Effects of different surgical procedures for meniscus injury on two-year clinical and radiological outcomes after anterior cruciate ligament reconstructions. -TMDU MAKS study.

BACKGROUND: The treatment of meniscus injuries combined with anterior cruciate ligament (ACL) reconstruction would be important to improve outcomes after ACL reconstruction. However, the effects of treatment methods for meniscus after ACL reconstruction have not been thoroughly investigated. The objective of this study was to investigate the effects of treatment methods for meniscus on clinical and radiological outcomes at 2 years after ACL reconstruction. METHODS: Three-hundred and eighteen patients with primary ACL reconstruction using autologous hamstring tendon registered in our multicenter study database and who were followed up for 2 years were included. They were then divided into 3 groups, the no meniscal lesion/untreated group (n = 149), the meniscal repair group (n = 139), and the meniscal resection group (n = 30). Patient-based subjective evaluations (Lysholm score, Knee injury and Osteoarthritis Outcome score and International Knee Documentation Committee subjective score), objective evaluations (Lachman test, pivot shift test and KT measurement), and radiological measurements (medial and lateral joint space width) were compared among the 3 groups preoperatively and at 2 years follow-up. RESULTS: All subjective scores and objective evaluations significantly improved in all groups without significant differences among the groups postoperatively. Regarding radiological findings, the medial joint space width significantly decreased only in the resection group during the 2-year period, and the medial joint space width in the resection group was significantly smaller than that of the other groups at the 2-year follow-up. Moreover, the medial joint space width significantly decreased during the 2-year period when MM was resected. CONCLUSIONS: In radiological findings, medial meniscus resection decreased medial joint space width two years after ACL reconstruction. On the other hand, treatment methods for meniscus neither significantly affected subjective nor objective findings until the 2-year follow-up. LEVEL OF EVIDENCE: Ⅱ, Cohort study.

Novel method for l-methionine determination using l-methionine decarboxylase and application of the enzyme for l-homocysteine determination.

For many years, clinical studies have suggested that blood levels of l-methionine and L-homocysteine correlate with health status or homocystinuria/hypermethioninemia. l-Methionine in a solution containing 0%, 10%, or 20% human serum was detected in 10-200 µM using l-methionine decarboxylase (MetDC). Spike and recovery tests showed that the enzymatic assay could accurately and reproducibly determine the increases in l-methionine in serum samples. These results suggest that our enzymatic method using MetDC is useful for primary screening of hypermethioninemia or homocystinuria based on serum l-methionine concentration. Additionally, we confirmed that l-methionine (100 nmol) in solution was degraded to less than the detection limit by incubation at 37ºC for 10 min using 2 U of MetDC. Therefore, l-homocysteine in serum samples can be detected with equivalent sensitivity using l-methionine γ-lyase (MGL), in solutions that either did not contain l-methionine or contained l-methionine preincubated with MetDC.Abbreviations: DTT: dithiothreitol; IPTG: isopropyl-ß-d-thiogalactopyranoside; KPB: potassium phosphate buffer; MBTH: 3-methyl-2-benzothiazolinonehydrazone; mdc: the gene coding l-methionine decarboxylase; MetDC: l-methionine decarboxylase; mgl: the gene coding l-methionine γ-lyase; MGL: l-methionine γ-lyase; PLP: pyridoxal 5'-phosphate.

Architecture of the TIM23 inner mitochondrial translocon and interactions with the matrix import motor.

