Publications by authors named "Takuya Umehara"

49 Publications

Amino acid activation analysis of primitive aminoacyl-tRNA synthetases encoded by both strands of a single gene using the malachite green assay.

Biosystems 2021 Jul 8;208:104481. Epub 2021 Jul 8.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

The Rodin-Ohno hypothesis postulates that two classes of aminoacyl-tRNA synthetases were encoded complementary to double-stranded DNA. Particularly, Geobacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS, belonging to class I) and Escherichia coli histidyl-tRNA synthetase (HisRS, belonging to class II) show high complementarity of the middle base of the codons in the mRNA sequence encoding each ATP binding site. Here, for the reported 46-residue peptides designed from the three-dimensional structures of TrpRS and HisRS, amino acid activation analysis was performed using the malachite green assay, which detects the pyrophosphate departing from ATP in the forward reaction of the first step of tRNA aminoacylation. A maltose-binding protein fusion with the 46 residues of TrpRS (TrpRS46mer) exhibited high activation capacity for several amino acids in the presence of ATP and amino acids, but the activity of an alanine substitution mutant of the first histidine in the HIGH motif (TrpRS46merH15A) was largely reduced. In contrast, pyrophosphate release by HisRS46mer in the histidine activation step was lower than that in the case of TrpRS46mer. Both HisRS46mer and the alanine mutant at the 113th arginine (HisRS46merR113A) showed slightly higher levels of pyrophosphate release than the maltose-binding protein alone. These results do not rule out the Rodin-Ohno hypothesis, but may suggest the necessity of establishing unique evolutionary models from different perspectives.
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http://dx.doi.org/10.1016/j.biosystems.2021.104481DOI Listing
July 2021

Physical Functions and Comorbidity Affecting Collapse at 4 or More Weeks after Admission in Patients with Osteoporotic Vertebral Fractures: A Prospective Cohort Study.

Asian Spine J 2021 May 6. Epub 2021 May 6.

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Study Design: A prospective cohort study.

Purpose: This study aimed to reveal physical functions and comorbidity affecting collapse at ≥4 weeks after hospital admission of patients with osteoporotic vertebral fracture.

Overview Of Literature: Only a few studies have investigated the influence of physical function and activity on collapse in patients with osteoporotic vertebral fractures.

Methods: This prospective cohort study analyzed patients with osteoporotic vertebral fractures admitted to the hospital between March 2018 and October 2019. Logistic regression analysis was performed to explore the predictors of vertebral collapse at >4 weeks after admission. Model 1 used basic medical information and physical functions at admission; model 2 used basic medical information and physical function and activity at >4 weeks after admission.

Results: In the model 1 results of logistic regression analysis, cardiovascular disease (odds ratio [OR], 12.27; 95% confidence interval [CI], 1.28-117.91) was extracted as a factor affecting vertebral collapse at ≥4 weeks after admission. In the model 2 results of logistic regression analysis, cardiovascular disease (OR, 34.57; 95% CI, 2.53-471.74), movement control during one leg standing at 4 weeks (OR, 7.25; 95% CI, 1.36-38.71), and Pain Catastrophizing Scale score at 4 weeks (OR, 1.11; 95% CI, 1.01-1.21) were extracted as factors affecting vertebral collapse at ≥4 weeks after admission.

Conclusions: Our results indicate that physical functions and comorbidity affect collapse at ≥4 weeks after admission in patients with osteoporotic vertebral fractures.
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http://dx.doi.org/10.31616/asj.2020.0285DOI Listing
May 2021

Factors Affecting Quality of Life at Discharge among Patients with Osteoporotic Vertebral Fractures.

South Med J 2021 Apr;114(4):252-259

From the Department of Rehabilitation, Faculty of Rehabilitation, Hiroshima International University, Hiroshima, the Department of Rehabilitation, Saiseikai Kure Hospital, Hiroshima, and the Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Objectives: This study aimed to identify factors, including physical functions and activities that affect quality of life (QOL) at discharge among patients with osteoporotic vertebral fractures.

Methods: Patients with osteoporotic vertebral fractures were included in our prospective cohort study. Multiple regression analysis was performed to determine the predictors of QOL at discharge using two models: model 1, basic medical information and physical functions at admission, and model 2, basic medical information, physical function, and activity after 4 weeks of admission.

Results: Multiple regression analysis (standard partial regression coefficients) using model 1 identified L2 to L4 bone mineral density (-0.2), Visual Analog Scale for pain during activity at admission (-0.31), and Revised Hasegawa Dementia Scale (HDS-R) score at admission (0.64) as factors affecting QOL at discharge. Multiple regression analysis using model 2 identified HDS-R at admission (0.64), Pain Catastrophizing Scale score at 4 weeks (-0.34), and knee extension muscle strength at 4 weeks (0.28) as factors affecting QOL at discharge.

