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BACKGROUND: This study aimed to evaluate the effects of the differences in initial graft tension (IGT) on patellofemoral alignment in the axial plane after anatomical anterior cruciate ligament reconstruction (ACLR). METHODS: A total of 102 patients who underwent primary anatomical ACLR using a bone-patellar-tendon-bone autograft were enrolled. The grafts were fixed with maximum manual force at full knee extension (higher graft tension; H group) and with 5-20 N at 20° knee flexion which corresponded to 80 N at full knee extension (lower graft tension; L group) pulls in 39 and 63 patients, respectively. All patients underwent computed tomography of the bilateral knee joints with knee extension 1 week postoperatively. The patellofemoral alignment (sulcus angle, lateral trochlear inclination angle, lateral patellofemoral angle (LPFA), condylar-patellar angle (CPA) (lateral facet, patellar tilt), congruence angle, and bisect offset index) on the axial computed tomography images were evaluated, and the side-to-side differences (SSDs) between the injured knee and the contralateral knee were calculated. RESULTS: Congruence angle SSD was remarkably higher in the H group than in the L group (3.8 ± 4.7 vs. 0.4 ± 5.7, P < 0.01). Furthermore, the LPFA SSD and CPA (patellar tilt) SSD were significantly higher in the H group (-1.8 ± 3.1 vs. -0.4 ± 2.7, P = 0.04 and -1.6 ± 2.8 vs. -0.3 ± 2.7, P = 0.04, respectively). CONCLUSIONS: A higher IGT during anatomical ACLR induces a lateral shift and tilt of the patella against the femur immediately after surgery compared with the lower IGT.
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Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.
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Purpose: The tibial anatomical anteroposterior (AP) axis "Akagi's line" was originally defined on computed tomography (CT) in total knee arthroplasty (TKA); however, its intraoperative reproducibility remains unknown. This study aimed to evaluate the intraoperative reproducibility of the Akagi's line and its effect on postoperative clinical outcomes. Methods: This prospective study included 171 TKAs. The rotational angle of the intraoperative Akagi's line relative to the original Akagi's line (RAA) defined on CT was measured. The RAA was calculated based on the tibial component rotational angles relative to the intraoperative Akagi's line measured using the navigation system and CT. The effects of RAA on postoperative clinical outcomes and rotational alignments of components were also evaluated. Results: The mean absolute RAA (standard deviation) value was 5.5° (3.9°). The range of RAA was 22° internal rotation to 16° external rotation. Intraoperative Akagi's line outliers (RAA > 10°) were observed in 14% of the knees (24 knees). In outlier analysis, the tibial component rotation angle was externally rotated 6.5° (5.6°) in the outlier group and externally rotated 3.7° (4.2°) in the nonoutlier group (≤10°), with a significant difference between the two groups. Additionally, the outlier group (RAA > 10°) showed lower postoperative clinical outcomes. Conclusion: The original Akagi's line defined on CT showed insufficient reproducibility intraoperatively. The poor intraoperative detection of Akagi's line could be the reason for the tibial component rotational error and worse postoperative clinical outcomes. Level of Evidence: Level IV, case series.
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Background: Previous studies have attempted to determine if certain risk factors can predict the occurrence of a lateral ankle sprain (LAS) in female soccer players. Unfortunately, there is limited evidence with regard to risk factors associated with an LAS in female soccer players. Purpose: To identify intrinsic risk factors for an LAS among young female soccer players. Study Design: Cohort study; Level of evidence, 2. Methods: Participants were 161 young female soccer players in Japan who were evaluated for LAS risk factors during a preseason medical assessment. The assessment included anthropometric, joint laxity, joint range of motion, muscle flexibility, muscle strength, and balance measurements. Each athlete's history of LASs was also collected. The participants were monitored during a single-yearseason for LASs, as diagnosed by physicians. Results: There were 26 instances of an LAS in 25 players (15.5%) during the season. Injured players were significantly more likely to have sustained a previous ankle sprain (P = .045) and demonstrated significantly worse balance than their peers without an LAS during the double- and single-leg balance tests (P = .008 for both). Athletes with lower hamstring-to-quadriceps muscle strength ratios were also significantly more likely to sustain an LAS (P = .02). Conclusion: Poor balance, a low hamstring-to-quadriceps ratio, and a history of ankle sprains were associated with an increased risk of LASs in young female soccer players in the current study. These findings may be useful for developing a program to prevent LASs in this population.
