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Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGDRQC+) or RQC uncoupled (NGDRQC−). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGDRQC− but not for NGDRQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGDRQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGDRQC−), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGDRQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not4 and deubiquitinated by Otu2 upon ribosomal subunit recycling. Despite its importance for translation efficiency the exact role and structural basis for this translational reset is poorly understood. Here, structural analysis by cryo-electron microscopy of native and reconstituted Otu2-bound ribosomal complexes reveals that Otu2 engages 40S subunits mainly between ribosome recycling and initiation stages. Otu2 binds to several sites on the intersubunit surface of the 40S that are not occupied by any other 40S-binding factors. This binding mode explains the discrimination against 80S ribosomes via the largely helical N-terminal domain of Otu2 as well as the specificity for mono-ubiquitinated eS7 on 40S. Collectively, this study reveals mechanistic insights into the Otu2-driven deubiquitination steps for translational reset during ribosome recycling/(re)initiation.

Cells can respond to stalled ribosomes by sensing ribosome collisions and employing quality control pathways. How ribosome stalling is resolved without collisions, however, has remained elusive. Here, focusing on noncolliding stalling exhibited by decoding-defective ribosomes, we identified Fap1 as a stalling sensor triggering 18S nonfunctional rRNA decay via polyubiquitination of uS3. Ribosome profiling revealed an enrichment of Fap1 at the translation initiation site but also an association with elongating individual ribosomes. Cryo-EM structures of Fap1-bound ribosomes elucidated Fap1 probing the mRNA simultaneously at both the entry and exit channels suggesting an mRNA stasis sensing activity, and Fap1 sterically hinders the formation of canonical collided di-ribosomes. Our findings indicate that individual stalled ribosomes are the potential signal for ribosome dysfunction, leading to accelerated turnover of the ribosome itself.

After translational stalls, colliding eukaryotic ribosomes are cleared through dissociation into subunits by the ribosome quality control trigger complex, RQT, by an unknown mechanism. Here we show that RQT requires accessible mRNA and the presence of a neighboring ribosome. Cryo-EM of several RQT-ribosome complexes revealed the structural basis of splitting: RQT engages the 40S subunit of the lead ribosome and can switch between two conformations. We propose a mechanistic model in which the Slh1 helicase subunit of RQT applies a pulling force on the mRNA, causing destabilizing conformational changes of the 40S subunit. The collided ribosome functions as a ram or giant wedge, ultimately resulting in subunit dissociation. Our findings provide a first conceptual framework for a helicase driven ribosomal splitting mechanism.One-Sentence SummaryRQT clears collided ribosomes by pulling mRNA to trigger destabilizing conformational transitions for subunit dissociation.

Ribosome-associated quality control (RQC) represents a rescue pathway in eukaryotic cells that is triggered upon translational stalling. Collided ribosomes are recognized for subsequent dissociation followed by degradation of nascent peptides. However, endogenous RQC-inducing sequences and the mechanism underlying the ubiquitin-dependent ribosome dissociation remain poorly understood. Here, we identified SDD1 messenger RNA from Saccharomyces cerevisiae as an endogenous RQC substrate and reveal the mechanism of its mRNA-dependent and nascent peptide-dependent translational stalling. In vitro translation of SDD1 mRNA enabled the reconstitution of Hel2-dependent polyubiquitination of collided disomes and, preferentially, trisomes. The distinct trisome architecture, visualized using cryo-EM, provides the structural basis for the more-efficient recognition by Hel2 compared with that of disomes. Subsequently, the Slh1 helicase subunit of the RQC trigger (RQT) complex preferentially dissociates the first stalled polyubiquitinated ribosome in an ATP-dependent manner. Together, these findings provide fundamental mechanistic insights into RQC and its physiological role in maintaining cellular protein homeostasis.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

Summary: 18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Dissociation of the non-functional 80S ribosome into 40S and 60S subunits is a prerequisite step for degradation of the non-functional 18S rRNA. However, the mechanisms by which the non-functional ribosome is recognized and dissociated into subunits remain elusive. Here, we report that the sequential ubiquitination of non-functional ribosomes is crucial for subunit dissociation. 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3 at the 212th lysine residue. Determination of the aberrant 18S rRNA levels in sucrose gradient fractions revealed that the subunit dissociation of stalled ribosomes requires sequential ubiquitination of uS3 by E3 ligases and ATPase activity of Slh1 (Rqt2), as well as Asc1 and Dom34. We propose that sequential uS3 ubiquitination of the non-functional 80S ribosome induces subunit dissociation by Slh1, leading to degradation of the non-functional 18S rRNA. : 18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Sugiyama et al. show that 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3. Subunit dissociation requires Asc1, Dom34, sequential ubiquitination of uS3, and ATPase activity of Slh1 (Rqt2). Keywords: non-functional rRNA decay, ribosome ubiquitination, sequential ubiquitination, RQT complex, subunit dissociation

Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.

