An anticodon-sensing T-boxzyme generates the elongator nonproteinogenic aminoacyl-tRNA in situ of a custom-made translation system for incorporation.

In the hypothetical RNA world, ribozymes could have acted as modern aminoacyl-tRNA synthetases (ARSs) to charge tRNAs, thus giving rise to the peptide synthesis along with the evolution of a primitive translation apparatus. We previously reported a T-boxzyme, Tx2.1, which selectively charges initiator tRNA with N-biotinyl-phenylalanine (BioPhe) in situ in a Flexible In-vitro Translation (FIT) system to produce BioPhe-initiating peptides. Here, we performed in vitro selection of elongation-capable T-boxzymes (elT-boxzymes), using para-azido-l-phenylalanine (PheAZ) as an acyl-donor. We implemented a new strategy to enrich elT-boxzyme-tRNA conjugates that self-aminoacylated on the 3'-terminus selectively. One of them, elT32, can charge PheAZ onto tRNA in trans in response to its cognate anticodon. Further evolution of elT32 resulted in elT49, with enhanced aminoacylation activity. We have demonstrated the translation of a PheAZ-containing peptide in an elT-boxzyme-integrated FIT system, revealing that elT-boxzymes are able to generate the PheAZ-tRNA in response to the cognate anticodon in situ of a custom-made translation system. This study, together with Tx2.1, illustrates a scenario where a series of ribozymes could have overseen aminoacylation and co-evolved with a primitive RNA-based translation system.

Walking ability after resection of soft tissue sarcoma of the thigh and the sciatic nerve.

Walking ability may be fairly well maintained after sciatic nerve resection combined with wide resection of soft tissue sarcoma, therefore, surgeons should not hesitate to perform sciatic nerve resection to achieve an adequate surgical margin.

Post-Transcriptional Methylation of Mitochondrial-tRNA Differentially Contributes to Mitochondrial Pathology.

Human mitochondrial tRNAs (mt-tRNAs), critical for mitochondrial biogenesis, are frequently associated with pathogenic mutations. These mt-tRNAs have unusual sequence motifs and require post-transcriptional modifications to stabilize their fragile structures. However, whether a modification that stabilizes a wild-type (WT) mt-tRNA structure would also stabilize its pathogenic variants is unknown. Here we show that the N 1 -methylation of guanosine at position 9 (m 1 G9) of mt-Leu(UAA), while stabilizing the WT tRNA, has an opposite and destabilizing effect on variants associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes). This differential effect is further demonstrated by the observation that demethylation of m 1 G9, while damaging to the WT tRNA, is beneficial to the major pathogenic variant, improving its structure and activity. These results have new therapeutic implications, suggesting that the N 1 -methylation of mt-tRNAs at position 9 is a determinant of pathogenicity and that controlling the methylation level is an important modulator of mt-tRNA-associated diseases.

Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Trends in prevalence, treatment use, and disease burden in patients with eosinophilic granulomatosis with polyangiitis in Japan: real-world database analysis.

OBJECTIVES: Report the prevalence of eosinophilic granulomatosis with polyangiitis (EGPA) and describe oral corticosteroid (OCS) use and disease burden before and after mepolizumab approval in 2018 for EGPA in Japan. METHODS: Two retrospective studies (GSK IDs: 218083; 218084) used two databases: 1) the JMDC insurer database (Japanese health insurer claims) was used to report annual EGPA prevalence and OCS use in mepolizumab-treated patients; 2) Medical Data Vision database was used to report annual treatment use, OCS dose, relapses, and healthcare resource utilization (HCRU) in patients with EGPA. RESULTS: EGPA prevalence (95% confidence interval) increased from 4.2 (0.1, 23.4) in 2005 to 58.6 (53.2, 64.5) per 1,000,000 in 2020. Median OCS dose (mg/day) decreased from a range of 4.8-7.7 during 2010-2017 to 4.5-4.8 during 2018-2020 (lowest dose in 2020). The proportion of patients with prednisolone-equivalent daily OCS dose >10 mg decreased from 2017 (11.9%) to 2020 (10.3%), while the median dose halved. The proportion of patients with EGPA relapses (64.3% to 41.6%) and hospitalisation (27.8% to 23.6%) decreased from 2010 to 2020. CONCLUSIONS: EGPA prevalence increased between 2005 and 2020. With the introduction of mepolizumab for EGPA in 2018, real-world OCS use, relapses and HCRU decreased.

