Tissue culture surface characteristics influence the expansion of human bone marrow cells

Human cell therapy applications in tissue engineering, such as the ex vivo production of hematopoietic cells for transplantation, have recently entered the clinic. Although considerable effort has been focused on the development of biological processes to generate therapeutic cells, little has been published on the design and manufacture of devices for implementation of these processes in a robust and reproducible fashion at a clinical scale. In this study, the effect of tissue culture surface chemistry and texture was assessed in human bone marrow (BM) mononuclear cell (MNC) and CD34-enriched cell cultures. Growth and differentiation was assessed by total, progenitor (CFU-GM), stromal (CFU-F), and primitive (LTC-IC) cell output. Tissue culture treated (TCT) plastic significantly increased MNC culture output as compared with non-TCT plastic, whereas CD34-enriched cell cultures gave lower output (than MNC cultures) that was unaffected by TCT plastic. Interestingly, the level of MNC culture output was significantly different on four commercial TCT surfaces, with the best performing surface giving output that was 1.6- to 2.8-fold greater than the worst one. The surface giving the highest output was the best at supporting development of a distinct morphological feature in the adherent layer (i.e. cobblestone area) indicative of primitive cells, and X-ray photoelectron spectroscopy (XPS) was used to characterize this surface. For custom injection molding of culture devices, the use of three different resins resulted in MNC culture output that was equivalent to commercial cultureware controls, whereas CD34-enriched cell cultures were highly sensitive to resins containing additives. When the texture of molded parts was roughened by sandblasting of the tool, MNC culture output was significantly reduced and higher spikes of IL-6 and G-CSF production were observed, presumably due to macrophage activation. In conclusion, the manufacture of BM MNC culture devices for clinical applications was optimized by consideration of plastic resin, surface treatment, and texture of the culture substratum. Although CD34-enriched cells were insensitive to surface treatment, they were considerably more sensitive to biocompatibility issues related to resin selection. The development of robust systems for BM MNC expansion will enable clinical trials designed to test the safety and efficacy of cells produced in this novel tissue engineering application.     

Assessment of conformational parameters as predictors of limited proteolytic sites in native protein structures

Despite the importance of limited proteolysis in biological systems it isoften difficult to rationalize why a proteinase hydrolyses a particularbond, given a simple sequence specificity alone. Understanding of thestructural properties limiting the proteolysis represents a first step onthe pathway to control and manipulation of this phenomena. An expanded setof nick-sites in proteins of known tertiary structure, cut by both narrowand broad specificity proteinases, has been generated yielding a robustdata set of strictly limited sites. A critical evaluation of an expandedset of conformational parameters revealed a strong correlation with limitedproteolytic sites, although they are only modest predictors in isolation.The overall predictive power is significantly improved when theconformational parameters are combined in a weighted predictive scheme thatpermits their relative importance to be compared via a Metropolis searchprotocol. A subset of the parameters performs equally well demonstratingthe key determinants of susceptibility. The derived predictive algorithmhas been made available via the internet. Its utility for predicting othersurface-correlated features is also discussed.     

Homology modelling of two subtilisin-like proteases from the hyperthermophilic archaea Pyrococcus furiosus and Thermococcus stetteri

The hyperthermophilic archaeon Pyrococcus furiosus produces an extracellular, glycosylated hyperthermostable subtilisin-like serine protease, termed pyrolysin (Voorhorst,W.G.B., Eggen,R.I.L., Geerling,A.C.M., Platteeuw,C., Siezen,R.J. and de Vos,W.M. (1996) J. Biol. Chem., 271, 20426-20431). Based on the pyrolysin coding sequence, a pyrolysin-like gene fragment was cloned and characterized from the extreme thermophilic archaeon Thermococcus stetteri. Like pyrolysin, the deduced sequence of this serine protease, designated stetterlysin, contains a catalytic domain with high homology with other subtilases, allowing homology modelling starting from known crystal structures. Comparison of the predicted three-dimensional models of the catalytic domain of stetterlysin and pyrolysin with the crystal structure of subtilases from mesophilic and thermophilic origin, i.e. subtilisin BPN' and thermitase, and the homology model of subtilisin S41 from psychrophilic origin, led to the identification of features that could be related to protein stabilization. Higher thermostability was found to be correlated with an increased number of residues involved in pairs and networks of charge-charge and aromatic-aromatic interactions. These highly thermostable proteases have several extra surface loops and inserts with a relatively high frequency of aromatic residues and Asn residues. The latter are often present in putative N-glycosylation sites. Results from modelling of known substrates in the substrate- binding region support the broad substrate range and the autocatalytic activation previously suggested for pyrolysin.      

