Impact of Cough Severity on the Diagnostic Yield of Endobronchial Ultrasonography Transbronchial Biopsy with Guide Sheath: A Retrospective Observational Study.

Bronchoscopy is an invasive procedure, and patient coughing during examination has been reported to cause patient distress. This study aimed to clarify the relationship between cough severity and diagnostic yield of endobronchial ultrasonography with guide sheath transbronchial biopsy (EBUS-GS-TBB). Data of patients who underwent bronchoscopy at Kyorin University Hospital between April 2019 and March 2022 were retrospectively evaluated. Bronchoscopists assessed the cough severity upon completion of the procedure using a four-point cough scale. Cough severity was included as a predictive factor along with those reportedly involved in bronchoscopic diagnosis, and their impact on diagnostic yield was evaluated. Predictors of cough severity were also examined. A total of 275 patients were enrolled in this study. In the multivariate analysis, the diagnostic group (n = 213) had significantly more 'within' radial endobronchial ultrasound findings (odds ratio [OR] 5.900, p < 0.001), a lower cough score (cough score per point; OR 0.455, p < 0.001), and fewer bronchial generations to target lesion(s) (OR 0.686, p < 0.001) than the non-diagnostic group (n = 62). The predictive factors for severe cough include the absence of virtual bronchoscopic navigation (VBN) and prolonged examination time. Decreased cough severity was a positive predictive factor for successful EBUS-GS-TBB, which may be controlled using VBN and awareness of the procedural duration.

Promotion of ABCG2 gene expression by neolignans from Piper longum L.

We focused on Piper longum L., a herbal drug produced in Myanmar, which has a renoprotective effect. Thus, we attempted to isolate and identify compounds that enhance the expression of the ABCG2 gene from the aerial parts of the plant except for the fruit. Among the various P. longum extracts, we isolated and identified the components. Using Caco-2 cells, the hABCG2 mRNA expression-enhancing effects of the isolated compounds were compared with the positive reference compound (3-methylcholanthrene [3MC]) using real-time polymerase chain reaction. Six compounds were isolated and identified from the methanol extract of P. longum. Among the isolated compounds, licarin A and neopomatene had lower toxicity and higher hABCG2 mRNA expression-enhancing effects in Caco-2 cells. Suppression of hAhR expression by siRNA reduced the activity of licarin A and neopomatene, as well as the hAhR agonist 3MC, suggesting that these 2 compounds may act as hAhR agonists to promote hABCG2 expression.

Molecular Evolutionary Analyses of the Spike Protein Gene and Spike Protein in the SARS-CoV-2 Omicron Subvariants.

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.

Molecular Diversity of Human Respiratory Syncytial Virus before and during the COVID-19 Pandemic in Two Neighboring Japanese Cities.

Human respiratory syncytial viruses (HRSVs) are divided into subgroups A and B, which are further divided based on the nucleotide sequence of the second hypervariable region (HVR) of the attachment glycoprotein (G) gene. Understanding the molecular diversity of HRSV before and during the coronavirus disease 2019 (COVID-19) pandemic can provide insights into the effects of the pandemic on HRSV dissemination and guide vaccine development. Here, we analyzed HRSVs isolated in Fukushima Prefecture from September 2017 to December 2021. Specimens from pediatric patients were collected at two medical institutions in neighboring cities. A phylogenetic tree based on the second HVR nucleotide sequences was constructed using the Bayesian Markov chain Monte Carlo method. HRSV-A (ON1 genotype) and HRSV-B (BA9 genotype) were detected in 183 and 108 specimens, respectively. There were differences in the number of HRSV strains within clusters prevalent at the same time between the two hospitals. The genetic characteristics of HRSVs in 2021 after the COVID-19 outbreak were similar to those in 2019. HRSVs within a cluster may circulate within a region for several years, causing an epidemic cycle. Our findings add to the existing knowledge of the molecular epidemiology of HRSV in Japan. IMPORTANCE Understanding the molecular diversity of human respiratory syncytial viruses during pandemics caused by different viruses can provide insights that can guide public health decisions and vaccine development.

Molecular Evolutionary Analyses of the RNA-Dependent RNA Polymerase (RdRp) Region and VP1 Gene in Human Norovirus Genotypes GII.P6-GII.6 and GII.P7-GII.6.

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.

Molecular evolutionary analyses of the fusion protein gene in human respirovirus 1.