Translocation of proteins from the cytosol across the mitochondrial inner membrane is driven by action of the matrix-localized multi-subunit import motor, which is associated with the TIM23 translocon. The architecture of the import apparatus is not well understood. Here, we report results of site-specific in vivo photocross-linking along with genetic and coimmunoprecipitation analyses dissecting interactions between import motor subunits and the translocon. The translocon is composed of the two integral membrane proteins Tim23 and Tim17, each containing four membrane-spanning segments. We found that Tim23 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane domains 1 and 2 (loop 1) cross-linked to Tim44. Alterations in this loop destabilized interaction of Tim44 with the translocon. Analogously, Tim17 having a photoactivatable cross-linker in the matrix exposed loop between transmembrane segments 1 and 2 (loop 1) cross-linked to Pam17. Alterations in this loop caused destabilization of the interaction of Pam17 with the translocon. Substitution of individual photoactivatable residues in Tim44 and Pam17 in regions we previously identified as important for translocon association resulted in cross-linking to Tim23 and Tim17, respectively. Our results are consistent with a model in which motor association is achieved via interaction of Tim23 with Tim44, which serves as a scaffold for association of other motor components, and of Tim17 with Pam17. As both Tim44 and Pam17 have been implicated as regulatory subunits of the motor, this positioning is conducive for responding to conformational changes in the translocon upon a translocating polypeptide entering the channel.

Older Age, Poor Preoperative Quadriceps Muscle Strength, and Residual Pain as Risk Factors for Poor Quadriceps Muscle Strength Recovery at 1 Year After ACL Reconstruction: A TMDU MAKS Study of 402 Patients.

Background: Poor postoperative quadriceps muscle strength recovery after anterior cruciate ligament reconstruction (ACLR) leads to delayed return to sports and lower patient satisfaction. Purpose/Hypothesis: The purpose of this study was to examine factors that affect quadriceps muscle strength 1 year after ACLR. It was hypothesized that older age, poor preoperative quadriceps muscle strength, and residual pain would be risk factors for poor quadriceps muscle strength recovery. Study Design: Case-control study; Level of evidence, 3. Methods: Included were patients from multiple institutions who underwent primary ACLR using autologous hamstring tendon grafts between August 1, 2013, and March 31, 2018, and who had at least 1 year of follow-up data. Patients with past ligamentous injuries in the affected knee, previous injuries or operations in the contralateral knee, accompanying ligament injuries of grade 2 or 3, or inflammatory or other types of osteoarthritis were excluded. Patients were categorized as having muscle strength ≥80% (good strength recovery) or <80% (poor strength recovery) compared with the contralateral leg at 1 year postoperatively. Multivariate logistic regression analysis was performed to investigate the factors influencing postoperative quadriceps muscle strength. In addition, a categorical analysis was conducted based on factors extracted by the multivariate logistic regression analysis. Results: A total of 402 patients were included. Multivariate logistic regression analysis revealed that age at surgery (P = .020), preoperative quadriceps muscle strength (P = .006), and postoperative Knee injury and Osteoarthritis Outcome Score (KOOS)-Pain score (P = .002) were significantly associated with quadriceps muscle strength at 1 year postoperatively. The odds of poor muscle strength recovery according to categorical analysis were 5.0-fold higher for patients aged >40 versus ≤20 years, 4.2-fold higher for those with preoperative quadriceps muscle index <60% versus ≥80%, and 7.7-fold higher for those with a postoperative KOOS-Pain score of <85 versus 100. Conclusion: Older age, poor preoperative quadriceps muscle strength, and low postoperative KOOS-Pain score were risk factors for poor quadriceps muscle strength 1 year after primary ACLR. Surgical indications, including age, preoperative active rehabilitation, and pain control, should be considered for optimization of postoperative quadriceps muscle strength recovery.

Ancient gene duplication provided a key molecular step for anaerobic growth of Baker's yeast.

Mitochondria are essential organelles required for a number of key cellular processes. As most mitochondrial proteins are nuclear encoded, their efficient translocation into the organelle is critical. Transport of proteins across the inner membrane is driven by a multicomponent, matrix-localized "import motor," which is based on the activity of the molecular chaperone Hsp70 and a J-protein cochaperone. In Saccharomyces cerevisiae, two paralogous J-proteins, Pam18 and Mdj2, can form the import motor. Both contain transmembrane and matrix domains, with Pam18 having an additional intermembrane space (IMS) domain. Evolutionary analyses revealed that the origin of the IMS domain of S. cerevisiae Pam18 coincides with a gene duplication event that generated the PAM18/MDJ2 gene pair. The duplication event and origin of the Pam18 IMS domain occurred at the relatively ancient divergence of the fungal subphylum Saccharomycotina. The timing of the duplication event also corresponds with a number of additional functional changes related to mitochondrial function and respiration. Physiological and genetic studies revealed that the IMS domain of Pam18 is required for efficient growth under anaerobic conditions, even though it is dispensable when oxygen is present. Thus, the gene duplication was beneficial for growth capacity under particular environmental conditions as well as diversification of the import motor components.

