High‐throughput instant quantification of protein expression and purity based on photoactive yellow protein turn off/on label

Quantifying the concentration and purity of a target protein is essential for high‐throughput protein expression test and rapid screening of highly soluble proteins. However, conventional methods such as PAGE and dot blot assay generally involve multiple time‐consuming tasks requiring hours or do not allow instant quantification. Here, we demonstrate a new method based on the Photoactive yellow protein turn Off/On Label (POOL) system that can instantly quantify the concentration and purity of a target protein. The main idea of POOL is to use Photoactive Yellow Protein (PYP), or its miniaturized version, as a fusion partner of the target protein. The characteristic blue light absorption and the consequent yellow color of PYP is absent when initially expressed without its chromophore, but can be turned on by binding its chromophore, p‐coumaric acid. The appearance of yellow color upon adding a precursor of chromophore to the co‐expressed PYP can be used to check the expression amount of the target protein via visual inspection within a few seconds as well as to quantify its concentration and purity with the aid of a spectrometer within a few minutes. The concentrations measured by the POOL method, which usually takes a few minutes, show excellent agreement with those by the BCA Kit, which usually takes ～1 h. We demonstrate the applicability of POOL in E. coli, insect, and mammalian cells, and for high‐throughput protein expression screening.     

Foraging area fidelity for Kemp's ridleys in the Gulf of Mexico

For many marine species, locations of key foraging areas are not well defined. We used satellite telemetry and switching state‐space modeling (SSM) to identify distinct foraging areas used by Kemp's ridley turtles (Lepidochelys kempii) tagged after nesting during 1998–2011 at Padre Island National Seashore, Texas, USA (PAIS; N = 22), and Rancho Nuevo, Tamaulipas, Mexico (RN; N = 9). Overall, turtles traveled a mean distance of 793.1 km (±347.8 SD) to foraging sites, where 24 of 31 turtles showed foraging area fidelity (FAF) over time (N = 22 in USA, N = 2 in Mexico). Multiple turtles foraged along their migratory route, prior to arrival at their “final” foraging sites. We identified new foraging “hotspots” where adult female Kemp's ridley turtles spent 44% of their time during tracking (i.e., 2641/6009 tracking days in foraging mode). Nearshore Gulf of Mexico waters served as foraging habitat for all turtles tracked in this study; final foraging sites were located in water <68 m deep and a mean distance of 33.2 km (±25.3 SD) from the nearest mainland coast. Distance to release site, distance to mainland shore, annual mean sea surface temperature, bathymetry, and net primary production were significant predictors of sites where turtles spent large numbers of days in foraging mode. Spatial similarity of particular foraging sites selected by different turtles over the 13‐year tracking period indicates that these areas represent critical foraging habitat, particularly in waters off Louisiana. Furthermore, the wide distribution of foraging sites indicates that a foraging corridor exists for Kemp's ridleys in the Gulf. Our results highlight the need for further study of environmental and bathymetric components of foraging sites and prey resources contained therein, as well as international cooperation to protect essential at‐sea foraging habitats for this imperiled species.     

Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N‐protected phenylalanine in near‐anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0–80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration‐induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration‐induced inactivation that takes place during the first activity measurement, (2) a phase of first‐order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:870–875, 2013     

An evolutionary computing approach for parameter estimation investigation of a model for cholera

We consider the problem of using time-series data to inform a corresponding deterministic model and introduce the concept of genetic algorithms (GA) as a tool for parameter estimation, providing instructions for an implementation of the method that does not require access to special toolboxes or software. We give as an example a model for cholera, a disease for which there is much mechanistic uncertainty in the literature. We use GA to find parameter sets using available time-series data from the introduction of cholera in Haiti and we discuss the value of comparing multiple parameter sets with similar performances in describing the data.     

An SEI model for sarcoptic mange among chamois

We consider a simple model to study the dynamics of sarcoptic mange in a population of chamois. The epidemiological patterns observed during an epidemic in Italy are reconstructed and key parameters of the model are estimated from field data. In particular, we calculate the basic reproductive ratio R ₀, a threshold value for chamois density for the occurrence of an epidemic and the speed of propagation of the epidemic wave. The model is then used to obtain indications on the effect of culling as a possible control measure in a closed population and extended to analyse the spatial diffusion of the epidemic. Our results are in agreement with mange epidemiology and observations, and suggest that intervention could be efficacious in reducing the impact of an epidemic.     

Application of Artificial Bee Colony Algorithm to Maximum Likelihood DOA Estimation

Maximum Likelihood (ML) method has an excellent performance for Direction-Of-Arrival (DOA) estimation, but a multidimensional nonlinear solution search is required which complicates the computation and prevents the method from practical use. To reduce the high computational burden of ML method and make it more suitable to engineering applications, we apply the Artificial Bee Colony (ABC) algorithm to maximize the likelihood function for DOA estimation. As a recently proposed bio-inspired computing algorithm, ABC algorithm is originally used to optimize multivariable functions by imitating the behavior of bee colony finding excellent nectar sources in the nature environment. It offers an excellent alternative to the conventional methods in ML-DOA estimation. The performance of ABC-based ML and other popular meta-heuristic-based ML methods for DOA estimation are compared for various scenarios of convergence, Signal-to-Noise Ratio (SNR), and number of iterations. The computation loads of ABC-based ML and the conventional ML methods for DOA estimation are also investigated. Simulation results demonstrate that the proposed ABC based method is more efficient in computation and statistical performance than other ML-based DOA estimation methods.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016