Conclusions: Our results suggest that if patients have high bone mineral density, intense pain, and low cognitive function at admission, then low QOL at discharge will be predicted; however, improvement of pain catastrophizing and knee extension muscle strength during first the 4 weeks of admission may be able to improve QOL at discharge. Because patients in this study were Japanese only, it is important to exercise caution when applying our results to other populations.
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http://dx.doi.org/10.14423/SMJ.0000000000001235DOI Listing
April 2021

Association between movement control during one-leg standing and femoral BMD in patients with hip fractures.

J Bone Miner Metab 2021 May 2;39(3):474-483. Epub 2021 Jan 2.

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Hiroshima Minami-ku, Hiroshima, Japan.

Introduction: Prior studies have focused only on the temporal component of one-leg standing, no reports have examined the relationship between the qualitative components of one-leg standing and femoral BMD. Thus, this study investigated whether quality (i.e., movement control) of one-leg standing also associated femoral BMD.

Materials And Methods: A total of 80 patients with unilateral hip fracture were included in a cross-sectional study. Basic and medical information and physical functions including movement control during one-leg standing were assessed at admission and 2 weeks after surgery, respectively. Hierarchical multiple regression analysis was performed to identify predictors of femoral BMDs on the non-fractured side. Dependent variables included femoral neck and total hip BMDs in models 1 and 2, respectively.

Results: Hierarchical multiple regression analysis (standardized partial regression coefficients) in model 1 identified age (- 0.18), sex (0.38), body mass index (BMI) (0.41), movement control during one-leg standing on the non-fractured side (0.19), and life-space assessment (0.17) as factors associating femoral neck BMD. Meanwhile, hierarchical multiple regression analysis (standardized partial regression coefficients) in model 2 identified age (- 0.12), sex (0.36), BMI (0.37), and movement control during one-leg standing on the non-fractured side (0.25) as factors associating total hip BMD. The coefficients of determination adjusted for degrees of freedom (R) were 0.529 and 0.470 for models 1 and 2, respectively.

Conclusion: Our results suggest that improving movement control during one-leg standing may be important for maintaining and improving femoral BMD on the non-fractured side.
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http://dx.doi.org/10.1007/s00774-020-01185-zDOI Listing
May 2021

Correction to: G:U‑Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl‑tRNA Synthetase: An Insight into the Evolution of Aminoacyl‑tRNA Synthetases.

J Mol Evol 2020 Dec;88(10):759-760

Department of Biological Science and Technology, Tokyo University of Science, 6‑3‑1 Niijuku, Katsushika‑ku, Tokyo, 125‑8585, Japan.

In the original version of this article, "A73" in Fig 6b was inadvertently labeled as "G73". The corrected Fig. 6 is given here.
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http://dx.doi.org/10.1007/s00239-020-09975-9DOI Listing
December 2020

Interactive effect of cognitive function and intervention on the walking independence of stroke patients: a retrospective cohort study.

J Exerc Rehabil 2020 Jun 30;16(3):242-248. Epub 2020 Jun 30.

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

The purpose of this study was to investigate the interactive effect of cognitive function and intervention on walking independence of stroke patients. Stroke patients (n=405) who admitted to convalescent rehabilitation ward, were classified as being walking independent or dependent. To examine the interaction between cognitive function and intervention, high cognitive function (functional independence measure score ≥20) and physical therapy and occupational therapy intervention delivered in 1 day (lasting >2 hr) were defined as cognition-intervention interaction and included as independent variables. The incidence of walking independence was calculated using Kaplan-Meier curves. Intergroup differences were estimated using log-rank test. Cox proportional hazards analysis was used to extract the predictors of walking independence. Survival analyses using Kaplan-Meier log-rank test showed that the probability of incidence of walking independence was significantly higher in the presence of a cognition-intervention interaction. The results of Cox proportional hazards analysis showed that age, left versus right cerebral damage, and cognition-intervention interaction significantly influenced walking independence at discharge from the hospital. The hazard ratios were 0.971 per year of age, 0.544 for left versus right cerebral damage, and 1.794 for cognition-intervention interaction. Walking independence was more likely to be achieved by stroke patients with high cognitive function who received therapy. In other words, the conditions that increase the likelihood of an effect of therapy intervention on walking independence were identified in this study.
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http://dx.doi.org/10.12965/jer.2040306.153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7365727PMC
June 2020

Efficient predictors for the decline of activities of daily living in patients with hip fracture one year after surgery: A multicenter prospective cohort study.

J Back Musculoskelet Rehabil 2020 ;33(4):553-560

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Background: Previous studies have examined when activities of daily living (ADL) recovery more than six months after surgery can be predicted, and how much accuracy the predictors have.

