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After the global spread of the SARS-CoV-2 Omicron BA.2, some BA.2 subvariants, including BA.2.9.1, BA.2.11, BA.2.12.1, BA.4, and BA.5, emerged in multiple countries. Our statistical analysis showed that the effective reproduction numbers of these BA.2 subvariants are greater than that of the original BA.2. Neutralization experiments revealed that the immunity induced by BA.1/2 infections is less effective against BA.4/5. Cell culture experiments showed that BA.2.12.1 and BA.4/5 replicate more efficiently in human alveolar epithelial cells than BA.2, and particularly, BA.4/5 is more fusogenic than BA.2. We further provided the structure of the BA.4/5 spike receptor-binding domain that binds to human ACE2 and considered how the substitutions in the BA.4/5 spike play roles in ACE2 binding and immune evasion. Moreover, experiments using hamsters suggested that BA.4/5 is more pathogenic than BA.2. Our multiscale investigations suggest that the risk of BA.2 subvariants, particularly BA.4/5, to global health is greater than that of original BA.2.
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Enzima Convertidora de Angiotensina 2 , COVID-19 , Anticuerpos Antivirales , Humanos , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismoRESUMEN
Soon after the emergence and global spread of the SARS-CoV-2 Omicron lineage BA.1, another Omicron lineage, BA.2, began outcompeting BA.1. The results of statistical analysis showed that the effective reproduction number of BA.2 is 1.4-fold higher than that of BA.1. Neutralization experiments revealed that immunity induced by COVID vaccines widely administered to human populations is not effective against BA.2, similar to BA.1, and that the antigenicity of BA.2 is notably different from that of BA.1. Cell culture experiments showed that the BA.2 spike confers higher replication efficacy in human nasal epithelial cells and is more efficient in mediating syncytia formation than the BA.1 spike. Furthermore, infection experiments using hamsters indicated that the BA.2 spike-bearing virus is more pathogenic than the BA.1 spike-bearing virus. Altogether, the results of our multiscale investigations suggest that the risk of BA.2 to global health is potentially higher than that of BA.1.
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COVID-19 , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Animales , COVID-19/virología , Cricetinae , Células Epiteliales , Humanos , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , Glicoproteína de la Espiga del Coronavirus/genéticaRESUMEN
IMPORTANCE: Most studies investigating the characteristics of emerging SARS-CoV-2 variants have been focusing on mutations in the spike proteins that affect viral infectivity, fusogenicity, and pathogenicity. However, few studies have addressed how naturally occurring mutations in the non-spike regions of the SARS-CoV-2 genome impact virological properties. In this study, we proved that multiple SARS-CoV-2 Omicron BA.2 mutations, one in the spike protein and another downstream of the spike gene, orchestrally characterize this variant, shedding light on the importance of Omicron BA.2 mutations out of the spike protein.
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Genoma Viral , Mutación , SARS-CoV-2 , Glicoproteína de la Espiga del Coronavirus , Humanos , COVID-19/virología , SARS-CoV-2/genética , SARS-CoV-2/patogenicidad , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Genoma Viral/genéticaRESUMEN
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the largest single-stranded RNA virus known to date. Its genome contains multiple accessory protein genes that act against host immune responses but are not required for progeny virus production. The functions of the accessory proteins in the viral life cycle have been examined, but their involvement in viral pathogenicity remains unclear. Here, we investigated the roles of the accessory proteins in viral immunopathogenicity. To this end, recombinant SARS-CoV-2 possessing nonsense mutations in the seven accessory protein open reading frames (ORFs) (ORF3a, ORF3b, ORF6, ORF7a, ORF8, ORF9b, and ORF10) was de novo generated using an early pandemic SARS-CoV-2 strain as a backbone. We confirmed that the resultant virus (termed ORF3-10 KO) did not express accessory proteins in infected cells and retained the desired mutations in the viral genome. In cell culture, the ORF3-10 KO virus exhibited similar virus growth kinetics as the parental virus. In hamsters, ORF3-10 KO virus infection resulted in mild weight loss and reduced viral replication in the oral cavity and lung tissue. ORF3-10 KO virus infection led to mild inflammation, indicating that an inability to evade innate immune sensing because of a lack of accessory proteins impairs virus growth in vivo and results in quick elimination from the body. Overall, we showed that SARS-CoV-2 accessory proteins are involved in immunopathogenicity.
