Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition.

Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.

In-process measurement of a keyhole using a low-coherence interferometer with a high repetition rate.

The shape of an instance hole (keyhole) created via a high-power laser was measured using a low-coherence interferometer with the following parameters: repetition rate, 10 MHz; center wavelength, 1550 nm; absolute spatial resolution, 10 µm; and measurement range, 5 mm. The keyhole was created on a 3-mm-thick stainless-steel plate using a high-power laser with 8-kW peak power and 1070-nm center wavelength. The cross-sectional area of the keyhole was measured to be 0.42 mm × 0.78 mm (width × depth) using the interferometer, and its side dimension was 0.46 mm × 0.78 mm (width × depth).

Expression of three isoforms of Na-K-2Cl cotransporter (NKCC2) in the kidney and regulation by dehydration.

Sodium reabsorption via Na-K-2Cl cotransporter 2 (NKCC2) in the thick ascending limbs has a major role for medullary osmotic gradient and subsequent water reabsorption in the collecting ducts. We investigated intrarenal localization of three isoforms of NKCC2 mRNA expressions and the effects of dehydration on them in rats. To further examine the mechanisms of dehydration, the effects of hyperosmolality on NKCC2 mRNA expression in microdissected renal tubules was studied. RT-PCR and RT-competitive PCR were employed. The expressions of NKCC2a and b mRNA were observed in the cortical thick ascending limbs (CAL) and the distal convoluted tubules (DCT) but not in the medullary thick ascending limbs (MAL), whereas NKCC2f mRNA expression was seen in MAL and CAL. Two-day dehydration did not affect these mRNA expressions. In contrast, hyperosmolality increased NKCC2 mRNA expression in MAL in vitro. Bradykinin dose-dependently decreased NKCC2 mRNA expression in MAL. However, dehydration did not change NKCC2 protein expression in membrane fraction from cortex and outer medulla and in microdissected MAL. These data show that NKCC2a/b and f types are mainly present in CAL and MAL, respectively. Although NKCC2 mRNA expression was stimulated by hyperosmolality in vitro, NKCC2 mRNA and protein expressions were not stimulated by dehydration in vivo. These data suggest the presence of the inhibitory factors for NKCC2 expression in dehydration. Considering the role of NKCC2 for the countercurrent multiplier system, NKCC2f expressed in MAL might be more important than NKCC2a/b.

Controlled morphology of aluminum alloy nanopillar films: from nanohorns to nanoplates.

Nanopillar films of Al-Nb alloys have been fabricated on substrates with a regular concave cell structure by oblique angle physical vapor deposition. The concave cell structure of the substrate increases the shadow region for the flux of depositing atoms, assisting the formation of an isolated nanopillar on each cell. Depending upon the alloy composition and deposition angle, the pillar shape changes from horn-like nanopillars through triangular nanoprisms to nanoplates. The Al-Nb alloy nanoplate films with wide gaps between plates are of interest as electrodes for capacitor applications. The dielectric oxide film formed on the nanoplate film showed a capacitance more than ten times larger than that on the respective flat film, due to the enlarged surface area.

Potency of double-layered poly L-lactic acid scaffold in tissue engineering of tendon tissue.

A successful scaffold for use in tendon tissue engineering requires a high affinity for living organisms and the ability to maintain its mechanical strength until maturation of the regenerated tissue. We compared two types of poly(L-lactic acid) (PLLA) scaffolds for use in tendon regeneration, a plain-woven PLLA fabric (fabric P) with a smooth surface only and a double layered PLLA fabric (fabric D) with a smooth surface on one side and a rough (pile-finished) surface on the other side. These two types of fabric were implanted into the back muscles of rabbits and evaluated at three and six weeks after implantation. Histological examination showed collagen tissues were highly regenerated on the rough surface of fabric D. On the other hand, liner cell attachment was seen in the smooth surface of fabric P and fabric D. The total DNA amount was significantly higher in fabric D. Additionally, mechanical examination showed fabric P had lost its mechanical strength by six weeks after implantation, while the strength of fabric D was maintained. Fabric D had more cell migration on one side and less cell adhesion on the other side and maintained its initial strength. Thus, a novel form of double-layered PLLA fabric has the potential to be used as a scaffold in tendon regeneration.

The effect of porosity and mechanical property of a synthetic polymer scaffold on repair of osteochondral defects.

