Rational design of oligonucleotides for enhanced in vitro transcription of small RNA.

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.

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs.

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.

A selective and sensitive detection system for 4-thiouridine modification in RNA.

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.

Genome-wide analysis of the in vivo tRNA structurome reveals RNA structural and modification dynamics under heat stress.

RNA structure plays roles in myriad cellular events including transcription, translation, and RNA processing. Genome-wide analyses of RNA secondary structure in vivo by chemical probing have revealed critical structural features of mRNAs and long ncRNAs. Here, we examine the in vivo secondary structure of a small RNA class, tRNAs. Study of tRNA structure is challenging because tRNAs are heavily modified and strongly structured. We introduce "tRNA structure-seq," a new workflow that accurately determines in vivo secondary structures of tRNA. The workflow combines dimethyl sulfate (DMS) probing, ultra-processive RT, and mutational profiling (MaP), which provides mutations opposite DMS and natural modifications thereby allowing multiple modifications to be identified in a single read. We applied tRNA structure-seq to E. coli under control and stress conditions. A leading folding algorithm predicts E. coli tRNA structures with only ∼80% average accuracy from sequence alone. Strikingly, tRNA structure-seq, by providing experimental restraints, improves structure prediction under in vivo conditions to ∼95% accuracy, with more than 14 tRNAs predicted completely correctly. tRNA structure-seq also quantifies the relative levels of tRNAs and their natural modifications at single nucleotide resolution, as validated by LC-MS/MS. Our application of tRNA structure-seq yields insights into tRNA structure in living cells, revealing that it is not immutable but has dynamics, with partial unfolding of secondary and tertiary tRNA structure under heat stress that is correlated with a loss of tRNA abundance. This method is applicable to other small RNAs, including those with natural modifications and highly structured regions.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase.

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.

Short-term clinical results of bicruciate-retaining total knee arthroplasty using personalized alignment.

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.

Risk factors for hamstring strain injury in male college American football players -a preliminary prospective cohort study.

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.

Postoperative quadriceps weakness and male sex are risk factors for patellofemoral articular cartilage lesions after anatomical anterior cruciate ligament reconstruction.

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.

Single-nucleotide control of tRNA folding cooperativity under near-cellular conditions.

RNA folding is often studied by renaturing full-length RNA in vitro and tracking folding transitions. However, the intracellular transcript folds as it emerges from the RNA polymerase. Here, we investigate the folding pathways and stability of numerous late-transcriptional intermediates of yeast and Escherichia coli transfer RNAs (tRNAs). Transfer RNA is a highly regulated functional RNA that undergoes multiple steps of posttranscriptional processing and is found in very different lengths during its lifetime in the cell. The precursor transcript is extended on both the 5' and 3' ends of the cloverleaf core, and these extensions get trimmed before addition of the 3'-CCA and aminoacylation. We studied the thermodynamics and structures of the precursor tRNA and of late-transcriptional intermediates of the cloverleaf structure. We examined RNA folding at both the secondary and tertiary structural levels using multiple biochemical and biophysical approaches. Our findings suggest that perhaps nature has selected for a single-base addition to control folding to the functional 3D structure. In near-cellular conditions, yeast tRNAPhe and E. coli tRNAAla transcripts fold in a single, cooperative transition only when nearly all of the nucleotides in the cloverleaf are transcribed by indirectly enhancing folding cooperativity. Furthermore, native extensions on the 5' and 3' ends do not interfere with cooperative core folding. This highly controlled cooperative folding has implications for recognition of tRNA by processing and modification enzymes and quality control of tRNA in cells.

Effects of a 1-mm difference in bearing thickness on intraoperative bearing movement and kinematics in Oxford unicompartmental knee arthroplasty.