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All kinds of RNA molecules can be produced by in vitro transcription using T7 RNA polymerase using DNA templates obtained by solid-phase chemical synthesis, primer extension, PCR, or DNA cloning. The oligonucleotide design, however, is a challenge to nonexperts as this relies on a set of rules that have been established empirically over time. Here, we describe a Python program to facilitate the rational design of oligonucleotides, calculated with kinetic parameters for enhanced in vitro transcription (ROCKET). The Python tool uses thermodynamic parameters, performs folding-energy calculations, and selects oligonucleotides suitable for the polymerase extension reaction. These oligonucleotides improve yields of template DNA. With the oligonucleotides selected by the program, the tRNA transcripts can be prepared by a one-pot reaction of the DNA polymerase extension reaction and the transcription reaction. Also, the ROCKET-selected oligonucleotides provide greater transcription yields than that from oligonucleotides selected by Primerize, a leading software for designing oligonucleotides for in vitro transcription, due to the enhancement of template DNA synthesis. Apart from over 50 tRNA genes tested, an in vitro transcribed self-cleaving ribozyme was found to have catalytic activity. In addition, the program can be applied to the synthesis of mRNA, demonstrating the wide applicability of the ROCKET software.
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Oligonucleotídeos , Software , Transcrição Gênica , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/síntese química , RNA Polimerases Dirigidas por DNA/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Catalítico/genética , RNA Catalítico/metabolismo , RNA Catalítico/química , Termodinâmica , RNA de Transferência/genética , RNA de Transferência/química , RNA de Transferência/metabolismo , Cinética , RNA Mensageiro/genética , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Background: Previous studies have aimed to determine the use of certain risk factors in predicting the occurrence of noncontact anterior cruciate ligament (ACL) injuries. Unfortunately, evidence regarding noncontact ACL injuries in male American football players is limited. This prospective cohort study aimed to identify intrinsic risk factors for noncontact ACL injury among male American football players. Methods: This study evaluated 152 male American football players in Japan for potential noncontact ACL injury risk factors during a preseason medical assessment, including anthropometric, joint laxity, and flexibility, muscle flexibility, muscle strength, and balance measurements. A total of 25 variables were examined. Participants were monitored during each season for noncontact ACL injury, as diagnosed by physicians. Results: Noncontact ACL injuries occurred in 11 knees of 11 players (prevalence; 7.1 %). Injured players were significantly more likely to have lightweight (P = 0.049). No statistically significant between-group differences were found for any other variables. Participants with a lower hamstring to quadriceps (H/Q) ratio (P = 0.04) were more likely to sustain noncontact ACL injuries. Conclusion: Lower H/Q ratio and lower body weight were significantly associated with new-onset noncontact ACL injury in male American football players. These findings will help develop strategies to prevent noncontact ACL injuries in male American football players.
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BACKGROUND: Bicruciate-retaining (BCR) prosthesis has been introduced to recreate normal knee movement by preserving both the anterior and posterior cruciate ligaments. However, the use of BCR total knee arthroplasty (TKA) is still debatable because of several disappointing reports. We have been performing BCR TKAs with personalized alignment (PA). This study aimed to reveal the limb alignment and soft tissue balance of FA-BCR TKAs and compare the clinical outcomes of FA-BCR TKAs with those of unicompartmental knee arthroplasty (UKA). METHODS: Fifty BCR TKAs and 58 UKAs were included in this study. The joint component gaps of BCR TKA were evaluated intraoperatively and the postoperative hip-knee-ankle (HKA) angle, medial proximal tibial angle (MPTA), and lateral distal femoral angle (LDFA) were measured using full-length standing radiography. The short-term clinical outcomes of BCR TKAs were compared with those of UKA using the scoring system of 2011 Knee Society Scoring (KSS) and the knee injury and osteoarthritis outcome score (KOOS) at an average of 2 years postoperatively (1-4yeras). RESULTS: The coronal alignment values of PA-BCR TKA were as follows: HKA angle, 177.9° ± 2.3°; MPTA, 85.4° ± 1.9°; and LDFA, 87.5° ± 1.9°. The joint component gaps at flexion angles of 10°, 30°, 60°, and 90° were 11.1 ± 1.2, 10.9 ± 1.4, 10.7 ± 1.3, and 11.2 ± 1.4 mm for the medial compartment and 12.9 ± 1.5, 12.6 ± 1.8, 12.5 ± 1.8 and 12.5 ± 1.7 mm for the lateral compartment, respectively. The patient expectation score and maximum extension angle of PA-BCR TKA were significantly better than those of UKAs. CONCLUSIONS: The short-term clinical outcomes of PA-BCR TKA were comparable or a slightly superior to those of UKAs.