We have made three types of poly (DL-lactide-co-glycolide) (PLG) scaffolds (porosity: scaffold I 80 +/- 0.9%, II 85 +/- 0.8%, III 92 +/- 0.7%; compression module determined with 10% strain: scaffold I 0.26 MPa, II 0.091 MPa, III 0.0047 MPa). Osteochondral defects made in the femoral condyle of rabbits were treated with these scaffolds and the possibilities of cartilage repair were investigated histologically. At post-operative weeks 6 and 12, histological scores in the groups of scaffolds II and III were significantly higher than the score in the group of scaffold I. Scaffolds II and III, which have higher porosity than scaffold I, allow better migration of bone marrow cells and better replacement of the scaffold with bone and cartilage than scaffold I. This study suggests that higher porosity allowing bone marrow cells to migrate to the scaffold is important in repairing osteochondral defects.

The effect of fibroblast growth factor-2 on autologous osteochondral transplantation.

In this study, we performed a mechanical analysis of the effect of fibroblast growth factor-2 (FGF-2) on autologous osteochondral transplantation in a rabbit model. A full-thickness cartilage defect (diameter: 5 mm; depth: 5 mm) made in the right femoral condyle was treated with osteochondral transplantation using an osteochondral plug (diameter: 6 mm; depth: 5 mm) taken from the left femoral condyle. The animals were divided into three groups: Group I, the defect was filled with 0.1 ml of gelatin hydrogel containing 1 microg of FGF-2; Group II, the defect was filled with 0.1 ml of gelatin hydrogel only; Group III, the defect was left untreated. Thereafter, osteochondral plugs were transplanted and the transplanted osteochondral grafts were evaluated mechanically and histologically at postoperative weeks 1, 3, 8 and 12. The structural property of the osteochondral graft was significantly greater in Group I than in Groups II and III at postoperative week 3. Histological analysis at 3 weeks revealed a tendency towards increased subchondral bone trabeculae in Group I compared with the other groups. Autologous osteochondral grafts transplanted with gelatin hydrogel containing FGF-2 acquired adequate stiffness at an early postoperative phase.

Stress fracture of the first rib in a high school weight lifter.

A 17-year-old boy, who played a weight lifting in high school, sustained stress fracture of the first rib without any causes. We successfully treated first rib stress fracture with limitation of using the upper extremity and with using low-intensity pulsed ultrasound. Key pointsStress fracture of the first rib in a weight lifter was successfully treated with limitation of sports activity and with LIPUS treatment.

Subcutaneous Peroneus Longus Tendon Rupture Associated with OS Peroneum Fracture.

We report a rare case of subcutaneous peroneus longus tendon rupture associated with os peroneum fracture. Three dimensional computed tomographic scan was useful to understand this disorder. We treated the patient with excision of fractured os peroneum and tenodesis of the proximal stump of the ruptured peroneus longus tendon to the lateral aspect of the calcaneus. Key pointsIn order to understand a rare case of subcutaneous peroneus longus tendon rupture associated with os peroneum fracture, three dimensional computed tomographic scan was useful.The patient was treated with excision of fractured os peroneum and tenodesis of the proximal stump of the ruptured peroneus longus tendon to the lateral aspect of the calcaneus.

Neglected fracture dislocation of the second and third carpometacarpal joints: a case report.

We report a rare case of neglected fracture dislocation of the second and third carpometacarpal joints, which was treated by arthrodesis of the involved joints.

An atypical phosphodiesterase capable of degrading haloalkyl phosphate diesters from Sphingobium sp. strain TCM1.

Sphingobium sp. strain TCM1 can degrade tris(2-chloroethyl) phosphate (TCEP) to inorganic phosphate and 2-chloroethanol. A phosphotriesterase (PTE), phosphodiesterase (PDE) and phosphomonoesterase (PME) are believed to be involved in the degradation of TCEP. The PTE and PME that respectively catalyze the first and third steps of TCEP degradation in TCM1 have been identified. However, no information has been reported on a PDE catalyzing the second step. In this study, we identified, purified, and characterized a PDE capable of hydrolyzing haloalkyl phosphate diesters. The final preparation of the enzyme had a specific activity of 29 µmol min-1 mg-1 with bis(p-nitrophenyl) phosphate (BpNPP) as the substrate. It also possessed low PME activity with p-nitrophenyl phosphate (pNPP) as substrate. The catalytic efficiency (k cat/K m) with BpNPP was significantly higher than that with pNPP, indicating that the enzyme prefers the organophosphorus diester to the monoester. The enzyme degraded bis(2,3-dibromopropyl) phosphate, bis(1,3-dichloro-2-propyl) phosphate and bis(2-chloroethyl) phosphate, suggesting that it is involved in the metabolism of haloalkyl organophosphorus triesters. The primary structure of the PDE from TCM1 is distinct from those of typical PDE family members and the enzyme belongs to the polymerase and histidinol phosphatase superfamily.