INTRODUCTION: The tightly controlled process of gene expression requires messenger RNAs (mRNAs), which represent DNA-derived blueprints for polypeptides, to be translated by the protein-producing machinery of the cell, the ribosomes. Therefore, protein levels depend largely on cellular mRNA levels, and the control of mRNA decay is one of the most critical processes for setting the overall level of gene expression. Half-lives of mRNAs vary greatly between different transcripts, and regulation of the mRNA decay rate is intimately connected to the elongation phase of mRNA translation. To that end, codon optimality has been established as a key parameter for determining mRNA half-life in multiple eukaryotic organisms. It has also been established that the timely decay of short-lived mRNAs enriched with nonoptimal codons requires the Ccr4-Not complex. Ccr4-Not is an essential protein complex, with its best understood role in mRNA degradation, where it serves as the major cytoplasmic 3′-poly(A)-tail deadenylase that initiates decay of most mRNAs. By deadenylation and subsequent activation of the mRNA decapping machinery, the Ccr4-Not complex renders mRNAs accessible to the major degrading exonucleases, such as Xrn1 on the 5′ end and the exosome on the 3′ end. The molecular mechanism underlying codon optimality monitoring and coordination with mRNA decay by the Ccr4-Not complex has remained elusive. RATIONALE: Because nonoptimal codons affect decoding kinetics of the ribosome and mRNA degradation occurs largely cotranslationally, it is highly plausible that codon optimality is directly monitored on the ribosome. In addition, a direct physical link between the participating Ccr4-Not complex and the ribosome has been suggested previously, and the Not4 subunit of the complex, an E3 ligase, ubiquitinates the eS7 protein of the 40S ribosomal subunit in yeast. Therefore, we set out to gain insights into the connection between the Ccr4-Not complex and the translation machinery in the context of mRNA homeostasis by combining cryo–electron microscopy (cryo-EM), ribosome profiling, and biochemical analysis. RESULTS: We used affinity-purified native Ccr4-Not–ribosome complexes from Saccharomyces cerevisiae for analysis by cryo-EM and found that recruitment of Ccr4-Not to the ribosome occurs via the Not5 subunit. The N terminus of Not5—in particular, a three α-helix bundle—interacted specifically with the ribosomal E-site, and deletion of the Not5 N-terminus resulted in the loss of stable ribosome association of the Ccr4-Not complex. However, ubiquitination of the small ribosomal subunit protein eS7 through the Not4 subunit still occurred. The Not5 interaction involved the ribosomal protein eS25 of the small subunit, in addition to transfer RNA (tRNA) and ribosomal RNAs (rRNAs). We found that Ccr4-Not interacts with both initiating and elongating ribosomes. In either case, Not5 engaged the E-site only when the ribosome adopted a distinct conformation lacking accommodated tRNA in the A-site, indicative of impaired decoding kinetics. Ribosome profiling revealed that low-optimality codons were enriched in the A-site in the Ccr4-Not–bound elongating ribosomes. This observation explained the low A-site tRNA occupancy observed with cryo-EM and suggested a link to codon optimality monitoring. Consistently, using mRNA stability assays, we found that loss of Not5 resulted in the inability of the mRNA degradation machinery to sense codon optimality. The observed dysregulation of mRNA half-life was detected upon Not5 deletion, Not5 N-terminal deletion, eS25 deletion, and loss of eS7 ubiquitination by Not4, which apparently serves as an upstream prerequisite for further Ccr4-Not activity on the ribosome. In addition, mRNA decapping was found to be impaired in these mutants, which confirmed that, in this pathway, Ccr4-Not triggers decapping downstream of optimality monitoring. CONCLUSION: Our analysis elucidates a direct physical link between the mRNA decay–mediating Ccr4-Not complex and the ribosome. Dependent on preceding ubiquitination of eS7 by the Not4 subunit, the Ccr4-Not complex binds (via the Not5 subunit) specifically to the ribosomal E-site when the A-site lacks tRNA because of slow decoding kinetics. This state of the ribosome occurs in the presence of nonoptimal codons in the A-site, which explains the shorter half-lives of transcripts enriched in nonoptimal codons. Thus, our findings provide mechanistic insights into the coordination of translation efficiency with mRNA stability through the Ccr4-Not complex.

Translation arrest by polybasic sequences induces ribosome stalling, and the arrest product is degraded by the ribosome-mediated quality control (RQC) system. Here we report that ubiquitination of the 40S ribosomal protein uS10 by the E3 ubiquitin ligase Hel2 (or RQT1) is required for RQC. We identify a RQC-trigger (RQT) subcomplex composed of the RNA helicase-family protein Slh1/Rqt2, the ubiquitin-binding protein Cue3/Rqt3, and yKR023W/Rqt4 that is required for RQC. The defects in RQC of the RQT mutants correlate with sensitivity to anisomycin, which stalls ribosome at the rotated form. Cryo-electron microscopy analysis reveals that Hel2-bound ribosome are dominantly the rotated form with hybrid tRNAs. Ribosome profiling reveals that ribosomes stalled at the rotated state with specific pairs of codons at P-A sites serve as RQC substrates. Rqt1 specifically ubiquitinates these arrested ribosomes to target them to the RQT complex, allowing subsequent RQC reactions including dissociation of the stalled ribosome into subunits., Several protein quality control mechanisms are in place to trigger the rapid degradation of aberrant polypeptides and mRNAs. Here the authors describe a mechanism of ribosome-mediated quality control that involves the ubiquitination of ribosomal proteins by the E3 ubiquitin ligase Hel2/RQT1.

Control of mRNA decay rate is intimately connected to translation elongation but the spatial coordination of these events is poorly understood. The Ccr4-Not complex initiates mRNA decay through deadenylation and activation of decapping. Using a combination of cryo-electron microscopy, ribosome profiling and mRNA stability assays we show recruitment of Ccr4-Not to the ribosome via specific interaction of the Not5 subunit with the ribosomal E-site. This interaction only occurs when the ribosome lacks accommodated A-site tRNA, indicative of low codon optimality. Loss of Not5 results in the inability of the mRNA degradation machinery to sense codon optimality. Our analysis elucidates a physical link between the Ccr4-Not complex and the ribosome providing mechanistic insight into the coupling of decoding efficiency with mRNA stability.

