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To better understand the molecular genetics of the Shiga toxin type 2 subunit A gene (stx2A gene), we collected many subtypes of stx2A genes and performed detailed molecular evolutionary analyses of the gene. To achieve the aim of the study, we used several bioinformatics technologies, including time-scaled phylogenetic analyses, phylogenetic distance analyses, phylodynamics analyses, selective pressure analyses, and conformational epitope analyses. A time-scaled phylogeny showed that the common ancestor of the stx2A gene dated back to around 18,600 years ago. After that, the gene diverged into two major lineages (Lineage 1 and 2). Lineage 1 comprised the stx2a–2d subtypes, while Lineage 2 comprised the stx2e, 2g, 2h, and 2o subtypes. The evolutionary rates of the genes were relatively fast. Phylogenetic distances showed that the Lineage 2 strains had a wider genetic divergence than Lineage 1. Phylodynamics also indicated that the population size of the stx2A gene increased after the 1930s and spread globally. Moreover, negative selection sites were identified in the Stx2A proteins, and these sites were diffusely distributed throughout the protein. Two negative selection sites were located adjacent to an active site of the common Stx2A protein. Many conformational epitopes were also estimated in these proteins, while no conformational epitope was found adjacent to the active site. The results suggest that the stx2A gene has uniquely evolved and diverged over an extremely long time, resulting in many subtypes. The dominance of the strains belonging to Lineage 1 suggests that differences in virulence may be involved in the prosperity of the offspring. Furthermore, some subtypes of Stx2A proteins may be able to induce effective neutralizing antibodies against the proteins in humans.

The human parainfluenza virus type 4 (HPIV4) can be classified into two distinct subtypes, 4a and 4b. The full lengths of the fusion gene (F gene) of 48 HPIV4 strains collected during the period of 1966–2022 were analyzed. Based on these gene sequences, the time-scaled evolutionary tree was constructed using Bayesian Markov chain Monte Carlo methods. A phylogenetic tree showed that the first division of the two subtypes occurred around 1823, and the most recent common ancestors of each type, 4a and 4b, existed until about 1940 and 1939, respectively. Although the mean genetic distances of all strains were relatively wide, the distances in each subtype were not wide, indicating that this gene was conserved in each subtype. The evolutionary rates of the genes were relatively low (4.41 × 10−4 substitutions/site/year). Moreover, conformational B-cell epitopes were predicted in the apex of the trimer fusion protein. These results suggest that HPIV4 subtypes diverged 200 years ago and the progenies further diverged and evolved.

Abstract Human behavioural changes are poorly understood, and this limitation has been a serious obstacle to epidemic forecasting. It is generally understood that people change their respective behaviours to reduce the risk of infection in response to the status of an epidemic or government interventions. We must first identify the factors that lead to such decision-making to predict these changes. However, due to an absence of a method to observe decision-making for future behaviour, understanding the behavioural responses to disease is limited. Here, we show that accommodation reservation data could reveal the decision-making process that underpins behavioural changes, travel avoidance, for reducing the risk of COVID-19 infections. We found that the motivation to avoid travel with respect to only short-term future behaviours dynamically varied and was associated with the outbreak status and/or the interventions of the government. Our developed method can quantitatively measure and predict a large-scale population’s behaviour to determine the future risk of COVID-19 infections. These findings enable us to better understand behavioural changes in response to disease spread, and thus, contribute to the development of reliable long-term forecasting of disease spread.

To better understand the evolution of the SARS-CoV-2 Omicron subvariants, we performed molecular evolutionary analyses of the spike (S) protein gene/S protein using advanced bioinformatics technologies. First, time-scaled phylogenetic analysis estimated that a common ancestor of the Wuhan, Alpha, Beta, Delta variants, and Omicron variants/subvariants diverged in May 2020. After that, a common ancestor of the Omicron variant generated various Omicron subvariants over one year. Furthermore, a chimeric virus between the BM.1.1.1 and BJ.1 subvariants, known as XBB, diverged in July 2021, leading to the emergence of the prevalent subvariants XBB.1.5 and XBB.1.16. Next, similarity plot (SimPlot) data estimated that the recombination point (breakpoint) corresponded to nucleotide position 1373. As a result, XBB.1.5 subvariants had the 5′ nucleotide side from the breakpoint as a strain with a BJ.1 sequence and the 3′ nucleotide side as a strain with a BM.1.1.1 sequence. Genome network data showed that Omicron subvariants were genetically linked with the common ancestors of the Wuhan and Delta variants, resulting in many amino acid mutations. Selective pressure analysis estimated that the prevalent subvariants, XBB.1.5 and XBB.1.16, had specific amino acid mutations, such as V445P, G446S, N460K, and F486P, located in the RBD when compared with the BA.4 and BA.5 subvariants. Moreover, some representative immunogenicity-associated amino acid mutations, including L452R, F486V, R493Q, and V490S, were also found in these subvariants. These substitutions were involved in the conformational epitopes, implying that these mutations affect immunogenicity and vaccine evasion. Furthermore, these mutations were identified as positive selection sites. These results suggest that the S gene/S protein Omicron subvariants rapidly evolved, and mutations observed in the conformational epitopes may reduce the effectiveness of the current vaccine, including bivalent vaccines such as mRNA vaccines containing the BA.4/BA.5 subvariants.

