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Coronavirus (CoV) possesses numerous functional cis-acting elements in its positive-strand genomic RNA. Although most of these RNA structures participate in viral replication, the functions of RNA structures in the genomic RNA of CoV in viral replication remain unclear. In this study, we investigated the functions of the higher-order RNA stem-loop (SL) structures SL5B, SL5C, and SL5D in the ORF1a coding region of Middle East respiratory syndrome coronavirus (MERS-CoV) in viral replication. Our approach, using reverse genetics of a bacterial artificial chromosome system, revealed that SL5B and SL5C play essential roles in the discontinuous transcription of MERS-CoV. In silico analyses predicted that SL5C interacts with a bulged stem-loop (BSL) in the 3' untranslated region, suggesting that the RNA structure of SL5C is important for viral RNA transcription. Conversely, SL5D did not affect transcription, but mediated the synthesis of positive-strand genomic RNA. Additionally, the RNA secondary structure of SL5 in the revertant virus of the SL5D mutant was similar to that of the wild-type, indicating that the RNA structure of SL5D can finely tune RNA replication in MERS-CoV. Our data indicate novel regulatory mechanisms of viral RNA transcription and replication by higher-order RNA structures in the MERS-CoV genomic RNA., (© 2024. The Author(s).)

We developed a surgical support system that visualises important microanatomies using artificial intelligence (AI). This study evaluated its accuracy in recognising the thoracic nerves during lung cancer surgery. Recognition models were created with deep learning using images precisely annotated for nerves. Computational evaluation was performed using the Dice index and the Jaccard index. Four general thoracic surgeons evaluated the accuracy of nerve recognition. Further, the differences in time lag, image quality and smoothness of movement between the AI system and surgical monitor were assessed. Ratings were made using a five-point scale. The computational evaluation was relatively favourable, with a Dice index of 0.56 and a Jaccard index of 0.39. The AI system was used for 10 thoracoscopic surgeries for lung cancer. The accuracy of thoracic nerve recognition was satisfactory, with a recall score of 4.5 ± 0.4 and a precision score of 4.0 ± 0.9. Though smoothness of motion (3.2 ± 0.4) differed slightly, nearly no difference in time lag (4.9 ± 0.3) and image quality (4.6 ± 0.5) between the AI system and the surgical monitor were observed. In conclusion, the AI surgical support system has a satisfactory accuracy in recognising the thoracic nerves., (© 2024. The Author(s).)

Platelet-rich plasma (PRP) promotes bone union through osteoinduction. We investigated whether adding demineralized bone matrix (DBM), derived naturally from biomaterial and with various growth factors, for osteoconductivity and bone marrow fluid for osteogenesis results in different bone unions. Eight-week-old male Sprague-Dawley rats were divided into four groups of five based on transplantation material: sham control (C group); DBM alone (D group); DBM + PRP (DP group); and DBM + PRP + bone marrow fluid (DPB group). After posterolateral fusion at L3-5, postoperative weekly CT imaging determined average number of bone union in facet joints (4 joints × 5 animals = 20 joints) and bone formation. Pathological evaluation and bone strength were assessed using 3-point bending two weeks postoperatively. Facet joint bone union at four weeks postoperatively was 4/20 (20%, DP group) and 8/20 (40%, DPB group) joints. Six weeks postoperatively, it was 7/20 (35%, D group), 12/20 (60%, DP group), and 16/20 (80%, DPB group). Eight weeks postoperatively, it was 13/20 (65%, D group), 17/20 (85%, DP group), and 20/20 (100%, DPB group), suggesting that DPB > DP > D > C. Bone formation and bone strength showed a similar DPB > DP > D > C group trend. Adding PRP and bone marrow fluid to DBM promotes bone union and strength., (© 2023. Springer Nature Limited.)

Conventional crop height measurements performed using aerial drone images require 3D reconstruction results of several aerial images obtained through structure from motion. Therefore, they require extensive computation time and their measurement accuracy is not high; if the 3D reconstruction result fails, several aerial photos must be captured again. To overcome these challenges, this study proposes a high-precision measurement method that uses a drone equipped with a monocular camera and real-time kinematic global navigation satellite system (RTK-GNSS) for real-time processing. This method performs high-precision stereo matching based on long-baseline lengths (approximately 1 m) during the flight by linking the RTK-GNSS and aerial image capture points. As the baseline length of a typical stereo camera is fixed, once the camera is calibrated on the ground, it does not need to be calibrated again during the flight. However, the proposed system requires quick calibration in flight because the baseline length is not fixed. A new calibration method that is based on zero-mean normalized cross-correlation and two stages least square method, is proposed to further improve the accuracy and stereo matching speed. The proposed method was compared with two conventional methods in natural world environments. It was observed that error rates reduced by 62.2% and 69.4%, for flight altitudes between 10 and 20 m respectively. Moreover, a depth resolution of 1.6 mm and reduction of 44.4% and 63.0% in the error rates were achieved at an altitude of 4.1 m, and the execution time was 88 ms for images with a size of 5472 × 3468 pixels, which is sufficiently fast for real-time measurement., (© 2023. The Author(s).)

Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO 2 · as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N 2 atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml -1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of β-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules., (© 2023. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs., (© 2022. The Author(s).)

This study aimed to investigate whether muscle synergy differs between swimmers with and without swimmer's shoulder in the butterfly technique. Muscle synergies, which can assess muscle coordination, were analyzed using surface electromyography. Twenty elite swimmers were included in this study (swimmer's shoulder: n = 8; control: n = 12). The motions involved in executing the butterfly technique were classified into the early pull-through, late pull-through, and recovery phases. Muscle synergy data analyzed using the nonnegative matrix factorization method were compared between the two groups.The swimming velocities were 1.66 ± 0.09 m・s -1 and 1.69 ± 0.06 m・s -1 for the control and swimmer's shoulder groups, respectively. Four muscle synergies in both groups were identified: synergy #1, which was involved in the early pull; synergy #2, involved in the late pull; synergy #3, involved in the early recovery; and synergy #4, involved in pre- and posthand entry. Compared to the control group, the swimmer's shoulder group had a small contribution from the pectoralis major (p = 0.032) and a high contribution from the rectus femoris during the early pull phase (p = 0.036). In the late pull phase, the contribution of the lower trapezius muscle in the swimmer's shoulder group was low (p = 0.033), while the contribution of the upper trapezius muscle in the pre- and postentry phases was high (p = 0.032). In the rehabilitation of athletes with swimmer's shoulder, it is therefore important to introduce targeted muscle rehabilitation in each phase., (© 2022. The Author(s).)

The COVID-19 pandemic has created urgent demand for rapid detection of the SARS-CoV-2 coronavirus. Herein, we report highly sensitive detection of SARS-CoV-2 nucleocapsid protein (N protein) using nanoparticle-enhanced surface plasmon resonance (SPR) techniques. A crucial plasmonic role in significantly enhancing the limit of detection (LOD) is revealed for exceptionally large gold nanoparticles (AuNPs) with diameters of hundreds of nm. SPR enhanced by these large nanoparticles lowered the LOD of SARS-CoV-2 N protein to 85 fM, resulting in the highest SPR detection sensitivity ever obtained for SARS-CoV-2 N protein., (© 2022. The Author(s).)

The aim of this study is to understand adaptive immunity to SARS-CoV-2 through the analysis of B cell epitope and neutralizing activity in coronavirus disease 2019 (COVID-19) patients. We obtained serum from forty-three COVID-19 patients from patients in the intensive care unit of Osaka University Hospital (n = 12) and in Osaka City Juso Hospital (n = 31). Most individuals revealed neutralizing activity against SARS-CoV-2 assessed by a pseudotype virus-neutralizing assay. The antibody production against the spike glycoprotein (S protein) or receptor-binding domain (RBD) of SARS-CoV-2 was elevated, with large individual differences, as assessed by ELISA. We observed the correlation between neutralizing antibody titer and IgG, but not IgM, antibody titer of COVID-19 patients. In the analysis of the predicted the linear B cell epitopes, hot spots in the N-terminal domain of the S protein were observed in the serum from patients in the intensive care unit of Osaka University Hospital. Overall, the analysis of antibody production and B cell epitopes of the S protein from patient serum may provide a novel target for the vaccine development against SARS-CoV-2.

A novel Asfarvirus-like virus is proposed as the etiological agent responsible for mass mortality in abalone. The disease, called abalone amyotrophia, originally was recognized in the 1980s, but efforts to identify a causative agent were unsuccessful. We prepared a semi-purified fraction by nuclease treatment and ultracentrifugation of diseased abalone homogenate, and the existence of the etiological agent in the fraction was confirmed by a challenge test. Using next-generation sequencing and PCR-based epidemiological surveys, we obtained a partial sequence with similarity to a member of the family Asfarviridae. BLASTP analysis of the predicted proteins against a virus database resulted in 48 proteins encoded by the novel virus with top hits against proteins encoded by African swine fever virus (ASFV). Phylogenetic analyses of predicted proteins of the novel virus confirmed that ASFV represents the closest relative. Comparative genomic analysis revealed gene-order conservation between the novel virus and ASFV. In situ hybridization targeting the gene encoding the major capsid protein of the novel virus detected positive signals only in tissue from diseased abalone. The results of this study suggest that the putative causative agent should be considered a tentative new member of the family Asfarviridae, which we provisionally designate abalone asfa-like virus (AbALV).