Dom34-Hbs1 plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). Here, we report a conserved function of human Pelota (hPelota) in mRNA quality control of nonstop mRNA. hPelota facilitated the expression of the nonstop products from GFP-Rz mRNA, which lacks a termination codon and a poly(A) tail, in exosome-defective mutant cells. hPelota promoted the dissociation of stalled ribosomes at the 3' end of GFP-Rz mRNA, and mutations in domain A diminished this activity. The hPelota-R45A mutant associated with ribosomes but was defective in peptide release. Finally, hPelota promoted the degradation of GFP-Rz mRNA and suppressed the sequential endonucleolytic cleavages caused by stalled ribosomes at the 3' end of mRNA in dom34∆ mutant cells.

The proteasome is a major proteolytic machine that regulates cellular proteostasis through selective degradation of ubiquitylated proteins1,2. A number of ubiquitin-related molecules have recently been found to be involved in the regulation of biomolecular condensates or membraneless organelles, which arise by liquid-liquid phase separation of specific biomolecules, including stress granules, nuclear speckles and autophagosomes3-8, but it remains unclear whether the proteasome also participates in such regulation. Here we reveal that proteasome-containing nuclear foci form under acute hyperosmotic stress. These foci are transient structures that contain ubiquitylated proteins, p97 (also known as valosin-containing protein (VCP)) and multiple proteasome-interacting proteins, which collectively constitute a proteolytic centre. The major substrates for degradation by these foci were ribosomal proteins that failed to properly assemble. Notably, the proteasome foci exhibited properties of liquid droplets. RAD23B, a substrate-shuttling factor for the proteasome, and ubiquitylated proteins were necessary for formation of proteasome foci. In mechanistic terms, a liquid-liquid phase separation was triggered by multivalent interactions of two ubiquitin-associated domains of RAD23B and ubiquitin chains consisting of four or more ubiquitin molecules. Collectively, our results suggest that ubiquitin-chain-dependent phase separation induces the formation of a nuclear proteolytic compartment that promotes proteasomal degradation.

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.

Dom34-Hbs1 stimulates degradation of aberrant mRNAs lacking termination codons by dissociating ribosomes stalled at the 3′ ends and plays crucial roles in Nonstop Decay (NSD) and No-Go Decay (NGD). In the dom34Δ mutant, nonstop mRNA is degraded by sequential endonucleolytic cleavages induced by a stalled ribosome at the 3′ end. Here, we report that ribosome-associated Asc1/RACK1 is required for the endonucleolytic cleavage of nonstop mRNA by stalled ribosome at the 3′ end of mRNA in dom34Δ mutant cells. Asc1/RACK1 facilitates degradation of truncated GFP-Rz mRNA in the absence of Dom34 and exosome-dependent decay. Asc1/RACK1 is required for the sequential endonucleolytic cleavages by the stalled ribosome in the dom34Δ mutant, depending on its ribosome-binding activity. The levels of peptidyl-tRNA derived from nonstop mRNA were elevated in dom34Δasc1Δ mutant cells and overproduction of nonstop mRNA inhibited growth of mutant cells. E3 ubiquitin ligase Ltn1 degrades the arrest products from truncated GFP-Rz mRNA in dom34Δ and dom34Δasc1Δ mutant cells and Asc1/RACK1 represses the levels of substrates for Ltn1-dependent degradation. These indicate that ribosome-associated Asc1/RACK1 facilitates endonucleolytic cleavage of nonstop mRNA by stalled ribosomes and represses the levels of aberrant products even in the absence of Dom34. We propose that Asc1/RACK1 acts as a fail-safe in quality control for nonstop mRNA.

Background. Ultrasound measurement of osteoarthritis cartilage thickness is not sufficiently accurate if constant sound velocity is used. The aim of this study was to investigate the applicability of estimating the sound velocity using echo reflectance from the cartilage surface. Methods. Change in measurement error of cartilage thickness was evaluated using collagenase-treated osteochondral plugs. Sound velocity was calculated from the reflectance ratio, and the reference thickness was measured using a needle method. Findings. The relative errors of thickness measurements using constant sound velocity were increased after collagenase treatment. The errors using calculated sound velocity showed no significant difference between intact and degenerated samples. Interpretation. The collagenase treatment-induced error was reduced from 7% to 2% in absolute value, suggesting the applicability of the correction method for ultrasound measurement.

The load-dependent frictional performance of vitamin E-blended ultrahigh molecular weight polyethylene (UHMWPE) in serum lubricant was examined. The UHMWPE specimens were slid against the surface of Co-28Cr-6Mo alloy by using a pin-on-disk friction test apparatus. The frictional performance of vitamin E-blended UHMWPE differed from that of virgin UHMWPE under 30 MPa loading, while little difference was observed under 3 MPa loading. The friction force of vitamin E-blended UHMWPE showed a lower value in initial friction and a higher value in steady state friction, and the variability of friction force in the transition process was relatively small. Frictional performance similar to that of the vitamin E-blended UHMWPE was observed as a result of applying vitamin E to the sliding surface of virgin UHMWPE. These results suggest that the frictional performance of vitamin E-blended UHMWPE in serum lubricant is affected by the presence of vitamin E on the sliding surface under high compressive load.