Pancreaticoduodenal Artery Aneurysm Rupture Presenting as Duodenal Obstruction Successfully Treated with Early Transcatheter Arterial Embolization: A Case Report of Suspected Segmental Arterial Mediolysis.

Visceral aneurysms are a rare but important form of abdominal vascular disease. Rupture of the aneurysms leads to serious symptoms, such as acute abdomen or abdominal bleeding. However, duodenal obstruction due to arterial rupture of an aneurysm is very rare. We herein report a 50-year-old woman with suspected segmental arterial mediolysis (SAM) who was first diagnosed with acute abdomen and duodenal obstruction. Rupture of a pancreaticoduodenal artery aneurysm was confirmed, and she was treated with transcatheter arterial embolization. In cases of acute abdomen, SAM is a rare but important possibility to consider as a differential diagnosis.

Restoration of mitochondrial function through activation of hypomodified tRNAs with pathogenic mutations associated with mitochondrial diseases.

Mutations in mitochondrial (mt-)tRNAs frequently cause mitochondrial dysfunction. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), and myoclonus epilepsy associated with ragged red fibers (MERRF) are major clinical subgroups of mitochondrial diseases caused by pathogenic point mutations in tRNA genes encoded in mtDNA. We previously reported a severe reduction in the frequency of 5-taurinomethyluridine (τm5U) and its 2-thiouridine derivative (τm5s2U) in the anticodons of mutant mt-tRNAs isolated from the cells of patients with MELAS and MERRF, respectively. The hypomodified tRNAs fail to decode cognate codons efficiently, resulting in defective translation of respiratory chain proteins in mitochondria. To restore the mitochondrial activity of MELAS patient cells, we overexpressed MTO1, a τm5U-modifying enzyme, in patient-derived myoblasts. We used a newly developed primer extension method and showed that MTO1 overexpression almost completely restored the τm5U modification of the MELAS mutant mt-tRNALeu(UUR). An increase in mitochondrial protein synthesis and oxygen consumption rate suggested that the mitochondrial function of MELAS patient cells can be activated by restoring the τm5U of the mutant tRNA. In addition, we confirmed that MTO1 expression restored the τm5s2U of the mutant mt-tRNALys in MERRF patient cells. These findings pave the way for epitranscriptomic therapies for mitochondrial diseases.

Microbial Burden Estimation of Food Items, Built Environments, and the International Space Station Using Film Media.

The use of film media involves considerably less preparation, waste, and incubator space than conventional agar-media-based assays and has proven in past studies to provide counts of cultivable microbes similar to those of traditional agar media. Film media also have the advantage of allowing sample volumes similar to those used in pour plates and, therefore, are well-suited for cultivable microbial counts in extremely low-biomass environments such as clean rooms or space habitats, particularly where the subsequent isolation of colonies is necessary. As the preparation of film media plates relies on water cohesion/adhesion rather than manual spreading, they may have future applications in low- or microgravity settings. In this study, cultivable microbial count performance was compared between agar media and film media in three kinds of samples: food items, surfaces in built environments on Earth (homes), and on the environmental surfaces of the International Space Station (ISS). Easy Plates (Kikkoman Corporation) and Petrifilm (3M) were compared with traditional agar plating for food and home surfaces, while only Easy Plates were compared with agar for ISS samples. For both food items and built environments on Earth, both types of film media performed comparably to agar media for bacterial counts, with R2 values of 0.94-0.96. Fungal counts for built-environment samples had a lower correlation between film and agar counts, with R2 values of 0.72-0.73. Samples from the ISS, which ranged from below detection to 103 CFU per 100 cm2, had R2 values of 0.80 for bacterial counts and 0.73 for fungal counts, partially due to multiple samples recording below the detection limit for agar or too numerous to count, and the growth of fungal species on R2A medium. The species compositions of isolates picked from agar vs. film media plates were similar; however, further phylogenetic analysis is needed to confirm the differential microbial diversity composition. Overall, film media such as Easy Plates and Petrifilm are viable alternatives to agar plates for low-biomass built environments as well as for food samples, and the two brands tested in this study performed equally well.