Objective: The purpose of this study was to determine the predictors of ADL decline and evaluate their accuracies one year post-operation for hip-fracture patients.

Methods: We studied patients who underwent hip fracture surgery and were able to walk independently pre-operatively. The predictors of ADL declined one year post-operation, as analyzed using data of the basic medical attributes of the patients, including pain, 30-s chair-stand test, dementia [using the Revised Hasegawa Dementia Scale (HDS-R)], and walking/mobility style [using Barthel Index (BI)]. Using a receiver operating curve (ROC) curve, the cut-off value for each significant predictor was determined in the logistic regression analysis. To calculate the cut-off values and diagnostic performances of each of the extraction factors.

Results: The data of 36 patients were collected over a period of one year. The prior probability of ADL decline at one year post-operation was 44.4%. The results of logistic regression analyses showed that the score of HDS-R at admission and the walking/mobility BI score at three weeks post-operation were significant predictors of the one year post-operative decline in ADL. The results of the ROC analyses showed that the cut-off value of the HDS-R score at admission was < 23 points. The posterior probability increased to 62.0%. In contrast, the cut-off value of the walking/mobility BI score was 0 points. The posterior probability increased to 91.0%.

Conclusion: The ADL decline of the patients who underwent hip fracture surgery at one year after surgery can be predicted at three weeks post-operation.
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http://dx.doi.org/10.3233/BMR-181126DOI Listing
December 2020

G:U-Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl-tRNA Synthetase: An Insight into the Evolution of Aminoacyl-tRNA Synthetases.

J Mol Evol 2020 08 7;88(6):501-509. Epub 2020 May 7.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan.

Nanoarchaeum equitans is a species of hyperthermophilic archaea with the smallest genome size. Its alanyl-tRNA synthetase genes are split into AlaRS-α and AlaRS-β, encoding the respective subunits. In the current report, we surveyed N. equitans AlaRS-dependent alanylation of RNA minihelices, composed only of the acceptor stem and the T-arm of tRNA. Combination of AlaRS-α and AlaRS-β showed a strong alanylation activity specific to a single G3:U70 base pair, known to mark a specific tRNA for charging with alanine. However, AlaRS-α alone had a weak but appreciable alanylation activity that was independent of the G3:U70 base pair. The shorter 16-mer RNA tetraloop substrate mimicking only the first four base pairs of the acceptor stem of tRNA, but with C3:G70 base pair, was also successfully aminoacylated by AlaRS-α. The end of the acceptor stem, including CCA-3' terminus and the discriminator A73, was able to function as a minimal structure for the recognition by the enzyme. Our findings imply that aminoacylation by N. equitans AlaRS-α may represent a vestige of a primitive aminoacylation system, before the appearance of the G3:U70 pair as an identity element for alanine.
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http://dx.doi.org/10.1007/s00239-020-09945-1DOI Listing
August 2020

RNA Aptamers for a tRNA-Binding Protein from with Homologous Counterparts Distributed Throughout Evolution.

Life (Basel) 2020 Feb 1;10(2). Epub 2020 Feb 1.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.

In the present selection study, we isolated and characterized RNA aptamers for a tRNA-binding protein (Trbp) from an extremophile archaeon . Trbp-like structures are frequently found not only in aminoacyl-tRNA synthetases but also in diverse types of proteins from different organisms. They likely arose early in evolution and have played important roles in evolution through interactions with key RNA structures. RNA aptamers specific for Trbp were successfully selected from a pool of RNAs composed of 60 nucleotides, including a random 30-nucleotide region. From the secondary structures, we obtained a shortened sequence composed of 21 nucleotides, of which the 3'-terminal single stranded CA nucleotides are essential for binding. This may be related to the initial evolutionary role of the universal CCA-3' terminus of tRNA in the interaction with Trbp-like structures.
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http://dx.doi.org/10.3390/life10020011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7175363PMC
February 2020

Factors affecting hospital readmission heart failure patients in Japan: a multicenter retrospective cohort study.

Heart Vessels 2020 Mar 13;35(3):367-375. Epub 2019 Sep 13.

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

The purpose of this study was to examine factors influencing readmission to hospital in patients with heart failure. This multicenter, retrospective cohort study analyzed 197 heart failure patients admitted to the research cooperation facilities between January 2017 and November 2017. We defined the readmission group as being readmitted to hospital in ≤ 6 months of discharge and the non-readmission group as > 6 months after discharge. Cox proportional hazards analysis was performed to explore the predictors of readmission. The incidence of readmission was calculated using Kaplan-Meier curves for the extracted factors. Intergroup differences were estimated using the log-rank test. The results of Cox proportional hazards analysis indicated that chronic renal dysfunction (hazard ratio (HR) = 4.729), dementia (HR = 7.105), HFrEF (HR = 8.138), walking without support (HR = 4.031), and walking with a cane (HR = 11.857) significantly contributed to the model. In the survival analyses using the Kaplan-Meier log-rank test, chronic renal dysfunction, dementia, and HFrEF were significant (P < 0.05), but walking without support and walking with a cane after discharge were not significant (P > 0.05). This study suggests that early involvement after discharge is important for lowering the readmission rates in patients with heart failure, even when their walking ability is good.
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http://dx.doi.org/10.1007/s00380-019-01500-3DOI Listing
March 2020

Effect of an intensive functional rehabilitation program on the recovery of activities of daily living after total knee arthroplasty: A multicenter, randomized, controlled trial.