We present a new method to reduce the insertion resistance for internal medical instruments by means of vibration. A rotating permanent magnet generates an oscillated magnetic field and vibrates the internal medical instruments from outside of the body. We investigated the relation between the frequency of vibration and the insertion resistance with a pin on flat test system in vitro. As a result, vibration is effective to reduce the friction and it is possible to keep a smooth insertion by vibration.

Therapeutic vascular catheterization techniques are sometimes hampered by the frictional forces between the blood vessel and the catheter, when contact points of the vessel are changing and deforming during the movement of the catheter. The goal of the present study was to characterize frictional interactions between the blood vessel wall and the catheter using experimental and numerical analysis. First, the frictional force was measured with an experimental apparatus that uses a ball and flattened porcine aorta to simulate frictional forces between the catheter and the vessel. Second, catheter motion was characterized by two-dimensional numerical calculations based on the experimental results. Experimental analysis demonstrated that slip occurred and that friction coefficient between the vessel and the catheter and the deformation of the specimen were small when the contact between the ball and the aorta occurred at a small angle. The compliance of the specimen in the normal direction obtained by the experiment was by far larger than that calculated according to the Hertzian contact theory. Numerical analysis shows that this difference of the parameter of the vessel, which must be determined accurately in surgical simulator, could affect the trajectory of the catheter.

The lubrication mechanism of articular cartilage is characterized by an efficient performance. In this work, friction of articular cartilage was evaluated with in-site images of articular surface. The images were captured with the laser light reflected at the interface between a prism and articular cartilage. The attenuation of reflectance was associated with the increase of the contact of collagen network of articular cartilage. The light reflectance and friction coefficient for short sliding presented a significant positive correlation. Friction tests were also carried out for short (30 s) and long (300 s) preloading times. The results indicate that depletion of fluid film is responsible for the increase of friction and the recovery of the fluid film was observed for the long preloading after the early stage of sliding.

We previously developed a novel ultrasound assessment system featuring wavelet transform to evaluate the material properties of articular cartilage. We aimed in this study to demonstrate the feasibility of quantitative evaluation of meniscus using ultrasound and to elucidate the relationships between its acoustic, mechanical, and biochemical properties. Meniscal disc specimens from mature pigs were assessed by ultrasound and compression testing, and their correlation was analyzed. A positive correlation was found between the ultrasound signal intensity and apparent Young's modulus (r=0.61). Subsequently, the porcine meniscal discs were treated with various enzymes and then characterized by ultrasound, by compression tests, by biochemical analyses, and by histology and immunohistochemistry. The signal intensity was decreased not by hyaluronidase but by collagenase treatment. Hyaluronidase-treated menisci showed a discrepancy between acoustic and mechanical properties, suggesting that the ultrasound reflection could not detect a reduction in proteoglycan content. Also, ultrasound signal intensity could only reflect superficial layers of the material. Several limitations exist at present, and further studies and improvements of the device are required. However, given the noninvasive nature and the requirement of only small equipment, this ultrasound assessment system will be an instrumental diagnostic tool for meniscal function in both research and clinical fields.

Effects of static magnetic field (SMF) on the vascularization in bone were evaluated using an ischemic bone model, where rat femoral artery was ligated. Magnetized and unmagnetized samarium–cobalt rods were implanted transcortically into the middle diaphysis of the ischemic femurs. Collateral circulation was evaluated by injection of microspheres into the abdominal aorta at the third week after ligation. It was found that the bone implanted with a magnetized rod showed a larger amount of trapped microspheres than that with an unmagnetized rod at the proximal and the distal region (P< 0.05 proximal region). There were no significant differences at the middle and the distal region. This tendency was similar to that of the bone mineral density in the SMF-exposed ischemic bone.

The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652–726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal.

We have developed catheter simulation system in blood vessels for surgical planning and intraoperative assistance. In this study, we evaluated the effect of the fundamental parameters of the guidewire and blood vessel models for the simulation system. The model of the guidewire was constructed with viscoelastic springs and segments to avoid complicated mathematical procedures as the dynamic deformation of a flexible structure. The vessel is an elastic circular cylinder, whose shape is defined by the centerline and the radii. Collisions between the guidewire and the vessel are calculated and the contact forces are determined according to the stiffness, damping and friction of the vessel wall. Using these models, the trajectory of the guidewire tip and the reaction force were calculated. When friction coefficient between the guidewire and the vessel is large, the guidewire tip vibrates. When deformation property of the vessel is changed, the trajectory of guidewire tip also changes. Bending of the guidewire tip decreases the reaction force at bending points of the vessel. When the guidewire becomes stiffer, the deformation of the vessel becomes large. These findings should facilitate determination of the parameters of the simulation system.

Although tribological function is the most important mechanical property of articular cartilage, few studies have examined this function in tissue-engineered cartilage. We investigated changes in the frictional properties of cartilage regenerated from the inoculation of rabbit chondrocytes into fibroin sponge. A reciprocating friction-testing apparatus was used to measure the friction coefficient of the regenerated cartilage under a small load. The specimen was slid against a stainless steel plate in a water vessel filled with physiological saline. The applied load was 0.03 N, the stroke length was 20 mm, and the mean sliding velocity was 0.8 mm/s. The friction coefficient of the regenerated cartilage decreased with increasing cultivation time, because a hydrophilic layer of synthesized extracellular matrix was formed on the fibroin sponge surface. The friction coefficient of the regenerated cartilage was as low as that of natural cartilage in the early stages of the sliding tests, but it increased with increasing duration of sliding owing to exudation of interstitial water from the surface layer.