Recombinant human BMP-7 effectively prevents non-union in both young and old rats.

The purpose of this study was to evaluate the influence of age on the effectiveness of rhBMP-7 treatment in a fracture with severe periosteal damage that is known to result in non-union formation. Closed stabilized femur fractures were produced in 3-month-old and 18-month-old rats. The fracture site was exposed and 2 mm of the periosteum cauterized circumferentially to impair normal fracture healing. The cauterized fracture site was immediately treated with either 100 microg rhBMP-7 (BMP-7 group), or with 25 microL of vehicle alone (control group). Fracture healing was evaluated with radiographs taken at 3 and 6 weeks. Animals were sacrificed at 3 and 6 weeks and specimens subjected to biomechanical and histological evaluation. In both age groups, none of the control animals healed throughout the 6 weeks experimental duration. All of the rhBMP-7-treated 3-month-old animals were radiographically healed at 3 weeks. In comparison, only 56% (9/16) of the rhBMP-7-treated 18-month-old animals were radiographically healed at 3 weeks. At 6 weeks, however, all of the 18-month-old rhBMP-7-treated animals had healed. Histology revealed slower healing in the 18-month-old animals. Treatment with rhBMP-7 significantly increased all of the biomechanical properties in both age groups. In the 3-month-old animals the mechanical strength approached that of the intact femur at 3 weeks, while in the 18-month-old animals this did not occur until 6 weeks. In conclusion, rhBMP-7 can effectively stimulate fracture repair in both young (3-month-old) and old (18-month-old) rats. However, the effect of rhBMP-7 on the rate of fracture healing is greater in young rats compared to old rats.

The structural properties of an osteochondral cylinder graft-recipient construct on autologous osteochondral transplantation.

PURPOSE: The purpose of this study was to investigate the changes in structural properties of an osteochondral cylinder graft-recipient construct after autologous osteochondral transplantation. METHODS: A full-thickness cylindrical osteochondral defect (5 mm in diameter and 3 mm in depth) was made on the femoral condyle of a mature female Japanese white rabbit using the Osteochondral Autograft Transfer System (OATS; Arthrex, Naples, FL). The defect was repaired with an osteochondral plug (6 mm in diameter and 3 mm in depth) taken from the contralateral femoral condyle using the OATS. The implanted osteochondral grafts were evaluated immediately after surgery and at postoperative weeks 1, 3, 8, and 12. The stiffness of articular cartilage was analyzed using a tactile sensor system (AXIOM, Fukushima, Japan), which measures stiffness based on changes in resonance frequency when a vibrating tactile sensor touches articular cartilage. The specimens were stained with hematoxylin and eosin and serial sections were examined microscopically. RESULTS: The cartilage stiffness of the graft immediately after surgery was 107,695.1 N/m, which was not statistically different from the normal cartilage stiffness (100,027.5 N/m). The stiffness at postoperative weeks 1, 3, 8, and 12 was 95,386.8, 92,899.3, 95,969.8, and 104,683.7 N/m, respectively. The stiffness at postoperative weeks 1, 3, and 8 was significantly lower than the normal cartilage stiffness and the stiffness at postoperative week 12 was the same as normal cartilage. A new bone formation with an increase of bone trabeculae between the osteochondral cylinder graft and the recipient was observed at postoperative weeks 1, 3, and 8. Thereafter, at postoperative week 12, bone trabeculae decreased to the same level as observed in a normal model due to the progress of bone remodeling. CONCLUSIONS: The stiffness of articular cartilage of the osteochondral graft was normal at the time the graft was initially placed and at postoperative week 12. However, the stiffness at postoperative weeks 1, 3, and 8 was lower than the normal cartilage stiffness. CLINICAL RELEVANCE: Care should be taken when planning the rehabilitation program at an early phase after osteochondral transplantation.

[Effect of recombinant human bone morphogenetic protein on preventing atrophic nonunion].