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Artroplastia do Joelho , Prótese do Joelho , Osteoartrite do Joelho , Ligamento Cruzado Posterior , Humanos , Artroplastia do Joelho/métodos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Joelho/cirurgia , Ligamento Cruzado Posterior/diagnóstico por imagem , Ligamento Cruzado Posterior/cirurgia , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/cirurgia , Estudos RetrospectivosRESUMO
There is a multitude of small (<100nt) RNAs that serve diverse functional roles in biology. Key amongst these is transfer RNA (tRNA), which is among the most ancient RNAs and is part of the translational apparatus in every domain of life. Transfer RNAs are also the most heavily modified class of RNAs. They are essential and their misregulation, due to mutated sequences or loss of modification, can lead to disease. Because of the severe phenotypes associated with mitochondrial tRNA defects in particular, the desire to deliver repaired tRNAs via droplets such as lipid nanoparticles or other compartments is an active area of research. Here we describe how to use our tRNA Structure-seq method to study tRNAs and other small RNAs in two different biologically relevant contexts, peptide-rich droplets and in vivo.
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RNA de Transferência , RNA , RNA de Transferência/genética , RNA/genéticaRESUMO
Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).
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Anticódon , RNA de Transferência , RNA de Transferência/metabolismo , Códon/genética , Biossíntese de Proteínas , tRNA Metiltransferases/genética , tRNA Metiltransferases/química , tRNA Metiltransferases/metabolismoRESUMO
PURPOSE: Patellofemoral (PF) compartment cartilage lesions are a frequent problem after anterior cruciate ligament (ACL) reconstruction. This study aimed to determine the factors that influence PF cartilage lesions after anatomical ACL reconstruction. METHODS: This study enrolled a total of 114 patients who did not manifest PF compartment cartilage lesions during anatomical ACL reconstruction and underwent second-look arthroscopy 18 months postoperatively. Arthroscopy using the International Cartilage Repair Society (ICRS) classification was used to assess cartilage lesions. The correlation between surgical findings, radiographic factors, and clinical factors and change of ICRS grade was analysed. Multivariate regression analysis was conducted to reveal the independent risk factors for PF cartilage lesions among patients' demographic data and parameters that correlated with the change of ICRS grade in the correlation analyses. RESULTS: ICRS grade changes in PF cartilage were significantly correlated with age, sex, quadriceps strength at 1 year postoperatively, hamstrings strength at pre- and 1 year postoperatively, and single leg hop test at 1 year postoperatively. However, no significant correlation was found between the time between injury and surgery, posterior tibial slope angle, pre- and postoperative Tegner activity scale, graft type, initial graft tension, meniscus injury, meniscus injury treatment, pre- and postoperative range of motion, anteroposterior laxity and preoperative quadriceps strength, and the change in ICRS grade. Multivariate regression analysis revealed male (P = 0.019) and quadriceps strength weakness at 1 year postoperatively (P = 0.009) as independent risk factors for PF cartilage lesions. CONCLUSIONS: Quadriceps strength weakness 1 year after ACL reconstruction and males were correlated with a new PF cartilage lesion after anatomical ACL reconstruction, with no significant correlation between bone-patellar tendon-bone autograft, initial graft tension, or extension deficit and new PF cartilage lesion. Rehabilitation that focuses on quadriceps strength after ACL reconstruction is recommended to prevent new PF cartilage lesions. LEVEL OF EVIDENCE: Level IV.