J Orthop Sci 2020 Mar 7;25(2):285-290. Epub 2019 Jun 7.

Department of Rehabilitation, Saiseikai Kure Hospital, Hiroshima, Japan.

Background: Postoperative exercise is effective for improving activities of daily living (ADL) in patients undergoing total knee arthroplasty (TKA), and exercise has been included in standard care after surgery provided in Japan. However, it is unknown whether standard care has room to develop for improving ADL. The goal of this study was to investigate the effect of an intensive functional rehabilitation (IFR) program in addition to standard care on the recovery of ADL following surgery.

Methods: A total of 104 inpatients with knee osteoarthritis undergoing TKA were randomly assigned to either the experimental or the control group. The experimental group (n = 57) were given standard care and participated in the IFR program immediately after surgery (e.g., stand up and sit down, strengthen the knee extensor muscle, climbing onto a platform, and walking laterally). The control group (n = 47) were given standard care only. Primary outcomes were the score of Functional Independence Measure (FIM) and the Barthel Index (BI) at 2 weeks postoperatively. Matching using the propensity score was performed to control the influence of the patient characteristic on the outcome.

Results: As a result of matching, 42 patients were extracted. The averages (standard deviation) of the FIM motor score were 79.0 (10.8) for the experimental group and 80.5 (9.4) for the control group. The BI scores were 88.1 (13.3) for the experimental group and 91.0 (10.1) for the control group. The experimental and control groups did not show significant intergroup differences in either score.

Conclusions: Our study shows that as compared to standard care, IFR program for 2 weeks in addition to standard care after surgery did not significantly improve ADL in patients undergoing TKA.
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http://dx.doi.org/10.1016/j.jos.2019.04.009DOI Listing
March 2020

Glycyl-tRNA synthetase from Nanoarchaeum equitans: The first crystal structure of archaeal GlyRS and analysis of its tRNA glycylation.

Biochem Biophys Res Commun 2019 04 13;511(2):228-233. Epub 2019 Feb 13.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

This study reports the X-ray crystallographic structure of the glycyl-tRNA synthetase (GlyRS) of Nanoarchaeum equitans - a hyperthermophilic archaeal species. This is the first archaeal GlyRS crystal structure elucidated. The GlyRS comprises an N-terminal catalytic domain and a C-terminal anticodon-binding domain with a long β-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNA was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNA with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNA elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNA G71 substitution mutants and a GlyRS mutant where Arg223 is mutated to alanine strengthen the possibility that the carbonyl oxygen at position 6 of G71 would hydrogen-bond with the guanidine nitrogen of Arg223 in N. equitans GlyRS.
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http://dx.doi.org/10.1016/j.bbrc.2019.01.142DOI Listing
April 2019

Effects of complementary loop composition in truncated R3C ligase ribozymes on kiss switch activation.

Biosystems 2019 Mar 11;177:9-15. Epub 2019 Jan 11.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

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http://dx.doi.org/10.1016/j.biosystems.2019.01.004DOI Listing
March 2019

Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in .

Genes (Basel) 2018 Sep 28;9(10). Epub 2018 Sep 28.

Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125-8585, Japan.

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which -acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in . The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.
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http://dx.doi.org/10.3390/genes9100473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6209979PMC
September 2018

Can the Amount of Interventions during the Convalescent Phase Predict the Achievement of Independence in Activities of Daily Living in Patients with Stroke? A Retrospective Cohort Study.

J Stroke Cerebrovasc Dis 2018 Sep 29;27(9):2436-2444. Epub 2018 May 29.

Department of Health Informatics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.

Background: This study aimed to evaluate the diagnostic performance of the amount of physical, occupational, and speech therapy intervention and optimal timing necessary for activities of daily living (ADL) independence in patients with stroke.

Method: Patients (N = 441) with stroke admitted to the convalescent rehabilitation ward were classified into an early intervention or a nonearly intervention group on the basis of the duration from the date of onset to date of hospital admission. Logistic regression model was used to identify factors influencing independence in ADL in both groups. Cutoff point, likelihood ratio, and posterior probabilities for ADL independence were calculated, and diagnostic accuracy was evaluated for extracted factors.

