Serum-free culture of rat keratinocytes

Summary Procedures for the serum-free culture of rat keratinocytes have been established. Basal cells prepared from epidermis of newborn rat were stored in liquid nitrogen and used for primary culture. Among the available media, MCDB 153, developed originally for human keratinocyte (HK) culture, was the best for the development of serum-free formulation. To grow rat keratinocytes, bovine serum albumin was arbitrarily substituted for the macromolecule supplements needed for HK culture, i.e. fetal bovine serum protein or bovine pituitary extract. Qualitative and quantitative adjustment of supplements was thereafter made to support rapid cell growth. Satisfactory cell growth was achieved in the optimized medium of MCDB 153 supplemented with growth factors and amino acids: insulin (10 μg/ml), hydrocortisone (0.1 μg/ml), epidermal growth factor (25 ng/ml), calcium chloride (0.2 mM), histidine (0.23 mM), isoleucine (0.05 mM), tryptophane (0.015 mM), threonine (1.25 mM), tyrosine (0.031 mM), alanine (4.08 mM), and albumin (2 mg/ml). This optimized culture system was superior to the original HK culture condition for rapid growth of rat keratinocytes. Under our condition, cells grew as a monolayer, becoming confluent, but without stratification, and were passaged 2 to 3 times without any changes in morphology. The serum-free formulation allows us to control more accurately the concentrations of biomolecules in the medium including lipids and hormones, and therefore will be suitable for the study focusing on lipid metabolism or hormonal regulation of rat keratinocytes.     

Defined media optimization for growth of recombinant Escherichia coli X90

An optimized, defined minimal medium was developed to support balanced growth of Escherichia coli X90 harboring a recombinant plasmid. Foreign protein expression was repressed in these studies. A pulse injection technique was used to identify the growth responses to nutrients in a chemostat. Once the nutrients essential for growth had been identified, the yield coefficients for individual medium components. These yield coefficients were used to develop an optimized, glucose-limited defined minimal medium that supports balanced cell growth in chemostat culture. The biomass and substrate concentrations follow the Monod chemostat model. The maximum specific growth rate determined in a washout experiment is 0.87 h(-1) for this strain in the optimized medium. the glucose yield factor is 0.42 g DCW/g glucose and the maintenance coefficient is zero in the glucose-limited chemostat culture. (c) 1993 John Wiley & Sons, Inc.     

谷氨酸棒杆菌代谢工程高效生产L-缬氨酸北大核心CSCD

L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。     

Estimating tree-based dynamic treatment regimes using observational data with restricted treatment sequences

A dynamic treatment regime (DTR) is a sequence of decision rules that provide guidance on how to treat individuals based on their static and time-varying status. Existing observational data are often used to generate hypotheses about effective DTRs. A common challenge with observational data, however, is the need for analysts to consider “restrictions” on the treatment sequences. Such restrictions may be necessary for settings where (1) one or more treatment sequences that were offered to individuals when the data were collected are no longer considered viable in practice, (2) specific treatment sequences are no longer available, or (3) the scientific focus of the analysis concerns a specific type of treatment sequences (eg, “stepped-up” treatments). To address this challenge, we propose a restricted tree–based reinforcement learning (RT-RL) method that searches for an interpretable DTR with the maximum expected outcome, given a (set of) user-specified restriction(s), which specifies treatment options (at each stage) that ought not to be considered as part of the estimated tree-based DTR. In simulations, we evaluate the performance of RT-RL versus the standard approach of ignoring the partial data for individuals not following the (set of) restriction(s). The method is illustrated using an observational data set to estimate a two-stage stepped-up DTR for guiding the level of care placement for adolescents with substance use disorder.     

Utilization of Response Surface Methodology in Optimization of Proline Extraction from Castanea sativa Honey

Proline constitutes approximately 85 % of the amino acid composition of honey. Therefore, the quantitative determination of this amino acid in honey samples is used by many national/international authorities to evaluate the quality of honey types. In this study, it was aimed to achieve maximum proline amino acid extraction from honey samples whose botanical origins were confirmed by melissopalynological analysis. For this reason, based on three different spectrophotometric methods used in the literature for proline analysis, proline extraction was optimized with the Response Surface Method (RSM) and Box-Behnken experimental design. Three independent variables were determined as treatment time (2, 6, and 10 min), treatment temperature (22, 46, and 70 °C), and cooling time (5, 25, and 45 min). As a result of the optimization, it was seen that only significantly effective independent variable on the proline content of honey was the processing temperature. The optimum conditions obtained as a result of the RSM were found to be 2 min for the treatment time, 70 °C for the treatment temperature and 45 min for the cooling time. The composite desirability of the optimum conditions (R²) was found to be 1.00. It was determined that the method proposed by International Honey Commission (IHC) is efficient for proline analysis, but it provides more proline extraction by reducing of time from 10 min to 2 min in hold time in boiling water bath only during the extraction step. As a result, the conditions to be used in order to achieve maximum proline extraction with different spectrophotometric methods were determined and optimum values were determined. In addition, since the botanical origin of honey samples significantly affects the proline content of honey, it can be suggested that this study be optimized for different monofloral honey samples as well.     