The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1–7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.

To understand the evolution of GII.P6-GII.6 and GII.P7-GII.6 strains, the prevalent human norovirus genotypes, we analysed both the RdRp region and VP1 gene in globally collected strains using authentic bioinformatics technologies. A common ancestor of the P6- and P7-type RdRp region emerged approximately 50 years ago and a common ancestor of the P6- and P7-type VP1 gene emerged approximately 110 years ago. Subsequently, the RdRp region and VP1 gene evolved. Moreover, the evolutionary rates were significantly faster for the P6-type RdRp region and VP1 gene than for the P7-type RdRp region and VP1 genes. Large genetic divergence was observed in the P7-type RdRp region and VP1 gene compared with the P6-type RdRp region and VP1 gene. The phylodynamics of the RdRp region and VP1 gene fluctuated after the year 2000. Positive selection sites in VP1 proteins were located in the antigenicity-related protruding 2 domain, and these sites overlapped with conformational epitopes. These results suggest that the GII.6 VP1 gene and VP1 proteins evolved uniquely due to recombination between the P6- and P7-type RdRp regions in the HuNoV GII.P6-GII.6 and GII.P7-GII.6 virus strains.

Despite the increasing evidence of the clinical impact of Pseudomonas-derived cephalosporinase (PDC) sequence polymorphisms, the molecular evolution of its encoding gene, blaPDC, remains elusive. To elucidate this, we performed a comprehensive evolutionary analysis of blaPDC. A Bayesian Markov Chain Monte Carlo phylogenetic tree revealed that a common ancestor of blaPDC diverged approximately 4660 years ago, leading to the formation of eight clonal variants (clusters A–H). The phylogenetic distances within clusters A to G were short, whereas those within cluster H were relatively long. Two positive selection sites and many negative selection sites were estimated. Two PDC active sites overlapped with negative selection sites. In docking simulation models based on samples selected from clusters A and H, piperacillin was bound to the serine and the threonine residues of the PDC active sites, with the same binding mode for both models. These results suggest that, in P. aeruginosa, blaPDC is highly conserved, and PDC exhibits similar antibiotic resistance functionality regardless of its genotype.

Abstract Esophageal intramural hematoma (EIH) is a condition which occurs as a result of hemorrhage within the esophageal wall including the submucosal layer. However, reports of EIH on achalasia patients are quite limited and per‐oral endoscopic myotomy (POEM) for achalasia with EIH has not been reported. This is the first case report that demonstrated a successful treatment of achalasia with EIH by POEM. In achalasia, since there is absence of lower esophageal sphincter relaxation, as barotraumatic pathogenesis, an increase in the intraesophageal pressure may cause EIH. As direct traumatic pathogenesis, the stasis of food may directly injure the esophageal wall resulting in EIH. After confirming the hematoma healed until it became an ulcer, POEM was performed on the posterior axis since the intramural hematoma was located anteriorly. The procedure was completed successfully without any occurrence of adverse events. On 2‐months follow‐up, improvement in dysphagia was noted, and complete epithelialization of the intramural hematoma region was seen on endoscopic examination. On 1‐year follow‐up, patient did not have recurrence of dysphagia and intramural hematoma. In summary, we reported a case of achalasia with EIH, which was treated by POEM. POEM procedure may be effective not only for the improvement of dysphagia but also for a better ulcer healing and prevention of intramural hematoma recurrence.