Results: Results of logistic regression analysis revealed that age and physical and occupational therapy intervention amount provided during convalescent phase and Functional Independent Measure (FIM) motor score at admission significantly influenced independence in ADL at discharge from the hospital in the early intervention group (hospitalization date was 30 days or less). The cutoff point was 168 hours; positive likelihood ratio was 1.74; negative likelihood ratio was .78; and the posterior probability for the time spent by the therapist was 81.0%. FIM motor score at admission was the only factor extracted for the nonearly intervention group (hospitalization date was 31 days or more).

Conclusion: The ADL independence in patients with stroke admitted to convalescent rehabilitation ward during their convalescent phase cannot be determined simply on the basis of the amount of physical and occupational therapy they receive.
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http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2018.04.038DOI Listing
September 2018

Effect of Continuous Compression Stimulation on Pressure-Pain Threshold and Muscle Spasms in Older Adults With Knee Osteoarthritis: A Randomized Trial.

J Manipulative Physiol Ther 2018 05;41(4):315-322

Department of Orthopedic Surgery, Saiseikai Kure Hospital, Hiroshima, Japan.

Objective: The purpose of this study was to assess the effect of continuous compression stimulation on pressure-pain threshold and muscle spasms in older adults with knee osteoarthritis.

Methods: Thirty-two older adults with knee osteoarthritis on outpatient visits were randomly divided into 2 groups. Those in the treatment group (n = 16) received 5-minute massage therapy (continuous compression stimulation), and those in the control group (n = 16) received sham massage therapy (touch without compression). Immediately before and after single-intervention sessions, the pressure-pain threshold, muscle spasm, and pain were quantified.

Results: The change in pain on walking in the treatment group exceeded 1.9 cm, corresponding to the minimum clinically important difference. In the treatment group, the pressure-pain threshold improved significantly for pain both at rest and while walking, but the improvement in muscle spasm was not significant.

Conclusions: Massage therapy resulted in minimal clinically important changes for pain relief. There was an increase in the pressure-pain threshold in the older adults with knee osteoarthritis. We propose that the improvements in pain may be related to the medial thigh muscle rather than knee osteoarthritis.
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http://dx.doi.org/10.1016/j.jmpt.2017.09.011DOI Listing
May 2018

The Kiss Switch Brings Inactive R3C Ligase Ribozyme Back to Life.

Biology (Basel) 2018 Jan 9;7(1). Epub 2018 Jan 9.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.

R3C ligase ribozyme catalyzes the nucleophilic attack by a 3'-hydroxyl on a 5'-α-phosphorus of triphosphates to form a 3'-5'-phosphodiester bond. In the present study, although the truncation of R3C ribozyme was accompanied by a large reduction in ligation activity (decrease by two orders of magnitude compared to that of the ligated product of full-length R3C ribozyme after 18.5 h at 23 °C), the introduction of complementary seven-membered kissing-loops served as a "switch" to reactivate the truncated R3C ribozyme with approximately one-fifth of the activity of the full-length R3C ribozyme. This reactivation occurred in a trans-manner, and the grip region and substrate-binding site of the truncated R3C ribozyme were necessary to locate the substrate in the proper position for ligation with the other molecule. Reactivation resulted from complex tertiary interactions between two ribozymes, including kissing-loop interaction-induced annealing and the formation of a stable duplex. The drastic increase of the activity of poorly active ribozymes through the kissing-loop interaction may provide an important clue into the acquisition of substantial activity during the evolution of the RNA world.
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http://dx.doi.org/10.3390/biology7010007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5872033PMC
January 2018

Effective exercise intervention period for improving body function or activity in patients with knee osteoarthritis undergoing total knee arthroplasty: a systematic review and meta-analysis.

Braz J Phys Ther 2018 Jul - Aug;22(4):265-275. Epub 2017 Nov 8.

Department of Rehabilitation, Hiroshima International University, Higashihiroshima, Japan.

Background: Various systematic reviews and/or meta-analyses examining the effects of pre- or postoperative exercise on body function or activity in patients undergoing total knee arthroplasty (TKA) have been published. However, the interventional period needed to at least improve outcomes is unknown.

Objective: The aim of this systematic review and meta-analysis was to investigate the exercise intervention period needed to effectively improve body function or activity before and after TKA in patients with knee osteoarthritis (OA).

Methods: Studies published until July 2017 were included in the review. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was applied to each meta-analysis to determine the quality of the evidence.

Results: Twenty-seven randomized controlled trials were identified. A meta-analysis indicated that exercises performed for 8 weeks after discharge in addition to standard postoperative intervention effectively improved body function as assessed using pain level; physical function, and stiffness on the Western Ontario and McMaster Universities Arthritis Index; extension strength; active knee flexion range of motion; timed up and go test; and gait speed.