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Lesões do Ligamento Cruzado Anterior , Reconstrução do Ligamento Cruzado Anterior , Cartilagem Articular , Humanos , Masculino , Cartilagem Articular/cirurgia , Lesões do Ligamento Cruzado Anterior/complicações , Lesões do Ligamento Cruzado Anterior/cirurgia , Músculo Quadríceps/cirurgia , Reconstrução do Ligamento Cruzado Anterior/efeitos adversos , Reconstrução do Ligamento Cruzado Anterior/reabilitação , Fatores de RiscoRESUMO
TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2'-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. Escherichia coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNA SerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyrand tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.
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Escherichia coli , Metiltransferases , Metiltransferases/genética , Metiltransferases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metilação , tRNA Metiltransferases/química , RNA de Transferência/química , Conformação de Ácido NucleicoRESUMO
Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Here, we detail next-generation sequencing (NGS) experiments performed in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Notably, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life.
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Nucleosídeos , Dobramento de RNA , Peptídeos , RNA/genética , Sequenciamento de Nucleotídeos em Larga EscalaRESUMO
BACKGROUND: Difference in the finite helical axis of the knee joints before and after anterior cruciate ligament-preserving knee arthroplasties such as unicompartmental knee arthroplasty and bi-cruciate-retaining total knee arthroplasty remains unknown. This study compared the knee finite helical axes before and after bi-cruciate-preserving knee arthroplasty. METHODS: Patients undergoing medial unicompartmental knee arthroplasty and bi-cruciate-retaining total knee arthroplasty were included. Under fluoroscopy, participants performed a deep knee bend before and after surgery. A two/three-dimensional registration technique was employed to measure tibiofemoral kinematics. Femoral finite helical axis was calculated in a flexion range of 0-120° using 30° windows (early-, mid-, late-, and deep-flexion phases). FINDINGS: In unicompartmental knee arthroplasty, the preoperative knee vertical angle was larger than the postoperative vertical angle in mid- and deep-flexion phases. The postoperative knee vertical angle was smaller in unicompartmental knee arthroplasty than in bi-cruciate-retaining total knee arthroplasty. In unicompartmental knee arthroplasty, the preoperative horizontal angle was smaller than the postoperative horizontal angle in the early-flexion phase. However, in bi-cruciate-retaining total knee arthroplasty, the preoperative horizontal angle was larger than the postoperative horizontal angle in mid- and deep-flexion phases. The horizontal angle was smaller before unicompartmental knee arthroplasty than that before bi-cruciate-retaining total knee arthroplasty in early-, mid-, and deep-flexion phases. However, the vertical angle was larger after unicompartmental knee arthroplasty than that after bi-cruciate-retaining total knee arthroplasty in the early-flexion phase. INTERPRETATION: The knee finite helical axes before and after unicompartmental knee arthroplasty differed from those before and after bi-cruciate-retaining total knee arthroplasty.
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Artroplastia do Joelho , Prótese do Joelho , Osteoartrite do Joelho , Humanos , Artroplastia do Joelho/métodos , Articulação do Joelho/cirurgia , Ligamento Cruzado Anterior/cirurgia , Joelho/cirurgia , Osteoartrite do Joelho/cirurgia , Amplitude de Movimento Articular , Fenômenos BiomecânicosRESUMO
BACKGROUND: Given the frequency of hamstring strain injuries (HSI) among male college American football players, several studies have attempted to determine whether certain risk factors can predict their occurrence. However, no consensus on modifiable risk factors for HSIs in male college American football players has yet been reached to prevent these injuries. This study aimed to clarify risk factors for HSI prospectively in college male American football players. METHODS: A total of 78 male college American football players, whose positions were limited to skill positions, were medically assessed for potential risk factors of HSI. The preseason medical assessment included anthropometric measurements, joint laxity and flexibility, muscle flexibility, muscle strength, and balance ability. RESULTS: HSI occurred in a total of 25 thighs from 25 players (32.1%). Injured players had significantly lower hamstring flexibility (p = 0.02) and hamstring to quadriceps strength ratio (H/Q) (p = 0.047) compared to uninjured players. Additionally, injured players had significantly lower general joint laxity scores, especially for the total (p = 0.04), hip (p = 0.007), and elbow (p = 0.04) scores, compared to uninjured players. CONCLUSIONS: Lower hamstring flexibility, lower hamstring to quadriceps strength ratio, and lower general joint laxity score were identified as risk factors for HSI in male college American football players placed in skill positions. The muscle flexibility and H/Q ratio could be useful in preventing HSI in such players.