In this study, we simulated a wide cortex separation from a cementless hip prosthesis using the bone resorption remodeling method that is based on the generation of high compressive stress around the distal cortical bone. Thereafter, we estimated the effect on late migration quantities of the hip prosthesis produced by the interface state arising from bone ingrowth. This was accomplished using cortical bone remodeling over a long period of time. Two-dimensional natural hip and implanted hip FEM models were constructed with each of the following interface statements between the bone and prosthesis: (1) non-fixation, (2) proximal 1/3, (3) proximal 2/3 and (4) full-fixation. The fixation interfaces in the fully and partially porous coated regions were rigidly fixed by bony ingrowth. The non-fixation model was constructed as a critical situation, with the fibrous or bony tissue not integrated at all into the implant surface. The daily load history was generated using the three loading cases of a one-legged stance as well as abduction and adduction motions. With the natural hip and one-legged stance, the peak compressive principal stresses were found to be under the criteria value for causing bone resorption, while no implant movement occurred. The migration magnitude of the stem of the proximal 1/3 fixation model with adduction motion was much higher, reaching 6%, 11%and 21%greater than those of the non-fixation, proximal 2/3 fixation and all-fixation models, respectively. The full-fixation model showed the lowest compressive principal stress and implant movement. Thus, we concluded that the late loosening and subsequent movement of the stem in the long term could be estimated with the cortical bone remodeling method based on a high compressive stress at the bone-implant interface. The change caused at the bone-prosthesis interface by bony or fibrous tissue ingrowth constituted the major factor in determining the extent of cortical bone resorption occurring with clinical loosening and subsequent implant movement.

In orthopedic field, various new treatments of articular cartilage defect, for example autogenous osteochondral grafts, have been developed. With the spread of these treatments, orthopedists began to focus on the mechanical properties of recovered articular cartilage. The quantitative evaluation of articular cartilage before and after these treatments gives orthopedists the important information to improve these treatments and develop new treatments. We have been investigating the non-contact ultrasonic evaluation for articular cartilage under arthroscopy. In this paper, it was hypothesized that the ultrasonic evaluation depended on the collagen fiber in cartilage. The enzymatically degradation of collagen fiber in cartilage surface was performed. The effect of the degradation on sound velocity, attenuation coefficient and signal intensity, which is the index of cartilage stiffness calculated from the proposed method, was measured. The numerical analysis was performed to clear the relation between the cartilage character and ultrasonic parameters. Experimental and numerical results suggest that the present method can be expanded the sensitive evaluation for cartilage disease in clinical field.

Mechanical stimulation has a great influence on articular cartilage regeneration. The objective of this study was to clarify the temporal sequences of spontaneous repair of weightless or immobilized joints.An osteochondral defect was created in the femoral patellar groove of F344 rats. A tail-suspension procedure was performed to control the mechanical environment of the hindlimbs. The experimental knee joints were classified into three conditions: CONT, normal gait; LLB, low load-bearing; LLI, low load and immobilized. The repair processes up to 4 weeks were evaluated histologically.The knee defects in the CONT and LLB conditions were repaired to a smooth surface with fibrous tissue and highly developed subchondral bone. The knees in the LLI condition had the lowest reformation rate of subchondral bone, although partial regeneration of hyaline cartilage-like tissue was seen at 4 weeks after the operation. Bulges of fibrous tissue from the defects were observed in the LLI condition.These results, combined with our previous report, suggest that dynamic compressive strain stimulates regeneration of the joint surface structures. They also suggest that the contact condition of the defect with surface cartilage may play a role in the hyaline cartilage repair.

Although numerous methods for regenerating articular cartilage have been investigated, the regenerated tissue showed various histological findings from hyaline-like cartilage to fibrous tissue. Without biopsy, we are unable to know whether the cartilage regeneration method was histologically successful or not. We developed a new ultrasonic evaluation system for articular cartilage using the maximum magnitude (MM) from ultrasonic analysis. The purpose of this study was to investigate the usefulness of ultrasonic judgment of the cartilage regeneration procedure. Using our system we quantitatively evaluated tissue-engineered cartilage in rabbit cartilage defects. The specimens were retrospectively divided into two groups on the basis of histological findings and investigated whether significant differences in ultrasonic analysis could be found between the two (group H: hyaline-like cartilage group, successful; group F: fibrous tissue group, failure). In the ultrasonic findings, the MM was 1.11 ± 0.32 in group H and 0.65 ± 0.18 in group F and these differences were significant ( P = 0.00061). Our results suggest that the ultrasonic evaluation system used in the present study is capable of judging the success or failure of cartilage regeneration procedures, and therefore, it could be a valuable tool arthroscopic diagnosis of cartilage regeneration.

Whiplash injuries are most common disorders in rear-end car accidents, while the injury mechanism is yet unknown. Many numerical and experimental approaches have conducted to investigate the cervical behaviors with solely two-dimensional analyses in the sagittal plane. In real accidents, however, as impacts may affect several directions, the cervical behaviors should be evaluated three-dimensionally. Therefore, we evaluated the cervical behaviors under assumption of the posterior-oblique impacts depending on the impact angles with 3-D FE analysis. In addition, we analyzed the stresses occurred in the facet joints considering the relationship with a whiplash disorders. The cervical behaviors showed complex motion combined with axial torsion and lateral bending. The bending angle peaked in the impact at the angle of 15°, and the peak compressive and shear stress on the facet cartilage at C6-C7 increased by 11% and 14%. In the impact at the angle of 30°, the torsion angle peaked at C2-C3, the peak shear stress in the facet cartilage increased by 27%. It showed that the torsion and lateral bending affected the cervical behaviors, and caused the increase of peak stresses on the soft tissues. It is assumed as one of important causes of whiplash injury.