Adaptive hunting management is commonly used for controlling the populations of overabundant large herbivores; however, induced behavioural changes can make the effective control of target populations difficult. However, few studies have compared the impact of different levels of hunting intensities on the activity patterns of ungulates before, during, and after a culling program. We investigated how different culling intensities affect the activity patterns of sika deer on Nakanoshima Island in Lake Toya, Hokkaido, Japan using camera-trap surveys comparing the period of treatment before, during, and after a culling program. We used the number of deer photographed per hour per camera as an index of activity. Sika deer showed consistent crepuscular activity patterns (i.e., dawn and dusk) during spring-summer and trimodal activity patterns (i.e., dawn, dusk, and midnight) in autumn throughout the study period. In response to increased culling intensity, the activity peaks shifted slightly towards the night. The shift towards nocturnal activity persisted during post-culling period. Understanding the changes in activity patterns in response to culling intensity could be used to facilitate population control and assist in establishing a night shooting program. Thus, wildlife managers should consider night shooting once hunting during day time has shifted the normal diurnal activity of deer to nocturnal activity.

In order to elucidate features of the denatured state ensembles that exist in equilibrium with the native state under physiological conditions, we performed 1.4-μs molecular dynamics (MD) simulations at 400 K and 450 K using the monomer subunits of three CutA1 mutants from Escherichia coli: an SH-free mutant (Ec0SH) with denaturation temperature (T d ) = 85.6 °C, a hydrophobic mutant (Ec0VV) with T d  = 113.3 °C, and an ionic mutant (Ec0VV_6) with T d  = 136.8 °C. The occupancy of salt bridges by the six substituted charged residues in Ec0VV_6 was 140.1% at 300 K and 89.5% at 450 K, indicating that even in the denatured state, salt bridge occupancy was high, approximately 60% of that at 300 K. From these results, we can infer that proteins from hyperthermophiles with a high ratio of charged residues are stabilized by a decrease in conformational entropy due to ion-ion interactions in the denatured state. The mechanism must be comparable to the stabilization conferred by disulfide bonds within a protein. This suggests that introduction of charged residues, to promote formation of salt bridges in the denatured state, would be a simple way to rationally design stability-enhanced mutants.

Complete removal of hepatitis B virus (HBV) DNA from nuclei is difficult by the current therapies. Recent reports have shown that a novel genome-editing tool using Cas9 with a single-guide RNA (sgRNA) system can cleave the HBV genome in vitro and in vivo. However, induction of a double-strand break (DSB) on the targeted genome by Cas9 risks undesirable off-target cleavage on the host genome. Nickase-Cas9 cleaves a single strand of DNA, and thereby two sgRNAs are required for inducing DSBs. To avoid Cas9-induced off-target mutagenesis, we examined the effects of the expressions of nickase-Cas9 and nuclease dead Cas9 (d-Cas9) with sgRNAs on HBV replication. The expression of nickase-Cas9 with a pair of sgRNAs cleaved the target HBV genome and suppressed the viral-protein expression and HBV replication in vitro. Moreover, nickase-Cas9 with the sgRNA pair cleaved the targeted HBV genome in mouse liver. Interestingly, d-Cas9 expression with the sgRNAs also suppressed HBV replication in vitro without cleaving the HBV genome. These results suggest the possible use of nickase-Cas9 and d-Cas9 with a pair of sgRNAs for eliminating HBV DNA from the livers of chronic hepatitis B patients with low risk of undesirable off-target mutation on the host genome.

It is well documented that a variety of viral quasispecies are found in the patients with chronic infection of hepatitis C virus (HCV). However, the significance of quasispecies in the specific infectivity to individual cell types remains unknown. In the present study, we analyzed the role of quasispecies of the genotype 2a clone, JFH1 (HCVcc), in specific infectivity to the hepatic cell lines, Huh7.5.1 and Hep3B. HCV RNA was electroporated into Huh7.5.1 cells and Hep3B/miR-122 cells expressing miR-122 at a high level. Then, we adapted the viruses to Huh7 and Hep3B/miR-122 cells by serial passages and termed the resulting viruses HCVcc/Huh7 and HCVcc/Hep3B, respectively. Interestingly, a higher viral load was obtained in the homologous combination of HCVcc/Huh7 in Huh7.5.1 cells or HCVcc/Hep3B in Hep3B/miR-122 cells compared with the heterologous combination. By using a reverse genetics system and deep sequence analysis, we identified several adaptive mutations involved in the high affinity for each cell line, suggesting that quasispecies of HCV participate in cell-specific infectivity.