让你的Android通知更有趣

正Android作为目前世界上最流行的手机操作系统,拥有最多的用户。然而千篇一律的界面并不能满足所有人的需求,长时间地面对一模一样的通知系统也会有些审美疲劳。怎样让我们的Android手机变得更有趣一些,让通知系统变得更强大一些,是我们可以考虑的事情。1拥有Windows Phone元素的Metro Notifications     

有限精度时间自动机的可达性检测

为了缓解状态空间爆炸问题,减小模型检测过程中生成的状态空间,加快模型检测速度,引入有限精度时间自动机(finite precision timed automata,简称FPTA)作为实时系统的形式模型,并提出了一种数据结构SDS(series of delay sequence)符号化表示状态空间中的状态集.FPTA只记录时钟变量的整数值及时钟变化的先后次序,从而减小生成的状态空间.在一定的时间约束下,Alur与Dill提出的时间自动机的可达性检测可简化为FPTA的可达性检测.举例描述了状态空间的生成过程和表示方法.最后,列出部分初步的实验结果,分析了SDS的特点及不足.     

Long-term inhalable particles and other air pollutants related to mortality in nonsmokers

Long-term ambient concentrations of inhalable particles less than 10 microm in diameter (PM10) (1973- 1992) and other air pollutants-total suspended sulfates, sulfur dioxide, ozone (O3), and nitrogen dioxide-were related to 1977-1992 mortality in a cohort of 6,338 nonsmoking California Seventh-day Adventists. In both sexes, PM10 showed a strong association with mortality for any mention of nonmalignant respiratory disease on the death certificate, adjusting for a wide range of potentially confounding factors, including occupational and indoor sources of air pollutants. The adjusted relative risk (RR) for this cause of death as associated with an interquartile range (IQR) difference of 43 d/yr when PM10 exceeded 100 microg/m3 was 1.18 (95% confidence interval [CI]: 1.02, 1.36). In males, PM10 showed a strong association with lung cancer deaths-RR for an IQR was 2.38 (95% CI: 1.42, 3.97). Ozone showed an even stronger association with lung cancer mortality for males with an RR of 4.19 (95% CI: 1.81, 9.69) for the IQR difference of 551 h/yr when O3 exceeded 100 parts per billion. Sulfur dioxide showed strong associations with lung cancer mortality for both sexes. Other pollutants showed weak or no association with mortality.     

Plasmalemmal pH-gradients in drug-sensitive and drug-resistant MCF-7 human breast carcinoma xenografts measured by 31P magnetic resonance spectroscopy

31p Magnetic resonance spectroscopy (MRS) was employed to investigate tumor pH in xenografts of drug-sensitive and drug-resistant MCF-7 human breast carcinoma cells. Measured extracellular pH values were found to be lower than the intracellular pH in all three tumor types investigated. The magnitude of this acid-outside plasmalemmal pH gradient increased with increasing tumor size in tumors of two drug-resistant variants of MCF-7 cells, but not in tumors of the parent (drug-sensitive) cells. The partitioning of weak-base or weak-acid drug molecules across the plasma membrane of a tumor cell is dependent upon the acid-dissociation constant (pKa) of the drug as well as the plasmalemmal pH gradient. A large acid-outside pH gradient, such as those seen in MCF-7 xenografts, can exert a protective effect on the cell from weak-base drugs such as anthracyclines and Vinca alkaloids, which have pKa values of 7.5 to 9.5. The possibility of enhancing the therapeutic efficacy of weak-base drugs by dietary or metabolic manipulation of the extracellular pH, in order to reduce or reverse the plasmalemmal pH gradient, deserves investigation.