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Traumatismos em Atletas , Futebol Americano , Músculos Isquiossurais , Instabilidade Articular , Doenças Musculares , Lesões dos Tecidos Moles , Humanos , Masculino , Futebol Americano/lesões , Estudos Prospectivos , Músculos Isquiossurais/fisiologia , Força Muscular/fisiologia , Fatores de Risco , Traumatismos em Atletas/epidemiologia , Traumatismos em Atletas/prevenção & controleRESUMO
Compartmentalization of RNA in biopolymer-rich membraneless organelles is now understood to be pervasive and critical for the function of extant biology and has been proposed as a prebiotically-plausible way to accumulate RNA. However, compartment-RNA interactions that drive encapsulation have the potential to influence RNA structure and function in compartment- and RNA sequence-dependent ways. Herein, we detail Next-Generation Sequencing (NGS) experiments performed for the first time in membraneless compartments called complex coacervates to characterize the fold of many different transfer RNAs (tRNAs) simultaneously under the potentially denaturing conditions of these compartments. Strikingly, we find that natural modifications favor the native fold of tRNAs in these compartments. This suggests that covalent RNA modifications could have played a critical role in metabolic processes at the origin of life. One Sentence Summary: We demonstrate that RNA folds into native secondary and tertiary structures in protocell models and that this is favored by covalent modifications, which is critical for the origins of life.
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4-Thiouridine (s4U) is a modified nucleoside, found at positions 8 and 9 in tRNA from eubacteria and archaea. Studies of the biosynthetic pathway and physiological role of s4U in tRNA are ongoing in the tRNA modification field. s4U has also recently been utilized as a biotechnological tool for analysis of RNAs. Therefore, a selective and sensitive system for the detection of s4U is essential for progress in the fields of RNA technologies and tRNA modification. Here, we report the use of biotin-coupled 2-aminoethyl-methanethiosulfonate (MTSEA biotin-XX) for labeling of s4U and demonstrate that the system is sensitive and quantitative. This technique can be used without denaturation; however, addition of a denaturation step improves the limit of detection. Thermus thermophilus tRNAs, which abundantly contain 5-methyl-2-thiouridine, were tested to investigate the selectivity of the MTSEA biotin-XX s4U detection system. The system did not react with 5-methyl-2-thiouridine in tRNAs from a T. thermophilus tRNA 4-thiouridine synthetase (thiI) gene deletion strain. Thus, the most useful advantage of the MTSEA biotin-XX s4U detection system is that MTSEA biotin-XX reacts only with s4U and not with other sulfur-containing modified nucleosides such as s2U derivatives in tRNAs. Furthermore, the MTSEA biotin-XX s4U detection system can analyze multiple samples in a short time span. The MTSEA biotin-XX s4U detection system can also be used for the analysis of s4U formation in tRNA. Finally, we demonstrate that the MTSEA biotin-XX system can be used to visualize newly transcribed tRNAs in S. cerevisiae cells.
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RNA , Tiouridina , RNA/metabolismo , Saccharomyces cerevisiae/genética , Biotina/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismoRESUMO
Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.
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Metiltransferases , tRNA Metiltransferases , Metiltransferases/genética , Mutação , RNA de Transferência/metabolismo , RNA de Transferência de Leucina , tRNA Metiltransferases/química , Bacillaceae/genética , Bacillaceae/metabolismoRESUMO
Bi-cruciate stabilized (BCS) total knee arthroplasty (TKA) was developed to approximate normal knee kinematics and is expected to improve clinical outcomes. However, the effects of soft tissue balance at the medial or lateral compartment on patient-reported outcome measures (PROMs) following BCS TKA are unclear. The purpose of this study was to clarify the relationship between the medial or lateral component gaps and PROMs following BCS TKA. One hundred seventeen knees with varus deformities which underwent BCS TKA were included in this study. They were divided into two groups according to the validated Japanese version of the Knee Injury and Osteoarthritis Outcome Score for each subscale of pain, symptoms, and activities of daily living (ADL) at 1 year postoperatively: group H consisted of patients with ≥90 points and group L consisted of patients with <90 points. Intraoperative medial and lateral joint laxities at 0°, 30°, 60°, 90°, and 120° flexion measured using a tensor device were compared between the two groups in each subscale. In the pain subscale, the medial joint laxities at 30° (p < 0.05) and 60° flexion (p < 0.05) in group H were significantly smaller than those in group L. In the ADL subscale, the medial joint laxity at 60° flexion in group H was significantly smaller than that in group L (p < 0.05). In the symptom subscale, the lateral joint laxity at 60° flexion in group H was significantly smaller than that in group L (p < 0.05). Surgeons should pay attention to the importance of both medial and lateral joint stabilities to achieve better postoperative PROMs following BCS TKA.
