Mechanism of strand displacement DNA synthesis by the coordinated activities of human mitochondrial DNA polymerase and SSB.

Many replicative DNA polymerases couple DNA replication and unwinding activities to perform strand displacement DNA synthesis, a critical ability for DNA metabolism. Strand displacement is tightly regulated by partner proteins, such as single-stranded DNA (ssDNA) binding proteins (SSBs) by a poorly understood mechanism. Here, we use single-molecule optical tweezers and biochemical assays to elucidate the molecular mechanism of strand displacement DNA synthesis by the human mitochondrial DNA polymerase, Polγ, and its modulation by cognate and noncognate SSBs. We show that Polγ exhibits a robust DNA unwinding mechanism, which entails lowering the energy barrier for unwinding of the first base pair of the DNA fork junction, by ∼55%. However, the polymerase cannot prevent the reannealing of the parental strands efficiently, which limits by ∼30-fold its strand displacement activity. We demonstrate that SSBs stimulate the Polγ strand displacement activity through several mechanisms. SSB binding energy to ssDNA additionally increases the destabilization energy at the DNA junction, by ∼25%. Furthermore, SSB interactions with the displaced ssDNA reduce the DNA fork reannealing pressure on Polγ, in turn promoting the productive polymerization state by ∼3-fold. These stimulatory effects are enhanced by species-specific functional interactions and have significant implications in the replication of the human mitochondrial DNA.

Primary modular total knee replacement in severe and unstable osteoarthritis. Predictive factors for failure.

PURPOSE: Severe cases of osteoarthritis with ligamentous instability require the use of a modular knee arthoplasty. To assess the survival, the complications, the clinical, radiological and functional outcomes, and the quality of life of those patients in whom a Optetrak Condylar Constrained Knee (CCK) had been implanted as a primary implant. To study how the pre-operative conditions (deformity, age, sex …) and the characteristics of the arthroplasty (stems, supplements, constrained component …) could change the survival or the clinical outcomes of the implants. METHODS: We performed an observational retrospective study of 105 CCKs implanted between 1999 and 2005. The mean follow-up was nine years (7, 13). Mean age was 70.5 years. The mortality was 6.9 %. There were 9.3 % of the patients lost during the follow-up. We studied all the medical files of the patients in order to assess the pre-operative, surgical and postoperative conditions. We used the Knee Society Score (KSS), both clinical and functional, to study the functional and clinical situation. We analysed the X-ray using the Knee Society roentgenographic evaluation. The quality of life was studied using the Oxford Knee Score (OKS). Then in order to find predictor conditions, we made statistical multivariable predictive studies attending to the preoperative factors and to the arthroplasty conditions to compare both outcomes and survival (Kaplan-Meier groups curves and Cox Multivariable Models [hazard ratio]). Processing and data analysis was performed using SPSS 15.0. RESULTS: The outcomes did not show differences between all the groups. While the mean result of the clinical KSS was 75.8, the mean functional KSS was 73.1. We did not find a condition associated with worst results of the knees (p > 0.05). The KSS obtained was excellent or good in 74.9 %. The global survival at 24 months was 93.8 % and at 96 months was 90.1 %. There were some conditions associated with poorest survival of the arthroplasties: patients younger than 70 years old, tibial tuberosity osteotomy, use of long stems and new surgery two months after arthoplasty (p < 0.05). The 11.1 % of the arthroplasties required more surgeries (3.3 % were soft tissue surgeries and 7.8 % were revisions). The mean OKS obtained was 34.78. Satisfaction was obtained by 86.2 % of the patients. CONCLUSIONS: All severe and unstable knees obtained similar and overall excellent-good outcomes and survival using the Optetrak CCK. Although we found that there are some factors that could change the survival of the total knee replacement, these did not change the outcomes.

Predictive factors for failure after total knee replacement revision.

PURPOSE: We used the Optetrak Condylar Constrained (CCK) implant, a modular and constrained knee implant as replacement for a failed primary arthroplasty, to assess the survivorship, the complications, the clinical, radiological, and functional situation, and the quality of life of those patients in whom a CCK had been implanted in recent years in order to find predictive pre-operative conditions of survival and clinical outcomes. METHODS: We performed a retrospective study of 125 CCK implanted between 1999 and 2005. The mean follow-up was nine years (range, seven to 13). Mean age was 73.6 years. A total of 78% of the revised TKA were cemented and 66% were CR. We assessed the pre-operative, the operative and the postoperative conditions studying the medical files of all the patients. In order to study the functional and clinical situation we used the Hospital for Special Surgery (HSS) score and the Knee Society score (KSS), both clinical and functional. We analysed all the X-rays using the Knee Society Roentgenographic evaluation. The quality of life was studied using the Oxford knee score (OKS). RESULTS: The mean results of the KSS clinical and the KSS functional were 68.24 and 63.85, respectively. There were not any conditions associated with poor results of the knees (p > 0.05). The global survival at 24 months was 92.7%, at 60 months 87.8% and at 96 months it was 87.8%. There were some conditions associated with poor survival of the knees, e.g. patients were younger than 70 years old, rheumatic diseases, kidney faliure, tibial tuberosity osteotomy, PS primary arthroplasty, revision before five years and septic loosening. CONCLUSIONS: Based on these results there are some pre-operative factors that change the survival of the total knee replacement revision.