Few evolutionary studies of the human respiratory virus (HRV) have been conducted, but most of them have focused on HRV3. In this study, the full-length fusion (F) genes in HRV1 strains collected from various countries were subjected to time-scaled phylogenetic, genome population size, and selective pressure analyses. Antigenicity analysis was performed on the F protein. The time-scaled phylogenetic tree using the Bayesian Markov Chain Monte Carlo method estimated that the common ancestor of the HRV1 F gene diverged in 1957 and eventually formed three lineages. Phylodynamic analyses showed that the genome population size of the F gene has doubled over approximately 80 years. Phylogenetic distances between the strains were short (< 0.02). No positive selection sites were detected for the F protein, whereas many negative selection sites were identified. Almost all conformational epitopes of the F protein, except one in each monomer, did not correspond to the neutralising antibody (NT-Ab) binding sites. These results suggest that the HRV1 F gene has constantly evolved over many years, infecting humans, while the gene may be relatively conserved. Mismatches between computationally predicted epitopes and NT-Ab binding sites may be partially responsible for HRV1 reinfection and other viruses such as HRV3 and respiratory syncytial virus.

Molecular Evolutionary Analyses of the Pseudomonas-Derived Cephalosporinase Gene.

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.

Rhinovirus Infection and Virus-Induced Asthma.

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.

Detailed Molecular Interactions between Respiratory Syncytial Virus Fusion Protein and the TLR4/MD-2 Complex In Silico.

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.

Molecular Evolution of the Pseudomonas aeruginosa DNA Gyrase gyrA Gene.

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.

Detailed Analyses of Molecular Interactions between Favipiravir and RNA Viruses In Silico.

There are currently no antiviral agents for human metapneumovirus (HMPV), respiratory syncytial virus (RSV), mumps virus (MuV), or measles virus (MeV). Favipiravir has been developed as an anti-influenza agent, and this agent may be effective against these viruses in vitro. However, the molecular mechanisms through which the agent affects virus replication remain to be fully elucidated. Thus, to clarify the detailed molecular interactions between favipiravir and the RNA-dependent RNA polymerase (RdRp) of HMPV, RSV, MuV, MeV, and influenza virus, we performed in silico studies using authentic bioinformatics technologies. As a result, we found that the active form of favipiravir (favipiravir ribofuranosyl-5'-triphosphate [F-RTP]) can bind to the RdRp active sites of HMPV, RSV, MuV, and MeV. The aspartic acid residue of RdRp active sites was involved in the interaction. Moreover, F-RTP was incorporated into the growing viral RNA chain in the presence of nucleotide triphosphate and magnesium ions. The results suggested that favipiravir shows two distinct mechanisms in various viruses: RdRp active site inhibition and/or genome replication inhibition.

Detailed Evolutionary Analyses of the F Gene in the Respiratory Syncytial Virus Subgroup A.

We performed evolution, phylodynamics, and reinfection-related antigenicity analyses of respiratory syncytial virus subgroup A (RSV-A) fusion (F) gene in globally collected strains (1465 strains) using authentic bioinformatics methods. The time-scaled evolutionary tree using the Bayesian Markov chain Monte Carlo method estimated that a common ancestor of the RSV-A, RSV-B, and bovine-RSV diverged at around 450 years ago, and RSV-A and RSV-B diverged around 250 years ago. Finally, the RSV-A F gene formed eight genotypes (GA1-GA7 and NA1) over the last 80 years. Phylodynamics of RSV-A F gene, including all genotype strains, increased twice in the 1990s and 2010s, while patterns of each RSV-A genotype were different. Phylogenetic distance analysis suggested that the genetic distances of the strains were relatively short (less than 0.05). No positive selection sites were estimated, while many negative selection sites were found. Moreover, the F protein 3D structure mapping and conformational epitope analysis implied that the conformational epitopes did not correspond to the neutralizing antibody binding sites of the F protein. These results suggested that the RSV-A F gene is relatively conserved, and mismatches between conformational epitopes and neutralizing antibody binding sites of the F protein are responsible for the virus reinfection.

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

Relationships between Viral Load and the Clinical Course of COVID-19.

To predict the clinical outcome of coronavirus disease-2019 (COVID-19), we examined relationships among epidemiological data, viral load, and disease severity. We examined viral loads of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) in fatal (15 cases), symptomatic/survived (133 cases), and asymptomatic cases (138 cases) using reverse transcription quantitative real-time PCR (RT-qPCR). We examined 5768 nasopharyngeal swabs (NPS) and attempted to detect the SARS-CoV-2 genome using RT-qPCR. Among them, the viral genome was detected using the method for the 370 NPS samples with a positive rate of 6.4%. A comparison of each age showed that the fatal case was higher than the survived case and asymptomatic patients. Survived cases were older than asymptomatic patients. Notably, the viral load in the fatal cases was significantly higher than in symptomatic or asymptomatic cases (p < 0.05). These results suggested that a high viral load of the SARS-CoV-2 in elderly patients at an early stage of the disease results in a poor outcome. We should, therefore, intervene early to prevent a severe stage of the disease in such cases.

