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The emergence and rapid spread of novel DS-1-like G1P[8] human rotaviruses in Japan were recently reported. More recently, such intergenogroup reassortant strains were identified in Thailand, implying the ongoing spread of unusual rotavirus strains in Asia. During rotavirus surveillance in Thailand, three DS-1-like intergenogroup reassortant strains having G3P[8] (RVA/Human-wt/THA/SKT-281/2013/G3P[8] and RVA/Human-wt/THA/SKT-289/2013/G3P[8]) and G2P[8] (RVA/Human-wt/THA/LS-04/2013/G2P[8]) genotypes were identified in fecal samples from hospitalized children with acute gastroenteritis. In this study, we sequenced and characterized the complete genomes of strains SKT-281, SKT-289, and LS-04. On whole genomic analysis, all three strains exhibited unique genotype constellations including both genogroup 1 and 2 genes: G3-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strains SKT-281 and SKT-289, and G2-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2 for strain LS-04. Except for the G genotype, the unique genotype constellation of the three strains (P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2) is commonly shared with DS-1-like G1P[8] strains. On phylogenetic analysis, nine of the 11 genes of strains SKT-281 and SKT-289 (VP4, VP6, VP1-3, NSP1-3, and NSP5) appeared to have originated from DS-1-like G1P[8] strains, while the remaining VP7 and NSP4 genes appeared to be of equine and bovine origin, respectively. Thus, strains SKT-281 and SKT-289 appeared to be reassortant strains as to DS-1-like G1P[8], animal-derived human, and/or animal rotaviruses. On the other hand, seven of the 11 genes of strain LS-04 (VP7, VP6, VP1, VP3, and NSP3-5) appeared to have originated from locally circulating DS-1-like G2P[4] human rotaviruses, while three genes (VP4, VP2, and NSP1) were assumed to be derived from DS-1-like G1P[8] strains. Notably, the remaining NSP2 gene of strain LS-04 appeared to be of bovine origin. Thus, strain LS-04 was assumed to be a multiple reassortment strain as to DS-1-like G1P[8], locally circulating DS-1-like G2P[4], bovine-like human, and/or bovine rotaviruses. Overall, the great genomic diversity among the DS-1-like G1P[8] strains seemed to have been generated through reassortment involving human and animal strains. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like intergenogroup reassortant strains having G3P[8] and G2P[8] genotypes that have emerged in Thailand. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] strains and related reassortant ones.

Objective Rapid eye movement (REM) obstructive sleep apnea (OSA) is associated with the risk of cardiovascular events. Arterial stiffness and carotid artery intima-media thickness (IMT) predict these events, but few relevant studies have been conducted. We compared long-term changes in arterial stiffness and IMT between patients with REM OSA and non-REM (NREM) OSA receiving continuous positive airway pressure (CPAP) or oral appliance (OA) therapy. Methods Newly diagnosed female patients with OSA received CPAP (n = 6) or OA (n = 7). Pulse wave velocity (PWV) and carotid artery ultrasound were performed before and 60 months after treatment. Results There were no differences in baseline characteristics (mean age: 56.0 vs. 61.3 years; mean body mass index: 22.6 vs. 21.7 kg/m2) between the REM OSA and non-REM OSA groups. The median apnea-hypopnea index was lower in the REM OSA group than in the non-REM OSA group. Increased PWV (12.92 ± 1.64 to 14.56 ± 2.73 m/s) and deteriorated glucose metabolism were observed in the REM OSA group after treatment. PWV, IMT, and cardiovascular risk factors were unaffected in the non-REM OSA group. Conclusion Arterial stiffness and glucose metabolism are deteriorated in patients with REM OSA compared with these parameters in patients with non-REM OSA after CPAP or OA treatment.

