Simulation von Umstellungsosteotomien an 3D-Finite Elemente Modellen des humanen Femurs

Diese Arbeit befaßt sich mit einer neuen Methode zur automatischen Erstellung von 3D-FE Modellen und deren Verwendung in einer voll parametrisierbaren und automatisierbaren Simulation von intertrochantären Umstellungsosteotomien. Die Methode der FE-Modellierung zeichnet sich dadurch aus, daß sie ein Hybridmodell erzeugt, das zum einen Teil aus einem individuellen, anhand von CT-Daten erstellten, Femurschaft und Schenkelhals (Voxelanteil) besteht, zum anderen Teil aus einem mit Hilfe des "Solid-Modelling" generierten, voll parametrisierbaren, Femurkopf mit Knorpelkappe und Kortikalisschicht (Tetraederanteil). Dieses Hybridmodell verbindet dadurch die Vorteile beider Modellierungstechniken, nämlich einfache Modellerstellung, Berücksichtigung individueller Patientendaten, glatte Kontakt- beziehungsweise Einleitungsflächen und hohe Elementenzahlen nur in den für die Auswertung interessanten Regionen. Die ersten Versuche einer Umstellungsosteotomie an einem Voxelmodell zeigten, daß die Technik funktionierte, aber die Auswertung durch Artefaktbildung nicht verwertbar war. Die in einem nächsten Schritt durchgeführten Vergleichsstudien mit einem nur auf Quaderelementen basierenden Voxelmodell und fünf verschieden konfigurierten Tetraedermodellen des Femurkopfes zeigten die große Bedeutung der Kortikalisschicht und der Knorpelkappe sowie deren Geometrie. Da es auf eine möglichst realitätsnahe Modellierung eben dieser drei Punkte ankommt, entfällt der weitere Einsatz der Modelle ohne die auslaufende Knorpelkappe und Kortikalisschicht. Die Verwendung des reinen Voxelmodelles entfällt alleine schon durch den notwendigen Mehraufwand bei der Modellerstellung, um ein Modell gleicher Qualität und Komplexität zu generieren. In den daraufhin durchgeführten Parameterstudien einer intertrochantären Umstellungsosteotomie zeigt sich die Verbindung des Osteotomiewinkel mit dem CE-Winkel als der wichtigste Faktor für die Belastung des Gelenkknorpels. Unter physiologischen Bedingungen, das heißt bei einem normalen CE-Winkel, ändert sich an den auftretenden Spannungen nur wenig, bei einer Hüftdysplasie (CE-Winkel=-15°) hingegen ist es möglich, durch eine varisierende 20° Osteotomie die maximal auftretende von-Mises-Vergleichsspannung am Knorpel von 25 MPa auf 11 MPa zu senken. Veränderungen der Parameter für die Osteotomiehöhe und den lateralen Versatz des proximalen Fragmentes zeigen dagegen nur einen minimalen Effekt auf die auftretenden Spannungen. Den größten Einfluß als alleiniger Faktor hat jedoch der CE-Winkel, der sich mit der hier simulierten Operation einer Umstellungsosteotomie des proximalen Femurs nicht beeinflussen läßt. Eine Weiterverwendung der bei dieser Arbeit entstandenen Werkzeuge an anderen Knochen des menschlichen Körpers ist ohne große Anpassungen möglich. Eine Weiterentwicklung zum Einsatz der Hybridtechnik bei der proximalen Tibiakopfumstellung ist bereits in Arbeit. Vor einem Praxiseinsatz der hier vorgestellten Methode steht noch die Notwendigkeit von größeren Studien mit dem Ziel, die Ergebnisse zu validieren, die Interpretierbarkeit zu verbessern und die Notwendigkeit der Individualisierung der einzelnen Parameter zu überprüfen

Analysis of MRE11's function in the 5′→3′ processing of DNA double-strand breaks

The resection of DNA double-strand breaks (DSBs) into 3′ single-strand tails is the initiating step of homology-dependent repair pathways. A key player in this process is the MRE11-RAD50-NBS1 complex, but its contribution to and mechanistic role in resection are not well understood. In this study, we took advantage of the Xenopus egg extract system to address these questions. We found that depletion of MRE11 caused a dramatic inhibition of 5′-resection, even for the first nucleotide at the 5′-end. Depletion of Xenopus CtIP also inhibited 5′-strand resection, but this inhibition could be alleviated by excess MRN. Both MRE11 and CtIP could be bypassed by a DNA that carried a 3′-ss-tail. Finally, using purified proteins, we found that MRN could stimulate both the WRN-DNA2-RPA pathway and the EXO1 pathway of resection. These findings provide important insights into the function of MRE11 in 5′-strand resection

