Comprehensive two-dimensional high-performance liquid chromatography for the isolation of overexpressed proteins and proteome mapping

A two-dimensional liquid chromatographic system is described here which uses size-exclusion liquid chromatography (SEC) followed by reversed-phase liquid chromatography (RPLC) to separate the mixture of proteins resulting from the lysis of Escherichia coli cells and to isolate the proteins that they produce. The size-exclusion chromatography can be conducted under either denaturing or nondenaturing conditions. Peaks eluting from the first dimension are automatically subjected to reversed-phase chromatography to separate similarly sized proteins on the basis of their various hydrophobicities. The RPLC also serves to desalt the analytes so that they can be detected in the deep ultraviolet region at 215 nm regardless of the SEC mobile phase used. The two-dimensional (2D) chromatograms produced in this manner then strongly resemble the format of stained 2D gels, in that spots are displayed on a X-Y axis and intensity represents quantity of analyte. Following chromatographic separation, the analytes are deposited into six 96-well (576 total) polypropylene microtiter plates via a fraction collector. Interesting fractions are analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) or electrospray mass spectrometry (ESI/MS) depending on sample concentration, which both yield accurate (2 to 0.02%) molecular weight information on intact proteins without any additional sample preparation, electroblotting, destaining, etc. The remaining 97% of a fraction can then be used for other analyses, such Edman sequencing, amino acid analysis, or proteolytic digestion and sequencing by tandem mass spectrometry. This 2D HPLC protein purification and identification system was used to isolate the src homology (SH2) domain of the nonreceptor tyrosine kinase pp60c-src and beta-lactamase, both inserted into E. coli, as well as a number of native proteins comprising a small portion of the E. coli proteome.     

Cyclic, low-dose total body irradiation for metastatic neuroblastoma

Total body irradiation (TBI) can be thought of as a systemic anticancer agent. It therefore might best be given like an adjuvant drug, i.e. in tolerable doses, cyclically. The therapeutic ration between normal bone marrow stem cells and suitably sensitive cancer cells should be widened by these means. Fourteen children with advanced (State IV) neuroblastomas were given 100-150 rad TBI in 50 rad daily fractions along with each three-week cycle of standard triple-agent chemotherapy, (vincristine, DTIC, cyclophosphamide). Two patients died of toxicity and one is still undergoing therapy. Four of the remaining 12 survive free of disease for 12+ to 31+ months. The regimen is well tolerated, but prolonged, pronounced bone marrow depression, especially thrombocytopenia, comonly occurs after doses of 300-450 rad.     

Simulation and analysis of a sugarcane juice evaporation system

The production of sugar and alcohol is the main objective of the sugarcane processing industry. The evaporation of sugarcane juice has a high energetic cost and is usually performed in multiple-effect evaporators. The loss of performance during operation due to fouling makes the process more complex. In this study, modeling, simulation, validation, and analysis were performed for a sugarcane juice industrial evaporation system (IES) composed of a falling film evaporator followed by three short vertical-tube evaporators arranged in parallel. The IES model was developed using a commercial process simulator and validated with data from the plant. The IES had marked performance losses in the first 14 days of operation, mainly due to fouling in the first effect, with a 30% decrease in the evaporation rate.     

Cyclic voltammetric analysis of antioxidant activity in cane sugars and palm sugars from Southeast Asia

Fourteen commonly available types of cane and palm sugar were analysed for antioxidant activity using cyclic voltammetry. Five of the sugars, dissolved in phosphate buffer, showed anodic current peaks which were indicative of antioxidant activity. The rank order of these sugars was: gula anau > gula merah > China rock honey sugar > soft brown sugar > raw sugar. Gula anau is an unrefined palm sugar, while the other four are from sugarcane. Soft brown sugar and raw sugar are coated brown sugars. The China rock honey sugar contained chrysanthemum flowers, and its antioxidant properties appear to be due to these flowers and not to the sugar per se. Pure white sugar, other rock sugars and rock honey sugars (all refined from cane) as well as gula Melaka, an unrefined rock sugar from palm trees, had no observable antioxidant activity. It is concluded that, from a nutritional point of view, using gula anau as a sweetener or ingredient in foods or drinks has an added benefit owing to its antioxidant content.     