Ruxolitinib inhibits poly(I:C) and type 2 cytokines-induced CCL5 production in bronchial epithelial cells: A potential therapeutic agent for severe eosinophilic asthma.

RATIONALE: Severe eosinophilic asthma is characterized by airway eosinophilia and corticosteroid-resistance, commonly overlapping with type 2 inflammation. It has been reported that chemokine (C-C motif) ligand 5 (CCL5) is involved in the exacerbation of asthma by RNA virus infections. Indeed, treatment with a virus-associated ligand and a T helper type 2 cell (Th2) cytokine can synergistically stimulate CCL5 production in bronchial epithelial cells. We aimed to evaluate the mechanisms underlying CCL5 production in this in vitro model and to assess the potential of Janus kinase 1 (JAK1) as a novel therapeutic target via the use of ruxolitinib. METHODS: We stimulated primary normal human bronchial epithelial (NHBE) cells and BEAS-2B cells with poly(I:C) along with interleukin-13 (IL-13) or IL-4, and assessed CCL5 production. We also evaluated the signals involved in virus- and Th2-cytokine-induced CCL5 production and explored a therapeutic agent that attenuates the CCL5 production. RESULTS: Poly(I:C) stimulated NHBE and BEAS-2B cells to produce CCL5. Poly(I:C) and IL-13 increased CCL5 production. Poly(I:C)-induced CCL5 production occurred via the TLR3-IRF3 and IFNAR/JAK1-phosphoinositide 3-kinase (PI3K) pathways, but not the IFNAR/JAK1-STATs pathway. In addition, IL-13 did not augment poly(I:C)-induced CCL5 production via the canonical IL-13R/IL-4R/JAK1-STAT6 pathway but likely via subsequent TLR3-IRF3-IFNAR/JAK1-PI3K pathways. JAK1 was identified to be a potential therapeutic target for severe eosinophilic asthma. The JAK1/2 inhibitor, ruxolitinib, was demonstrated to more effectively decrease CCL5 production in BEAS-2B cells than fluticasone propionate. CONCLUSION: We have demonstrated that JAK1 is a possible therapeutic target for severe corticosteroid-resistant asthma with airway eosinophilia and persistent Th2-type inflammation, and that ruxolitinib has potential as an alternative pharmacotherapy.

Detailed Molecular Interactions of Favipiravir with SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza Virus Polymerases In Silico.

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5'-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.

Editorial for the Special Issue: Molecular Epidemiology, Diagnostics and Management of Respiratory Virus Infections.

In Japan, there is a proverb that the common cold is associated with all diseases [...].

Pseudomonas aeruginosa-derived flagellin stimulates IL-6 and IL-8 production in human bronchial epithelial cells: A potential mechanism for progression and exacerbation of COPD.

Background and purpose of the study:Pseudomonas aeruginosa commonly colonizes the airway of patients with chronic obstructive pulmonary disease (COPD) and exacerbates their symptoms. P. aeruginosa carries flagellin that stimulates toll-like receptor (TLR)-5; however, the role of flagellin in the pathogenesis of COPD remains unclear. The aim of the study was to evaluate the mechanisms of the flagellin-induced innate immune response in bronchial epithelial cells, and to assess the effects of anti-inflammatory agents for treatment. Materials and methods: We stimulated BEAS-2B cells with P. aeruginosa-derived flagellin, and assessed mRNA expression and protein secretion of interleukin (IL)-6 and IL-8. We also used mitogen-activated protein kinases (MAPK) inhibitors to assess the signaling pathways involved in flagellin stimulation, and investigated the effect of clinically available anti-inflammatory agents against flagellin-induced inflammation. Results: Flagellin promoted protein and mRNA expression of IL-6 and IL-8 in BEAS-2B cells and induced phosphorylation of p38, ERK, and JNK; p38 phosphorylation-induced IL-6 production, while IL-8 production resulted from p38 and ERK phosphorylation. Fluticasone propionate (FP) and dexamethasone (DEX) suppressed IL-6 and IL-8 production in BEAS-2B cells, but clarithromycin (CAM) failed to do so. Conclusions:P. aeruginosa-derived flagellin-induced IL-6 and IL-8 production in bronchial epithelial cells, which partially explains the mechanisms of progression and exacerbation of COPD. Corticosteroids are the most effective treatment for the suppression of flagellin-induced IL-6 and IL-8 production in the bronchial epithelial cells.