Supplementary Tibial Fixation in Anterior Cruciate Ligament Reconstruction With Bone-Tendon-Bone Graft.

Good to excellent results at long-term follow-up have been published for anterior cruciate ligament reconstruction with bone-tendon-bone graft. Despite improvements in fixation devices, concerns regarding the stability of graft fixation on the tibial side remain. We present supplementary tibial fixation for anterior cruciate ligament reconstruction with bone-tendon-bone graft using a transosseous technique that is simple and inexpensive and avoids the risk of symptomatic hardware.

Multiple mutations in the Kras gene in colorectal cancer: review of the literature with two case reports.

PURPOSE: Kras mutations are negative predictors of anti-EGFR therapy, occurring in 40% of colorectal carcinomas (CRCs). Point substitutions in codon 12 or 13 are the most frequent mutations in Kras, but multiple mutations (MMs) in other codons can also develop. Few data exist on MMs with regard to their frequency and the codons and amino acids that are affected. We report two cases of Kras double mutations in codons 12 and 13 and review Kras MMs in primary CRC in PubMed databases. CASE REPORT: A 53-year-old woman and a 70-year-old man presented with deep, invasive, moderately differentiated CRC at an advanced clinical stage. The former had regional lymph node involvement and vaginal wall neoplastic implantation, and the latter had liver metastasis. Primary tumors were examined for Kras mutations by pyrosequencing, which were confirmed by direct sequencing. Both tumors had a mutation in codons 12 and 13, wherein codon 12 was mutated to GAT, and codon 13 became GAC. CONCLUSIONS: We identified 69 reported cases of Kras MMs and reported two other cases, representing 2.1% of all mutated tumors; the incidence of such mutations is 1.0% in CRC patients. In most cases (59%), MMs develop in a single codon, usually codon 12. Codons 12 and 13 are affected simultaneously in only 27% of cases. These findings add information about the impact of specific amino acid changes in the Kras gene.

Midterm results of Optetrak posterior-stabilized total knee system after 7 to 12 years in a university hospital.

A clinical study has been carried out on 434 posterior-stabilized knee prostheses (Optetrak; Exactech, Gainesville, Fla) implanted between 1995 and 2000 in a university general hospital by 23 surgeons. At a mean follow-up of 8.8 years, 297 knees in 249 patients were available for review. Average patient age at surgery was 71.8 years. Average body mass index was 28.8 kg/m(2). Mean flexion range was 108° .The average knee score (Hospital for Special Surgery) increased from 48 points preoperatively to 86 points (60-97 points) at the final review. Of the patients, 81% had an excellent or good result, 14.9% had a fair result, and 4.1% had a poor result. Using the Kaplan-Meier method, we obtained a 91.3% predicted implant survival at 12 years including septic and aseptic revision in the best-case scenario.

Diagnosis of infection in hip and knee revision surgery: intraoperative frozen section analysis.

UNLABELLED: In this study we validated intraoperatively the analysis of polymorphonuclear leucocyte frozen sections for diagnosis of infection in hip and knee revisions. Between 1996 and 2002 we examined sections and cultured periprosthetic tissues in prosthetic revision in 170 cases, including 146 cases (83 hips and 63 knees). We assessed sensitivity (SE), specificity (SP), positive predictive value (PPV), negative predictive value (NPV), Youden index, positive likelihood ratio (PLR) and negative likelihood ratio (NLR). We compared intraoperative findings and paraffin-embedded samples. The results in the knee group were SE=66.7%, SP=89.7% [95% confidence interval (CI)], PPV=81% (95% CI), NPV=81.4% (95% CI), Youden index=0.56, PLR=6.5 (95% CI) and NLR=0.4 (95% CI). In the paraffin-embedded samples the results were SE=91%, SP=87% (95% CI), PPV=81% (95% CI), NPV=94% (95% CI), PLR=7 (95% CI) and NLR=8.7 (95% CI). We found a significant difference. The results in the hip group were SE=50%, SP=100% (95% CI), PPV=100% (95% CI), NPV=94.9% (95% CI), Youden index=0.5 and PLR=0.5 (95% CI). In hip and knee prosthetic revision surgery the presence of polymorphonuclear cells correlates with infection, but their absence does not exclude it. It is a quick and inexpensive test that should be included in the diagnostic protocol in revision surgery. LEVEL OF EVIDENCE: diagnostic Study (investigating a diagnostic test), level I. See instructions to authors for a complete description of levels of evidence.

Peripheral primitive neuroectodermal tumor after radiotherapy.

A 41-year-old man had a peripheral neuroectodermal tumor develop at the distal third of the fibula 4 years after radiotherapy for relapsed villonodular synovitis. This type of sarcoma usually is classified into the heterogeneic group of small round-cell bone tumors as a subdivision of Ewing's sarcomas. The immuno-staining positivity of the neoplastic cells for the neuron-specific enolase allowed the authors to make the diagnosis of a tumor with neuroectodermal origin. When the histologic study confirmed the diagnosis, the patient was treated with chemotherapy, surgical excision of the tumor, and adjuvant radiotherapy. Radiotherapy is thought to be involved in the genesis of osteogenic sarcomas as it has been shown in several reports, but there is no evidence in the literature of a peripheral neuroectodermal tumor developing after radiotherapy.