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COVID-19 , Sistemas de Lectura Abierta , SARS-CoV-2 , Replicación Viral , Animales , SARS-CoV-2/genética , SARS-CoV-2/inmunología , SARS-CoV-2/fisiología , SARS-CoV-2/patogenicidad , COVID-19/virología , COVID-19/inmunología , Humanos , Pulmón/virología , Pulmón/inmunología , Pulmón/patología , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Células Vero , Cricetinae , Chlorocebus aethiops , Mesocricetus , Genoma Viral , Codón sin Sentido , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
PURPOSE: Periostin, an extracellular matrix protein closely related to mechanical stress, inflammation, and ageing, has been implicated in intervertebral disc degeneration (IVDD) in basic research. However, it has not been examined in clinical cases. This study aimed to evaluate the association between IVDD severity and serum periostin concentration as well as to analyse potential associations between IVDD and clinical and demographic factors. METHODS: This retrospective cohort study included 198 patients who underwent lumbar disc herniation and lumbar canal stenosis between January 2020 and December 2022. The severity of IVDD was evaluated using the Pfirrmann grading, whereas serum periostin levels were measured using ELISA kits. Clinical demographics, including age, sex, body mass index, comorbidities, psoas muscle index, and spinal disease, were also recorded. RESULTS: This study demonstrated a significant correlation between high serum periostin levels and IVDD severity, as indicated by a high cumulative Pfirrmann score. Serum periostin levels were identified as an independent risk factor for IVDD in a multivariate regression model. Correlation analysis showed a correlation between periostin levels and Pfirrmann grade at each lumbar level (ρ = 0.458-0.550, p < 0.001) and a strong correlation with cumulative Pfirrmann score (ρ = 0.690, p < 0.001). CONCLUSION: The higher the serum periostin level, the higher the cumulative Pfirrmann score. Multivariate analysis showed that serum periostin was an independent risk factor for IVDD. Periostin levels may be a clinically suitable and useful biomarker for diagnosing IVDD, estimating disease progression and activity, providing prognostic information, and evaluating treatment options.
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Moléculas de Adhesión Celular , Degeneración del Disco Intervertebral , Índice de Severidad de la Enfermedad , Humanos , Masculino , Moléculas de Adhesión Celular/sangre , Femenino , Degeneración del Disco Intervertebral/sangre , Persona de Mediana Edad , Estudios Retrospectivos , Adulto , Vértebras Lumbares , Anciano , Biomarcadores/sangre , Desplazamiento del Disco Intervertebral/sangre , PeriostinaRESUMEN
Thermophilic Geobacillus kaustophilus HTA426 genome possesses a monoacylglycerol lipase (MAGL) gene. MAGLs can synthesize emulsifiers for use in the food and pharmaceutical industries from fatty acids and glycerol. They can also be used to analyze monoacylglycerol (MAG) levels in serum and food. The MAGL gene from strain HTA426 was artificially synthesized and heterologously expressed in Escherichia coli BL21(DE3). The recombinant His-tag fused MAGL (GkMAGL) was purified using a Ni2+-affinity column. The purified enzyme showed a temperature optimum at 65 °C and was stable up to 75 °C after 30 min incubation. In addition, the enzyme exhibited a pH optimum of 7.5 and was stable from pH 5.0 to 11.0. The enzyme hydrolyzed monoacylglycerols and showed the highest activity toward 1-monolauroylglycerol. The enzyme was stable in the presence of various organic solvents and detergents. The addition of Triton X-100 significantly increased GkMAGL activity. The thermal stability of the enzyme was higher than that of thermostable MAGL from Geobacillus sp. 12AMOR1 (12AMOR1_MAGL). Circular dichroism spectral analysis showed that the conformational stability of the GkMAGL was higher than that of 12AMOR1_MAGL at higher temperatures. These results indicate that the GkMAGL has useful features that can be used for various biotechnological applications.