BACKGROUND: The choice of mobile bearing (MB) thickness is essential for obtaining successful results after mobile-bearing Oxford unicompartmental knee arthroplasty (UKA). This study aimed to investigate the effects of a 1-mm difference in bearing thickness on intraoperative MB movement and intraoperative knee kinematics in Oxford UKAs. METHODS: We prospectively investigated the effects of a 1-mm difference in bearing thickness on intraoperative MB movement and knee kinematics in 25 patients who underwent Oxford UKAs when surgeons didn't know which bearing thickness to choose with 1-mm difference. A trial tibial component that was scaled every 2 mm was used to measure the intraoperative MB movement, and the tibial internal rotation relative to the femur and the knee varus angle was simultaneously evaluated using the navigation system as the knee kinematics. We separately evaluated sets of two MB thicknesses with 1-mm differences, and we compared the intraoperative parameters at maximum extension; 30º, 45º, 60º, and 90º flexion; and maximum flexion between the thicker MB (thick group) and the thinner MB (thin group). RESULTS: The MB in the thin group was located significantly posteriorly at 90º flexion compared with that in the thick group; however, there were no differences at the other flexion angles. There was significantly less tibial internal rotation in the thin group at 90º flexion than that in the thick group; however, there were no differences at the other flexion angles. The knee varus angles in the thick group were significantly smaller than those in the thin group by approximately one degree at all angles other than at 30º and 45º flexion. CONCLUSION: The thicker MB could bring the less posterior MB movement and the more tibial internal rotation at 90º flexion, additionally the valgus correction angle in the thicker MB should be paid attention. These results could help surgeons to decide the thickness of MBs when they wonder the thickness of MB.

Femoral rollback at high-flexion during squatting is related to the improvement of sports activities after bicruciate-stabilized total knee arthroplasty: an observational study.

BACKGROUND: In bicruciate-stabilized total knee arthroplasty (BCS-TKA), the relationship between the postoperative kinematics and sports subscales in patient-reported outcome measures (PROMs) remains unknown. The purpose of this study was to determine the relationship between kinematics and sports subscales using the PROMs after BCS-TKA. METHODS: Sixty-one patients with severe knee osteoarthritis were examined at 13.5 ± 7.8 months after BCS-TKA. The patients performed squats under single fluoroscopic surveillance in the sagittal plane. Range of motion of the knee, axial rotation of the femur relative to the tibial component, and anteroposterior (AP) translation of the medial and lateral femorotibial contact points were measured using a 2D-to-3D registration technique. In addition, the relationship between the kinematics and improvement of the sports subscales in the Knee Injury and Osteoarthritis Outcome Score (KOOS) was evaluated. RESULTS: In rotation angle, the femoral external rotation was observed from 0 to 50° of flexion. The amount of femoral external rotation did not correlate with PROMs-SP. In medial AP translation, posterior translation was observed from 0 to 20° and 80-110° of flexion. Mild anterior translation was observed from 20 to 80° of flexion. Beyond 80° of flexion, posterior translation was positively correlated with squatting. In lateral AP translation, posterior translation was observed from 0 to 20° and 80-110° of flexion. Beyond 80° of flexion, posterior translation was positively correlated with running, jumping, twisting/pivoting, and kneeling. CONCLUSION: Femoral rollback at high flexion during squatting may be important to improve sports performance after BCS-TKA.

The higher patient-reported outcome measure group had smaller external rotation of the femur in bicruciate-stabilized total knee arthroplasty.

PURPOSE: The hypothesis of this study was that the kinematics of patients with higher patient-reported outcome measures (PROMs) differ from those of patients with lower PROMs after bicruciate-stabilized total knee arthroplasty (BCS-TKA). METHODS: A total of 32 patients with severe knee osteoarthritis were examined 11.2 ± 3.2 months after BCS-TKA. The patients performed squats under single fluoroscopic surveillance in the sagittal plane. To estimate the spatial position and orientation of the femoral and tibial components, a 2D-to-3D registration technique was used. This technique uses a contour-based registration algorithm, single-view fluoroscopic images and 3D computer-aided design models. Knee range of motion, varus-valgus alignment, axial rotation of the femur relative to the tibial component, anteroposterior translation of the medial and lateral femorotibial contact points, kinematic paths, and anterior and posterior post-cam engagement were measured. The patients were divided into two groups using hierarchical cluster analysis based on the 1-year postoperative Knee Injury and Osteoarthritis Outcome Score and 2011 Knee Society Score. RESULTS: The femoral component had significantly more external rotation in the low-score group than in the high-score group (5.1 ± 1.8° vs. 2.2 ± 2.7°, p = 0.02). The high-score group had a medial pivot pattern from 0 to 20° of flexion, without significant movement from 20 to 70°, and final bicondylar rollback beyond 70°. The low-score group had a medial pivot from 0 to 70° of flexion and bicondylar rollback beyond 70°. There were no significant between-group differences in the varus-valgus angle or post-cam engagement. CONCLUSION: The higher PROM group had smaller external rotation of the femur after BCS-TKA. LEVEL OF EVIDENCE: Level III.

Required Elements in tRNA for Methylation by the Eukaryotic tRNA (Guanine-N2-) Methyltransferase (Trm11-Trm112 Complex).

