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991.
Michal Dabros Michael Amrhein Dominique Bonvin Ian W. Marison Urs von Stockar 《Biotechnology progress》2009,25(2):578-588
Real‐time data reconciliation of concentration estimates of process analytes and biomass in microbial fermentations is investigated. A Fourier‐transform mid‐infrared spectrometer predicting the concentrations of process metabolites is used in parallel with a dielectric spectrometer predicting the biomass concentration during a batch fermentation of the yeast Saccharomyces cerevisiae. Calibration models developed off‐line for both spectrometers suffer from poor predictive capability due to instrumental and process drifts unseen during calibration. To address this problem, the predicted metabolite and biomass concentrations, along with off‐gas analysis and base addition measurements, are reconciled in real‐time based on the closure of mass and elemental balances. A statistical test is used to confirm the integrity of the balances, and a non‐negativity constraint is used to guide the data reconciliation algorithm toward positive concentrations. It is verified experimentally that the proposed approach reduces the standard error of prediction without the need for additional off‐line analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
992.
João Dias Filipa Pardelha Mário Eusébio Maria A. M. Reis Rui Oliveira 《Biotechnology progress》2009,25(2):390-398
In this work, an algorithm for on‐line adaptive metabolic flux analysis (MFA) is proposed and applied to polyhydroxybutyrate (PHB) production by mixed microbial cultures (MMC). In this process, population dynamics constitutes an important source of perturbation to MFA calculations because some stoichiometric and energetic parameters of the underlying metabolic network are continuously changing over time. The proposed algorithm is based on the application of the observer‐based estimator (OBE) to the central MFA equation, whereby the role of the OBE is to force the accumulation of intracellular metabolites to converge to zero by adjusting the values of unknown network parameters. The algorithm was implemented in a reactor equipped with on‐line analyses of dissolved oxygen and carbon dioxide through respirometric and titrimetric measurements. The oxygen and carbon dioxide fluxes were measured directly, whereas acetate, PHB, and sludge production fluxes were estimated indirectly using a projection of latent structures model calibrated a priori with off‐line measurements. The algorithm was implemented in a way that the network parameters associated with biosynthesis were adjusted on‐line. The algorithm proofed to converge exponentially with the steady state error always below 1 mmol/L. The estimated fluxes passed the consistency index test for experimental error variances as low as 1%. The comparison of measured and estimated respiratory coefficient and of the theoretical and estimated yield of sludge on acetate further confirmed the metabolic consistency of the parameters that were estimated on‐line. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 相似文献
993.
Gengxian Shi Yulan Jin Zhuangzhi Wang Wei Cui Yin Liu Xun Wang Liping Zhu 《中国科学:生命科学英文版》2001,44(4):365-372
The effect of transduction with a reverse fragment to a cDNA encoding human 6A8 ?-mannosidase on apoptosis induction of human B cell line SKW6 by anti-Fas antibody was tested. Apoptosis-inducer of anti-Fas monoclonal antibody was used to induce apoptosis in SKW6 cells. Giemsa’s staining, Annexin-V-FLUOS staining and DNA ladder test were used to determine the events of apoptosis. Indirect immunofluorescent staining with anti-Fas antibody was performed to detect the surface Fas expression. In a time-course test of 12, 24 and 36 h for apoptosis induction by anti-Fas antibody, DNA ladder was observed in the wild-type SKW6 cells in a time-dependent fashion. Mock transduction had no effect on DNA ladder production. However, no DNA ladder was detected in the rAAV-antisense 6A8 cDNA-transduced SKW6. Results from Annexin-V-FLUOS staining on anti-Fas antibody-treated cells revealed that the staining-positive rate in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells was decreased in comparison to that in the wild-type and the mock-transduced cells. Giemsa’s staining observation showed that the number of dying (with apoptotic bodies) and dead cells was reduced in the rAAV-antisense 6A8 cDNA-transduced SKW6 cells in comparison with that in the wild-type and the mock-transduced cells upon anti-Fas antibody induction. The transduction did not affect the expression of Fas molecular on cell surface. 100% cells in all the groups showed Fas expression. The SKW6 cells became resistant to apoptosis induction by anti-Fas antibody upon transduction with a reverse fragment to a cDNA encoding human 6A8 a-mannosidase. The transduction did not affect the expression of Fas molecule on cells. 相似文献
994.
