Long-term effects of non-weight bearing and immobilization after anterior cruciate ligament reconstruction on joint contracture formation in rats.

PURPOSE: Non-weight bearing improves and immobilization worsens contracture induced by anterior cruciate ligament reconstruction (ACLR), but effect persistence after reloading and remobilization remains unclear, and the combined effects of these factors on ACLR-induced contracture are unknown. We aimed to determine 1) whether the effects of short-term (2-week) non-weight bearing or immobilization after ACLR on contracture would be sustained by reloading or remobilization during a 10-week observation period, and 2) how the combination of both interventions compared to the outcome of either alone. METHODS: We divided 88 ACL-reconstructed male rats into four groups: non-intervention, non-weight bearing, joint immobilization, and both interventions. Interventions were performed for 2 weeks, followed by rearing without intervention. Twelve untreated rats were used as controls. At 2, 4, and 12 weeks post-surgery, we assessed range of motion (ROM) and histological changes. RESULTS: ACLR resulted in persistent loss of ROM, accompanied by synovial shortening, capsule thickening, and osteophyte formation. Two weeks of non-weight bearing increased ROM and reduced osteophyte size, but the beneficial effects disappeared within 10 weeks after reloading. Two-week immobilization decreased ROM and facilitated synovial shortening. After remobilization, ROM partially recovered but remained below non-intervention levels at 12 weeks. When both interventions were combined, ROM was similar to immobilization alone. CONCLUSIONS: The beneficial effects of 2-week non-weight bearing on contracture diminished within 10 weeks after reloading. The adverse effects of 2-week immobilization on contracture persisted after 10 weeks of remobilization. The effects of the combined use of both interventions on contracture were primarily determined by immobilization.

Movement control during one-leg standing is important for the bone mineral density maintenance or improvement.

INTRODUCTION: Both hip fracture and bone mineral density (BMD) decline on the non-fractured side are more likely to occur within 1 year. However, there are no longitudinal study reports on the factors associated with BMD maintenance or improvement within the first year after hip fracture. This study aimed to investigate the factors influencing the neck BMD maintenance or improvement in patients with hip fractures from within 2 weeks-6 months after surgery. MATERIALS AND METHODS: Patients were hip fracture after surgery and were divided into two groups: Among neck BMD changes (6 months minus 2 weeks after surgery) were calculated. Based on among neck BMD change, patients were classified into the BMD maintenance or improvement (change ≥ 0) and the BMD decrease groups (change < 0). Propensity score matching was performed to adjust for confounding factors. To predict the factors affecting neck BMD, hierarchical logistic regression analysis was performed. The dependent variable was the BMD maintenance or improvement group and the BMD decrease group. The independent variables were basic and medical information, and physical functions. RESULTS: The hierarchical logistic regression analysis results showed that movement control during one-leg standing affected femoral neck BMD independently from age, sex. The odds ratio for movement control during one-leg standing was 8.22. The discrimination rate of the model was 69.7%. CONCLUSION: This study suggested that adequate movement control during one-leg standing is important to maintain or improve neck BMD.

Association of muscle mass and quality with hand grip strength in elderly patients with heart failure.

In patients with heart failure, it is unknown whether the extracellular water (ECW)/intracellular water (ICW) ratio is associated with muscle strength, and thus, it is not well understood whether poor muscle quality contributes to muscular weakness. This study examined the relationship among hand grip strength, skeletal muscle mass index (SMI), and upper limb ECW/ICW ratio in patients with heart failure. This study followed a cross-sectional design. Demographic data, medical information, and hand grip strength were collected. The SMI and ECW/ICW ratio were measured using bio-impedance analysis (BIA). Hierarchical multiple regression analysis was conducted to identify factors associated with hand grip strength. 51 patients with heart failure were analyzed for this study (mean age 84.58 ± 7.18). Hierarchical multiple regression analysis identified SMI as well as upper limb ECW/ICW ratio as factors associated with hand grip strength, independent of age, sex, body mass index, and Life Space Assessment scores. Standardized partial regression coefficients representing the magnitude of involvement of each independent variable were 0.33 and - 0.16. The coefficient of determination adjusted for degrees of freedom (R2), representing the contribution rate of the regression equation, was 0.830. We revealed that loss of hand grip strength in patients with heart failure is associated with not only with a decrease in skeletal muscle mass, but also with a decline in muscle quality, characterized by an increased upper limb ECW/ICW ratio. BIA is a simple and useful method to measure the ECW/ICW ratio, and in turn, the muscle quality, in patients with heart failure.