The emergence and rapid spread of unusual DS-1-like G1P[8] rotaviruses in Japan have been recently reported. During rotavirus surveillance in Thailand, three DS-1-like G1P[8] strains (RVA/Human-wt/THA/PCB-180/2013/G1P[8], RVA/Human-wt/THA/SKT-109/2013/G1P[8], and RVA/Human-wt/THA/SSKT-41/2013/G1P[8]) were identified in stool specimens from hospitalized children with severe diarrhea. In this study, we sequenced and characterized the complete genomes of strains PCB-180, SKT-109, and SSKT-41. On whole genomic analysis, all three strains exhibited a unique genotype constellation including both genogroup 1 and 2 genes: G1-P[8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. This novel genotype constellation is shared with Japanese DS-1-like G1P[8] strains. Phylogenetic analysis revealed that the G/P genes of strains PCB-180, SKT-109, and SSKT-41 appeared to have originated from human Wa-like G1P[8] strains. On the other hand, the non-G/P genes of the three strains were assumed to have originated from human DS-1-like strains. Thus, strains PCB-180, SKT-109, and SSKT-41 appeared to be derived through reassortment event(s) between Wa-like G1P[8] and DS-1-like human rotaviruses. Furthermore, strains PCB-180, SKT-109, and SSKT-41 were found to have the 11-segment genome almost indistinguishable from one another in their nucleotide sequences and phylogenetic lineages, indicating the derivation of the three strains from a common origin. Moreover, all the 11 genes of the three strains were closely related to those of Japanese DS-1-like G1P[8] strains. Therefore, DS-1-like G1P[8] strains that have emerged in Thailand and Japan were assumed to have originated from a recent common ancestor. To our knowledge, this is the first report on whole genome-based characterization of DS-1-like G1P[8] strains that have emerged in an area other than Japan. Our observations will provide important insights into the evolutionary dynamics of emerging DS-1-like G1P[8] rotaviruses.

Cellular senescence is a state of irreversible growth arrest induced either by telomere shortening (replicative senescence) or by telomere-independent signals (stress-induced senescence). The alveolar epithelium is often injured by a variety of inhaled toxins, including cigarette smoke (CS). In the present study, we investigated whether exposure to CS induces senescence of alveolar epithelial cells. In vitro experiments showed that exposure of A549 cells or normal human alveolar epithelial cells to sublethal concentrations of aqueous CS extracts induced cellular senescence. The senescence was characterized by a dose- and time-dependent increase in senescence-associated β-galactosidase activity, senescence-associated changes in cell morphology, an increase in cell size and lysosomal mass, accumulation of lipofuscin, overexpression of p21CIP1/WAF1/Sdi1 protein, and irreversible growth arrest. In vivo experiments in Institute for Cancer Research mice showed that inhalation of CS for 2 wk induced increases in senescence-associated β-galactosidase activity, lipofuscin accumulation, and p21CIP1/WAF1/Sdi1 protein expression in alveolar epithelial cells. These results suggest that CS induces a phenotype that is indistinguishable from that of senescence in alveolar epithelial cells. The induction of cellular senescence by CS may contribute to impaired re-epithelialization, leading to CS-related chronic lung diseases.

Botulinum neurotoxins produced by Clostridium botulinum cause flaccid paralysis by inhibiting neurotransmitter release at peripheral nerve terminals. Previously, we found that neurons derived from the murine P19 embryonal carcinoma cell line exhibited high sensitivity to botulinum neurotoxin type C. In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of the gene encoding beta-1,4 N-acetylgalactosaminyltransferase 1 in P19 cells using the clustered regularly interspaced short palindromic repeats combined with Cas9 (CRISPR/Cas9) system. By using this system, knockout cells could be generated more easily than with previous methods. The sensitivity of the generated beta-1,4 N-acetylgalactosaminyltransferase 1-depleted P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. In particular, addition of a mixture of these three ganglioside more effectively recovered the sensitivity of knockout cells compared to independent addition of GD1a, GD1b, or GT1b. Consequently, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins.