The Saccharomyces cerevisiae Trm11 and Trm112 complex (Trm11-Trm112) methylates the 2-amino group of guanosine at position 10 in tRNA and forms N2-methylguanosine. To determine the elements required in tRNA for methylation by Trm11-Trm112, we prepared 60 tRNA transcript variants and tested them for methylation by Trm11-Trm112. The results show that the precursor tRNA is not a substrate for Trm11-Trm112. Furthermore, the CCA terminus is essential for methylation by Trm11-Trm112, and Trm11-Trm112 also only methylates tRNAs with a regular-size variable region. In addition, the G10-C25 base pair is required for methylation by Trm11-Trm112. The data also demonstrated that Trm11-Trm112 recognizes the anticodon-loop and that U38 in tRNAAla acts negatively in terms of methylation. Likewise, the U32-A38 base pair in tRNACys negatively affects methylation. The only exception in our in vitro study was tRNAValAAC1. Our experiments showed that the tRNAValAAC1 transcript was slowly methylated by Trm11-Trm112. However, position 10 in this tRNA was reported to be unmodified G. We purified tRNAValAAC1 from wild-type and trm11 gene deletion strains and confirmed that a portion of tRNAValAAC1 is methylated by Trm11-Trm112 in S. cerevisiae. Thus, our study explains the m2G10 modification pattern of all S. cerevisiae class I tRNAs and elucidates the Trm11-Trm112 binding sites.

High initial graft tension increases external tibial rotation on the axial plane after anatomical anterior cruciate ligament reconstruction.

PURPOSE: The purpose of this study was to clarify the effects of applying different amounts of initial graft tension on the femorotibial positional relationship on the axial plane after anatomical ACL reconstruction. METHODS: Eighty patients who underwent isolated ACL reconstructions using bone-patellar tendon-bone grafts were included in this study. In 40 of the 80 patients, the grafts were fixed at full knee extension with maximum manual force (high graft tension; Group H), whereas in the other 40 patients, the grafts were fixed at full knee extension with force of 80 N (low graft tension; Group L). One week postoperatively, all patients underwent computed tomography (CT) on bilateral knee joints with knee extension. The femorotibial positional relationship in axial CT images were retrospectively evaluated. Side-to-side differences (the surgical side minus the unaffected side) were calculated in these variables. RESULTS: The side-to-side differences in anterior tibial translation distances were - 1.8 ± 2.1 mm in Group H and - 1.9 ± 2.0 mm in Group L, with no significant difference between the two groups. The side-to-side differences in tibial lateral shifts were - 0.2 ± 1.5 mm in Group H and 0.0 ± 1.4 mm in Group L, with no significant difference between the two groups. The side-to-side differences in tibial external rotation angles were 2.7 ± 4.5° in Group H and 0.3 ± 3.3° in Group L, with a significant difference between the two groups (P < 0.01). CONCLUSION: Applying high initial graft tension (maximum manual force) resulted in the external rotation of the tibia against the femur just after anatomical ACL reconstruction. In contrast, applying low initial graft tension (80 N at full knee extension) did not change the femorotibial rotational relationship.

Functional Roles of Chelated Magnesium Ions in RNA Folding and Function.

RNA regulates myriad cellular events such as transcription, translation, and splicing. To perform these essential functions, RNA often folds into complex tertiary structures in which its negatively charged ribose-phosphate backbone interacts with metal ions. Magnesium, the most abundant divalent metal ion in cells, neutralizes the backbone, thereby playing essential roles in RNA folding and function. This has been known for more than 50 years, and there are now thousands of in vitro studies, most of which have used ≥10 mM free Mg2+ ions to achieve optimal RNA folding and function. In the cell, however, concentrations of free Mg2+ ions are much lower, with most Mg2+ ions chelated by metabolites. In this Perspective, we curate data from a number of sources to provide extensive summaries of cellular concentrations of metabolites that bind Mg2+ and to estimate cellular concentrations of metabolite-chelated Mg2+ species, in the representative prokaryotic and eukaryotic systems Escherichia coli, Saccharomyces cerevisiae, and iBMK cells. Recent research from our lab and others has uncovered the fact that such weakly chelated Mg2+ ions can enhance RNA function, including its thermodynamic stability, chemical stability, and catalysis. We also discuss how metabolite-chelated Mg2+ complexes may have played roles in the origins of life. It is clear from this analysis that bound Mg2+ should not be simply considered non-RNA-interacting and that future RNA research, as well as protein research, could benefit from considering chelated magnesium.