In order to improve the manipulation ability of endoscope, we have developed a new tactile sensor system using image processing. As this system uses an infrared (IR) cut pattern, it is possible to install the sensor in the tip of an existing endoscope easily. In addition, software has also been developed to convert the image into the tactile force. We tested the performance of this system with the trial device. The tactile force can be determined from the image of IR cut pattern. The effects of the noises can be predicted and eliminated. And the results of this study confirm that this system is useful for endoscopic surgery.

Objective Although numerous studies have been performed to determine whether there are histologic, biochemical, biomechanical, and metabolic differences between knee and ankle cartilage, none have investigated the presence of such differences in living human cartilage. We previously developed an ultrasonic evaluation system for articular cartilage that analyzes the A-mode echogram using wavelet transformation. The current study was undertaken to determine whether the acoustic properties of living human cartilage differ between knee and ankle joints. Methods Twenty-eight patients were subjected to ultrasonic evaluation under arthroscopy. After arthroscopic grading, the cartilage was measured using an ultrasonic probe. Two quantitative parameters were used, i.e., the maximum magnitude and the echo duration at the 95% interval of the maximum magnitude. Results In intact cartilage, the maximum magnitude and echo duration did not differ between the knee and the ankle. In lesional cartilage, in contrast, the maximum magnitude was higher, and the echo duration was shorter, in the ankle than in the knee. These differences were statistically significant. Conclusion Ultrasound findings could be used to judge the degree of early cartilage degeneration in vivo on the basis of objective data such as the maximum magnitude and echo duration. Because we were unable to quantitatively analyze the biochemical and biomechanical properties of the cartilage in this study, our biochemical and biomechanical findings are based only on qualitative assessment. Nevertheless, the results indicate that this ultrasonic evaluation system may be useful for elucidating the processes of articular cartilage degeneration in osteoarthritis.

The purpose of this study is to predict with the use of FEA, the differing predisposition to cortical bone resorption and subsequent distal migration of an un-cemented femoral hip replacement stem subjected to long term biomechanical high compressive stresses, while varying the load angles, the material properties of the stem, and the stem length. A two-dimensional hip model was constructed to estimate the minimum principle stresses (P3) and migration magnitudes. Bone remodeling at the interface between the bone and the prosthesis was performed by comparison of the local compressive stress to physiological stress values governing bone resorption. With respect to load angles, migrations of the hip prosthesis did not occur with load angles between 63° and 74° load angle in relation to the longitudinal axis of the bony femur, as the compressive stress generated on the cortical bone was under the criteria threshold for bone resorption (-50MPa). In addition, the magnitude of migration (17%decrease) was relatively more sensitive to changes in stem length than those (92%decrease) of changes of material properties. In conclusion, using an FEA model for bone remodeling, based on the high compressive stresses exerted on distal cortical bone, it is possible to estimate migration magnitudes of cementless hip prostheses in the long term. The load angles have been shown to be an important parameter affecting the migration magnitudes and furthermore, it can be demonstrated that the stiffer materials and reduction of stem length can decrease the migration of cementless hip prosthesis in the long term.

Objective. To evaluate regenerated articular cartilage quantitatively by introducing an ultrasonic probe into the knee joint under arthroscopy and analysing the A-mode echogram by means of wavelet transformation. Methods. Three experimental rabbit models (spontaneous repair model, large cartilage defect model, treatment model) were examined using our ultrasonic evaluation system and a histological grading scale. From resulting wavelet map, the percentage of maximum magnitude was selected as the quantitative index of the ultrasonic evaluation system. Results. The percentage maximum magnitude in the spontaneous repair model was 61.1%, that in the large defect model was 29.8% and that in the treatment model was 36.3%. There was modest correlation between the percentage maximum magnitude and the histological grading scale (r ¼� 0.594) Conclusion. Our findings indicate that ultrasound analysis can predict the microstructure of regenerated cartilage. Numerous attempts to induce the healing of large articular cartilage defects have been made. Each treatment has its own strong and weak points, and it remains difficult to choose among treatments. There are no quantitative non-invasive methods to evaluate the regenerated cartilage clinically [1]. Generally, arthroscopy can be performed to evaluate regenerated cartilage. However, although arthroscopy can identify cartilage surface changes, it cannot reveal the microstructure of the regenerated cartilage. Brismar et al. [2] reported on observer reliability in arthroscopic grading of osteoarthritis of the knee, and concluded that observer-specific disagreement was evident both within and between observers and that arthroscopic grading of early osteoarthritic lesions was inexact. Therefore, arthroscopic judgement cannot exclude the potential for observer bias in the evaluation of the cartilage surface. For the strict assessment of regenerated cartilage, histological and biochemical analyses are performed from experimental and clinical aspects. However, these analyses require collection of the regenerated tissue, and the biopsy could possibly induce the progression of osteoarthritis. Therefore, these evaluation methods should be avoided. We developed an intra-articular ultrasonic probe and a new evaluation system for articular cartilage in the human knee, and revealed that this system was able to quantitatively evaluate cartilage degeneration [3, 4]. However, it remains to be shown whether this system can accurately evaluate regenerated cartilage. The purpose of this study was to determine the efficacy of our system for evaluating regenerated cartilage. To this end, we quantitatively evaluated regenerated cartilage using experimental rabbit models.