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To deal with the broad spectrum of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that threaten human health, it is essential to not only drugs develop that target viral proteins but also consider drugs that target host proteins/cellular processes to protect them from being hijacked for viral infection and replication. To this end, it has been reported that autophagy is deeply involved in coronavirus infection. In this study, we used airway organoids to screen a chemical library of autophagic modulators to identify compounds that could potentially be used to fight against infections by a broad range of coronaviruses. Among the 80 autophagy-related compounds tested, cycloheximide and thapsigargin reduced SARS-CoV-2 infection efficiency in a dose-dependent manner. Cycloheximide treatment reduced the infection efficiency of not only six SARS-CoV-2 variants but also human coronavirus (HCoV)-229E and HCoV-OC43. Cycloheximide treatment also reversed viral infection-induced innate immune responses. However, even low-dose (1 µM) cycloheximide treatment altered the expression profile of ribosomal RNAs; thus, side effects such as inhibition of protein synthesis in host cells must be considered. These results suggest that cycloheximide has broad-spectrum anti-coronavirus activity in vitro and warrants further investigation.
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COVID-19 , Coronavirus Humano 229E , Humanos , SARS-CoV-2 , Cicloheximida/farmacología , AutofagiaRESUMEN
Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.
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COVID-19 , Humanos , Enzima Convertidora de Angiotensina 2/genética , Peptidil-Dipeptidasa A/genética , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2/metabolismo , Fumar/efectos adversosRESUMEN
PURPOSE: This study evaluated the population pharmacokinetics of daptomycin in nonobese elderly patients with hypoalbuminemia and chronic kidney disease (CKD) using the glomerular filtration rate estimated from cystatin C (eGFRcys) and estimated its optimal dose. METHODS: We performed population pharmacokinetic analysis of the unbound concentrations of daptomycin. The probability of target attainment of 90% for achieving an area under the concentration-time curve of unbound daptomycin at steady state/ minimum inhibitory concentration ratio of ≥66.6 was stochastically simulated. RESULTS: In the population pharmacokinetic analysis of 25 patients aged ≥65 years, the two-compartment model using eGFRcys and age as covariates of clearance in central compartment of unbound daptomycin were optimal. The unbound fraction rate (fu) was 0.05-0.14. According to the Monte Carlo simulation, the optimal doses for patients with eGFRcys of 20-60 mL/min and aged 65-95 years were calculated as 200-500 mg q24h. CONCLUSION: These results suggest that establishing the dose using total concentrations may result in under- or overestimation caused by alterations in fu. The optimal dose for nonobese elderly patients with hypoalbuminemia and CKD depends on eGFRcys and age, and a standard dose may be insufficient for some patients.
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Antibacterianos/sangre , Cistatina C/sangre , Daptomicina/sangre , Hipoalbuminemia/sangre , Método de Montecarlo , Insuficiencia Renal Crónica/sangre , Anciano , Anciano de 80 o más Años , Antibacterianos/administración & dosificación , Antibacterianos/farmacocinética , Cistatina C/administración & dosificación , Cistatina C/farmacocinética , Daptomicina/administración & dosificación , Daptomicina/farmacocinética , Relación Dosis-Respuesta a Droga , Femenino , Tasa de Filtración Glomerular/efectos de los fármacos , Tasa de Filtración Glomerular/fisiología , Humanos , Hipoalbuminemia/tratamiento farmacológico , Masculino , Estudios Prospectivos , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Insuficiencia Renal Crónica/tratamiento farmacológicoRESUMEN
Spinal subdural hematoma is uncommon but may become more prevalent with increasing anticoagulant use. Early diagnosis from symptoms like lower back pain and leg paralysis is crucial for timely intervention.
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Many methods have been developed to measure the neutralizing capacity of antibodies to SARS-CoV-2. However, these methods are low throughput and can be difficult to quickly modify in response to emerging variants. Therefore, an experimental system for rapid and easy measurement of the neutralizing capacity of antibodies against various variants is needed. In this study, we developed an experimental system that can efficiently measure the neutralizing capacity of sera by using a GFP-carrying recombinant SARS-CoV-2 with spike proteins of multiple variants (B.1.1, BA.5, or XBB.1.5). For all 3 recombinant chimeric genomes generated, neutralizing antibody titers determined by measuring GFP fluorescence intensity correlated significantly with those calculated from viral RNA levels measured by RT-qPCR in the supernatant of infected cells. Furthermore, neutralizing antibody titers determined by visually assessing GFP fluorescence using microscopy were also significantly correlated with those determined by RT-qPCR. By using this high-throughput method, it is now possible to quickly and easily determine the neutralizing capacity of antibodies against SARS-CoV-2 variants.
