Directed Evolution of a Cp*RhIII-Linked Biohybrid Catalyst Based on a Screening Platform with Affinity Purification

Directed evolution of Cp*Rh^III-linked nitrobindin (NB), a biohybrid catalyst, was performed based on an in vitro screening approach. A key aspect of this effort was the establishment of a high-throughput screening (HTS) platform that involves an affinity purification step employing a starch-agarose resin for a maltose binding protein (MBP) tag. The HTS platform enables efficient preparation of the purified MBP-tagged biohybrid catalysts in a 96-well format and eliminates background influence of the host E. coli cells. Three rounds of directed evolution and screening of more than 4000 clones yielded a Cp*Rh^III-linked NB(T98H/L100K/K127E) variant with a 4.9-fold enhanced activity for the cycloaddition of acetophenone oximes with alkynes. It is confirmed that this HTS platform for directed evolution provides an efficient strategy for generating highly active biohybrid catalysts incorporating a synthetic metal cofactor.     

Integration of imitation learning using GAIL and reinforcement learning using task-achievement rewards via probabilistic graphical model

The integration of reinforcement learning (RL) and imitation learning (IL) is an important problem that has long been studied in the field of intelligent robotics. RL optimizes policies to maximize the cumulative reward, whereas IL attempts to extract general knowledge about the trajectories demonstrated by experts, i.e, demonstrators. Because each has its own drawbacks, many methods combining them and compensating for each set of drawbacks have been explored thus far. However, many of these methods are heuristic and do not have a solid theoretical basis. This paper presents a new theory for integrating RL and IL by extending the probabilistic graphical model (PGM) framework for RL, control as inference. We develop a new PGM for RL with multiple types of rewards, called probabilistic graphical model for Markov decision processes with multiple optimality emissions (pMDP-MO). Furthermore, we demonstrate that the integrated learning method of RL and IL can be formulated as a probabilistic inference of policies on pMDP-MO by considering the discriminator in generative adversarial imitation learning (GAIL) as an additional optimality emission. We adapt the GAIL and task-achievement reward to our proposed framework, achieving significantly better performance than policies trained with baseline methods.     

Silver(I)-Ion-Mediated Cytosine-Containing Base Pairs: Metal Ion Specificity for Duplex Stabilization and Susceptibility toward DNA Polymerases

Spectroscopic characterization of Ag^I-ion-mediated C-Ag^I-A and C-Ag^I-T base pairs found in primer extension reactions catalyzed by DNA polymerases was conducted. UV melting experiments revealed that C-A and C-T mismatched base pairs in oligodeoxynucleotide duplexes are specifically stabilized by Ag^I ions in 1:1 stoichiometry in the same manner as a C-C mismatched base pair. Although the stability of the mismatched base pairs in the absence of Ag^I ions is in the order C-A≈C-T>C-C, the stabilizing effect of Ag^I ions follows the order C-C>C-A≈C-T. However, the comparative susceptibility of dNTPs to Ag^I-mediated enzymatic incorporation into the site opposite templating C is dATP>dTTP≫dCTP, as reported. The net charge, as well as the size and/or shape complementarity of the metal-mediated base pairs, or the stabilities of mismatched base pairs in the absence of metal ions, would be more important than the stability of the metallo-base pairs in the replicating reaction catalyzed by DNA polymerases.     

Geosynthetic-sheet pile reinforced foundation for mitigation of earthquake and tsunami induced damage of breakwater

Earthquake and tsunami impose great threats on the stability of a breakwater. Foundation of the breakwater is weakened by these forces, and it may result in collapse of the breakwater. Lateral flow of seabed soils take place beneath the breakwater, and excess pore water pressure is generated in the foundation by an earthquake that precedes tsunami. These factors may lead to excessive settlement and horizontal displacement of the breakwater. Tsunami introduces additional instability to the deformed breakwater. Due to water level difference between seaside and harborside of the breakwater during a tsunami, seepage occurs through its foundation, and it may cause pipping of seabed soils. Tsunami induced scouring of mound is also a big problem for the stability of the breakwater foundation. Finally, these result in failure of the breakwater foundation. Due to failure of its foundation, the breakwater may collapse and cannot block the tsunami. It results in entering of the tsunami in coastal areas. In order to make a breakwater resilient against earthquake and tsunami induced damage, reinforcing countermeasures were developed for foundation of a breakwater. Geogrid, gabions and sheet piles were used for reinforcing a foundation model. The effectiveness of the model is evaluated through physical modeling for mitigating the earthquake and tsunami induced damage. Shaking table tests were performed to determine the effectiveness of the reinforced model under different earthquake loadings. Tsunami overflow test was conducted on the same deformed model in order to see the effects of tsunami on the model. Comparisons were made between the unreinforced and reinforced foundations, and it was observed during the tests that the reinforced foundation performed well in reducing the damage of the breakwater brought by the earthquake and tsunami. Overall, this study is useful for practice engineering, and the reinforced foundation model can be adopted for designing a breakwater foundation to reduce damage triggered by an earthquake and tsunami in the future.     