Interplay of Mre11 Nuclease with Dna2 plus Sgs1 in Rad51-Dependent Recombinational Repair

The Mre11/Rad50/Xrs2 complex initiates IR repair by binding to the end of a double-strand break, resulting in 5′ to 3′ exonuclease degradation creating a single-stranded 3′ overhang competent for strand invasion into the unbroken chromosome. The nuclease(s) involved are not well understood. Mre11 encodes a nuclease, but it has 3′ to 5′, rather than 5′ to 3′ activity. Furthermore, mutations that inactivate only the nuclease activity of Mre11 but not its other repair functions, mre11-D56N and mre11-H125N, are resistant to IR. This suggests that another nuclease can catalyze 5′ to 3′ degradation. One candidate nuclease that has not been tested to date because it is encoded by an essential gene is the Dna2 helicase/nuclease. We recently reported the ability to suppress the lethality of a dna2Δ with a pif1Δ. The dna2Δ pif1Δ mutant is IR-resistant. We have determined that dna2Δ pif1Δ mre11-D56N and dna2Δ pif1Δ mre11-H125N strains are equally as sensitive to IR as mre11Δ strains, suggesting that in the absence of Dna2, Mre11 nuclease carries out repair. The dna2Δ pif1Δ mre11-D56N triple mutant is complemented by plasmids expressing Mre11, Dna2 or dna2K1080E, a mutant with defective helicase and functional nuclease, demonstrating that the nuclease of Dna2 compensates for the absence of Mre11 nuclease in IR repair, presumably in 5′ to 3′ degradation at DSB ends. We further show that sgs1Δ mre11-H125N, but not sgs1Δ, is very sensitive to IR, implicating the Sgs1 helicase in the Dna2-mediated pathway

Mechanistic analysis of Xenopus EXO1's function in 5′-strand resection at DNA double-strand breaks

The processing of DNA double-strand breaks (DSBs) into 3′ single-stranded tails is the first step of homology-dependent DSB repair. A key player in this process is the highly conserved eukaryotic exonuclease 1 (EXO1), yet its precise mechanism of action has not been rigorously determined. To address this issue, we reconstituted 5′-strand resection in cytosol derived from unfertilized interphase eggs of the frog Xenopus laevis. Xenopus EXO1 (xEXO1) was found to display strong 5′→3′ dsDNA exonuclease activity but no significant ssDNA exonuclease activity. Depletion of xEXO1 caused significant inhibition of 5′ strand resection. Co-depletion of xEXO1 and Xenopus DNA2 (xDNA2) showed that these two nucleases act in parallel pathways and by distinct mechanisms. While xDNA2 acts on ssDNA unwound mainly by the Xenopus Werner syndrome protein (xWRN), xEXO1 acts directly on dsDNA. Furthermore, xEXO1 and xWRN are required for both the initiation stage and the extension stage of resection. These results reveal important novel information on the mechanism of 5′-strand resection in eukaryotes

Identification of the Xenopus DNA2 protein as a major nuclease for the 5′→3′ strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes

Minimal stress shielding with a Mallory-Head titanium femoral stem with proximal porous coating in total hip arthroplasty