Anatomical Repair of a Femoral-Sided Avulsion Fracture of the Anterior Cruciate Ligament: A Case Report.

CASE: We report a rare case of femoral-sided avulsion fracture of both the anteromedial and posterolateral bundle attachments of the anterior cruciate ligament (ACL). We performed an arthroscopic double-bundle pull-out repair. At the 1-year follow-up, the patient had no deformity, laxity of the knee, and no limitations when engaging in various sports activities. CONCLUSION: An avulsion fracture of both the anteromedial and posterolateral bundle attachments is a rare injury. Arthroscopically assisted reduction and fixation demonstrated successful achievement of both bone union and good ACL function.

Structural basis for substrate specificity of l-methionine decarboxylase.

l -Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B6 -dependent enzyme and catalyzes the non-oxidative decarboxylation of l -methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.

Gene cloning, recombinant expression, purification and characterization of l-methionine decarboxylase from Streptomyces sp. 590.

l-Methionine decarboxylase (MetDC) from Streptomyces sp. 590 depends on pyridoxal 5'-phosphate and catalyzes the non-oxidative decarboxylation of l-methionine to produce 3-methylthiopropylamine and carbon dioxide. MetDC gene (mdc) was determined to consist of 1,674 bp encoding 557 amino acids, and the amino acid sequence is similar to that of l-histidine decarboxylases and l-valine decarboxylases from Streptomyces sp. strains. The mdc gene was cloned and recombinant MetDC was heterologously expressed by Escherichia coli. The purification of recombinant MetDC was carried out by DEAE-Toyopearl and Ni-NTA agarose column chromatography. The recombinant enzyme was homodimeric with a molecular mass of 61,000 Da and showed optimal activity between 45 to 55 °C and at pH 6.6, and the stability below 30 °C and between pH 4.6 to 7.0. l-Methionine and l-norleucine were good substrates for MetDC. The Michaelis constants for l-methionine and l-norleucine were 30 and 73 mM, respectively. The recombinant MetDC (0.50 U/ml) severely inhibited growth of human tumour cells A431 (epidermoid ovarian carcinoma cell line) and MDA-MB-231 (breast cancer cell line), however showed relatively low cytotoxicity for human normal cell NHDF-Neo (dermal fibroblast cell line from neonatal foreskin). This study revealed the properties of the gene and the protein sequence of MetDC for the first time.

Genetic analysis of complex interactions among components of the mitochondrial import motor and translocon in Saccharomyces cerevisiae.

A highly conserved, Hsp70-based, import motor, which is associated with the translocase on the matrix side of the inner mitochondrial membrane, is critical for protein translocation into the matrix. Hsp70 is tethered to the translocon via interaction with Tim44. Pam18, the J-protein co-chaperone, and Pam16, a structurally related protein with which Pam18 forms a heterodimer, are also critical components of the motor. Their N termini are important for the heterodimer's translocon association, with Pam18's and Pam16's N termini interacting in the intermembrane space and the matrix, respectively. Here, using the model organism Saccharomyces cerevisiae, we report the identification of an N-terminal segment of Tim44, important for association of Pam16 with the translocon. We also report that higher amounts of Pam17, a nonessential motor component, are found associated with the translocon in both PAM16 and TIM44 mutants that affect their interaction with one another. These TIM44 and PAM16 mutations are also synthetically lethal with a deletion of PAM17. In contrast, a deletion of PAM17 has little, or no genetic interaction with a PAM18 mutation that affects translocon association of the Pam16:Pam18 heterodimer, suggesting a second role for the Pam16:Tim44 interaction. A similar pattern of genetic interactions and enhanced Pam17 translocon association was observed in the absence of the C terminus of Tim17, a core component of the translocon. We suggest the Pam16:Tim44 interaction may play two roles: (1) tethering the Pam16:Pam18 heterodimer to the translocon and (2) positioning the import motor for efficient engagement with the translocating polypeptide along with Tim17 and Pam17.