Carbon radicals generated by solid polymers: Electron spin resonance spectroscopy for detection of species in water

Radicals generated in plastic medical devices (solid phase) by γ-rays or electron-beam irradiation during sterilization are known to cause oxidation of protein drugs, resulting in a loss or reduction in drug efficacy. The generation of radical species in water by the radical species in solid polymers has not been proved. Using electron spin resonance (ESR) spectroscopy, we confirm the generation of new radicals in water by γ-ray irradiated cyclic olefin polymers (COP). ESR measurements are obtained using 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) as a spin probe and 5-(2,2-dimethyl-1,3-propoxycyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) as a spin trap, in which the irradiated COP was immersed. The ESR signals indicate the TEMPOL radicals decline over time, suggesting the generation of new radicals. Conversely, the characteristic ESR signals of the adduct formed by the reaction between CYPMPO and the hydroxyl radical are observed. Thus, hydroxyl radicals are generated because of the migration of the radicals from COP to water. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020 , 137, 48604.     

Molecular Features and Clinical Management of Hereditary Gynecological Cancers

Hereditary gynecological cancers are caused by several inherited genes. Tumors that arise in the female reproductive system, such as ovaries and the uterus, overlap with hereditary cancers. Several hereditary cancer-related genes are important because they might lead to therapeutic targets. Treatment of hereditary cancers should be updated in line with the advent of various new methods of evaluation. Next-generation sequencing has led to rapid, economical genetic analyses that have prompted a concomitant and significant paradigm shift with respect to hereditary cancers. Molecular tumor profiling is an epochal method for determining therapeutic targets. Clinical treatment strategies are now being designed based on biomarkers based on tumor profiling. Furthermore, the National Comprehensive Cancer Network (NCCN) guidelines significantly changed the genetic testing process in 2020 to initially consider multi-gene panel (MGP) evaluation. Here, we reviewed the molecular features and clinical management of hereditary gynecological malignancies, such as hereditary breast and ovarian cancer (HBOC), and Lynch, Li–Fraumeni, Cowden, and Peutz–Jeghers syndromes. We also reviewed cancer-susceptible genes revealed by MGP tests.     

Analysis of Transglucosylation Products of Aspergillus niger α-Glucosidase that Catalyzes the Formation of α-1,2- and α-1,3-Linked Oligosaccharides

According to whole-genome sequencing, Aspergillus niger produces multiple enzymes of glycoside hydrolases (GH) 31. Here we focus on a GH31 α-glucosidase, AgdB, from A. niger . AgdB has also previously been reported as being expressed in the yeast species, Pichia pastoris ; while the recombinant enzyme (rAgdB) has been shown to catalyze tranglycosylation via a complex mechanism. We constructed an expression system for A. niger AgdB using Aspergillus nidulans . To better elucidate the complicated mechanism employed by AgdB for transglucosylation, we also established a method to quantify glucosidic linkages in the transglucosylation products using 2D NMR spectroscopy. Results from the enzyme activity analysis indicated that the optimum temperature was 65 °C and optimum pH range was 6.0–7.0. Further, the NMR results showed that when maltose or maltopentaose served as the substrate, α-1,2-, α-1,3-, and small amount of α-1,1-β-linked oligosaccharides are present throughout the transglucosylation products of AgdB. These results suggest that AgdB is an α-glucosidase that serves as a transglucosylase capable of effectively producing oligosaccharides with α-1,2-, α-1,3-glucosidic linkages.