In order to examine the initiation mechanism of delamination in ultrahigh molecular weight polyethylene (UHMWPE) knee components, a bi-directional sliding fatigue test was performed for three types of UHMWPE specimens: nonirradiated, gamma-irradiated (25 kGy) and gamma-irradiated (25 kGy) with 0.1% vitamin E added. Sliding surfaces of post-tested UHMWPE specimens were observed using an optical microscope and a scanning electron microscope. Also, surface roughness was measured at the sliding surfaces of UHMWPE specimens. Delamination was observed only in gamma-irradiated specimens. A networked structure of surface asperity that resembled grain boundary was observed prior to delamination in gamma-irradiated specimens. Surface roughness in the gamma-irradiated specimens, higher than in any other specimen, showed a rapid increase prior to delamination. Detailed observation using an optical microscope and a scanning electron microscope showed microscopic crack initiation along subsurface grain boundaries in gamma-irradiated specimens. These results suggest that subsurface crack initiation is a precursor of delamination and is accelerated by oxidative degradation due to gamma irradiation. Of the three types of specimens, UHMWPE with vitamin E added showed the lowest surface roughness values at all measuring points. The addition of vitamin E is effective in improving wear resistance and fatigue performance of UHMWPE.

Ultra-high molecular weight polyethylene (UHMWPE) is a heterogeneous material composed of a networked substructure of grain boundary and grain aggregation. A new numerical model based on the discrete element method (DEM) was proposed to examine microscopic defect formation and propagation in UHMWPE. Numerical simulations were carried out using this model under two types of loading condition: unidirectional repetitive compression (simple loading) and bidirectional repetitive compression (switched loading). Subsurface defects were initiated and propagated in the vicinity of grain boundaries under both loading conditions. The defect propagation behavior was especially sensitive to grain boundary allocation under switched loading. An increase in defects was more rapid under switched loading than under simple loading. These numerical results showed qualitatively good agreement with experimental ones. It is suggested that the newly developed numerical method based on the DEM is a promising method to investigate fatigue behavior of a heterogeneous material such as UHMWPE under complicated loading conditions.

The purpose of this study is to evaluate correlation between extracellular matrix and mechanical properties of articular cartilage. Collagenase and hyaluronidase were used to degrade colloagen fibril and proteoglycan respectively. Indentation test, dynamic visco-elasticity measurement and tensile test were carried out to evaluate mechanical properties before and after the enzyme treatment. Acoording to indentation test, maximum strain was increased and recovery ratio (=recovery strain/maximum strain) was decreased with treatment time for both enzyme treatments. There were no significant differences in tensile strength before and after the hyaluronidase treatment, where as the strength was decreased by the collgenase treatment. Result of dynamic visco-elasticity measurement showed that peak value and peak frequency of tan δ were shifted to a higher value with treatment time after the hyaluronidase treatment. These results suggest that compressive modulus and visco-elastic properties are much affected by collagen-proteoglycan network and tensile strength is affected mainly by collagen fibers.

Objective. To develop a new diagnostic technology to evaluate articular cartilage quantitatively by introducing an ultrasonic probe into the knee joint under arthroscopy and analyzing the A-mode echogram by means of wavelet transformation. Design. Quantitative evaluation using comparison of two indices on the wavelet map and macroscopic evaluation using the Outerbridge classification. As the quantitative indices on the wavelet map, the maximum magnitude and the echo duration which was defined as the length of time that included 95% of echo signal were selected. Background. Quantitative evaluation of articular cartilage in situ is required for new tissue-engineered cartilage but an evaluation system that fully meets this requirement has yet to be established for clinical use. Methods. Human articular cartilage specimens were analyzed using an ultrasonic probe after macroscopic evaluation and the cartilage characteristics on the echo duration–maximum magnitude graph were examined. Results. There were significant differences between grade 1 and 3, and grade 2 and 3 in the echo duration and maximum magnitude. The cartilage specimens had a L-shaped distribution in echo duration–maximum magnitude graph. Conclusions. The present study is the new quantitative evaluation system of the articular cartilage in situ and a clinical trial under arthroscopy is presently underway. The ultrasonic measurement is highly reproducible, so we can expect this system to be suitable for in situ reliable examination under arthroscopy. Relevance Precise evaluation of articular cartilage is of particular importance for longitudinal clinical trials for determination of best surgical option or effect of new chondroprotective drugs in arthritic disease and rheumatoid arthritis.

While alumina is the only material applied to all-ceramic joint prostheses, zirconia has high potential too, because it has high strength and fracture toughness. However, wear of zirconia/zirconia combination is too high for clinical use. It is likely that a combination of different materials may improve wear properties. In this study, wear tests were carried out with an end-face wear testing apparatus for four material combinations: zirconia/zirconia (Z/Z), zirconia/alumina (Z/A), alumina/zirconia (A/Z), and alumina/alumina (A/A). Mean contact pressures were 5 and 10 MPa, and sliding velocity was 40 mm/s. Wear tests continued for 72 h and total sliding distance was 10 km. After the test, the wear factor defined by (wear volume)/(applied load×sliding distance) was calculated. Worn surfaces were observed with a scanning electron microscope and analyzed by means of Raman spectroscopy. When zirconia was combined with zirconia counterface, the wear factor of zirconia under 5 MPa was about 2.5×10−7 mm3/N m and that under 10 MPa was about 1.9×10−5 mm3/N m. In the case of Z/Z combination, the wear factor was very high due to adhesive wear and the phase transformation. When zirconia was combined with alumina counterface, the wear factor of zirconia under 5 MPa was about 1.7×10−9 mm3/N m and that under 10 MPa was about 1.7×10−7 mm3/N m. In the case of Z/A and A/Z combinations, the wear factor of zirconia decreased drastically as compared with Z/Z combination, because adhesive wear was controlled. Micro cracking and third body abrasion were predominant in this case.