Reduction in asthma exacerbation rate after mepolizumab treatment initiation in patients with severe asthma: A real-world database study in Japan.

OBJECTIVE: To investigate the changes in asthma exacerbation, as well as in oral corticosteroid (OCS) use, exacerbation-related healthcare resource utilization (HRU), and healthcare costs before and after mepolizumab treatment initiation in patients with severe asthma who started treatment with mepolizumab in a real-world clinical setting in Japan. METHODS: A retrospective, observational, self-controlled study was conducted in Japan using a hospital-based administrative claims database. Patients who were diagnosed with asthma and who were new users of mepolizumab were included in the study. The primary outcome was the incidence rate of any asthma exacerbation/patient-year during the 12-month period before (baseline period) and after (follow-up period) the first mepolizumab prescription. Secondary outcome measures included the proportion of patients with ≥1 any asthma exacerbation, patients with exacerbation requiring hospitalization, the incidence rate of exacerbations requiring hospitalization/patient-year, the median daily OCS dose (OCS sparing effect), exacerbation-related HRU (hospitalization length, the proportion of patients with emergency visits, and the number of emergency/outpatient visits), and associated costs. RESULTS: Of the 377 patients included, 56.2% were ≥65 years of age. Following the first mepolizumab prescription, incidence rates for any asthma exacerbation were reduced by 40.6% (4.00/patient-year to 2.38/patient-year; the incidence rate ratio [95% confidence interval]: 0.60 [0.53-0.67]; p < 0.0001) from the baseline to follow-up periods. The incidence rate of exacerbations requiring hospitalization was reduced by 55.8% (0.37/patient-year to 0.16/patient-year) from the baseline to follow-up periods. The proportion of patients experiencing any exacerbation decreased from 84.4% to 57.8% and those requiring hospitalization decreased from 23.9% to 10.3% both from the baseline to follow-up periods. The median daily OCS dose decreased by 44.6% (median [interquartile range]: 6.7 [4.7-9.9] mg/day to 3.3 [0.9-5.6] mg/day) from the last baseline quarter to the 4th quarter of the follow-up period. All exacerbation-related HRUs decreased from the baseline to follow-up periods. Inpatient cost reduced by >50% (123,279 Japanese Yen [JPY]/patient-year vs. 57,283 JPY/patient-year), reducing the total cost by 80,716 JPY from the baseline to follow-up periods. CONCLUSION: Mepolizumab was effective in treating patients with severe asthma by reducing the incidence rates of exacerbations and exacerbation requiring hospitalization, OCS dose, exacerbation-related HRU, and cost in routine clinical practice in Japan.

Mass spectrometric analysis of mRNA 5' terminal modifications.

The 7-methylguanosine (m7G) cap structure, an essential epitranscriptomic mark at the 5' terminus of eukaryotic mRNAs, plays critical roles in mRNA stability, export, and translation. Following the cap structure, the first and second nucleotides at the 5' ends of mRNAs are frequently methylated to give more diverse modifications, especially in vertebrates. To understand the biological roles of the cap structures, precise analyses of the 5' terminal modifications are necessary. Here, we describe a detailed protocol for mass spectrometric analysis of 5' terminal fragments of mRNAs.

N6-methyladenosine (m6A) is an endogenous A3 adenosine receptor ligand.