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COVID-19 , Humanos , COVID-19/diagnóstico , SARS-CoV-2/genética , Ensayos Analíticos de Alto Rendimiento , Anticuerpos Neutralizantes , Anticuerpos AntiviralesRESUMEN
Introduction: Perioperative cerebrovascular accidents (CVAs) related to spine surgery, although rare, can lead to significant disabilities. More studies on spine surgeries are required to identify those at risk of perioperative CVAs. The characteristics and outcomes of patients that experienced CVAs during spine surgery were assessed through a retrospective descriptive study and meta-analysis. Methods: Patients aged ≥18 years who underwent spine surgery under general anesthesia at a hospital between April 2011 and March 2023 were examined. Of the 2,391 initially identified patients, 2,346 were included after excluding 45 who underwent debridement for surgical site infections. Subsequently, a meta-analysis including the present retrospective descriptive study was conducted. Databases such as PubMed and Google Scholar were searched for original peer-reviewed articles written in English. Results: Of the 2,346 patients, 4 (0.17%) (three men, one woman) exhibited perioperative CVAs associated with spine surgery. The CVAs were diverse in nature: one case of cerebral hemorrhage resulting from dural injury during posterior occipitocervical fusion, two cases of cerebral infarctions after lumbar laminectomy and anterior thoracic fusion due to anticoagulant discontinuation, and one case of posterior reversible encephalopathy syndrome following microscopic lumbar discectomy due to gestational hypertension. The subsequent meta-analysis included three studies (n=186,860). It showed several risk factors for perioperative CVAs, including cervical level (pooled odds ratio [OR]=1.33), hypertension (pooled OR=2.27), atrial fibrillation (pooled OR=8.78), history of heart disease (pooled OR=2.47), and diabetes (pooled OR=2.13). Conclusions: It was speculated that the potential risk factors for the four perioperative CVA cases of spine surgery in this retrospective descriptive study were intraoperative dural injury, preoperative anticoagulant discontinuation, and gestational hypertension history. The meta-analysis revealed that cervical spine surgery, hypertension, atrial fibrillation, heart disease, and diabetes increased the CVA risk. This highlights the need for risk assessment, preoperative optimization, and postoperative care to reduce spine surgery-associated perioperative CVAs.
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Monitoring in vivo viral dynamics can improve our understanding of pathogenicity and tissue tropism. Because the gene size of RNA viruses is typically small, NanoLuc is the primary choice for accommodation within viral genome. However, NanoLuc/Furimazine and also the conventional firefly luciferase/D-luciferin are known to exhibit relatively low tissue permeability and thus less sensitivity for visualization of deep tissue including lungs. Here, we demonstrated in vivo sufficient visualization of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection using the pair of a codon-optimized Akaluc and AkaLumine. We engineered the codon-optimized Akaluc gene possessing the similar GC ratio of SARS-CoV-2. Using the SARS-CoV-2 recombinants carrying the codon-optimized Akaluc, we visualized in vivo infection of respiratory organs, including the tissue-specific differences associated with particular variants. Additionally, we could evaluate the efficacy of antivirals by monitoring changes in Akaluc signals. Overall, we offer an effective technology for monitoring viral dynamics in live animals.