Design of highly active double-pseudoknotted ribozymes: a combined computational and experimental study.

Design of RNA sequences that adopt functional folds establishes principles of RNA folding and applications in biotechnology. Inverse folding for RNAs, which allows computational design of sequences that adopt specific structures, can be utilized for unveiling RNA functions and developing genetic tools in synthetic biology. Although many algorithms for inverse RNA folding have been developed, the pseudoknot, which plays a key role in folding of ribozymes and riboswitches, is not addressed in most algorithms. For the few algorithms that attempt to predict pseudoknot-containing ribozymes, self-cleavage activity has not been tested. Herein, we design double-pseudoknot HDV ribozymes using an inverse RNA folding algorithm and test their kinetic mechanisms experimentally. More than 90% of the positively designed ribozymes possess self-cleaving activity, whereas more than 70% of negative control ribozymes, which are predicted to fold to the necessary structure but with low fidelity, do not possess it. Kinetic and mutation analyses reveal that these RNAs cleave site-specifically and with the same mechanism as the WT ribozyme. Most ribozymes react just 50- to 80-fold slower than the WT ribozyme, and this rate can be improved to near WT by modification of a junction. Thus, fast-cleaving functional ribozymes with multiple pseudoknots can be designed computationally.

Intraoperative rotational kinematics and its influence on postoperative clinical outcomes differ according to age in Unicompartmental knee Arthroplasty.

BACKGROUND: Although Oxford unicompartmental knee arthroplasty (UKA) is used in patients of wide age ranges, there is no clear information regarding the age differences in terms of intraoperative femorotibial rotational kinematics and its influence on clinical outcomes. Therefore, this study was conducted to examine the age differences in terms of intraoperative rotational kinematics and postoperative clinical outcomes and to analyze their relationship with classification according to the age group. METHODS: We investigated 111 knees of patients who underwent Oxford UKA using a navigation system and divided them into two groups: elderly (aged ≥75 years; 48 knees) and nonelderly (aged < 75 years; 63 knees). Intraoperative tibial internal rotational angles relative to the femur during passive knee flexion were measured using a navigation system, and clinical outcomes were evaluated using knee range of motion, the Knee Injury and Osteoarthritis Outcome Score (KOOS), and the Knee Society Functional Score at 2 years postoperatively. The relationships between intraoperative tibiofemoral rotational angles and clinical outcomes were also evaluated in the two groups. RESULTS: The intraoperative tibial internal rotational angle relative to the femur during knee flexion was significantly larger in the nonelderly group (13.5°) than in the elderly group (9.0°). The intraoperative tibial internal rotational angle showed a positive correlation with the pain subscale of KOOS only in the nonelderly group. CONCLUSION: Intraoperative rotational kinematics and its influence on clinical outcomes were different between elderly and nonelderly patients, and the tibial internal rotational angle could be a more important factor for successful UKA in nonelderly patients.

Influence of surgical factors on patient satisfaction after bi-cruciate stabilized total knee arthroplasty: retrospective examination using multiple regression analysis.

BACKGROUND: One of the causes of dissatisfaction following total knee arthroplasty (TKA) is abnormal knee kinematics. A newly designed bi-cruciate stabilized (BCS) TKA system has been developed to produce close-to-normal kinematics because of its anatomic tibiofemoral articular geometry and cam-post mechanism. Although BCS TKA is expected to improve patient satisfaction, no reports have described the appropriate technique or soft tissue handling required to achieve excellent satisfaction with BCS TKA. This study is to identify intraoperative surgical predictors of patient satisfaction after BCS TKA. METHODS: We studied 104 knees with primary varus knee osteoarthritis that underwent BCS TKA with a navigation system retrospectively. Surgical parameters including coronal, sagittal and axial alignment and joint laxity in each compartment that affected patient satisfaction was evaluated. Satisfaction score was evaluated with use of the 2011 Knee Society Scoring system. The multivariate regression analysis included age, gender, body mass index and intraoperative parameters that correlated with satisfaction scores in the univariate analysis (P < 0.05). The current study focused on the patient satisfaction score at 1 year postoperatively and didn't evaluate the long term clinical results nor survivorship. RESULTS: The postoperative satisfaction score was 28.6 ± 8.1. Multivariate analysis showed that medial joint laxity at 30° flexion (P = 0.003), tibial excessive external rotation alignment (P = 0,009) and tibial varus alignment (P = 0.029) were predictors of poor satisfaction score. CONCLUSIONS: When performing BCS TKA, surgeons should pay attention to maintaining proper stability of the medial compartment at mid flexion range and should avoid tibial varus and excessive external rotational alignment.

