Optimization of ultrasound-assisted water extraction of flavonoids from Psidium guajava leaves by response surface analysis

Psidium guajava leaves are rich in health-promoting flavonoids compounds. For better utilization of the resource, the ultrasound-assisted aqueous extraction was investigated using Box-Behnken design under response surface methodology. A high coefficient of determination (R²?=?97.8%) indicated good agreement between the experimental and predicted values of flavonoids yield. The optimal extraction parameters to obtain the highest total flavonoids yield were ultrasonic power of 407.41?W, extraction time of 35.15?min, and extraction temperature of 72.69?°C. The average extraction rate of flavonoids could reach 5.12% under the optimum conditions. Besides, HPLC analysis and field emission scanning electron microscopy indicated that the ultrasonic treatment did not change the main component of flavonoids during extraction process and the higher flavonoids content was attributed by the disruption of the cell walls of guava particles. Thus, the extraction method could be applied successfully for large-scale extraction of total flavonoids from guava leaves.     

根癌农杆菌介导的高效大豆遗传转化体系的建立

利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。     

The emerging role of mass spectrometry-based proteomics in molecular pharming practices

Molecular pharming relies on the integration of foreign genes into a plant system for production of the desired recombinant protein. The speed, scalability, and lack of contaminating human pathogens highlights plants as an enticing and feasible system to produce diverse protein-based products, including vaccines, antibodies, and enzymes. However, limitations of expression levels, host defense responses, and production irregularities underscore distinct areas for improvement within the molecular pharming pipeline. Within the past five years, mass spectrometry-based proteomics has begun to address these critical areas and show promise in advancing our understanding of the complex biological systems driving molecular pharming. Further, opportunities to leverage comprehensive proteome profiling have surfaced to meet good manufacturing practice regulations and move biopharmaceuticals derived from plants into mainstream production.     

Optimization of compressive loading parameters to mimic in vivo cervical spine kinematics in vitro

The human cervical spine supports substantial compressive load in vivo. However, the traditional in vitro testing methods rarely include compressive loads, especially in investigations of multi-segment cervical spine constructs. Previously, a systematic comparison was performed between the standard pure moment with no compressive loading and published compressive loading techniques (follower load – FL, axial load – AL, and combined load – CL). The systematic comparison was structured a priori using a statistical design of experiments and the desirability function approach, which was chosen based on the goal of determining the optimal compressive loading parameters necessary to mimic the segmental contribution patterns exhibited in vivo. The optimized set of compressive loading parameters resulted in in vitro segmental rotations that were within one standard deviation and 10% of average percent error of the in vivo mean throughout the entire motion path. As hypothesized, the values for the optimized independent variables of FL and AL varied dynamically throughout the motion path. FL was not necessary at the extremes of the flexion–extension (FE) motion path but peaked through the neutral position, whereas, a large negative value of AL was necessary in extension and increased linearly to a large positive value in flexion. Although further validation is required, the long-term goal is to develop a “physiologic” in vitro testing method, which will be valuable for evaluating adjacent segment effect following spinal fusion surgery, disc arthroplasty instrumentation testing and design, as well as mechanobiology experiments where correct kinematics and arthrokinematics are critical.     

Development of a novel MATLAB-based framework for implementing mechanical joint stability constraints within OpenSim musculoskeletal models

The Static Optimization (SO) solver in OpenSim estimates muscle activations and forces that only equilibrate applied moments. In this study, SO was enhanced through an open-access MATLAB interface, where calculated muscle activations can additionally satisfy crucial mechanical stability requirements. This Stability-Constrained SO (SCSO) is applicable to many OpenSim models and can potentially produce more biofidelic results than SO alone, especially when antagonistic muscle co-contraction is required to stabilize body joints. This hypothesis was tested using existing models and experimental data in the literature. Muscle activations were calculated by SO and SCSO for a spine model during two series of static trials (i.e. simulation 1 and 2), and also for a lower limb model (supplementary material 2). In simulation 1, symmetric and asymmetric flexion postures were compared, while in simulation 2, various external load heights were compared, where increases in load height did not change the external lumbar flexion moment, but necessitated higher EMG activations. During the tasks in simulation 1, the predicted muscle activations by SCSO demonstrated less average deviation from the EMG data (6.8% −7.5%) compared to those from SO (10.2%). In simulation 2, SO predicts constant muscle activations and forces, while SCSO predicts increases in the average activations of back and abdominal muscles that better match experimental data. Although the SCSO results are sensitive to some parameters (e.g. musculotendon stiffness), when considering the strategy of the central nervous system in distributing muscle forces and in activating antagonistic muscles, the assigned activations by SCSO are more biofidelic than SO.     