Volatile sulphur compounds composition of monovarietal white wines

The sulphur compounds composition of wines produced experimentally from six white cultivars (Alvarinho, Loureiro, Trajadura, Pedernã, Azal Branco and Avesso) was evaluated during two consecutive vintages. Results show that wines could be differentiated according to their sulphur compounds content. In general, Loureiro, Trajadura and Pedernã cultivars led to wines with low concentrations of sulphur compounds; however, Loureiro wines were characterised by significant amounts of dimethyl sulphone, whereas Trajadura wines possessed a high content of 3-(methylthio)propyl acetate and 4-(methylthio)-1-butanol. Alvarinho and Avesso wines showed high levels of S-methyl thioacetate, 3-mercapto-1-propanol, 3-(ethylthio)-1-propanol and 3-methylthiopropionic acid. Significant amounts of 2-methyltetrahydrothiophen-3-one, cis- and trans-2-methyltetrahydrothiophen-3-ol were also found in Avesso wines. Azal Branco wines were low in 3-(methylthio)propyl acetate and 2-(methylthio)ethanol, and high contents in S-methyl thioacetate, 3-mercapto-1-propanol and 2-mercaptoethanol. A linear discriminant analysis of sulphur compounds levels showed a differentiation of wines according to their varietal origin.     

Species,roasting degree and decaffeination influence the antibacterial activity of coffee against Streptococcus mutans

Coffee beverage has been associated with antibacterial activity against Streptococcus mutans, a cariogenic bacterium. This study aimed at identifying natural compounds in coffee that contribute to such activity and investigate the influence of species, roasting and decaffeination on it. Coffee chemical compounds and aqueous extracts of green and roasted regular and decaffeinated Coffea arabica and Coffea canephora beans were tested. MIC, biofilm inhibition and biofilm reduction results were correlated with the concentration of coffee compounds in the extracts. 5-Caffeoylquinic acid, trigonelline and caffeic acid solutions showed bacteriostatic activity (MIC = 0.8 mg/mL). Lighter and regular extracts showed higher inhibitory activity than darker and decaffeinated extracts, with an inverse correlation between bacterial colony-forming units and roasting degree. Only regular C. canephora extracts showed biofilm formation inhibition. The joint effect of chlorogenic acids, trigonelline and caffeine or other compounds removed by decaffeination seems to be one of the causes for coffee antibacterial activity against S. mutans.     

Improving the Fitness of High-Dimensional Biomechanical Models via Data-Driven Stochastic Exploration

The field of complex biomechanical modeling has begun to rely on Monte Carlo techniques to investigate the effects of parameter variability and measurement uncertainty on model outputs, search for optimal parameter combinations, and define model limitations. However, advanced stochastic methods to perform data-driven explorations, such as Markov chain Monte Carlo (MCMC), become necessary as the number of model parameters increases. Here, we demonstrate the feasibility and, what to our knowledge is, the first use of an MCMC approach to improve the fitness of realistically large biomechanical models. We used a Metropolis-Hastings algorithm to search increasingly complex parameter landscapes (3, 8, 24, and 36 dimensions) to uncover underlying distributions of anatomical parameters of a ldquotruth modelrdquo of the human thumb on the basis of simulated kinematic data (thumbnail location, orientation, and linear and angular velocities) polluted by zero-mean, uncorrelated multivariate Gaussian ldquomeasurement noise.rdquo Driven by these data, ten Markov chains searched each model parameter space for the subspace that best fit the data (posterior distribution). As expected, the convergence time increased, more local minima were found, and marginal distributions broadened as the parameter space complexity increased. In the 36-D scenario, some chains found local minima but the majority of chains converged to the true posterior distribution (confirmed using a cross-validation dataset), thus demonstrating the feasibility and utility of these methods for realistically large biomechanical problems.     

MESFET DC model parameter extraction using Quantum Particle Swarm Optimization

This paper presents two techniques for DC model parameter extraction for a Gallium Arsenide (GaAs) based MEtal Semiconductor Field Effect Transistor (MESFET) device. The proposed methods uses Particle Swarm Optimization (PSO) and Quantum Particle Swarm Optimization (QPSO) methods for optimizing the difference between measured data and simulated data. Simulated data are obtained by using four different popular DC models. These techniques avoid complex computational steps involved in traditional parameter extraction techniques. The performance comparison in terms of quality of solution and execution time of classical PSO and QPSO to extract the model parameters are presented. The validity of this approach is verified by comparing the simulated and measured results of a fabricated GaAs MESFET device with gate length of 0.7 μm and gate width of 600 μm (4 × 150). Simulation results indicate that both the technique based on PSO and QPSO accurately extracts the model parameters of MESFET.