Severe periosteal and soft tissue disruption at the time of fracture may result in the formation of an atrophic nonunion. The purpose of this study was to evaluate whether the immediate application of recombinant human bone morphogenetic protein (BMP) to the fracture site could rescue the healing process in this nonunion model. In this atrophic nonunion model, we have demonstrated that the immediate application of BMP at the time of injury may rescue the fracture healing process and prevent the development of atrophic nonunion. Early application of BMP may obviate the need for additional surgical treatment, such as autogenous bone grafting, in situations where there is significant periosteal disruption.

A Case Report of Sleeve Fracture of the Patella in a Shield.

INTRODUCTION: Sleeve fractures of the patella are rare fractures that only occur in children. The diagnosis is difficult both clinically and radiologically since the distal bony fragment may be too small to be detectable by radiography. A high-riding patella and hemarthrosis are important signs of sleeve fractures. CASE REPORT: A 12-year-old boy was admitted to the emergency room after having felt a severe pain in his left knee on kicking the ground while skateboarding earlier that day. Knee swelling, tense hemarthrosis, and periarticular tenderness were noted. On physical examination, an extension lag of 15 was observed. The active range of motion of the injured knee was 45-90° of flexion. Radiography showed an avulsion fracture of the lower pole of the patella and a high-riding patella. At the next day after the injury, we performed open reduction and internal fixation surgery. Open reduction with transosseous tunneling and cerclage wiring was performed because the distal bony fragment was too small for tension band wiring to be used. At 9 months after surgery, there was no extension lag, and the active range of motion of the injured knee was 0-140° of flexion. Callus formation over the fracture site and bone union was confirmed, and the cerclage wire was removed. To date, he had no further symptoms and has been able to carry out all types of physical activities, including skateboarding. CONCLUSION: Although sleeve fractures in children are uncommon, it should be considered a possibility in children with a chief complaint of pain around the knee. Open reduction and internal fixation was effective in the treatment of sleeve fracture of the patella.

Prevention of atrophic nonunion development by recombinant human bone morphogenetic protein-7.

Severe periosteal and soft tissue disruption at the time of fracture may result in the formation of an atrophic nonunion. We have developed a reproducible atrophic nonunion in an animal model. The purpose of this study was to evaluate whether the immediate application of recombinant human BMP-7 to the fracture site could rescue the healing process in this nonunion model. A total of 56 three month old Fisher 344 rats were utilized. A 1.25 mm diameter K-wire was inserted into the femur in a retrograde fashion, and a mid-diaphyseal closed transverse fracture was created using a standard three point bending device. To create a nonunion, the fracture site was exposed and 2 mm of the periosteum was cauterized on each side of the fracture. The fracture site was immediately treated with either the application of rhBMP-7 50 microg in 25 microl of rat tail tendon collagen buffer (BMP-7 group), or with 25 microl of rat tail tendon collagen buffer only (Control group). Fracture healing was evaluated with serial radiographs every two weeks for an eight weeks period. Specimens at four and eight weeks were subjected to biomechanical and histological evaluation. None of the Control group healed throughout the eight weeks experimental duration. At four weeks 63% of the BMP-7 group had healed, and all had healed by six weeks. This investigation showed pronounced differences between the BMP-7 group and the Control group both histologically and biomechanically. In conclusion, we have demonstrated that the immediate application of BMP-7 may rescue the fracture healing process and prevent the development of nonunion following severe periosteal disruption.

Evaluation of an interference screw for tendon reattachment to small bones.

OBJECTIVES: To compare the strength of interference screw fixation of the tendon to suture button fixation and staple fixation methods. DESIGN: Biomechanical study. METHODS: The femurs and flexor digitorum tendon of twelve rabbits were used. The tendon was attached to the medial condyle of the femur using one of the following three surgical procedures: (a) interference screw fixation: tendon-junction screw was used as an interference screw, (b) suture button fixation: the tendon was inserted through a hole and attached with a button on the opposite side, and (c) staple fixation: the tendon was attached using two staples. Strengths of the attached tendons were then measured using a universal testing machine. RESULTS: The ultimate failure load of interference screw fixation was 62.8 +/- 9.0 N, whereas that of suture button fixation was 13.9 +/- 3.8 N and staple fixation was 23.9 +/- 2.6 N. CONCLUSION: Interference screw fixation provides significant initial fixation strength (p < 0.05).