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Artroplastia do Joelho , Instabilidade Articular , Osteoartrite do Joelho , Humanos , Instabilidade Articular/etiologia , Instabilidade Articular/cirurgia , Instabilidade Articular/diagnóstico , Osteoartrite do Joelho/cirurgia , Atividades Cotidianas , Articulação do Joelho/cirurgia , Amplitude de Movimento Articular , Fenômenos Biomecânicos , Medidas de Resultados Relatados pelo PacienteRESUMO
BACKGROUND: Medial-pivot type total knee arthroplasty is designed to reproduce physiological femoral rotational kinematics during knee flexion; however, its rotational kinematics in valgus knees remain unknown. This study's hypothesis is that its kinematics show the similar medial pivot motion in valgus knees as in varus knees. METHODS: This retrospective study included 50 cases of primary medial-pivot type total knee arthroplasty performed with navigation for knee osteoarthritis. Cases were grouped as valgus (n = 20) or varus (n = 30). In valgus knees, surgeons used preoperative manual testing to confirm that alignment was correctable. They evaluated femoral rotational kinematics at maximum extension, 30°, 60°, 90°, and maximum flexion, using a navigation system, both before and after implantation. Finally, intraoperative rotational kinematics, postoperative patient-reported outcomes, and knee range of motion were compared between the two groups. FINDINGS: Before implantation, both valgus and varus knees displayed external femoral rotation relative to the tibia during knee flexion. The rotation magnitude was significantly larger in varus knees at 60°, 90°, and maximum flexion angles compared to valgus knees (P < 0.05). In contrast, after implantation, both groups displayed external femoral rotation of 12-13° during knee flexion with no significant differences between the two groups at any knee flexion angle tested (P > 0.05). Short-term clinical outcomes were comparable between valgus and varus knees. INTERPRETATION: Postoperatively, medial-pivot type total knee arthroplasty for knee osteoarthritis resulted in external femoral rotation during knee flexion. Notably, this occurred even in valgus knees with good short-term clinical outcomes.
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Artroplastia do Joelho , Osteoartrite do Joelho , Humanos , Artroplastia do Joelho/métodos , Osteoartrite do Joelho/cirurgia , Fenômenos Biomecânicos/fisiologia , Estudos Retrospectivos , Articulação do Joelho/cirurgia , Amplitude de Movimento Articular/fisiologiaRESUMO
Although an anterior cruciate ligament (ACL) deficiency induces knee osteoarthritis, particularly in the medial compartment, the kinematics remains partially unclear. This study investigated the in vivo knee kinematics of ACL-deficient and normal knees by comparing them during squatting. This prospective comparative study included 17 ACL-deficient knees and 20 normal knees. The kinematics was investigated under fluoroscopy using a two- to three-dimensional registration technique. The anteroposterior (AP) translation of the medial and lateral sides of the femur, axial rotation of the femur relative to the tibia, and kinematic pathways were evaluated and compared. At first, the medial AP position of the femur translated anteriorly from 0° to midflexion, afterward it translated posteriorly in both ACL-deficient and normal knees. However, the medial AP position of the femur in the ACL-deficient knees was located significantly posteriorly compared with normal knees at 0-110° flexion. The lateral AP position of the femur translated posteriorly from 0° to 150° flexion in both ACL-deficient and normal knees. The lateral AP position of the femur in the ACL-deficient knees was located significantly posteriorly compared with that in normal knees at 0-10° flexion. The femur showed external rotation from 0° to 150° flexion in both ACL-deficient and normal knees. A medial pivot motion and subsequent bicondylar rollback were observed in both knees in the kinematic pathway. In conclusion, the AP position of the femur relative to the tibia was altered due to ACL deficiency, particularly in the medial compartment.