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BACKGROUND: Although several SARS-CoV-2-related coronaviruses (SC2r-CoVs) were discovered in bats and pangolins, the differences in virological characteristics between SARS-CoV-2 and SC2r-CoVs remain poorly understood. Recently, BANAL-20-236 (B236) was isolated from a rectal swab of Malayan horseshoe bat and was found to lack a furin cleavage site (FCS) in the spike (S) protein. The comparison of its virological characteristics with FCS-deleted SARS-CoV-2 (SC2ΔFCS) has not been conducted yet. METHODS: We prepared human induced pluripotent stem cell (iPSC)-derived airway and lung epithelial cells and colon organoids as human organ-relevant models. B236, SARS-CoV-2, and artificially generated SC2ΔFCS were used for viral experiments. To investigate the pathogenicity of B236 in vivo, we conducted intranasal infection experiments in hamsters. FINDINGS: In human iPSC-derived airway epithelial cells, the growth of B236 was significantly lower than that of the SC2ΔFCS. A fusion assay showed that the B236 and SC2ΔFCS S proteins were less fusogenic than the SARS-CoV-2 S protein. The infection experiment in hamsters showed that B236 was less pathogenic than SARS-CoV-2 and even SC2ΔFCS. Interestingly, in human colon organoids, the growth of B236 was significantly greater than that of SARS-CoV-2. INTERPRETATION: Compared to SARS-CoV-2, we demonstrated that B236 exhibited a tropism toward intestinal cells rather than respiratory cells. Our results are consistent with a previous report showing that B236 is enterotropic in macaques. Altogether, our report strengthens the assumption that SC2r-CoVs in horseshoe bats replicate primarily in the intestinal tissues rather than respiratory tissues. FUNDING: This study was supported in part by AMED ASPIRE (JP23jf0126002, to Keita Matsuno, Kazuo Takayama, and Kei Sato); AMED SCARDA Japan Initiative for World-leading Vaccine Research and Development Centers "UTOPIA" (JP223fa627001, to Kei Sato), AMED SCARDA Program on R&D of new generation vaccine including new modality application (JP223fa727002, to Kei Sato); AMED SCARDA Hokkaido University Institute for Vaccine Research and Development (HU-IVReD) (JP223fa627005h0001, to Takasuke Fukuhara, and Keita Matsuno); AMED Research Program on Emerging and Re-emerging Infectious Diseases (JP21fk0108574, to Hesham Nasser; JP21fk0108493, to Takasuke Fukuhara; JP22fk0108617 to Takasuke Fukuhara; JP22fk0108146, to Kei Sato; JP21fk0108494 to G2P-Japan Consortium, Keita Matsuno, Shinya Tanaka, Terumasa Ikeda, Takasuke Fukuhara, and Kei Sato; JP21fk0108425, to Kazuo Takayama and Kei Sato; JP21fk0108432, to Kazuo Takayama, Takasuke Fukuhara and Kei Sato; JP22fk0108534, Terumasa Ikeda, and Kei Sato; JP22fk0108511, to Yuki Yamamoto, Terumasa Ikeda, Keita Matsuno, Shinya Tanaka, Kazuo Takayama, Takasuke Fukuhara, and Kei Sato; JP22fk0108506, to Kazuo Takayama and Kei Sato); AMED Research Program on HIV/AIDS (JP22fk0410055, to Terumasa Ikeda; and JP22fk0410039, to Kei Sato); AMED Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125008 to Keita Matsuno); AMED CREST (JP21gm1610005, to Kazuo Takayama; JP22gm1610008, to Takasuke Fukuhara; JST PRESTO (JPMJPR22R1, to Jumpei Ito); JST CREST (JPMJCR20H4, to Kei Sato); JSPS KAKENHI Fund for the Promotion of Joint International Research (International Leading Research) (JP23K20041, to G2P-Japan Consortium, Keita Matsuno, Takasuke Fukuhara and Kei Sato); JST SPRING (JPMJSP2108 to Shigeru Fujita); JSPS KAKENHI Grant-in-Aid for Scientific Research C (22K07103, to Terumasa Ikeda); JSPS KAKENHI Grant-in-Aid for Scientific Research B (21H02736, to Takasuke Fukuhara); JSPS KAKENHI Grant-in-Aid for Early-Career Scientists (22K16375, to Hesham Nasser; 20K15767, to Jumpei Ito); JSPS Core-to-Core Program (A. Advanced Research Networks) (JPJSCCA20190008, to Kei Sato); JSPS Research Fellow DC2 (22J11578, to Keiya Uriu); JSPS Research Fellow DC1 (23KJ0710, to Yusuke Kosugi); JSPS Leading Initiative for Excellent Young Researchers (LEADER) (to Terumasa Ikeda); World-leading Innovative and Smart Education (WISE) Program 1801 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) (to Naganori Nao); Ministry of Health, Labour and Welfare (MHLW) under grant 23HA2010 (to Naganori Nao and Keita Matsuno); The Cooperative Research Program (Joint Usage/Research Center program) of Institute for Life and Medical Sciences, Kyoto University (to Kei Sato); International Joint Research Project of the Institute of Medical Science, the University of Tokyo (to Terumasa Ikeda and Takasuke Fukuhara); The Tokyo Biochemical Research Foundation (to Kei Sato); Takeda Science Foundation (to Terumasa Ikeda and Takasuke Fukuhara); Mochida Memorial Foundation for Medical and Pharmaceutical Research (to Terumasa Ikeda); The Naito Foundation (to Terumasa Ikeda); Hokuto Foundation for Bioscience (to Tomokazu Tamura); Hirose Foundation (to Tomokazu Tamura); and Mitsubishi Foundation (to Kei Sato).