Weight-bearing knee flexion angle better correlates with patient-reported outcome measures than non-weight-bearing condition in total knee arthroplasty: a three-dimensional analysis study.

BACKGROUND: This study aims to elucidate and compare the relationship between the knee flexion angle and patient-reported outcome measures (PROM) in both non-weight-bearing (NWB) and weight-bearing (WB) conditions. METHODS: This retrospective cohort study included 61 knees (47 patients) who underwent total knee arthroplasty. The knee flexion angle was measured by three conditions: NWB in manual goniometer, NWB in fluoroscopic three-dimensional (3D) analysis and WB in the fluoroscopic 3D analysis. The PROM was evaluated by postoperative 2011 Knee Society Score (2011 KSS) and Knee injury and Osteoarthritis Outcome Score (KOOS). Correlations between the knee flexion angle and PROM was analyzed using Spearman's correlation coefficient. Additionally, whether the angular difference between NWB and WB correlated with the PROM or not was evaluated. RESULTS: The NWB knee flexion angle in a goniometer, NWB in 3D analysis, and WB in 3D analysis were 124.6° ± 8.4°, 118.0° ± 10.5°, and 109.5° ± 13.3°, respectively. The angular difference was 8.5° ± 12.8°. No PROM correlation existed in NWB using a goniometer. Moreover, significant positive correlations in 2011 KSS symptoms (r = 0.35) and 2011 KSS functional activities (r = 0.27) were noted in NWB using 3D analysis. Significant positive correlations existed in 2011 KSS symptoms (r = 0.32), 2011 KSS functional activities (r = 0.57), KOOS pain (r = 0.37), KOOS activity of daily living (ADL; r = 0.45), KOOS sports (r = 0.42), and KOOS quality of life (r = 0.36) in WB using 3D analysis. Significant negative correlations were noted in 2011 KSS functional activities (r = - 0.45), KOOS ADL (r = - 0.30), and KOOS sports (r = - 0.38) in angular difference. CONCLUSIONS: The WB knee flexion angle better correlated with PROM compared with NWB by evaluation of 3D analysis. The larger the angular difference existed between NWB and WB, the lower the PROM score.

Both intraoperative medial and lateral soft tissue balances influence intraoperative rotational knee kinematics in bi-cruciate stabilized total knee arthroplasty: A retrospective investigation.

BACKGROUND: Tibial internal rotation following total knee arthroplasty (TKA) is important in achieving favorable postoperative clinical outcomes. Studies have reported the effect of intraoperative soft tissue balance on tibial internal rotation in conventional TKA, no studies have evaluated the effects of soft tissue balance at medial or lateral compartments separately on tibial internal rotation in bi-cruciate stabilized (BCS) TKA. The purpose of this study was to clarify the relationship between medial or lateral component gaps and rotational knee kinematics in BCS TKA. METHODS: One hundred fifty-eight knees that underwent BCS TKA were included in this study. The intraoperative medial and lateral joint laxities which was defined as the value of component gap minus the thickness of the tibial component were firstly divided into two groups, respectively: Group M-stable (medial joint laxity, ≤ 2 mm) or Group M-loose (medial joint laxity, ≥ 3 mm) and Group L-stable (lateral joint laxity, ≤ 3 mm) or Group L-loose (lateral joint laxity, ≥ 4 mm). And finally, the knees enrolled in this study were divided into four groups based on the combination of Group M and Group L: Group A (M-stable and L-stable), Group B (M-stable and L-loose), Group C (M-loose and L-stable), and Group D (M-loose and L-loose). The intraoperative rotational knee kinematics were compared between the four Groups at 0°, 30°, 60°, and 90° flexion, respectively. RESULTS: The rotational angular difference between 0° flexion and maximum flexion in Group B at 30° flexion was significantly larger than that in Group A at 30° flexion (*p < 0.05). The rotational angular difference between 30° flexion and maximum flexion in Group B at 30° flexion was significantly larger than that in Group D at 30° flexion (*p < 0.05). The rotational angular differences between 30° or 90° flexion and maximum flexion in Group B at 60° flexion were significantly larger than those in Group A at 60° flexion (*p < 0.05). CONCLUSION: Surgeons should pay attention to the importance of medial joint stability at midflexion and lateral joint laxities at midflexion and 90° flexion on a good tibial internal rotation in BCS TKA.