Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

Track D Social Science, Human Rights and Political Science

Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/138414/1/jia218442.pd

Thermal Hydraulic Behavior of the First ITER CS Module

The ITER Central Solenoid (CS) modules are under fabrication by the US ITER organization and its subcontractors. US ITER will supply seven modules to the ITER Organization (IO), six of which will be assembled in a stack that forms the ITER Central Solenoid, the last one being spare. The first module, namely CSM 1 was manufactured by General Atomics (GA) and went through Factory Acceptance Tests (FAT) including high voltage testing, Paschen testing and then cold test at 4.5 K and up to 40.0 kA in order to demonstrate compliance with coil performance requirements. The paper focuses on the results of the first CS Module thermal hydraulic characterization without current (and field). Pressure drops, transit time, and thermal coupling between pancakes are presented. Analysis results and tests are also compared

AC Losses in the Second Module of the ITER Central Solenoid

The ITER Central Solenoid (CS) will be realized by assembling a stack of six modules. Each module is a solenoid consisting of 40 pancakes wound with a Nb3Sn Cable in Conduit Conductor (CICC). The tests of the second module (CSM#2) were performed at the General Atomics (GA) facility in Poway (US). During the test campaign, the CS Module is submitted to dumps of the transport current from different initial values (10, 15, 20, 25, 30, 35, 40 kA) to 0 kA, which allow measuring the AC losses in the coil. In this paper we present the results on AC losses during the dumps from different initial transport currents, as computed with two different methods. The first method is based on the observation that the dumps determine a very fast pressure rise of the supercritical helium embedded in the module, which undergoes an isochoric transformation. This approach is therefore based on the computation of the variation of internal energy of the helium during the pressure rise itself. The second method is based instead on a calorimetric procedure aimed at estimating the enthalpy variation of the supercritical helium due to the thermal power deposited during the current dumps. The main contribution to the losses during these dumps is due to the coupling losses. As for the hysteresis losses in the magnet, the results obtained through slow current cycle tests are also presented

Testing of the ITER CS Module #4

The ITER Central Solenoid is under fabrication by the US ITER organization and its subcontractors. US ITER will supply seven modules to ITER IO, six of which will be assembled in a stack that forms the ITER Central Solenoid (CS). All CS modules were or will be tested at 40 kA in the Final Test facility at General Atomics, Poway, CA. Testing included high voltage, as well as Paschen testing in the vacuum and global leak tests before and after the cooldown to 4.5 K and EM cycling to 40 kA. In the paper we present the results of the CS Module 4 performance, after modifications to the test facility to improve reliability and instrumentation. We measured critical temperatures in several pancakes, AC losses before and after 10 cycles to 40 kA, joints resistance and hydraulic characteristics of the coils. We also measured displacements of the coil height and vertical strain of the CSM (central solenoid module) to verify structural mechanical characteristics of the coil along with cooldown shrinkage of the coil. We studied performance of the cowound quench detectors, confirmed their effectiveness in suppression of the inductive noise, but also developed a plan to improve sensitivity of the quench detection in ITER CS. This information is necessary for verification of the stack behavior of CS in ITER operation. The test results, preliminary analyses, comparisons to the other tested modules are presented and discussed

Testing of the ITER Central Solenoid Modules

The ITER Central Solenoid is under fabrication by the U.S. ITER organization and its subcontractors. U.S. ITER will supply seven modules to ITER IO, six of which will be assembled in a stack that forms the ITER Central Solenoid. The first modules that were built by GA at their facility, went into high voltage testing, including Paschen testing in the vacuum, and then they were tested at 4.5 K and up to 40 kA to demonstrate compliance of the coil with the ITER requirements. In this article, we present the Test Plan and results of the central solenoid (CS) module's performance, especially at the full current. We measured critical temperatures in several pancakes, we measured ac losses, joint resistance, and hydraulic characteristics of the coils. We also measured displacements of the coil height and hoop strain of the CS module (CSM) to verify the structural mechanical characteristics of the coil along with the cooldown shrinkage of the coil. We studied the performance of the cowound quench detectors and confirmed their effectiveness in the suppression of inductive noise. This information is necessary for verification of the stack behavior of CS in ITER operation. The test results and preliminary analyses are presented, compared to expectations, and discussed

First ITER CS module test results

The ITER Central Solenoid (CS) is under fabrication by the US ITER organization and its subcontractors. US ITER will supply seven modules to ITER IO, six of which will be assembled in a stack that forms the ITER Central Solenoid, with one as a spare. The first module fabrication has been completed by General Atomics (GA) at their facility and has begun testing including high voltage testing, Paschen testing in the vacuum and then testing at 4.5 K and up to 40 kA in order to demonstrate compliance of the coil to ITER requirements. In the paper we present the Test Plan and results of the CS Module performance tests, especially at 40 kA current. AC losses, joint resistances and hydraulic characteristics of the coil are all measured. Displacements of the coil height and hoop strain of the CS Module are also measured to verify structural and mechanical characteristics of the coil along with cooldown shrinkage of the coil. This information is used for verification of the stack behavior of CS in ITER operation. The test results and preliminary analyses results are presented, compared to expectations, and discussed. All measured parameters suggest that the CS module will perform well in ITER machine

Electrical insulation testing for ITER fusion tokamak

The uniqueness of the ITER fusion project drives purpose and scope here to fulfil (functional) Procurement Arrangement (PA) requirements for the Central Solenoid (CS) and Correction Coil (CC) magnets' electrical insulation [1-3]; as used and undertaken for covering high voltage testing operations of the magnet coil winding pack (WP) vacuum pressure insulation (VPI) beam qualification, followed in 2016 by start of magnets series production in the USA and China [1, 2]. Useful to be considered common state-of-the-art electrical power components testing practices [3, 4] are presented here as performed to verify that the integrity and insulation of the various components are within acceptable limits during different phases of the fabrication and to certify acceptance on delivery of the complete coils. Initial measurement plans were complemented into electrical tests with typical high voltage engineering implemented measures as a pre-requisite to successfully validate produced results. The above permitted the first quantitative validation of the obtained final production, including life time behavior