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COVID-19 , Quirópteros , SARS-CoV-2 , Animales , SARS-CoV-2/genética , SARS-CoV-2/fisiología , Humanos , COVID-19/virología , Quirópteros/virología , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/genética , Organoides/virología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/virología , Cricetinae , Furina/metabolismo , Células Epiteliales/virología , Células Vero , Chlorocebus aethiopsRESUMEN
Circulation of SARS-CoV-2 Omicron XBB has resulted in the emergence of XBB.1.5, a new Variant of Interest. Our phylogenetic analysis suggests that XBB.1.5 evolved from XBB.1 by acquiring the S486P spike (S) mutation, subsequent to the acquisition of a nonsense mutation in ORF8. Neutralization assays showed similar abilities of immune escape between XBB.1.5 and XBB.1. We determine the structural basis for the interaction between human ACE2 and the S protein of XBB.1.5, showing similar overall structures between the S proteins of XBB.1 and XBB.1.5. We provide the intrinsic pathogenicity of XBB.1 and XBB.1.5 in hamsters. Importantly, we find that the ORF8 nonsense mutation of XBB.1.5 resulted in impairment of MHC suppression. In vivo experiments using recombinant viruses reveal that the XBB.1.5 mutations are involved with reduced virulence of XBB.1.5. Together, our study identifies the two viral functions defined the difference between XBB.1 and XBB.1.5.
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COVID-19 , Animales , Cricetinae , Humanos , Codón sin Sentido , Filogenia , SARS-CoV-2/genética , BioensayoRESUMEN
Nucleotide repeat expansion of GGGGCC (G4C2) in the non-coding region of C9orf72 is the most common genetic cause underlying amyotrophic lateral sclerosis and frontotemporal dementia. Transcripts harboring this repeat expansion undergo the translation of dipeptide repeats via a non-canonical process known as repeat-associated non-AUG (RAN) translation. In order to ascertain the essential components required for RAN translation, we successfully recapitulated G4C2-RAN translation using an in vitro reconstituted translation system comprising human factors, namely the human PURE system. Our findings conclusively demonstrate that the presence of fundamental translation factors is sufficient to mediate the elongation from the G4C2 repeat. Furthermore, the initiation mechanism proceeded in a 5' cap-dependent manner, independent of eIF2A or eIF2D. In contrast to cell lysate-mediated RAN translation, where longer G4C2 repeats enhanced translation, we discovered that the expansion of the G4C2 repeats inhibited translation elongation using the human PURE system. These results suggest that the repeat RNA itself functions as a repressor of RAN translation. Taken together, our utilization of a reconstituted RAN translation system employing minimal factors represents a distinctive and potent approach for elucidating the intricacies underlying RAN translation mechanism.
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Proteína C9orf72 , Biosíntesis de Proteínas , Extensión de la Cadena Peptídica de Translación , Factores de Elongación de Péptidos/metabolismo , Humanos , Proteína C9orf72/genética , Sistema de Lectura Ribosómico , Iniciación de la Cadena Peptídica Traduccional , Técnicas In Vitro , Células HeLa , Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genéticaRESUMEN
Winter geometrid moths show striking sexual dimorphism by having female-specific flightless morphs. The evolutionary grades of wing reduction in winter geometrid moths vary and range from having short wings, vestigial wings, to being wingless. Although the ontogenetic processes underlying the development of the wingless or short-wing morphs in Lepidoptera has been well studied, the mechanisms underlying the development of vestigial wing morphs in winter geometrid moths during metamorphosis are poorly understood. In the winter geometrid moth Sebastosema bubonaria Warren, 1896, the males have functional wings, but the females have vestigial wings. Here, we studied the ontogenetic processes affecting wing reduction in the winter geometrid moth S. bubonaria using light microscopy and transmission electron microscopy, and compared the ontogenetic process of wing reduction in this species with that in another species of the wingless-female winter moth that we investigated previously. Our results showed that, in the vestigial-wing morphs, the loss of pupal wing epithelium was terminated in the middle of the wing degeneration process, whereas in the wingless morph, the pupal wing epithelium disappeared almost completely and the final appearance of the wings differed slightly among flightless morphs. We propose that the extent and location of cell death in the pupal wing play an important role in the various patterns of reduced wings that are observed in flightless moths.