Conclusion: Overall, we found low- to moderate-quality evidence that an 8-week exercise period was needed after discharge to improve body function and activity in patients with knee OA undergoing TKA.
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http://dx.doi.org/10.1016/j.bjpt.2017.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6095047PMC
January 2019

A step into the RNA world: Conditional analysis of hydrogel formation of adenosine 5'-monophosphate induced by cyanuric acid.

Biosystems 2017 Dec 5;162:53-58. Epub 2017 Sep 5.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan; Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan. Electronic address:

Nucleotide polymerization occurs by the nucleophilic attack of 3'-oxygen of the 3'-terminal nucleotide on the α-phosphorus of the incoming nucleotide 5'-triphosphate. The π-stacking of mononucleotides is an important factor for prebiotic RNA polymerization in terms of attaining the proximity of two reacting moieties. Adenosine and adenosine 5'-monophosphate (AMP) are known to form hydrogel in the presence of cyanuric acid at neutral pH. However, we observed that other canonical ribonucleotides did not gel under the same condition. The π-stacking-induced hydrogel formation of AMP was destroyed at pH 2.0, suggesting that the protonation of N at position 1 of adenine abolished hydrogen bonding with the NH of cyanuric acid and resulted in the deformation of the hexad of adenine and cyanuric acid. A liquid-like gel was formed in the case of adenosine with cyanuric acid and boric acid, whereas AMP caused the formation of a solid gel, implying that the negative charge inherent to AMP prevented the formation of esters of boric acid with the cis-diols of ribose. Cyanuric acid-driven oligomerizations of AMP might have been the first crucial event in the foundation of the RNA world.
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http://dx.doi.org/10.1016/j.biosystems.2017.09.004DOI Listing
December 2017

Binding Properties of Split tRNA to the C-terminal Domain of Methionyl-tRNA Synthetase of Nanoarchaeum equitans.

J Mol Evol 2017 06 6;84(5-6):267-278. Epub 2017 Jun 6.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan.

The C-terminal domain of methionyl-tRNA synthetase (MetRS-C) from Nanoarchaeum equitans is homologous to a tRNA-binding protein consisting of 111 amino acids (Trbp111) from Aquifex aeolicus. The crystal structure of MetRS-C showed that it existed as a homodimer, and that each monomer possessed an oligonucleotide/oligosaccharide-binding fold (OB-fold). Analysis using a quartz crystal microbalance indicated that MetRS-C freshly isolated from N. equitans was bound to tRNA. However, binding of the split 3'-half tRNA species was stronger than that of the 5'-half species. The T-loop and the 3'-end regions of the split 3'-half tRNA were found to be responsible for the binding. The minimum structure for binding to MetRS-C might be a minihelix-like stem-loop with single-stranded 3'-terminus. After successive duplications of such a small hairpin structure with the assistance of a Trbp-like structure, the interaction of the T-loop region of the 3'-half with a Trbp-like structure could have been evolutionarily replaced by RNA-RNA interactions, along with many combinational tertiary interactions, to form the modern tRNA structure.
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http://dx.doi.org/10.1007/s00239-017-9796-6DOI Listing
June 2017

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea.

mBio 2017 05 9;8(3). Epub 2017 May 9.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA

The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA is first acylated with phosphoserine (Sep) by -phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA by Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN, , TACK, and Asgard) and two groups of bacteria (" Parcubacteria" and ). Bacterial SepRS and SepCysS charged bacterial tRNA species with cysteine Homologs of SepCysE, a scaffold protein facilitating SepRS⋅SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea and bacteria. We discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes. Comprehensive analyses of all genomic and metagenomic protein sequence data in public databases revealed the distribution and evolution of an alternative cysteine-encoding system in diverse archaea and bacteria. The finding that the SepRS-SepCysS-SepCysE- and the selenocysteine-encoding systems are shared by the class I methanogens, the AK8/W8A-19 group, and an Asgard archaeon suggests that ancient archaea may have used both systems. In contrast, bacteria may have obtained the SepRS-SepCysS system from archaea. The SepRS-SepCysS system sometimes coexists with a pyrrolysine-encoding system in both archaea and bacteria. Our results provide additional bioinformatic evidence for the contribution of the SepRS-SepCysS system for sulfur assimilation and diverse metabolisms which require vast amounts of iron-sulfur enzymes and proteins. Among these biological activities, methanogenesis, methylamine metabolism, and organohalide respiration may have local and global effects on earth. Taken together, uncultured bacteria and archaea provide an expanded record of the evolution of the genetic code.
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http://dx.doi.org/10.1128/mBio.00561-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5424206PMC
May 2017

A genomically modified Escherichia coli strain carrying an orthogonal E. coli histidyl-tRNA synthetase•tRNA pair.