Structural Optimization,Fungicidal Activities Evaluation,DFT Study and Structure-Activity Relationship of Dopamine Derivatives with Benzothiazole Fragment from Polyrhachis dives

In our previous work, two dopamine derivatives with benzothiazole fragment were isolated and identified from Polyrhachis dives (P. dives). Based on their characteristic structure, we used them as lead compound to carry out structural optimization and subsequent fungicidal evaluation. Here 20 dopamine derivatives with benzothiazole fragment were designed and synthesized by a facile method, and their structures were characterized by ¹H-NMR, ¹³CNMR and HMRS. In bioassays, most of the title compounds possess potential fungicidal activities against Altenaia alternala (A. alternala) and Botrytis cinerea (B. cinerea). Especially, (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(p-tolyl)acrylamide and (E)-N-(2-(benzo[d]thiazol-6-yl)ethyl)-3-(4-(trifluoromethyl)phenyl)acrylamide displayed 29.3 mg/L and 10.7 mg/L EC₅₀ value against A. alternala, respectively, which possessed equivalent fungicidal activities level to hymexazol.     

EFFECTS OF VARIATION IN TEMPERATURE. I. ON THE BIOCHEMICAL COMPOSITION OF EIGHT SPECIES OF MARINE PHYTOPLANKTON1

The influence of temperature on the biochemical composition of eight species of marine phytoplankton was investigated. Thalassiosira pseudonana Hasle and Heim-dal, Phaeodactylum tricornutum Bohlin and, Pavlova lutheri Droop (three of eight species studied) had minimum values of carbon and nitrogen quotas at intermediate temperatures resulting in a broad U-shaped response in quotas over the temperature range of 10 to 25°C. Protein per cell also had minimum values at intermediate temperatures for six species. For T. pseudonana, P. tricornutum, and P. lutheri, patterns of variation in carbon, nitrogen, and protein quotas as a function of temperature were similar. Over all species, lipid and carbohydrate per cell showed no consistent trends with temperature. Only chlorophyll a quotas and the carbon: chlorophyll a ratios (θ) showed consistent trends across all species. Chlorophyll a quotas were always lower at 10°C than at 25°C. Carbon: chlorophyll a ratios (θ) were always higher at 10°C than at 25°C. We suggest that although θ consistently increases at lower temperatures, the relationship between temperature and θ ranges from linear to exponential and is species specific. Accordingly, the interspecific variance in θ that results from species showing a range of possible responses to temperature increases as temperature declines and reaches a maximum at low temperatures. High photon flux densities appear to increase the potential interspecific variance in the carbon: chlorophyll a ratio and therefore exacerbate these trends.     

适用于获取最优化配方的一种算法

本文应用印楝种仁提取物（F3）与敌敌畏混配为例,以斜纹夜蛾（Spodopteralitura）为目标害虫,介绍一种适用于获取最优化配方的算法,在二次通用回归旋转组合设计的基础上,经参数辨识,获取二次回归方程,经失拟性、回归显著性检验,本方程基本能够反映杀虫剂用量与斜纹夜蛾幼虫死亡机率值之间的关系．在害虫防治实践中,要求在防治费用最小的基础上,目标害虫有最大的死亡率.因此,以防治目标害虫的费用作为优化算法的目标函数,以害虫死亡机率值最大作为约束条件,有如下的一组优化算式为目标函数约束条件式中a1,a2分别为参试杀虫剂1,2最低用量,b1,b2则为相应的最高用量．C1,C2分别为杀虫剂1,2的单价,N1,N2为杀虫剂l,2的用量．Y为目标害虫死亡机率值回归方程．本文所依据的试验设计中,以对数函数关系变换编码值与使用浓度之间的关系,所以应用拉格朗日求极值原理求取最优化配方．由计算所得的混配比例与其他方法所获结果一致．     

Parametric sensitivity of stoichiometric flux balance models applied to wild-type Escherichia coli metabolism

The esterification of lauric acid with geraniol catalyzed by the commercially immobilized lipase preparation from Mucor miehei, Lipozyme(R), was studied in well-stirred flasks. The enzyme support was characterized in terms of its internal and external surface area, protein location, and protein content. It was found that the enzyme was mainly located on the external surface of the support, therefore, internal diffusional limitations were not important. It was also shown that the protein content of the support depends on the size of the particle, with smaller particles containing higher amounts of protein per unit weight. Under the conditions studied, the reaction was not under external mass transfer limitations, and the initial reaction rate depended on the size of the support particles. This was mainly due to the different protein contents on the support as a function of particle size and not to internal or external mass transfer limitations. Also, it was found that the inhibition exerted by water was predominantly a physical effect due to its accumulation around the enzyme. It was also found that the reaction was substrate inhibited by lauric acid, but not by geraniol. (c) 1995 John Wiley & Sons, Inc.