Intra-articular injections of bone morphogenetic protein-7 retard progression of existing cartilage degeneration.

We investigated the effect of weekly intra-articular injections of bone morphogenetic protein-7 (BMP-7) on prevention of progression of existing cartilage degeneration in an osteoarthritis model in rabbits. An anterior cruciate ligament transection (ACLT) model was used to create a progressive osteoarthritis model. BMP-7 was intra-articular injected weekly into the right knee and PBS into the left knee from 4 weeks after ACLT. Both sides of the knees were compared macroscopically, histologically, immunohistochemically, and by micro CT. Macroscopically, fibrillation in the femoral condyle was observed 4 weeks after ACLT. In the control knees, cartilage degeneration further progressed throughout the 12-week period. In the BMP-7 treated knee, osteoarthritis progression was milder than in the control knees. Histologically, safranin-O staining was decreased in the surgical knees at 4 weeks. Obvious erosions in both medial and lateral condyles were revealed in the control knees at 12 weeks, while cartilage matrix was predominantly retained in the BMP-7 treated knees. The macroscopic and microscopic OA score in the BMP-7 treated knee was better than that in the control in each rabbit. Immunohistochemical analysis demonstrated that both type II collagen and BMP-7 were more expressed in cartilage treated with BMP-7. Micro CT analysis showed that osteophytes were smaller in the BMP-7 treated knee compared to that of the control. Weekly intra-articular injections of BMP-7 inhibited progression of existing cartilage degeneration.

Periodic knee injections of BMP-7 delay cartilage degeneration induced by excessive running in rats.

Strenuous running of rats enhances mechanical stress on the knee, thereby inducing degeneration of articular cartilage. Bone morphogenetic protein-7 (BMP-7) has an inhibitory effect on cartilage degeneration, suggesting its usefulness for human osteoarthritis patients. However, its mode of administration should be investigated. We examined whether weekly knee injections of BMP-7 delayed the progression of cartilage degeneration. Wistar rats were forced to run 30 km in 6 weeks on a rodent treadmill, and BMP-7 was injected weekly into the knee. Macroscopically and histologically, this strenuous running regimen induced cartilage degeneration. Weekly injections of 250 ng BMP-7 delayed the progression of cartilage degeneration. Immunohistochemically, in the control knee, type II collagen expression decreased, while BMP-7 expression in chondrocytes slightly increased. Interestingly, weekly injection of BMP-7 increased BMP-7 expression even 9 days after the final injection. Disulfate disaccharide keratan sulfate in serum transiently increased in the control group, while it remained at a low level in the BMP-7 group. Weekly BMP-7 injection increased BMP-7 expression in chondrocytes and its effect seemed to last more than 7 days. The effect of BMP-7 could be monitored by serum keratan sulfate concentration. Periodical injections of BMP-7 delayed progression of cartilage degeneration induced by excessive running in rats.

Interaction of the J-protein heterodimer Pam18/Pam16 of the mitochondrial import motor with the translocon of the inner membrane.

Import of proteins across the inner mitochondrial membrane through the Tim23:Tim17 translocase requires the function of an essential import motor having mitochondrial 70-kDa heat-shock protein (mtHsp70) at its core. The heterodimer composed of Pam18, the J-protein partner of mtHsp70, and the related protein Pam16 is a critical component of this motor. We report that three interactions contribute to association of the heterodimer with the translocon: the N terminus of Pam16 with the matrix side of the translocon, the inner membrane space domain of Pam18 (Pam18(IMS)) with Tim17, and the direct interaction of the J-domain of Pam18 with the J-like domain of Pam16. Pam16 plays a major role in translocon association, as alterations affecting the stability of the Pam18:Pam16 heterodimer dramatically affect association of Pam18, but not Pam16, with the translocon. Suppressors of the growth defects caused by alterations in the N terminus of Pam16 were isolated and found to be due to mutations in a short segment of TIM44, the gene encoding the peripheral membrane protein that tethers mtHsp70 to the translocon. These data suggest a model in which Tim44 serves as a scaffold for precise positioning of mtHsp70 and its cochaperone Pam18 at the translocon.