The effect of graft sizing on osteochondral transplantation.

PURPOSE: The goal of this study was to analyze the effect of graft sizing on the histologic property of articular cartilage in autologous osteochondral transplantation. TYPE OF STUDY: Histologic analysis using an animal model. METHODS: Eighteen skeletally mature female Japanese white rabbits were used in this study and divided into 2 groups based on the surgical procedure. Group I: A cylindrical fragment (7 mm in diameter and 5 mm in depth) was harvested at the femoral condyle using the Osteochondral Autograft Transfer System (Arthrex, Naples, FL), then precisely returned to the defect from which the fragment was originally harvested. This surgical model supposes that the articular surface geometry of the osteochondral graft exactly matches that of the cartilage lesion; however, the osteochondral graft is not the same size as the defect in this model because of the surrounding gap that corresponds to the blade thickness of the chisel used to take the graft. Group II: A 1-mm larger osteochondral fragment (8 mm in diameter and 5 mm in depth) taken from the contralateral femoral condyle was transplanted to the osteochondral defect made as in group I, achieving a tight fit. Specimens were stained with Safranin-O fast green and analyzed microscopically at 4, 12, and 24 weeks after surgery. RESULTS: In group I, histologic examination revealed an increase in cartilage thickness and cell density during the implantation period. Round and polygonal hypertrophic clusters of chondrocytes with cytoplasmic vacuoles were observed. By contrast, in group II, the thickness of the articular cartilage was almost the same as that of the normal adjacent cartilage with no significant change observed. CONCLUSIONS: It is suggested that sizing of the implanted osteochondral fragment plays an important role in preserving the histologic properties of cartilage. CLINICAL RELEVANCE: This study suggests that osteochondral graft stability may influence the histologic properties of the repaired cartilage. It is preferable to implant the slightly oversized graft into the cartilage lesion in autologous osteochondral transplantation to preserve the histologic properties of cartilage.

Analysis of the thickness and curvature of articular cartilage of the femoral condyle.

PURPOSE: To develop an optimal autologous osteochondral transplantation technique, we analyzed the thickness and curvature of the femoral condyle. TYPE OF STUDY: Basic research for surgical technique, anatomic study. METHODS: Eight cadaveric femoral condyles were sectioned sagittally into 8 slices. Photographs of each slice were digitized, and the thickness and curvature of the overlaying cartilage were analyzed using a National Institutes of Health image program. RESULTS: The average thickness of the cartilage was 1.69 mm. The minimum and maximum values were measured at 0.22 mm and 3.65 mm, respectively, with the thinnest part seen in the sulcus terminalis (the groove for meniscus). The average curvature of the cartilage was 4.4 m(-1). The minimum and maximum values were measured at -20.0 m(-1) and 27.2 m(-1), respectively. CONCLUSIONS: When the chondral lesion is assumed at the weight bearing area of the medial or lateral femoral condyle, the anterior portion of the sulcus terminalis (the groove for meniscus) in the middle section of the lateral condyle is considered to be the optimal donor site.

The effect of the small and unstable autologous osteochondral graft on repairing the full-thickness large articular cartilage defect in a rabbit model.

We have investigated the effect of the insufficient autologous osteochondral graft on healing of the large articular cartilage defect using a rabbit model. An osteochondral defect, 7 mm in diameter, was made on the patellar groove of the femoral condyle and repaired with two surgical procedures: Group I, the osteochondral fragment as half as the defect was grafted. The graft was unstable and the size of the graft was smaller than the defect. This is a model of the insufficient autologous osteochondral graft to the large articular cartilage defect; Group II, the defect was left empty. At 2, 4, 12, and 24 weeks after the surgery, the specimens were analyzed macroscopically and histologically. To evaluate the microscopic morphology, a histologic grading scale composed of 5 categories was used. In Group I, although the graft sank a little, a grafted cartilage survived and the reparative fibrous tissue filled the defect covering implanted cartilage. In contrast, in Group II, the defect was only partially covered by fibrocartilaginous tissue with a faintly staining matrix. Throughout the entire observed periods, the scores of the repaired cartilage in Group I are significantly higher than those in Group II. Even the half size of osteochondral graft has an effect to reduce the size of the cartilage defect such as the spacer and leads to better healing compared to the cartilage defect untreated. In case that it is hard to transplant an optimal osteochondral graft because of large cartilage lesion, even the small and unstable osteochondral plug should be transplanted.