基因重组大肠杆菌表达血红素加氧酶-1及培养条件优化

【背景】血红素加氧酶-1 (HO-1)具有抗氧化应激、抗凋亡和抗纤维化等多种生理效应,有望成为一种新型药物应用于临床疾病的治疗。【目的】构建表达HO-1的基因重组大肠杆菌(Escherichiacoli),并优化其表达培养条件,实现HO-1高产率的表达。【方法】PCR法克隆集胞藻(Synechocystissp.)PCC6803的HO-1基因(ho1),构建重组质粒pET-28a-ho1,转化大肠杆菌BL21(DE3)菌株,单因素实验优化表达培养基的种类、诱导剂添加时间、诱导培养时间、诱导剂浓度和诱导培养温度。【结果】构建了表达HO-1的基因重组大肠杆菌BL21(DE3)/pET-28a-ho1菌株,用甘油(GY)培养基培养至菌体浓度OD_(600)约为0.8时,加入终浓度为0.1 mmol/L的IPTG诱导,30°C诱导培养6 h,HO-1的表达量最高,Ni-NTA柱分离纯化得到的HO-1收率占细胞总蛋白的10.9%。【结论】获得了可溶性表达HO-1的基因重组大肠杆菌及其较佳的培养条件,为进一步研究集胞藻来源的HO-1的酶学性质和应用奠定了基础。     

Optimization of biochemical systems by linear programming and general mass action model representations

A new method is proposed for the optimization of biochemical systems. The method, based on the separation of the stoichiometric and kinetic aspects of the system, follows the general approach used in the previously presented indirect optimization method (IOM) developed within biochemical systems theory. It is called GMA-IOM because it makes use of the generalized mass action (GMA) as the model system representation form. The GMA representation avoids flux aggregation and thus prevents possible stoichiometric errors. The optimization of a system is used to illustrate and compare the features, advantages and shortcomings of both versions of the IOM method as a general strategy for designing improved microbial strains of biotechnological interest. Special attention has been paid to practical problems for the actual implementation of the new proposed strategy, such as the total protein content of the engineered strain or the deviation from the original steady state and its influence on cell viability.     

Response surface methodology for the optimization of ubiquinone self-nanoemulsified drug delivery system

The aim of the present study was to prepare and evaluate an optimized, self-nanoemulsified drug delivery system of ubiquinone. A 3-factor, 3-level Box-Behnken design was used for the optimization procedure with the amounts of Polyoxyl 35 castor oil (X₁), medium-chain mono- and diglyceride (X₂), and lemon oil (X₃) as the independent variables. The response variable was the cumulative percentage of ubiquinone emulsified in 10 minutes. Different ubiquinone release rates were obtained. The amount released ranged from 11% to 102.3%. Turbidity profile revealed 3 regions that were used to describe the progress of emulsion formation: lag phase, pseudolinear phase, and plateau turbidity. An increase in the amount of surfactant decreased turbidity values and caused a delay in lag time. Addition of cosurfactant enhanced the release rates. Increasing the amount of the eutectic agent was necessary to overcome drug precipitation especially at higher loading of surfactants and cosurfactants. Mathematical equations and response surface plots were used to relate the dependent and independent variables. The regression equation generated for the cumulative percentage emulsified in 10 minutes was Y1=90.9–22.1X₁+5.03X₂+13.95X₃+12.13X₁X₂+15.13X₁X₃-14.40X₁ ²-6.25X₃ ². The optimization model predicted a 93.4% release with X₁, X₂, and X₃ levels of 35, 35, and 30 respectively. The observed responses were in close agreement with the predicted values of the optimized formulation. This demonstrated the reliability of the optimization procedure in predicting the dissolution behavior of a self-emulsified drug delivery system. Published: February 8, 2002.     

N-acetyl- -arginine ethyl ester synthesis catalysed by bovine trypsin in organic media

The bovine trypsin-catalysed synthesis of N-acetyl- -arginine ethyl ester from N-acetyl- -arginine and ethanol was studied in various organic solvents (dimethyl sulfoxide, dioxane, dimethylformamide, acetonitrile, acetone, tetrahydrofuran, chloroform, toluene, carbon tetrachloride, cyclohexane and n-hexane). The highest yield was achieved in acetonitrile after incubation for 6 or 24 h. The optimal conditions for ester synthesis in acetonitrile for 6 h were as follows: 5.0 mM N-acetyl- -arginine, 10.0 M ethanol, 7.2 mg trypsin, 2.87% water, total volume 10.3 ml, pH 7.0 and 30°C. The hydrolytic activity of trypsin was determined after incubation for 6 days, when 87.7% of the original activity remained, suggesting that acetonitrile caused little inactivation of the enzyme. The synthetic reaction resulted in a maximal 79.3% conversion under optimized conditions after incubation for 48 h.     

Mathematical modeling of citric acid production by repeated batch culture

A mathematical model has been created for the process of citric acid biosynthesis by yeast (mutant strain Yarrowia lipolytica) cultivated by the repeated batch (RB) method on ethanol under conditions of nitrogen limitation. The model accounts for cell growth as a function of nitrogen concentration in the culture liquid; nitrogen uptake by growing cells; citric acid production; pH control in the fermentor by means of NaOH addition; and changes in system volume. The model represents a system of five nonlinear differential equations. Experimental measurements of cell concentration, citric acid concentration, and cultivation broth volume were used with the least squares method to determine the values of eight model parameters. The parameter values obtained were consistent with literature data and general concepts of cell growth and citric acid biosynthesis. The model has been used to predict optimum RB culture conditions.