Biochim Biophys Acta Gen Subj 2017 Nov 10;1861(11 Pt B):3009-3015. Epub 2017 Mar 10.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520, USA; Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan. Electronic address:

Background: Development of new aminoacyl-tRNA synthetase (aaRS)•tRNA pairs is central for incorporation of novel non-canonical amino acids (ncAAs) into proteins via genetic code expansion (GCE). The Escherichia coli and Caulobacter crescentus histidyl-tRNA synthetases (HisRS) evolved divergent mechanisms of tRNA recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNA pair is a good candidate for GCE, its use in C. crescentus is limited by the lack of established genetic selection methods and by the low transformation efficiency of C. crescentus.

Methods: E. coli was genetically engineered to use a C. crescentus HisRS•tRNA pair. Super-folder green fluorescent protein (sfGFP) and chloramphenicol acetyltransferase (CAT) were used as reporters for read-through assays. A library of 313 ncAAs coupled with the sfGFP reporter system was employed to investigate the specificity of E. coli HisRS in vivo.

Results: A genomically modified E. coli strain (named MEOV1) was created. MEVO1 requires an active C. crescentus HisRS•tRNA pair for growth, and displays a similar doubling time as the parental E. coli strain. sfGFP- and CAT-based assays showed that the E. coli HisRS•tRNA pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNA elevated its suppression efficiency by 2-fold.

Conclusions: The C. crescentus HisRS•tRNA pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNA is orthogonal in MEOV1 cells. E. coli tRNA is an efficient amber suppressor in MEOV1.

General Significance: We developed a platform that allows protein engineering of E. coli HisRS that should facilitate GCE in E. coli. This article is part of a Special Issue entitled "Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O'Donoghue.
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http://dx.doi.org/10.1016/j.bbagen.2017.03.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5592127PMC
November 2017

Clinical Prediction Rule for Declines in Activities of Daily Living at 6 Months After Surgery for Hip Fracture Repair.

Arch Phys Med Rehabil 2016 12 13;97(12):2076-2084. Epub 2016 Aug 13.

Department of Rehabilitation, Hiroshima International University, Higashihiroshima, Japan.

Objectives: To develop and assess a clinical prediction rule (CPR) to predict declines in activities of daily living (ADL) at 6 months after surgery for hip fracture repair.

Design: Prospective, cohort study.

Setting: From hospital to home.

Participants: Patients (N=104) with hip fractures after surgery.

Interventions: Not applicable.

Main Outcome Measure: ADL were assessed using the Barthel Index at 6 months after surgery.

Results: At 6 months after surgery, 86 patients (82.6%) were known to be alive, 1 patient (1.0%) had died, and 17 (16.3%) were lost to follow-up. Thirty-two patients (37.2%) did not recover their ADL at 6 months after surgery to levels before fracture. The classification and regression trees methodology was used to develop 2 models to predict a decline in ADL: (1) model 1 included age, type of fracture, and care level before fracture (sensitivity=75.0%, specificity=81.5%, positive predictive value=70.6%, positive likelihood ratio=4.050); and (2) model 2 included the degree of independence 2 weeks postsurgery for ADL chair transfer, ADL ambulation, and age (sensitivity=65.6%, specificity=87.0%, positive predictive value=75.0%, positive likelihood ratio=5.063). The areas under the receiver operating characteristic curves of both CPR models were .825 (95% confidential interval, .728-.923) and .790 (95% confidence interval, .683-.897), respectively.

Conclusions: CPRs with moderate accuracy were developed to predict declines in ADL at 6 months after surgery for hip fracture repair.
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http://dx.doi.org/10.1016/j.apmr.2016.07.016DOI Listing
December 2016

Development of a Functionally Minimized Mutant of the R3C Ligase Ribozyme Offers Insight into the Plausibility of the RNA World Hypothesis.

Biology (Basel) 2014 Jul 29;3(3):452-65. Epub 2014 Jul 29.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.

The R3C ligase ribozyme is an artificial ligase ribozyme produced by modification of the ribozyme that lacks cytidine. Here, we attempted to modify the original R3C ribozyme (73 nucleotides) by reducing the number of nucleotides while maintaining the maximum possible catalytic efficiency. By partially deleting both the "grip" (P4 + P5) and "hammer" (P3) stem-loops, we found the critical border to retain activity comparable to that of full-length R3C. The three-way junction structure was necessary to maintain enzymatic function and the stability of the "grip" (P4 + P5) stem had a large influence on the catalytic activity of R3C. The final minimized ribozyme we obtained comprised ~50 nucleotides, comparable to the estimated length of prebiotically synthesized RNA. Our findings suggest that the autocatalytic function in ribozymes is indeed possible to obtain using sequence lengths achievable with prebiotic synthesis.
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http://dx.doi.org/10.3390/biology3030452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4192621PMC
July 2014

Proline might have been the first amino acid in the primitive genetic code.