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Mariposas Nocturnas , Masculino , Femenino , Animales , Metamorfosis Biológica , Muerte Celular , Evolución Biológica , Alas de AnimalesRESUMEN
Introduction: We examined the neutralizing antibody production efficiency of the second and third severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine doses (2nd- and 3rd-dose) and neutralizing activity on mutant strains, including, the Ancestral, Beta and Omicron strains using green fluorescent protein-carrying recombinant SARS-CoV-2, in living-donor liver transplantation (LDLT) recipients. Methods: The patients who were administered vaccines other than Pfizer- BioNTechBNT162b2 and who had coronavirus disease 2019 in this study period were excluded. We enrolled 154 LDLT recipients and 50 healthy controls. Result: The median time were 21 days (between 1st and 2nd vaccination) and 244 days (between 2nd and 3rd vaccination). The median neutralizing antibody titer after 2nd-dose was lower in LDLT recipients than in controls (0.46 vs 1.00, P<0.0001). All controls had SARS-CoV-2 neutralizing antibodies, whereas 39 LDLT recipients (25.3%) had no neutralizing antibodies after 2nd-dose; age at vaccination, presence of ascites, multiple immunosuppressive treatments, and mycophenolate mofetil treatment were significant risk factors for nonresponder. The neutralizing activities of recipient sera were approximately 3-fold and 5-fold lower than those of control sera against the Ancestral and Beta strains, respectively. The median antibody titer after 3rd-dose was not significantly different between recipients and controls (1.02 vs 1.22, p=0.0758); only 5% recipients was non-responder. The neutralizing activity after third dose to Omicron strains were enhanced and had no significant difference between two groups. Conclusion: Only the 2nd-dose was not sufficiently effective in recipients; however, 3rd-dose had sufficient neutralizing activity against the mutant strain and was as effective as that in healthy controls.
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COVID-19 , Trasplante de Hígado , Humanos , SARS-CoV-2/genética , Vacuna BNT162 , COVID-19/prevención & control , Donadores Vivos , Anticuerpos Neutralizantes , Anticuerpos Antivirales , VacunaciónRESUMEN
Abnormal expansions of GGGGCC repeat sequence in the noncoding region of the C9orf72 gene is the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). The expanded repeat sequence is translated into dipeptide repeat proteins (DPRs) by noncanonical repeat-associated non-AUG (RAN) translation. Since DPRs play central roles in the pathogenesis of C9-ALS/FTD, we here investigate the regulatory mechanisms of RAN translation, focusing on the effects of RNA-binding proteins (RBPs) targeting GGGGCC repeat RNAs. Using C9-ALS/FTD model flies, we demonstrated that the ALS/FTD-linked RBP FUS suppresses RAN translation and neurodegeneration in an RNA-binding activity-dependent manner. Moreover, we found that FUS directly binds to and modulates the G-quadruplex structure of GGGGCC repeat RNA as an RNA chaperone, resulting in the suppression of RAN translation in vitro. These results reveal a previously unrecognized regulatory mechanism of RAN translation by G-quadruplex-targeting RBPs, providing therapeutic insights for C9-ALS/FTD and other repeat expansion diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Humanos , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Demencia Frontotemporal/patología , ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ARN/genética , Drosophila/genéticaRESUMEN
The unremitting emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants necessitates ongoing control measures. Given its rapid spread, the new Omicron subvariant BA.5 requires urgent characterization. Here, we comprehensively analyzed BA.5 with the other Omicron variants BA.1, BA.2, and ancestral B.1.1. Although in vitro growth kinetics of BA.5 was comparable among the Omicron subvariants, BA.5 was much more fusogenic than BA.1 and BA.2. Airway-on-a-chip analysis showed that, among Omicron subvariants, BA.5 had enhanced ability to disrupt the respiratory epithelial and endothelial barriers. Furthermore, in our hamster model, in vivo pathogenicity of BA.5 was slightly higher than that of the other Omicron variants and less than that of ancestral B.1.1. Notably, BA.5 gains efficient virus spread compared with BA.1 and BA.2, leading to prompt immune responses. Our findings suggest that BA.5 has low pathogenicity compared with the ancestral strain but enhanced virus spread /inflammation compared with earlier Omicron subvariants.