While the aetiology of asthma is unclear, the onset and/or exacerbation of asthma may be associated with respiratory infections. Virus-induced asthma is also known as virus-associated/triggered asthma, and the reported main causative agent is rhinovirus (RV). Understanding the relationship between viral infections and asthma may overcome the gaps in deferential immunity between viral infections and allergies. Moreover, understanding the complicated cytokine networks involved in RV infection may be necessary. Therefore, the complexity of RV-induced asthma is not only owing to the response of airway and immune cells against viral infection, but also to allergic immune responses caused by the wide variety of cytokines produced by these cells. To better understand RV-induced asthma, it is necessary to elucidate the nature RV infections and the corresponding host defence mechanisms. In this review, we attempt to organise the complexity of RV-induced asthma to make it easily understandable for readers.

Molecular interactions between respiratory syncytial virus (RSV) fusion protein (F protein) and the cellular receptor Toll-like receptor 4 (TLR4) and myeloid differentiation factor-2 (MD-2) protein complex are unknown. Thus, to reveal the detailed molecular interactions between them, in silico analyses were performed using various bioinformatics techniques. The present simulation data showed that the neutralizing antibody (NT-Ab) binding sites in both prefusion and postfusion proteins at sites II and IV were involved in the interactions between them and the TLR4 molecule. Moreover, the binding affinity between postfusion proteins and the TLR4/MD-2 complex was higher than that between prefusion proteins and the TLR4/MD-2 complex. This increased binding affinity due to conformational changes in the F protein may be able to form syncytium in RSV-infected cells. These results may contribute to better understand the infectivity and pathogenicity (syncytium formation) of RSV.

Background and study aims Image-enhanced magnifying endoscopy allows optimization of the detection and diagnosis of lesions found in the gastrointestinal tract. Current organ-specific classifications are well-accepted by specialized endoscopists but may pose confusion for general gastroenterologists. To address this, our group proposed the Unified Magnifying Endoscopic Classification (UMEC) which can be applied either in esophagus, stomach, or colon. The aim of this study was to evaluate the diagnostic performance and clinical applicability of UMEC. Patients and methods A single-center, feasibility pilot study was conducted. Two endoscopists with experience in magnifying narrow band imaging (NBI), blinded to white-light and non-magnifying NBI findings as well as histopathological diagnosis, independently reviewed and diagnosed all images based on UMEC. In brief, UMEC is divided into three categories: non-neoplasia, intramucosal neoplasia, and deep submucosal invasive cancer. The diagnostic performance of UMEC was assessed while using the gold standard histopathology as a reference. Results A total of 303 gastrointestinal lesions (88 esophageal squamous lesions, 90 gastric lesions, 125 colonic lesions) were assessed. The overall accuracy for both endoscopists in the diagnosis of esophageal squamous cell cancer, gastric cancer, and colorectal cancer were 84.7 %, 89.5 %, and 83.2 %, respectively. The interobserver agreement for each organ, Kappa statistics of 0.51, 0.73, and 0.63, was good. Conclusions UMEC appears to be a simple and practically acceptable classification, particularly to general gastroenterologists, due to its good diagnostic accuracy, and deserves further evaluation in future studies.

Objectives: The variable-stiffness colonoscope is reportedly useful for making colonoscope insertion easier. However, this function is not associated with all colonoscopes. We developed a variable-stiffness stylet that can be inserted into the endoscope instrumentation channel to change the rigidity of the endoscope. Methods: We developed a stylet with adjustable stiffness and investigated its utility in colonoscope insertion using an ex-vivo model. Four endoscopists performed 24 colonoscope insertions, alternating between using the stylet (Stylet method) and the conventional method. We assessed insertion rate, rate of applying abdominal compression, and insertion time between the two groups. Results: In all procedures, the endoscope was inserted up to the cecum. There were significantly fewer external abdominal compressions with the Stylet method (1/12, 8.3%) compared to the conventional method (6/12, 50%). The insertion time was shorter with the Stylet method (140.9 ± 53.7 s) compared to the conventional method (181.3 ± 64.9 s). Conclusions: Using the variable-stiffness stylet, currently under development, resulted in significantly fewer external abdominal compressions and tended to have shorter insertion time.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonasaeruginosa. The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.