Fresh platelet-rich plasma (PRP) accelerates bone union in rat model. However, fresh PRP has a short half-life. We suggested freeze-dried PRP (FD-PRP) prepared in advance and investigated its efficacy in vivo. Spinal posterolateral fusion was performed on 8-week-old male Sprague-Dawley rats divided into six groups based on the graft materials (n = 10 per group): sham control, artificial bone (A hydroxyapatite-collagen composite) -alone, autologous bone, artificial bone + fresh-PRP, artificial bone + FD-PRP preserved 8 weeks, and artificial bone + human recombinant bone morphogenetic protein 2 (BMP) as a positive control. At 4 and 8 weeks after the surgery, we investigated their bone union-related characteristics including amount of bone formation, histological characteristics of trabecular bone at remodeling site, and biomechanical strength on 3-point bending. Comparable radiological bone union was confirmed at 4 weeks after surgery in 80% of the FD-PRP groups, which was earlier than in other groups (p < 0.05). Histologically, the trabecular bone had thinner and more branches in the FD-PRP. Moreover, the biomechanical strength was comparable to that of autologous bone. FD-PRP accelerated bone union at a rate comparable to that of fresh PRP and BMP by remodeling the bone with thinner, more tangled, and rigid trabecular bone.

The H-type of atypical bovine spongiform encephalopathy (H-BSE) was serially passaged in bovinized transgenic (TgBoPrP) mice. At the fourth passage, most challenged mice showed a typical H-BSE phenotype with incubation periods of 223 ± 7.8 days. However, a different phenotype of BSE prion with shorter incubation periods of 109 ± 4 days emerged in a minor subset of the inoculated mice. The latter showed distinct clinical signs, brain pathology, and abnormal prion protein profiles as compared to H-BSE and other known BSE strains in mice. This novel prion was transmitted intracerebrally to cattle, with incubation periods of 14.8 ± 1.5 months, with phenotypes that differed from those of other bovine prion strains. These data suggest that intraspecies transmission of H-BSE in cattle allows the emergence of a novel BSE strain. Therefore, the continuation of feed ban programs may be necessary to exclude the recycling of H-BSE prions, which appear to arise spontaneously, in livestock. Such measures should help to reduce the risks from both novel and known strains of BSE.

Hepatitis E virus (HEV) causes not only endemics via a fecal-oral route but also sporadic cases via zoonotic transmission or blood transfusion. HEV-like particles (HEV-LP) produced by using a baculovirus expression system are considered a candidate for mucosal vaccines for HEV infection. In this study, we attempted to produce a chimeric HEV-LP presenting various foreign epitopes on its surface. Expression of the recombinant capsid proteins carrying a myc- or FLAG-tag inserted between amino acid residues 488 and 489, which are located in the exterior loop on the protruding domain of the HEV capsid, resulted in the production of recombinant HEV-LP. Although expression of the recombinant capsid protein carrying the HA-tag inserted at the same site failed to produce any particles, co-expression with the myc-tagged capsid protein successfully yielded a chimeric HEV-LP consisting of both recombinant capsid proteins. Immunoprecipitation analyses confirmed that the chimeric particles present these foreign epitopes on the surface. Similar results were obtained for the expression of the recombinant capsid proteins carrying neutralizing epitopes of Japanese encephalitis virus. These results suggest the chimeric HEV-LP system provides a novel vaccine carrier that can accommodate multiple neutralizing epitopes on its surface.

Sodium taurocholate cotransporting polypeptide (NTCP) has been reported as a functional receptor for hepatitis B virus (HBV) infection. However, HBV could not efficiently infect HepG2 cells expressing NTCP (NTCP-HepG2 cells) under adherent monolayer-cell conditions. In this study, NTCP was mainly detected in the basolateral membrane region, but not the apical site, of monolayer NTCP-HepG2 cells. We hypothesized that non-adherent cell conditions of infection would enhance HBV infectivity. Non-adherent NTCP-HepG2 cells were prepared by treatment with trypsin and EDTA, which did not degrade NTCP in the membrane fraction. HBV successfully infected NTCP-HepG2 cells at a viral dose 10 times lower in non-adherent phase than in adherent phase. Efficient infection of non-adherent NTCP-HepG2 cells with blood-borne or cell-culture-derived HBV was observed and was remarkably impaired in the presence of the myristoylated preS1 peptide. HBV could also efficiently infect HepaRG cells under non-adherent cell conditions. We screened several compounds using our culture system and identified proscillaridin A as a potent anti-HBV agent with an IC50 value of 7.2 nM. In conclusion, non-adherent host cell conditions of infection augmented HBV infectivity in an NTCP-dependent manner, thus providing a novel strategy to identify anti-HBV drugs and investigate the mechanism of HBV infection.

