抑食肼在浓度为 2 .5mg/L和 10mg/L时 ,微核率与对照组相比 ,无显著性差异 (p >0 .0 5) ;当浓度增至 40mg/L时 ,微核与对照组相比 ,有极显著性差异 (p <0 .0 1) ;吡虫啉与抑食肼各浓度组对青蛙红细胞的DNA损伤与阴性对照组相比 ,都有极显著性差异 (p <0 .0 1) ,且具有明显的剂量 -效应关系 (r =0 .960 ,r=0 .990 )。     

Optimization of the first dimension for separation by two-dimensional gel electrophoresis of basic proteins from human brain tissue

A major cause of poor resolution in the alkaline pH range of two-dimensional electrophoresis (2-DE) gels is unsatisfactory separation of basic proteins in the first dimension. We have compared methods for the separation of basic proteins in the isoelectric focusing dimension of human brain proteins. The combined use of anodic cup-loading and the hydroxyethyldisulphide containing solution (DeStreak) produced better resolution in both analytical and micropreparative protein loaded 2-DE gels than the other methods investigated.     

Comparative analysis of virulence factors secreted by Bacillus anthracis Sterne at host body temperature

Aims: For the analysis of virulence factors produced and secreted by Bacillus anthracis vegetative cells during mammalian host infection, we evaluated the secretome of B. anthracis Sterne exposed to host‐specific factors specifically to host body temperature. Methods and Results: We employed a comparative proteomics‐based approach to analyse the proteins secreted by B. anthracis Sterne under host‐specific body temperature conditions. A total of 17 proteins encoded on a single chromosome and the pXO1 plasmid were identified by peptide mass fingerprinting. Multiple algorithms were used to predict the secretion mechanisms of the detected proteins in B. anthracis. Conclusions: Several putative virulence factors and known factors responsible for sporulation were differentially regulated, including CodY, pXO1‐130 and BA1952, revealing insights into temperature cues in the B. anthracis secretome. Significance and Impact of the Study: This study identified temperature‐regulated proteins. Further studies aimed at understanding the physical and functional roles of these proteins in infection and control by elevated temperatures will contribute to detection, diagnostics and prophylaxis.     

Guanylate Cyclase-Activating Protein-2 Undergoes Structural Changes upon Binding to Detergent Micelles and Bicelles

GCAPs are neuronal Ca^2 +-sensors playing a central role in light adaptation. GCAPs are N-terminally myristoylated membrane-associated proteins. Although, the myristoylation of GCAPs plays an important role in light adaptation its structural and physiological roles are not yet clearly understood. The crystal-structure of GCAP-1 shows the myristoyl moiety inside the hydrophobic core of the protein, stabilizing the protein structure; but ²H-solid-state NMR investigations on the deuterated myristoyl moiety of GCAP-2 in the presence of liposomes showed that it is inserted into the lipid bilayer. In this study, we address the question of the localization of the myristoyl group of Ca^2 +-bound GCAP-2, and the influence of CHAPS-, DPC-micelles and DMPC/DHPC-bicelles on the structure, and on the localization of the myristoyl group, of GCAP-2 by solution-state NMR. We also carried out the backbone assignment. Characteristic chemical shift differences have been observed between the myristoylated and the non-myristoylated forms of the protein. Our results support the view that in the absence of membrane forming substances the myristoyl moiety is buried inside a hydrophobic pocket of GCAP-2 similar to the crystal structure of GCAP-1. Addition of CHAPS-micelles and DMPC/DHPC-bicelles cause specific structural changes localized in and around the myristoyl binding pocket. We interpret these changes as an indication for the extrusion of the myristoyl moiety from its binding pocket and its insertion into the hydrophobic interior of the membrane mimic. On the basis of the backbone chemical shifts, we propose a structural model of myristoylated GCAP-2 in the presence of Ca^2 + and membrane mimetics.     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

高速逆流色谱法分离纯化茶黄素

首次应用高速逆流色谱法分离纯化茶黄素单体成分,溶剂系统为乙酸乙酯－正己烷－甲醇－水（３：１：１：６）,优化了分离茶黄素的条件。同时与ＳｅｐｈａｄｅｘＬＨ－２０柱色谱法梯度洗脱对比,结果表明,高速逆流色谱法分离时间相对较短,可进行较大量的分离制备。高速逆流色谱法较之ＳｅｐｈａｄｅｘＬＨ－２０柱色谱法还有一个突出的优点,即无不可逆吸附污染及不会导致样品化学变性。