An Automated Robotic Interface for Assays: Facilitating Machine Learning in Drug Discovery by the Automation of Physicochemical Property Assays.

Measuring the physicochemical properties of molecules is an iterative but integral process in the drug development process. A strategy to overcome the challenges in maximizing assay throughput relies on the usage of in silico machine learning (ML) prediction models trained on experimental data. Consequently, the performance of these in silico models are dependent on the quality of the utilized experimental data. To improve the data quality, we have designed and implemented an automated robotic system to prepare and run physicochemical property assays (Automated Robotic Interface for Assays, ARIA) with an increase in sample throughput of 6 to10-fold. Through this process, we overcame major challenges and achieved consistent reproducible assay data compared to semiautomated assay preparation.

MASSpy: Building, simulating, and visualizing dynamic biological models in Python using mass action kinetics.

Mathematical models of metabolic networks utilize simulation to study system-level mechanisms and functions. Various approaches have been used to model the steady state behavior of metabolic networks using genome-scale reconstructions, but formulating dynamic models from such reconstructions continues to be a key challenge. Here, we present the Mass Action Stoichiometric Simulation Python (MASSpy) package, an open-source computational framework for dynamic modeling of metabolism. MASSpy utilizes mass action kinetics and detailed chemical mechanisms to build dynamic models of complex biological processes. MASSpy adds dynamic modeling tools to the COnstraint-Based Reconstruction and Analysis Python (COBRApy) package to provide an unified framework for constraint-based and kinetic modeling of metabolic networks. MASSpy supports high-performance dynamic simulation through its implementation of libRoadRunner: the Systems Biology Markup Language (SBML) simulation engine. Three examples are provided to demonstrate how to use MASSpy: (1) a validation of the MASSpy modeling tool through dynamic simulation of detailed mechanisms of enzyme regulation; (2) a feature demonstration using a workflow for generating ensemble of kinetic models using Monte Carlo sampling to approximate missing numerical values of parameters and to quantify biological uncertainty, and (3) a case study in which MASSpy is utilized to overcome issues that arise when integrating experimental data with the computation of functional states of detailed biological mechanisms. MASSpy represents a powerful tool to address challenges that arise in dynamic modeling of metabolic networks, both at small and large scales.

Fractionation of radiocesium in soil, sediments, and aquatic organisms in Lake Onuma of Mt. Akagi, Gunma Prefecture using sequential extraction.

The Fukushima Daiichi Nuclear Power Plant (FDNPP) accident has resulted in the contamination of the environment in Gunma Prefecture with radioisotope cesium (radio-Cs, 134Cs and 137Cs). Concentrations of radio-Cs >500Bqkg-1 were found in wakasagi (Hypomesus nipponensis) in Lake Onuma at the top of Mount (Mt.) Akagi in August 2011. To explain the mechanism of this contamination, monitoring studies have been conducted around Lake Onuma by measuring radio-Cs concentrations in samples of fish, aquatic plants, plankton, lake water, lake sediments, and surrounding soil. The leachability of radio-Cs was evaluated using sequential extraction by Tessier et al. The total concentration of radio-Cs in Lake Onuma ecosystems decreased gradually with time. In the brown forest soil, radio-Cs concentrations of 2000 to 6000Bqkg-1 were detected. The abundance ratio of the easy-elution form (exchangeable and carbonate forms) in the samples was <10%. The concentrations in phytoplankton samples were 3-6 times higher than those in wakasagi samples. The ratios of easy-elution forms increased by the rank in the food chain; 37% in phytoplankton, 78% in zooplankton, and 97% in wakasagi. It is likely that the lower ratio of the easy-elution form in phytoplankton is related to the adsorption of radio-Cs on suspended substances in the lake, as suggested by the analyses of aluminum and titanium in the phytoplankton, zooplankton, and wakasagi samples. The high concentrations of radio-Cs in wakasagi would be related also to the characteristics of closed mountain lakes.

Development of a genetic system in chitinase-producing streptomyces and the application of an allosamidin-insensitive chitinase gene to homologous overexpression.

A transformation system for Streptomyces sp. AJ9463 strain (allosamidin producer) was successfully developed using protoplasts and a PEG-mediated method. To prepare protoplasts, the concentration of glycine and sucrose in YEME medium were optimized to 0.5% (w/v) and 34.0% (w/v), respectively. When the protoplasts of Streptomyces sp. AJ9463 were transformed with pUWL-KS, transformants could be obtained at a high efficiency of 7.0 x 10(4) transformants per microg DNA. To ensure that the transformation system worked properly, we then constructed a constitutive expression vector pYK1, in which the ermE* promoter drives transcription of the allosamidin-insensitive chitinase gene, chiIS. Although no transformant could be obtained by the genetic system using pYK1 isolated from Escherichia coli DH5alpha, pYK1 isolated from the methylase-deficient mutant E. coli SCS110, could be introduced into Streptomyces sp. AJ9463. This indicates that Streptomyces sp. AJ9463 has a methylation-specific restriction system, and that the chiIS and/or ermE* promoter region of pYK1 includes a restriction site of its endonuclease. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that pYK1 in Streptomyces sp. AJ9463 started to obviously express ChiIS from 14-h. Moreover, the pYK1-introduced strain gave a five-fold higher chitinase activity than the wild-type, suggesting that this system can be widely applied for the overexpression and gene functional analysis.

Design and synthesis of the potent, orally available, brain-penetrable arylpyrazole class of neuropeptide Y5 receptor antagonists.

Novel arylpyrazole derivatives were synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Compound (-)-7, which features a novel chiral 2,3-dihydro-1H-cyclopenta[a]naphthalene moiety, showed good binding affinity and antagonistic activity for the Y5 receptor. After intracerebroventricular administration in SD rats, (-)-7 significantly inhibited food intake that was induced by the centrally administered Y5-preferring agonist, bovine pancreatic polypeptide, but had only a negligible effect on NPY-induced feeding.