The chaperone system is known to be exploited by viruses for their replication. In the present study, we identified the cochaperone FKBP6 as a host factor required for hepatitis C virus (HCV) replication. FKBP6 is a peptidyl prolyl cis-trans isomerase with three domains of the tetratricopeptide repeat (TPR), but lacks FK-506 binding ability. FKBP6 interacted with HCV nonstructural protein 5A (NS5A) and also formed a complex with FKBP6 itself or FKBP8, which is known to be critical for HCV replication. The Val(121) of NS5A and TPR domains of FKBP6 were responsible for the interaction between NS5A and FKBP6. FKBP6 was colocalized with NS5A, FKBP8, and double-stranded RNA in HCV-infected cells. HCV replication was completely suppressed in FKBP6-knockout hepatoma cell lines, while the expression of FKBP6 restored HCV replication in FKBP6-knockout cells. A treatment with the FKBP8 inhibitor N-(N', N'-dimethylcarboxamidomethyl)cycloheximide impaired the formation of a homo- or hetero-complex consisting of FKBP6 and/or FKBP8, and suppressed HCV replication. HCV infection promoted the expression of FKBP6, but not that of FKBP8, in cultured cells and human liver tissue. These results indicate that FKBP6 is an HCV-induced host factor that supports viral replication in cooperation with NS5A.

Although the thermodynamics of protein denaturation at temperatures over 100 °C is essential for the rational design of highly stable proteins, it is not understood well because of the associated technical difficulties. We designed certain hydrophobic mutant proteins of CutA1 from Escherichia coli, which have denaturation temperatures (Td) ranging from 101 to 113 °C and show a reversible heat denaturation. Using a hydrophobic mutant as a template, we successfully designed a hyperthermostable mutant protein (Td = 137 °C) by substituting six residues with charged ones. Thermodynamic analyses of these mutant proteins indicated that the hydrophobic mutants were stabilized by the accumulation of denaturation enthalpy (ΔH) with no entropic gain from hydrophobic solvation around 100 °C, and that the stabilization due to salt bridges resulted from both the increase in ΔH from ion-ion interactions and the entropic effect of the electrostatic solvation over 113 °C. This is the first experimental evidence that has successfully overcome the typical technical difficulties.

A non-classical nuclear localization signal (ncNLS) of influenza A virus nucleoprotein (NP) is critical for nuclear import of viral genomic RNAs that transcribe and replicate in the nucleus of infected cells. Here we report a 2.3 Å resolution crystal structure of mouse importin-α1 in complex with NP ncNLS. The structure reveals that NP ncNLS binds specifically and exclusively to the minor NLS-binding site of importin-α. Structural and functional analyses identify key binding pockets on importin-α as potential targets for antiviral drug development. Unlike many other NLSs, NP ncNLS binds to the NLS-binding domain of importin-α weakly with micromolar affinity. These results suggest that a modest inhibitor with low affinity to importin-α could have anti-influenza activity with minimal cytotoxicity.

This study aimed to quantify the flux of radiocesium in the Abukuma Basin (5,172 km(2)), the largest river system affected by fallout from the Fukushima Daiichi Nuclear Power Plant (FDNPP) event. In the period from 10 August 2011 to 11 May 2012 an estimated 84 to 92% of the total radiocesium transported in the basin's fluvial system was carried in particulate form. During this monitoring period Typhoon Roke (September 2011) was observed to induce a significant and temporally punctuated redistribution of radiocesium. The storm-mobilised radiocesium was an estimated 6.18 Terabecquerels corresponding to 61.4% of the total load delivered to the coastal zone during the observation period. The total flux of radiocesium into the Pacific Ocean estimated at the outlet station (basin area 5,172 km(2)) was 5.34 TBq for (137)Cs, and 4.74 TBq for (134)Cs, corresponding to 1.13% of the total estimated radiocesium fallout over the basin catchment (890 TBq). This was equivalent to the estimated amount of direct leakage from FDNPP to the ocean during June 2011 to September 2012 of 17 TBq and the Level 3 Scale Leakage on 21 August 2013 (24 TBq).