Yi Zhang Peixian Tang Ying Jin Xiusen Li Shuangxi Zhang Ying Wu Ning Mao 《中国科学:生命科学英文版》2001,44(5):461-468
To elucidate the effect of gene transfected marrow stromal cell on expansion of human cord blood CD34+ cells, a culture system was established in which FL and TPO genes were transfected into human stromal cell line HFCL. To
establish gene transfected stromal cells co-culture system, cord blood CD34+ cells were purified by using a magnetic beads sorting system. The number of all cells and the number of CD34+ cells and CFC (CFU-GM and BFU-E) were counted in different culture systems. The results showed that in all 8 culture systems,
SCF+IL-3+HFT manifested the most potent combination, with the number of total nucleated cells increasing by (893.3 ±52.1)-fold,
total progenitor cells (CFC) by (74.5 ±5.2)-fold and CD34+ cells by 15.7-fold. Maximal expansions of CFC and CD34+ cells were observed at the end of the second week of culture. Within 14 days of culture, (78.1 ± 5.5)-fold and (57.0 ± 19.7)-fold
increases in CFU-GM and BFU-E were obtained. Moreover, generation of LTC-IC from amplified CD34+ cells within 28 days was found only in two combinations, i.e. SCF+IL-3+FL+TPO and SCF+IL-3+HFT, and there was no significant
difference between these two groups statistically. These results suggest that human umbilical cord blood CD34+ cells can be extensively expandedex vivo by using gene transfected stromal cells along with cytokines. 相似文献
995.
van Berloo Ralph Aalbers Hanneke Werkman Arjen Niks Rients E. 《Molecular breeding : new strategies in plant improvement》2001,8(3):187-195
Near-isogenic lines (NILs) differing with regard to disease QTLs provide valuable material for a more detailed study into the genetic basis of quantitative resistance. Previously obtained information on QTLs that show an effect on leaf rust (Puccinia hordei) in barley was used in a marker-assisted backcross programme. The genome origin in backcross plants was controlled through AFLP marker analysis and graphical genotyping. Plants obtained after the third generation of backcrossing sufficiently resembled the recurrent parent. For one QTL, BC3S1 plants were evaluated in a disease test and genotyped. NILs containing the desired QTL in homozygous condition in a recipient background were finally obtained. A disease test and verification of the marker genotype confirmed the identity of the NILs. Simultaneous with the backcross programme a simulation study on efficiency of marker-assisted backcrossing was performed. 相似文献
996.
用乙硫氨酸为筛选剂,通过幼苗和组织培养筛选得到乙硫氨酸抗性愈伤组织。该愈伤组织在含乙硫氨酸的培养基上表现出较高的半抑制剂量和相对生长量。作为天门冬氨酸途径的产物,甲硫氨酸、异亮氨酸和赖氨酸在所筛选的愈伤组织中分别增加到对照的两倍多,但苏氨酸保持正常水平,另外酪氨酸、半胱氨酸和亮氨酸也有所增加,而在所筛选的愈伤组织中缬氨酸浓度却下降。说明在所筛选愈伤组织中存在一个以上与氨基酸合成相关的酶发生改变。同工酶分析表明,该愈伤组织中出现对照中没有的分子量为44kD的超氧化物歧化酶和分子量为45kD的酯酶谱带。 相似文献
997.
Laura E. Brown Celine Fuchs Martin W. Nicholson F. Anne Stephenson Alex M. Thomson Jasmina N. Jovanovic 《Journal of visualized experiments : JoVE》2014,(93)
Inhibitory neurons act in the central nervous system to regulate the dynamics and spatio-temporal co-ordination of neuronal networks. GABA (γ-aminobutyric acid) is the predominant inhibitory neurotransmitter in the brain. It is released from the presynaptic terminals of inhibitory neurons within highly specialized intercellular junctions known as synapses, where it binds to GABAA receptors (GABAARs) present at the plasma membrane of the synapse-receiving, postsynaptic neurons. Activation of these GABA-gated ion channels leads to influx of chloride resulting in postsynaptic potential changes that decrease the probability that these neurons will generate action potentials. During development, diverse types of inhibitory neurons with distinct morphological, electrophysiological and neurochemical characteristics have the ability to recognize their target neurons and form synapses which incorporate specific GABAARs subtypes. This principle of selective innervation of neuronal targets raises the question as to how the appropriate synaptic partners identify each other. To elucidate the underlying molecular mechanisms, a novel in vitro co-culture model system was established, in which medium spiny GABAergic neurons, a highly homogenous population of neurons isolated from the embryonic striatum, were cultured with stably transfected HEK293 cell lines that express different GABAAR subtypes. Synapses form rapidly, efficiently and selectively in this system, and are easily accessible for quantification. Our results indicate that various GABAAR subtypes differ in their ability to promote synapse formation, suggesting that this reduced in vitro model system can be used to reproduce, at least in part, the in vivo conditions required for the recognition of the appropriate synaptic partners and formation of specific synapses. Here the protocols for culturing the medium spiny neurons and generating HEK293 cells lines expressing GABAARs are first described, followed by detailed instructions on how to combine these two cell types in co-culture and analyze the formation of synaptic contacts. 相似文献
998.