Fenofibrate (FF), a peroxisome proliferator‐activated receptor‐alpha (PPAR‐α) agonist and a lipid‐lowering agent, can decrease experimental pulmonary fibrosis. However, the mechanisms underlying the antifibrotic effect of FF remain unknown. Hence, this study was conducted to evaluate the effects of FF on transforming growth factor‐beta (TGF‐β)‐induced myofibroblast differentiation and activation in lung fibroblasts. The results showed that FF inhibited alpha‐smooth muscle actin (α‐SMA) and connective tissue growth factor expression, collagen production, cell motility, SMAD3 phosphorylation and nuclear translocation, and metabolic reprogramming in TGF‐β‐exposed cells. The inhibitory effect of FF did not decrease with the addition of a PPAR‐α antagonist. Moreover, the inhibitory effect given by FF could not be reproduced with the addition of an alternative PPAR‐α agonist. FF inhibited mitochondrial respiration. However, rotenone, a complex I inhibitor, did not suppress TGF‐β‐induced myofibroblast differentiation. Furthermore, the TGF‐β‐induced nuclear reduction of protein phosphatase, Mg2+/Mn2+‐dependent 1A (PPM1A), a SMAD phosphatase, was inhibited by FF. These results showed that FF suppressed TGF‐β‐induced myofibroblast differentiation and activation independent of PPAR‐α activation and impaired mitochondrial respiration. In conclusion, this study provides information on the effects of FF on anti‐TGF‐β mechanisms. Fenofibrate, a peroxisome proliferator‐activated receptor‐α (PPAR‐α) agonist and a lipid‐lowering agent, inhibits transforming growth factor‐β (TGF‐β)‐induced myofibroblast differentiation via PPAR‐α‐independent mechanisms, which probably include the upregulation of protein phosphatase, Mg2+/Mn2+‐dependent 1A, a SMAD phosphatase, leading to export of nuclear SMAD3 to cytoplasm. This study provides evidence on the effects of fenofibrate on anti‐TGF‐β actions.

An unusual rotavirus strain, SKT-27, with the G6P[14] genotypes (RVA/Human-wt/THA/SKT-27/2012/G6P[14]), was identified in a stool specimen from a hospitalized child aged eight months with severe diarrhea. In this study, we sequenced and characterized the complete genome of strain SKT-27. On whole genomic analysis, strain SKT-27 was found to have a unique genotype constellation: G6-P[14]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The non-G/P genotype constellation of this strain (I2-R2-C2-M2-A3-N2-T6-E2-H3) is commonly shared with rotavirus strains from artiodactyls such as cattle. Phylogenetic analysis indicated that nine of the 11 genes of strain SKT-27 (VP7, VP4, VP6, VP2-3, NSP1, NSP3-5) appeared to be of artiodactyl (likely bovine) origin, while the remaining VP1 and NSP2 genes were assumed to be of human origin. Thus, strain SKT-27 was found to have a bovine rotavirus genetic backbone, and thus is likely to be of bovine origin. Furthermore, strain SKT-27 appeared to be derived through interspecies transmission and reassortment events involving bovine and human rotavirus strains. Of note is that the VP7 gene of strain SKT-27 was located in G6 lineage-5 together with those of bovine rotavirus strains, away from the clusters comprising other G6P[14] strains in G6 lineages-2/6, suggesting the occurrence of independent bovine-to-human interspecies transmission events. To our knowledge, this is the first report on full genome-based characterization of human G6P[14] strains that have emerged in Southeast Asia. Our observations will provide important insights into the origin of G6P[14] strains, and into dynamic interactions between human and bovine rotavirus strains.

Here we describe a simple affinity purification method for Shiga toxin 2e (Stx2e), a major causative factor of edema disease in swine. Escherichia coli strain MV1184 transformed with the expression plasmid pBSK-Stx2e produced Stx2e when cultivated in CAYE broth containing lincomycin. Stx2e bound to commercial D-galactose gel, containing α-D-galactose immobilized on agarose resin via a divinyl sulfone linker, and was eluted with phosphate-buffered saline containing 4.5 M MgCl2. A small amount of Stx2e bound to another commercial α-galactose-immobilized agarose resin, but not to β-galactose-immobilized resin. In addition, Stx2e bound to thiophilic adsorbent resin containing β-mercaptoethanol immobilized on agarose resin via a divinyl sulfone, and was purified in the same manner as from D-galactose gel, but the Stx2e sample contained some contamination. These results indicate that Stx2e bound to D-galactose gel mainly through the divinyl sulfone group on the resin and to a lesser extent through α-D-galactose. With these methods, the yields of Stx2e and attenuated mutant Stx2e (mStx2e) from 1 L of culture were approximately 36 mg and 27.7 mg, respectively, and the binding capacity of the D-galactose gel and thiophilic adsorbent resin for Stx2e was at least 20 mg per 1 ml of resin. In addition, using chimeric toxins with prototype Stx2 which did not bind to thiophilic adsorbent resin and some types of mutant Stx2e and Stx2 which contained inserted mutations in the B subunits, we found that, at the least, asparagine (amino acid 17 of the B subunits) was associated with Stx2e binding to the divinyl sulfone group. The mStx2e that was isolated exhibited vaccine effects in ICR mice, indicating that these methods are beneficial for large-scale preparation of Stx2e toxoid, which protects swine from edema disease.