Crystal structure of Nanoarchaeum equitans tyrosyl-tRNA synthetase and its aminoacylation activity toward tRNATyr with an extra guanosine residue at the 5'-terminus.

tRNATyr of Nanoarchaeum equitans has a remarkable feature with an extra guanosine residue at the 5'-terminus. However, the N. equitans tRNATyr mutant without extra guanosine at the 5'-end was tyrosylated by tyrosyl-tRNA synthase (TyrRS). We solved the crystal structure of N. equitans TyrRS at 2.80 Å resolution. By comparing the present solved structure with the complex structures TyrRS with tRNATyr of Thermus thermophilus and Methanocaldococcus jannaschii, an arginine substitution mutant of N. equitans TyrRS at Ile200 (I200R), which is the putative closest candidate to the 5'-phosphate of C1 of N. equitans tRNATyr, was prepared. The I200R mutant tyrosylated not only wild-type tRNATyr but also the tRNA without the G-1 residue. Further tyrosylation analysis revealed that the second base of the anticodon (U35), discriminator base (A73), and C1:G72 base pair are strong recognition sites.

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

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 (R2) 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.

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

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.

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

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-ß, 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 tRNAAla. Combination of AlaRS-α and AlaRS-ß 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 tRNAAla, 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.

Correction to: G:U­Independent RNA Minihelix Aminoacylation by Nanoarchaeum equitans Alanyl­tRNA Synthetase: An Insight into the Evolution of Aminoacyl­tRNA Synthetases.

In the original version of this article, "A73" in Fig 6b was inadvertently labeled as "G73". The corrected Fig. 6 is given here.

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

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.

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

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.

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

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 ß-sheet inserted between these domains. An unmodified transcript of the wild-type N. equitans tRNAGly was successfully glycylated using GlyRS. Substitution of the discriminator base A73 of tRNAGly with any other nucleotide caused a significant decrease in glycylation activity. Mutational analysis of the second base-pair C2G71 of the acceptor stem of tRNAGly elucidated the importance of the base-pair, especially G71, as an identity element for recognition by GlyRS. Glycylation assays using tRNAGly 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.

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.

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.

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

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.

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

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.

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

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 tRNAHis recognition that prevent their cross-reactivity. Although the E. coli HisRS•tRNAHis 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•tRNAHis 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•tRNAHis 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•tRNAHis pair is orthogonal in MEOV1 cells. A mutation in the anticodon loop of E. coli tRNAHisCUA elevated its suppression efficiency by 2-fold. CONCLUSIONS: The C. crescentus HisRS•tRNAHis pair functionally complements an E. coli ΔhisS strain. The E. coli HisRS•tRNAHis is orthogonal in MEOV1 cells. E. coli tRNAHisCUA 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.

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

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.

Pyrrolysyl-tRNA synthetase-tRNA(Pyl) structure reveals the molecular basis of orthogonality.

Pyrrolysine (Pyl), the 22nd natural amino acid, is genetically encoded by UAG and inserted into proteins by the unique suppressor tRNA(Pyl) (ref. 1). The Methanosarcinaceae produce Pyl and express Pyl-containing methyltransferases that allow growth on methylamines. Homologous methyltransferases and the Pyl biosynthetic and coding machinery are also found in two bacterial species. Pyl coding is maintained by pyrrolysyl-tRNA synthetase (PylRS), which catalyses the formation of Pyl-tRNA(Pyl) (refs 4, 5). Pyl is not a recent addition to the genetic code. PylRS was already present in the last universal common ancestor; it then persisted in organisms that utilize methylamines as energy sources. Recent protein engineering efforts added non-canonical amino acids to the genetic code. This technology relies on the directed evolution of an 'orthogonal' tRNA synthetase-tRNA pair in which an engineered aminoacyl-tRNA synthetase (aaRS) specifically and exclusively acylates the orthogonal tRNA with a non-canonical amino acid. For Pyl the natural evolutionary process developed such a system some 3 billion years ago. When transformed into Escherichia coli, Methanosarcina barkeri PylRS and tRNA(Pyl) function as an orthogonal pair in vivo. Here we show that Desulfitobacterium hafniense PylRS-tRNA(Pyl) is an orthogonal pair in vitro and in vivo, and present the crystal structure of this orthogonal pair. The ancient emergence of PylRS-tRNA(Pyl) allowed the evolution of unique structural features in both the protein and the tRNA. These structural elements manifest an intricate, specialized aaRS-tRNA interaction surface that is highly distinct from those observed in any other known aaRS-tRNA complex; it is this general property that underlies the molecular basis of orthogonality.

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

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.

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

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.