The stent implantation is a low invasive and effective method to treat the coronary artery blockage. The problem of stenting is restenosis because of initimal thickening, which occurs in 20%. Restenosis rate is related to the radial stiffness and bending stiffness of a stent. In order to prevent the recurrences of coronary stenosis, it is necessary to investigate the mechanical properties of a stent. However, test method about these properties is little known. The purpose of this study is to propose a method to estimate the flexibility of a stent. To achieve the aim, we developed a test apparatus by use of four points bending and measured the bending stiffness of 4 stent structures. The result showed that the link stent structure was more flexible than the tube one. Considering a link configuration, S shaped link stent structure was flexible. The flexible stent deformed smoothly by bending moment. On the other hand, the low flexible stents deformed with buckling. This feature was clear in compressive side when stent was bent.

The present study was carried out to improve the corrosion resistance of pure magnesium in Hank's Balanced Salt Solution (HBSS) through the surface modification. Three kinds of alkaline compounds, such as sodium hydrogen carbonate, sodium carbonate, and lithium hydroxide, were used for treatment. Only magnesium treated with aqueous sodium hydrogen carbonate solution gave a good corrosion resistance in HBSS solution at 25°C up to 75 days, while almost no effect in sodium carbonate and lithium hydroxide. It was likely that hydrogen carbonate ions were essential for the surface improvement of magnesium. X-ray diffraction patterns of modified magnesium showed new peaks of magnesium carbonate and others on the surface. New surface structure of needle-shaped crystals was observed by scanning electron microscopy (SEM). Both elemental mapping, and energy dispersive (EDX) techniques in SEM showed the precipitation of calcium magnesium phosphate (low crystallinity whitlockite:(Ca,Mg)3(PO 4 ) 2 ) on the surface of magnesium specimen. It could be concluded that surface modification with sodium hydrogen carbonate made it possible to apply metal magnesium in biomedical, dental, and other industrial usage.

This paper presents a simple, cheap and effective sheet exchanger composed of four rolling cylinders because it is a troublesome task to change a bed sheet for a bedridden person. To change a bed sheet, while a cylinder wind the used sheet, the new sheet wound around another cylinder is spread under the body with advance of the device. The system composed of the exchanger, a person lying on the bed and an operator was investigated. It is found that the energy for driving dissipates immediately during operation. According to the result, necessary torque to drive the device is less than 4 Nm. Therefore, it can be driven easily by hands or by a small electric motor.

Objective . The objective of this study was to evaluate load transmission in the cancellous bone of the tibia under static and impact load. Background . Abnormal transmission of loads may result in osteoarthritis and fractures. However, the role of the cancellous bone in these processes is not well understood. Methods . The compressive stresses in the subchondral bone, epiphysis and diaphysis of the tibia of porcine knees were measured under static and impact load using mini-pressure transducers. The tests were performed using a drop-tower type testing machine in neutral, varus and valgus alignments. Tests were repeated after meniscectomy and again after removing the articular cartilage. Results . In the intact knee in all alignments, the highest stress on the medial side was found in the epiphysis, and in the subchondral bone on the lateral side. After meniscectomy, a significant increase was observed in the stress in the subchondral bone on both sides. After the articular cartilage was also removed, the stress in the subchondral bone increased again, but slightly. Conclusion . The importance of the meniscus in load transmission is supported by this study. The contribution of the articular cartilage to load transmission is less than that of the meniscus. Relevance . The patterns of static and dynamic load transmission observed in this study correlate with the sites of insufficiency fractures of the medial tibial plateau and traumatic lateral tibial plateau fractures observed clinically. Examination of the load transmission is key to a more complete understanding of the causes of osteoarthritis and tibial plateau fractures.

Attempts have been made to develop an artificial articular cartilage on the basis of a new viewpoint of joint biomechanics in which the lubrication and load-bearing mechanisms of natural and artificial joints are compared. Polyvinyl alcohol hydrogel (PVA-H), ‘a rubber-like gel’, was investigated as an artificial articular cartilage and the mechanical properties of this gel were improved through a new synthetic process.In this article the biocompatibility and various mechanical properties of the new improved PVA-H is reported from the perspective of its usefulness as an artifical articular cartilage. As regards lubrication, the changes in thickness and fluid pressure of the gap formed between a glass plate and the specimen under loading were measured and it was found that PVA-H had a thicker fluid film under higher pressures than polyethylene (PE) did. The momentary stress transmitted through the specimen revealed that PVA-H had a lower peak stress and a longer duration of sustained stress than PE, suggesting a better damping effect. The wear factor of PVA-H was approximately five times that of PE. Histological studies of the articular cartilage and synovial membranes around PVA-H implanted for 8–52 weeks showed neither inflammation nor degenerative changes. The artificial articular cartilage made from PVA-H could be attached to the underlying bone using a composite osteochondral device made from titanium fibre mesh.In the second phase of this work, the damage to the tibial articular surface after replacement of the femoral surface in dogs was studied. Pairs of implants made of alumina, titanium or PVA-H on titanium fibre mesh were inserted into the femoral condyles. The two hard materials caused marked pathological changes in the articular cartilage and menisci, but the hydrogel composite replacement caused minimal damage. The composite osteochondral device became rapidly attached to host bone by ingrowth into the supporting mesh.The clinical implications of the possible use of this material in articular resurfacing and joint replacement are discussed.