<p>Abstract</p> <p>Background</p> <p>As longevity of cementless femoral components enters the third decade, concerns arise with long-term effects of fixation mode on femoral bone morphology. We examined the long-term consequences on femoral remodeling following total hip arthroplasty with a porous plasma-sprayed tapered titanium stem.</p> <p>Methods</p> <p>Clinical data and radiographs were reviewed from a single center for 97 randomly selected cases implanted with the Mallory-Head Porous femoral component during primary total hip arthroplasty. Measurements were taken from preoperative and long-term follow-up radiographs averaging 14 years postoperative. Average changes in the proximal, middle and diaphyseal zones were determined.</p> <p>Results</p> <p>On anteroposterior radiographs, the proximal cortical thickness was unchanged medially and the lateral zone increased 1.3%. Middle cortical thickness increased 4.3% medially and 1.2% laterally. Distal cortical thickness increased 9.6% medially and 1.9% laterally. Using the anteroposterior radiographs, canal fill at 100 mm did not correlate with bony changes at any level (Spearman's rank correlation coefficient of -0.18, 0.05, and 0.00; p value = 0.09, 0.67, 0.97). On lateral radiographs, the proximal cortical thickness increased 1.5% medially and 0.98% laterally. Middle cortical thickness increased 2.4% medially and 1.3% laterally. Distal cortical thickness increased 3.5% medially and 2.1% laterally. From lateral radiographs, canal fill at 100 mm correlated with bony hypertrophy at the proximal, mid-level, and distal femur (Spearman's rank correlation coefficient of 0.85, 0.33, and 0.28, respectively; p value = 0.001, 0.016, and 0.01, respectively).</p> <p>Conclusion</p> <p>Stress shielding is minimized with the Mallory-Head titanium tapered femoral stem with circumferential proximal plasma-sprayed coating in well-fixed and well-functioning total hip arthroplasty. Additionally, the majority of femora demonstrated increased cortical thickness in all zones around the stem prosthesis. Level of Evidence: Therapeutic Level III.</p

MRE11 Function in Response to Topoisomerase Poisons Is Independent of its Function in Double-Strand Break Repair in Saccharomyces cerevisiae

Camptothecin (CPT) and etoposide (ETP) trap topoisomerase-DNA covalent intermediates, resulting in formation of DNA damage that can be cytotoxic if unrepaired. CPT and ETP are prototypes for molecules widely used in chemotherapy of cancer, so defining the mechanisms for repair of damage induced by treatment with these compounds is of great interest. In S. cerevisiae, deficiency in MRE11, which encodes a highly conserved factor, greatly enhances sensitivity to treatment with CPT or ETP. This has been thought to reflect the importance of double-strand break (DSB) repair pathways in the response to these to agents. Here we report that an S. cerevisiae strain expressing the mre11-H59A allele, mutant at a conserved active site histidine, is sensitive to hydroxyurea and also to ionizing radiation, which induces DSBs, but not to CPT or ETP. We show that TDP1, which encodes a tyrosyl-DNA phosphodiesterase activity able to release both 5′- and 3′-covalent topoisomerase-DNA complexes in vitro, contributes to ETP-resistance but not CPT-resistance in the mre11-H59A background. We further show that CPT- and ETP-resistance mediated by MRE11 is independent of SAE2, and thus independent of the coordinated functions of MRE11 and SAE2 in homology-directed repair and removal of Spo11 from DNA ends in meiosis. These results identify a function for MRE11 in the response to topoisomerase poisons that is distinct from its functions in DSB repair or meiotic DNA processing. They also establish that cellular proficiency in repair of DSBs may not correlate with resistance to topoisomerase poisons, a finding with potential implications for stratification of tumors with specific DNA repair deficiencies for treatment with these compounds

Affimer proteins for F-actin: novel affinity reagents that label F-actin in live and fixed cells

Imaging the actin cytoskeleton in cells uses a wide range of approaches. Typically, a fluorescent derivative of the small cyclic peptide phalloidin is used to image F-actin in fixed cells. Lifeact and F-tractin are popular for imaging the cytoskeleton in live cells. Here we characterised novel affinity reagents called Affimers that specifically bind to F-actin in vitro to determine if they are suitable alternatives as eGFP-fusion proteins, to label actin in live cells, or for labeling F-actin in fixed cells. In vitro experiments showed that 3 out of the 4 Affimers (Affimers 6, 14 and 24) tested bind tightly to purified F-actin, and appear to have overlapping binding sites. As eGFP-fusion proteins, the same 3 Affimers label F-actin in live cells. FRAP experiments suggest that eGFP-Affimer 6 behaves most similarly to F-tractin and Lifeact. However, it does not colocalize with mCherry-actin in dynamic ruffles, and may preferentially bind stable actin filaments. All 4 Affimers label F-actin in methanol fixed cells, while only Affimer 14 labels F-actin after paraformaldehyde fixation. eGFP-Affimer 6 has potential for use in selectively imaging the stable actin cytoskeleton in live cells, while all 4 Affimers are strong alternatives to phalloidin for labelling F-actin in fixed cells