J Mol Evol 2014 Jun 28;78(6):310-2. Epub 2014 Jun 28.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo, 125-8585, Japan.

Stereochemical assignment of amino acids and corresponding codons or anticodons has not been successful so far. Here, we focused on proline and GGG (anticodon of tRNA(Pro)) and investigated their mutual interaction. Circular dichroism spectroscopy revealed that guanosine nucleotides (GG, GGG) formed G-quartet structures. The structures were destroyed by adding high concentrations of proline. We propose that the possibility of the reversible proline/G-quartet interaction could have contributed to the specific assignment of proline on GGG and that this coding could have been the first in the genetic code.
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http://dx.doi.org/10.1007/s00239-014-9629-9DOI Listing
June 2014

Pyrrolysyl-tRNA synthetase variants reveal ancestral aminoacylation function.

FEBS Lett 2013 Oct 28;587(19):3243-8. Epub 2013 Aug 28.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Pyrrolysyl-tRNA synthetase (PylRS) is a class IIc aminoacyl-tRNA synthetase that is related to phenylalanyl-tRNA synthetase (PheRS). Genetic selection provided PylRS variants with a broad range of specificity for diverse non-canonical amino acids (ncAAs). One variant is a specific phenylalanine-incorporating enzyme. Structural models of the PylRSamino acid complex show that the small pocket size and π-interaction play an important role in specific recognition of Phe and the engineered PylRS active site resembles that of Escherichia coli PheRS.
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http://dx.doi.org/10.1016/j.febslet.2013.08.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778162PMC
October 2013

Glycols modulate terminator stem stability and ligand-dependency of a glycine riboswitch.

Biosystems 2013 Aug 27;113(2):59-65. Epub 2013 May 27.

Department of Biological Science and Technology, Tokyo University of Science, 6-3-1 Niijuku, Katsushika-ku, Tokyo 125-8585, Japan.

The Bacillus subtilis glycine riboswitch comprises tandem glycine-binding aptamers and a putative terminator stem followed by the gcvT operon. Gene expression is regulated via the sensing of glycine. However, we found that the riboswitch behaves in a "glycine-independent" manner in the presence of polyethylene glycol (PEG) and ethylene glycol. The effect is related to the formation of a terminator stem within the expression platform under such conditions. The results revealed that increasing PEG stabilized the structure of the terminator stem. By contrast, the addition of ethylene glycol destabilized the terminator stem. PEG and ethylene glycol have opposite effects on transcription as well as on stable terminator stem formation. The glycine-independency of the riboswitch and the effects of such glycols might shed light on the evolution of riboswitches.
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http://dx.doi.org/10.1016/j.biosystems.2013.05.004DOI Listing
August 2013

Self-enhancement of hepatitis C virus replication by promotion of specific sphingolipid biosynthesis.

PLoS Pathog 2012 16;8(8):e1002860. Epub 2012 Aug 16.

Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.
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http://dx.doi.org/10.1371/journal.ppat.1002860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3420934PMC
December 2012

RNA tetraplex as a primordial peptide synthesis scaffold.

Biosystems 2012 Aug 21;109(2):145-50. Epub 2012 Mar 21.

Research Institute for Science and Technology, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan.

Peptide bond formation at the peptidyl transferase center on the ribosome is a crucial phenomenon in life systems. In this study, we conceptually propose possible roles of the RNA tetraplex as a scaffold for two aminoacyl minihelices that enable peptide bond formation. The basic rationale of this model is that "parallel" complementary templates composed of only 10-mer nucleotides can position two amino acids in close proximity, which is conceptually and essentially similar to the situation observed in ribosomes. Using supportive experimental data, we discuss the origin and evolution of peptide bond formation in early biological systems.
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http://dx.doi.org/10.1016/j.biosystems.2012.03.003DOI Listing
August 2012

N-acetyl lysyl-tRNA synthetases evolved by a CcdB-based selection possess N-acetyl lysine specificity in vitro and in vivo.

FEBS Lett 2012 Mar 28;586(6):729-33. Epub 2012 Jan 28.

Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520-8114, USA.

Posttranslational modifications play a crucial role in modulating protein structure and function. Genetic incorporation of unnatural amino acids into a specific site of a protein facilitates the systematic study of protein modifications including acetylation. We here report the directed evolution of pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei to create N-acetyl lysyl-tRNA synthetases (AcKRSs) using a new selection system based on the killing activity of the toxic ccdB gene product. The amino acid specificity of these and of published AckRSs was tested in vitro and in vivo, and the enzyme-kinetic properties of the AckRSs were evaluated for the first time.
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http://dx.doi.org/10.1016/j.febslet.2012.01.029DOI Listing
March 2012
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