Weekly intra-articular injections of bone morphogenetic protein-7 inhibits osteoarthritis progression.

INTRODUCTION: We investigated the ability of a weekly intra-articular injection of bone morphogenetic protein (BMP)-7 to prevent osteoarthritis in rabbits with anterior cruciate ligament transections. METHODS: First, 36 knee joints were randomly divided into four groups: 50, 500, 5,000 ng BMP-7, and control. Knee cartilage was evaluated at 4, 8, and 12 weeks. Then, in order to control for individual differences, 500 ng BMP-7 was injected into one knee and phosphate-buffered saline (PBS) into the other, and the two knees were compared at 4, 8, and 12 weeks (n = 5). For pharmacokinetic analysis, cartilage was harvested at 1 hour and 1, 2, 4, and 7 days after knee injection of 500 ng BMP-7 or PBS (n = 3). RESULTS: Histological scores in the 500 and 5,000 ng BMP-7 groups were significantly better than those in the other groups at 12 weeks. Matched pair analysis demonstrated that both macroscopic and histological scores in the 500 ng BMP-7 group were better than those in the control group. Immunohistochemical analysis revealed higher BMP-7 expression by chondrocytes in the BMP-7 injected knees. Histology of whole knee and quantitative micro computed tomography analysis showed that weekly injections of 500 ng BMP-7 did not induce synovial fibrosis, ectopic bone, or osteophyte formation. As detected by enzyme-linked immunosorbent assay, BMP-7 concentration in the cartilage tissue was still higher in the BMP-7 treated group 7 days after the injection. CONCLUSIONS: Weekly intra-articular injections of BMP-7 inhibited progression of osteoarthritis. Obvious adverse effects were not observed. BMP-7 concentration and expression in cartilage were still higher 7 days after injection.

Local adherent technique for transplanting mesenchymal stem cells as a potential treatment of cartilage defect.

INTRODUCTION: Current cell therapy for cartilage regeneration requires invasive procedures, periosteal coverage and scaffold use. We have developed a novel transplantation method with synovial mesenchymal stem cells (MSCs) to adhere to the cartilage defect. METHODS: For ex vivo analysis in rabbits, the cartilage defect was faced upward, filled with synovial MSC suspension, and held stationary for 2.5 to 15 minutes. The number of attached cells was examined. For in vivo analysis in rabbits, an autologous synovial MSC suspension was placed on the cartilage defect, and the position was maintained for 10 minutes to adhere the cells to the defect. For the control, either the same cell suspension was injected intra-articularly or the defects were left empty. The three groups were compared macroscopically and histologically. For ex vivo analysis in humans, in addition to the similar experiment in rabbits, the expression and effects of neutralizing antibodies for adhesion molecules were examined. RESULTS: Ex vivo analysis in rabbits demonstrated that the number of attached cells increased in a time-dependent manner, and more than 60% of cells attached within 10 minutes. The in vivo study showed that a large number of transplanted synovial MSCs attached to the defect at 1 day, and the cartilage defect improved at 24 weeks. The histological score was consistently better than the scores of the two control groups (same cell suspension injected intra-articularly or defects left empty) at 4, 12, and 24 weeks. Ex vivo analysis in humans provided similar results to those in rabbits. Intercellular adhesion molecule 1-positive cells increased between 1 minute and 10 minutes, and neutralizing antibodies for intercellular adhesion molecule 1, vascular cell adhesion molecule 1 and activated leukocyte-cell adhesion molecule inhibited the attachment. CONCLUSION: Placing MSC suspension on the cartilage defect for 10 minutes resulted in adherence of >60% of synovial MSCs to the defect, and promoted cartilage regeneration. This adherent method makes it possible to adhere MSCs with low invasion, without periosteal coverage, and without a scaffold.

Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes.

Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (PhiBL) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in PhiBL was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and PhiBL to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and PhiBL in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of PhiBL.