Juwon Park Eun-Kyung Bae Chansu Lee Jee-Hye Choi Woo June Jung Kwang-Sung Ahn Sung-Soo Yoon 《BMB reports》2014,47(5):274-279
Bortezomib has been known as the most promising anti-cancer drug for multiple myeloma (MM). However, recent studies reported that not all MM patients respond to bortezomib. To overcome such a stumbling-block, studies are needed to clarify the mechanisms of bortezomib resistance. In this study, we established a bortezomib-resistant cell line (U266/velR), and explored its biological characteristics. The U266/velR showed reduced sensitivity to bortezomib, and also showed crossresistance to the chemically unrelated drug thalidomide. U266/velR cells had a higher proportion of CD138 negative subpopulation, known as stem-like feature, compared to parental U266 cells. U266/velR showed relatively less inhibitory effect of prosurvival NF-κB signaling by bortezomib. Further analysis of RNA microarray identified genes related to ubiquitination that were differentially regulated in U266/velR. Moreover, the expression level of CD52 in U266 cells was associated with bortezomib response. Our findings provide the basis for developing therapeutic strategies in bortezomib-resistant relapsed and refractory MM patients. [BMB Reports 2014; 47(5): 274-279] 相似文献
999.
One major challenge currently facing the biopharmaceutical industry is to understand how MAb microheterogeneity affects therapeutic efficacy, potency, immunogenicity, and clearance. MAb micro‐heterogeneity can result from post‐translational modifications such as sialylation, galactosylation, C‐terminal lysine cleavage, glycine amidation, and tryptophan oxidation, each of which can generate MAb charge variants; such heterogeneity can affect pharmacokinetics (PK) considerably. Implementation of appropriate on‐line quality control strategies may help to regulate bioprocesses, thus enabling more homogenous material with desired post‐translational modifications and PK behavior. However, one major restriction to implementation of quality control strategies is the availability of techniques for obtaining on‐line or at‐line measurements of these attributes. In this work, we describe the development of an at‐line assay to separate MAb charge variants in near real‐time, which could ultimately be used to implement on‐line quality control strategies for MAb production. The assay consists of a 2D‐HPLC method with sequential in‐line Protein A and WCX‐10 HPLC column steps. To perform the 2D‐HPLC assay at‐line, the two columns steps were integrated into a single method using a novel system configuration that allowed parallel flow over column 1 or column 2 or sequential flow from column 1 to column 2. A bioreactor system was also developed such that media samples could be removed automatically from bioreactor vessels during production and delivered to the 2D‐HPLC for analysis. With this at‐line HPLC assay, we have demonstrated that MAb microheterogeneity occurs throughout the cell cycle whether the host cell line is grown under different or the same nominal culture conditions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:249–255, 2014 相似文献
1000.
摘要 目的:分析信迪利单抗联合白紫+替吉奥(S-1)二线在人表皮生长因子受体2(Her-2)阴性胃癌治疗中的近远期疗效及其安全性。方法:根据随机数字表法将2018年1月~2021年1月在本院接受治疗的76例Her-2阴性胃癌患者分为对照组与观察组,每组各38例,对照组给予白紫+替吉奥二线治疗,观察组给予白紫+替吉奥二线+信迪利单抗治疗;观察两组患者的近期疗效和不良反应发生率,并在随访24个月后记录两组患者的远期疗效,Logistic多因素分析影响患者达到中位OS、PFS的独立危险因素。结果:与对照组比较,观察组ORR、DCR率较高(P<0.05);与对照组比较,观察组中位OS、PFS较高(P<0.05);Logistic多因素分析结果显示,治疗方法(白紫+替吉奥二线)是影响HER-2阴性胃癌达到中位PFS和OS的独立危险因素(P<0.05);两组不良反应发生率比较无差异(P>0.05)。结论:白紫、替吉奥二线与信迪利单抗联合治疗不仅能保障Her-2阴性胃癌治疗的安全性,还能进一步提升患者的临床治疗效果,并延长其生存期。 相似文献