About 150 post-transcriptional RNA modifications have been identified in all kingdoms of life. During RNA catabolism, most modified nucleosides are resistant to degradation and are released into the extracellular space. In this study, we explored the physiological role of these extracellular modified nucleosides and found that N6-methyladenosine (m6A), widely recognized as an epigenetic mark in RNA, acts as a ligand for the human adenosine A3 receptor, for which it has greater affinity than unmodified adenosine. We used structural modeling to define the amino acids required for specific binding of m6A to the human A3 receptor. We also demonstrated that m6A was dynamically released in response to cytotoxic stimuli and facilitated type I allergy in vivo. Our findings implicate m6A as a signaling molecule capable of activating G protein-coupled receptors (GPCRs) and triggering pathophysiological responses, a previously unreported property of RNA modifications.

Mass Spectrometric Analysis of Mitochondrial RNA Modifications.

Mitochondrial RNAs are modified posttranscriptionally. These modifications are required for proper functioning of RNA molecules, and thereby contribute to essential mitochondrial processes. Herein, we describe our latest mass spectrometry-based platform for analysis of posttranscriptional modifications of mitochondrial tRNAs, and measuring the in vitro activity of mitochondrial RNA-modifying enzymes.

Associated Factors and Comorbidities of Airflow Limitation in Subjects Undergoing Comprehensive Health Examination in Japan - Survey of Chronic Obstructive Pulmonary Disease Patients Epidemiology in Japan (SCOPE- J).

Purpose: To identify associated factors of having at least one of the airflow limitation, chronic cough/phlegm, and currently treated respiratory diseases in health examinees, and to describe the characteristics of each subgroup classified by comorbidities. Subjects and Methods: This was an observational cross-sectional survey carried out in multiple regions of Japan. Subjects aged 40 years older, undergoing comprehensive health examination, were recruited. Airflow limitation was defined as having forced expiratory volume in 1 s/forced vital capacity lower than 70%. Associated factors of having at least one of the airflow limitation, chronic cough/phlegm, and currently treated respiratory diseases were examined by logistic regression analysis. Subgroup classification by comorbidity patterns was conducted by hierarchical cluster analysis. Results: In a total of 22,293 subjects, 1520 (6.8%) had at least one of the airflow limitation, chronic cough/phlegm, and currently treated respiratory diseases. With this objective variable, the following explanatory variables were significantly associated: older age, higher total score in the chronic obstructive pulmonary disease assessment test (CAT) and coexistence of lung cancer (common in ever-smokers and never-smokers), higher pack-years, lower body mass index, higher C-reactive protein, without coexistence of diabetes mellitus (specific in ever-smokers), male sex, coexistence of anxiety, and sleep disorder (specific in never-smokers). Among the 1520 subjects, 1512 subjects with smoking history data were classified by comorbidity patterns into subgroups of "no comorbidities," "mixed comorbidities," "inflammatory comorbidities," "overweight," "underweight," and "chronic kidney disease." "Inflammatory comorbidities" were specific in ever-smokers, and "underweight" was specific in never-smokers. Conclusion: Several factors were identified as associated factors of having at least one of airflow limitation, chronic cough/phlegm, and currently treated respiratory diseases and they were different between ever-smokers and never-smokers. Different comorbidity patterns were observed by smoking history. These findings could provide information to assist the management of subjects with COPD or at risk for COPD in the general population.

Complete chemical structures of human mitochondrial tRNAs.

Mitochondria generate most cellular energy via oxidative phosphorylation. Twenty-two species of mitochondrial (mt-)tRNAs encoded in mtDNA translate essential subunits of the respiratory chain complexes. mt-tRNAs contain post-transcriptional modifications introduced by nuclear-encoded tRNA-modifying enzymes. They are required for deciphering genetic code accurately, as well as stabilizing tRNA. Loss of tRNA modifications frequently results in severe pathological consequences. Here, we perform a comprehensive analysis of post-transcriptional modifications of all human mt-tRNAs, including 14 previously-uncharacterized species. In total, we find 18 kinds of RNA modifications at 137 positions (8.7% in 1575 nucleobases) in 22 species of human mt-tRNAs. An up-to-date list of 34 genes responsible for mt-tRNA modifications are provided. We identify two genes required for queuosine (Q) formation in mt-tRNAs. Our results provide insight into the molecular mechanisms underlying the decoding system and could help to elucidate the molecular pathogenesis of human mitochondrial diseases caused by aberrant tRNA modifications.

