During the emergence of novel coronavirus 2019 (nCoV) outbreak in Wuhan city, China at the end of 2019, there was movement of many airline travelers between Wuhan and Japan, suggesting that the ...Japanese population was at high risk of infection by the virus. Hence, we urgently developed diagnostic systems for detection of 2019 nCoV. Two nested RT-PCR and two real-time RT-PCR assays were adapted for use in Japan. As of February 8, 2020, these assays have successfully detected 25 positive cases of infection in Japan.
A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many ...countries globally. Here, we show that a TMPRSS2- expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.
Proteins involve motions over a wide range of spatial and temporal scales. While the large conformational changes, such as folding and functioning, are slow and appear to occur in a highly ...cooperative manner, how the hierarchical dynamics over different time scales play a role during these slow transitions has been of great interest over the decades. Here we study the folding mechanism of the villin headpiece subdomain (HP35) to understand the molecular mechanism behind this prototypical fast-folding protein. The ∼400 μs molecular dynamics (MD) trajectories obtained by Piana et al. Piana, S. ; Lindorff-Larsen, K. ; Shaw, D. E. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17845 are analyzed in detail. By extracting the slowest mode from the trajectories, which is responsible for the folding/unfolding transitions, and by analyzing the transition events along this mode, we find that the transitions occur in a heterogeneous manner. Detailed analysis of the individual transition events shows that the folding/unfolding transitions occur via two qualitatively different pathways, i.e., the unfolding triggered from the C-terminal (α3 helix) and from the N-terminal (α1-α2 loop). Non-native contacts are also found to contribute in slowing down the transitions. The folding of HP35 thus proceeds in a segmental manner rather than cooperatively at the submicrosecond time scale. The Lys→Nle mutation is found to speed up the transitions by rigidifying the α3 helix, i.e., suppressing one transition pathway. The analysis of the microsecond dynamics in the single-molecule Förster resonance energy transfer efficiency trajectories, which are calculated from the MD data, reveals that the folding/unfolding transitions in the NleNle mutant can be fitted with a two-state model, whereas those in WT appear to be more complex and involves multiple time scales. This is due to the coupling between the folding/unfolding transitions and conformational transitions within the unfolded and intermediate states. The present study demonstrates that a protein as small as HP35 already involves heterogeneous characters during folding/unfolding transitions when the hierarchical dynamics at the molecular level is considered, thus heterogeneity can be a general characteristic in protein folding.
Free energy surfaces have played a central role in studying protein conformational changes and enzymatic reactions over decades. Yet, free energy barriers and kinetics are highly dependent on the ...coordinates chosen to define the surface, and furthermore, the dynamics during the reactions are often overlooked. Our recent study on the Pin1-catalyzed isomerization reaction has indicated that the isomerization transition events remarkably deviate from the free energy path, highlighting the need to understand the reaction dynamics in more detail. To this end, here we investigate the reaction coordinates that describe the transition states of the free energy and transition pathways by minimizing the cross-entropy function. We show that the isomerization transition events can be expressed by the concerted changes in the improper torsion angle ζ and nearby backbone torsional angles of the ligand, whereas the transition state of the free energy surface involves changes in a broad range of coordinates including multiple protein–ligand interactions. The current result supports the previous finding that the isomerization transitions occur quickly from the conformational excited states, which is in sharp contrast to the slow and collective changes suggested from the free energy path. Our results further indicate that the coordinates derived from the transition trajectories are not sufficient for finding the transition states on the free energy surfaces due to the lack of information from conformational excited states.
The coronavirus induced disease 2019 (COVID-19) outbreak caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Wuhan (China) in December 2019 is currently spreading rapidly ...worldwide. We recently reported a laboratory protocol for the diagnosis of SARS-CoV-2 based on real-time reverse transcriptase PCR (RT-PCR) assays using two primer sets, N and N2. On January 30–31, 2020, the protocol and the reagents for these assays were distributed to the local public health institutes and quarantine depots in Japan nationwide, and thereafter SARS-CoV-2 diagnostic testing was initiated. For further validation, the assays were compared with the commercially available kits using the SARS-CoV-2 viral RNA and clinical specimens obtained from COVID19-suspected individuals. The LightMix Modular SARS and Wuhan CoV E-gene (LM S&W-E) assay was highly sensitive for the SARS-CoV-2, as was the N2 set, as both the assays showed consistent results for the clinical specimens. While the LM S&W-E set targets the highly conserved region of E gene in the SARS-CoV and SARS-CoV-2, the N2 set was designed to target specifically the unique region in the SARS-CoV-2 N gene. Therefore, the N2 set exhibits high specificity and sensitivity for SARS-CoV-2 detection. These results indicate that the protocol using the N and N2 sets is comparable to the commercially available kits, and thus is reliable for laboratory diagnosis of COVID-19.
Concentrations of PM2.5 in Tokyo, a densely populated megacity, often increase because of NH4NO3 formation under low-wind conditions during winter. To obtain source information of NH3 as a NH4NO3 ...precursor, hourly NH3 and NH4+ concentrations were measured at an urban site in Tokyo in December 2017. Results show that PM2.5 and NH4+ concentrations increased simultaneously under low-wind and low-temperature conditions along with NH3, NOx, CO, and optical black carbon (OBC) concentrations. The remaining NH4+ (herein, ΔNH4+) subtracted from observed NH4+ to equivalent SO42− concentration correlated well with NO3− in PM2.5, indicating the existence of fine NH4NO3 particles. Regression analysis of hourly NH3 + ΔΝH4+ concentrations with CO, NOx, and OBC showed significant correlation. Virtual emission rates (VER) of NH3 per motor vehicle in Tokyo were estimated using observed relations between NH3 + ΔΝH4+ and OBC, etc. with data from vehicular statistics: they were 3.7–32 mg/km. The regression of NH3 + ΔΝH4+ with OBC concentrations indicated an intercept of about 3.2 ppb, which is about half of the monthly average in December. This result implies that the non-vehicular source strength was nearly the same strength as the bulk vehicular emissions of NH3 during winter in Tokyo.
•Considering excess NH4+ concentrations over SO42− was useful to study urban NH3.•Virtual emission rates of NH3 per vehicle in Tokyo were 3.7–32 mg/km.•Non-vehicular NH3 sources contributed almost equally to winter vehicular emissions.
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