Background Predicting the incidence of chemotherapy‐triggered acute exacerbation of interstitial lung disease (AE‐ILD) in patients with lung cancer is important because AE‐ILD confers a poor prognosis. The Glasgow prognostic score (GPS), which is an inflammation‐based index composed of serum levels of C‐reactive protein and albumin, predicts prognosis in patients with small cell lung cancer (SCLC) without ILD. In this study, we investigated AE‐ILD and survival outcome based on the GPS in patients with ILD associated with SCLC who were receiving chemotherapy. Methods Medical records of patients who received platinum‐based first‐line chemotherapy between June 2010 and May 2019 were retrospectively reviewed to compare the incidence of AE‐ILD and overall survival (OS) between GPS 0, 1, and 2. Results Among our cohort of 31 patients, six (19.3%) experienced chemotherapy‐triggered AE‐ILD. The AE‐ILD incidence increased from 9.5% to 25.0% and 50.0% with increase in GPS of 0, 1, and 2, respectively. Univariate and multivariate analyses revealed remarkable associations between GPS 2 and both AE‐ILD (odds ratio for GPS 2, 18.69; p = 0.046) and prognosis (hazard ratio of GPS 2, 13.52; p = 0.002). Furthermore, median OS in the GPS 0, 1, and 2 groups was 16.2, 9.8, and 7.1 months, respectively (p < 0.001). Conclusions Our results suggest that GPS 2 is both a predictor of risk of chemotherapy‐triggered AE‐ILD and a prognostic indicator in patients with ILD associated with SCLC. We propose that GPS may be used as a guide to distinguish chemotherapy‐tolerant patients from those at high risk of AE‐ILD. The Glasgow prognostic score (GPS) tends to be associated with incidence of acute exacerbation of interstitial lung disease (AE‐ILD) (p = 0.082). GPS 2 is an independent risk factor for chemotherapy‐triggered AE‐ILD and prognosis in SCLC patients. GPS may be used as a guide to distinguish chemotherapy‐tolerant patients from those at high risk of AE‐ILD.

Background Interstitial lung disease (ILD) in patients with non‐small cell lung cancer (NSCLC) worsens the prognosis for overall survival (OS) due to chemotherapy‐triggered acute exacerbation (AE)‐ILD. The Glasgow Prognostic Score (GPS), which is based on serum C‐reactive protein and albumin levels, has been suggested as a reliable prognostic tool for mortality in cancer patients, including NSCLC. In this study, we investigated whether GPS is a predictor for chemotherapy‐triggered AE‐ILD and the prognosis in patients with NSCLC and pre‐existing ILD. Methods We conducted a retrospective review on 56 NSCLC and ILD patients at our hospital who received platinum agent‐based treatment as first‐line chemotherapy between June 2010 and May 2019. We categorized these patients according to their GPS (0–2) and compared the incidence of chemotherapy‐triggered AE‐ILD and OS. Results The GPS 0, 1, and 2 groups included 31, 16, and nine patients, respectively, out of 56. A total of 12 (21.4%) patients showed chemotherapy‐triggered AE‐ILD. The median OS was at 11.5 months (95% confidence interval: 8.0–15.1). The incidence of chemotherapy‐triggered AE‐ILD within the first year of chemotherapy in the GPS 0, 1, and 2 groups was three (9.6%), four (25.0%), and five (55.5%), and the median OS time was 16.9, 9.8 and 7.6 months, respectively. Univariate and multivariate analyses indicated that only GPS 2 could predict both chemotherapy‐triggered AE‐ILD and OS (P < 0.05). Conclusions GPS assessment of patients with NSCLC and pre‐existing ILD is a valuable prognostic tool for predicting chemotherapy‐triggered AE‐ILD and OS. Key points Significant findings of the study We found that GPS 2 was an independent risk factor for chemotherapy‐triggered AE‐ILD and prognosis in patients with ILD associated with NSCLC. What this study adds GPS may potentially enable the discrimination of patients tolerant of chemotherapy from those at an increased risk of AE‐ILD and predict the prognosis in patients with NSCLC and ILD receiving chemotherapy. We assessed GPS and AE‐ILD/prognosis in patients with NSCLC and ILD.