Clinical characteristics, treatment patterns, disease burden, and persistence/adherence in patients with asthma initiating inhaled triple therapy: real-world evidence from Japan.

Objectives: To help optimize triple therapy use, treatment patterns and disease burden were investigated in patients in Japan with persistent asthma who initiated multi-inhaler triple therapy (inhaled corticosteroid/long-acting ß2-agonist/long-acting muscarinic antagonist; ICS/LABA/LAMA).Methods: This retrospective, observational cohort study using health insurance claims data included adults with persistent asthma who initiated triple therapy in 2016. Patients who were prescribed ICS/LABA in 2016 were included as an ICS/LABA-matched cohort. Patients were stratified into those with asthma only and those with asthma and chronic obstructive pulmonary disease (COPD) codes (asthma-COPD overlap [ACO]). Patient data from 1-year prior to 1 year post index date were analyzed.Results: For patients with asthma only in the triple therapy and ICS/LABA cohorts, baseline demographics were similar. A higher proportion of the triple-therapy cohort than the ICS/LABA cohort was receiving high-dose ICS at index (68.2% and 27.6%, respectively), and had experienced an exacerbation in the last year (64.0% and 29.4%, respectively). The proportion of patients with asthma only who developed any exacerbation was lower in the year following initiation of triple therapy compared with the year prior to initiation of triple therapy (45.8% vs 64.0%, respectively). For asthma only patients receiving triple therapy, the mean (standard deviation) proportion of days covered and medication possession ratio was 0.51 (0.36) and 0.86 (0.16), respectively. Similar trends were seen in patients with ACO in the triple-therapy and ICS/LABA cohorts.Conclusion: Evidence from this study may serve as a reference for the use of inhaled triple therapy for asthma.

Chronic Cough and Phlegm in Subjects Undergoing Comprehensive Health Examination in Japan - Survey of Chronic Obstructive Pulmonary Disease Patients Epidemiology in Japan (SCOPE-J).

Purpose: The purpose of this study was to estimate the prevalence of subjects with chronic cough and phlegm and describe their characteristics including the presence or absence of airflow limitation among the general population in Japan. Subjects and Methods: This was an observational cross-sectional survey targeting multiple regions of Japan. Subjects aged 40 years or above who were undergoing comprehensive health examination were recruited. The existence of chronic cough and phlegm, airflow limitation, and treatment for respiratory diseases were examined. Chronic cough and phlegm were defined as having both symptoms for at least 3 months of the year and for at least 2 consecutive years, or as receiving any treatment for chronic bronchitis at the time of recruitment. Airflow limitation was defined as forced expiratory volume in 1 s/forced vital capacity (FEV1/FVC) less than 0.7. Results: In a total of 22,293 subjects, 380 subjects (1.7%) had chronic cough and phlegm. Among these 380 subjects, 21.8% received treatment for a respiratory disease, and 11.6% had airflow limitation. Compared to subjects without both chronic cough and phlegm but with airflow limitation, subjects with chronic cough and phlegm without airflow limitation were younger, more likely to be current smokers (39.6%), and had higher total scores on a chronic obstructive pulmonary disease (COPD) assessment test (CAT). Scores of CAT questions 1-4 (cough, phlegm, chest tightness, breathlessness, respectively) were higher in subjects with chronic cough and phlegm regardless of airflow limitation. Conclusion: This study demonstrated that subjects identified to have chronic cough and phlegm in comprehensive health examination settings were symptomatic, while most of them did not receive any treatment for respiratory diseases and did not have airflow limitation. Screening subjects for chronic cough and phlegm in a comprehensive health examination followed by a detailed examination of screened subjects could be an effective approach for better management of chronic cough and phlegm. Smoking cessation should be included in the management, in consideration that around 40% of subjects with chronic cough and phlegm were current smokers.