Nuclear power plants are equipped with a hardened containment venting system and a filtered containment venting system to avoid a primary containment vessel (PCV) break by over-pressurization. Considering the gas release from these venting systems, it is desirable to delay venting start time or to avoid operation of the systems by ensuring sufficient PCV cooling in view of the time needed to evacuate residents and the half-life of the radioactive materials. We have been developing a new drywell cooler specialized for severe accidents (DWC_SA) as one additional PCV cooling method. The DWC_SA can cool the PCV by continuous steam condensation using downward flow induced by natural force without any electrical devices in the PCV. We have already conducted element tests using a small tube bundle to confirm the occurrence of continuous downward flow with the DWC_SA configuration and to measure heat transfer rate of condensation with non-condensable gas (NCG). The heat transfer model was validated using the element test data. Here, to estimate plant performance of a representative advanced boiling water reactor (ABWR) applying the DWC_SA, we carried out a severe accident analysis using the MAAP code. We originally installed the heat transfer rate map of the DWC_SA as a function of pressure and NCG ratio in the code. The velocities using in the heat transfer model were estimated from CFD analysis. According to the severe accident analysis results, the DWC_SA can extend venting start time sufficiently to allow evacuation of residents in the accident scenario (LOCA with large pipe failure + ECCS failure + SBO) with the external water injection into the PCV and it can avoid operation of the containment venting systems in the case of internal water circulation in the PCV.

Background and study aims: The clinical benefit of endoscopic submucosal dissection (ESD) for superficial duodenal neoplasias has not yet been verified. The aims of this study were to validate the feasibility and long-term outcomes of ESD for nonampullary superficial duodenal neoplasias, larger than 20 mm. Patients and methods: 41 patients, with 41 nonampullary large superficial duodenal neoplasias, who underwent ESD between April 2005 to March 2013 were included in the study. The short- and long-term outcomes were retrospectively evaluated, related to tumor size, resection size, histological type, invasion depth, complete resection rate, operation time, perforation rate, delayed bleeding rate, local recurrence rate, distant metastasis, and survival rate. Results: ESD was successfully completed in 38 of 41 patients (92.7 %). The median sizes of tumors and resected specimens were, respectively, 26 mm (range 20 – 70 mm) and 36.5 mm (range 23 – 80 mm). Histopathological findings showed 13 adenocarcinomas (12 mucosal, 1 submucosal) and 28 adenomas (26 high grade dysplasia, 2 low grade dysplasia). The complete resection rate was 89.5 % (34 /38 tumors). The mean operation time was 128.3minutes. Perforation during ESD occurred in 39 % and delayed bleeding occurred in 18.4 %. The median observation period was 48 months (range 3.2 – 94.1 months), and no local recurrence or distant metastasis was seen during this time, representing a disease-free survival rate of 100 %. Conclusions: ESD for nonampullary large superficial duodenal adenocarcinoma/adenoma is feasible with favorable long-term outcomes. However, because of the high complication rate due to its technical difficulty, experience and skill of the operator, along with adequate perioperative management, are essential.

Background: Anti-arrhythmic drugs (AADs) are often administered following catheter ablation (CA) for persistent atrial fibrillation (peAF) to maintain sinus rhythm (SR). It remains unclear whether AADs can be withdrawn in patients showing no recurrence after CA. Method: We administered hybrid therapy consisting of CA and AAD (bepridil) in 75 patients with peAF. Withdrawal of AADs was attempted in patients who had no recurrence of AF for ≥6 months. We followed them for 22±16 months. Results: Patients received 1.2±0.4 sessions of CA and a mean bepridil dose of 131±46 mg/day. After a 3-month “blanking period,” 62 (83%) patients maintained SR without recurrence of tachyarrhythmia for ≥6 months. AADs were discontinued in 41 patients who agreed to medication withdrawal. Ten of these (24%) experienced a relapse of tachycardia, and these patients had a higher incidence of residual inducibility of tachyarrhythmia at the end of the CA procedure (70% vs. 32%; P=0.03) and required a higher dose of bepridil to maintain SR (170±48 mg vs. 106±30 mg; P

A 67-year-old man with poor left ventricular function due to a dilated cardiomyopathy was implanted with an implantable cardioverter defibrillator for secondary prevention. Eight years later, he was admitted to our hospital with worsening of heart failure. An electrocardiogram showed a repetitive nonreentrant ventriculoatrial synchrony (RNRVAS). RNRVAS is a device-related arrhythmia that can occur when a dual-chamber pacemaker does not sense a retrograde P wave within the postventricular atrial refractory period, resulting in a repetitive ventricular pacing and noncaptured atrial pacing after the retrograde P wave. We suspected that ventricular pacing caused his heart failure and used the noncompetitive atrial pacing algorithm, which was programmed to prevent RNRVAS. The algorithm was able to prevent the arrhythmia, and the patient successfully recovered from heart failure.