Resultados de investigación

[Introduction] Tissue engineering has emerged as an innovative approach to treat critical-size bone defects using biocompatible scaffolds, thus avoiding complex distraction surgeries or limited stock grafts. Continuous regeneration monitoring is essential in critical-size cases due to the frequent appearance of non-unions. This work evaluates the potential clinical use of gait analysis for the mechanical assessment of a tissue engineering regeneration as an alternative to the traditional and hardly conclusive manual or radiological follow-up., [Materials and methods] The 15-mm metatarsal fragment of eight female merino sheep was surgically replaced by a bioceramic scaffold stabilized with an external fixator. Gait tests were performed weekly by making the sheep walk on an instrumented gangway. The evolution of different kinematic and dynamic parameters was analyzed for all the animal’s limbs, as well as asymmetries between limbs. Finally, potential correlation in the recovery of the gait parameters was evaluated through the linear regression models., [Results] After surgery, the operated limb has an altered way of carrying body weight while walking. Its loading capacity was significantly reduced as the stance phases were shorter and less impulsive. The non-operated limbs compensated for this mobility deficit. All parameters were normalizing during the consolidation phase while the bone callus was simultaneously mineralizing. The results also showed high levels of asymmetry between the operated limb and its contralateral, which exceeded 150% when analyzing the impulse after surgery. Gait recovery significantly correlated between symmetrical limbs., [Conclusions] Gait analysis was presented as an effective, low-cost tool capable of mechanically predicting the regeneration of critical-size defects treated by tissue engineering, as comparing regeneration processes or novel scaffolds. Despite the progressive normalization as the callus mineralized, the bearing capacity reduction and the asymmetry of the operated limb were more significant than in other orthopedic alternatives., Peer reviewed

OBJ of the geometry of the scaffold designed from CT scans of an intermediate 13-mm ovine metatarsal bone fragment. The specific modifications made to the scaffold for stability and regenerative response are included., Peer reviewed

Ground reaction force data from the recorded stance phases in the non-operated control sheep of the study (n = 3)., Peer reviewed

Complete set of all experimental data for all analyzed gait parameters (GRFpeak, GRFmean, t and Imp) in all animals in the study., Peer reviewed

Complete set of all data asymmetries in the analyzed gait parameters (GRFpeak, GRFmean, t and Imp) calculated between the hind- and forelimbs., Peer reviewed

Eukaryotic chromosome segregation relies on the assembly of a bipolar machinery based on microtubules (MTs), named the mitotic spindle. Formation of the mitotic spindle follows a force balance mechanism that ensures the proper capture and separation of sister chromatids. Many proteins have been involved in the establishment of this force balance, although kinesin 5 is well recognized as the major outward pushing force generator, since its inactivation results in monopolar, non-functional spindles. In order to find additional players in the force balance mechanism, we have performed a suppressor screen using a conditional allele of the fission yeast kinesin 5 ortholog Cut7. This screen identified that the lack of the PP6 phosphatase partially suppresses cut7 phenotypes, at least by defective translation of MT regulators, such as the minus end-directed kinesin Klp2, the MT stabilizer Alp7 and the MT bundler Ase1, impacting on the force balance mechanism. Additionally, our data show that the Elongator complex, a target activated by PP6 for efficient tRNA modification, also contributes to the force balance, albeit to a lesser extent. Importantly, this complex has recently been implicated in direct MT polymerization in metazoans, a role not shared by its fission yeast counterpart., Peer reviewed

Eukaryotic chromosome segregation relies on the assembly of a bipolar machinery based on microtubules (MTs), named the mitotic spindle. Formation of the mitotic spindle follows a force balance mechanism that ensures the proper capture and separation of sister chromatids. Many proteins have been involved in the establishment of this force balance, although kinesin 5 is well recognized as the major outward pushing force generator, since its inactivation results in monopolar, non-functional spindles. In order to find additional players in the force balance mechanism, we have performed a suppressor screen using a conditional allele of the fission yeast kinesin 5 ortholog Cut7. This screen identified that the lack of the PP6 phosphatase partially suppresses cut7 phenotypes, at least by defective translation of MT regulators, such as the minus end-directed kinesin Klp2, the MT stabilizer Alp7 and the MT bundler Ase1, impacting on the force balance mechanism. Additionally, our data show that the Elongator complex, a target activated by PP6 for efficient tRNA modification, also contributes to the force balance, albeit to a lesser extent. Importantly, this complex has recently been implicated in direct MT polymerization in metazoans, a role not shared by its fission yeast counterpart., Peer reviewed

A: Serial dilution assay of the wild type, ekc1∆, cut7–24 and cut7–24 ekc1∆ strains incubated for 3 days at the indicated temperatures. B: SDS PAGE analysis of Cut7-3xHA levels on wild type or ppe1∆ cells. The upper panel corresponds to the blot incubated with an anti-HA antibody, while the bottom panel corresponds to the blot incubated with an anti-tubulin antibody. C: Plot showing the total Cut7-3xHA levels relative to the corresponding tubulin levels in wild type and ppe1∆ cells. n for wild type: 3; n for ppe1∆: 3. a.u.: arbitrary units. Statistical significance was determined using a T-test (p = 0.086). D: Upper panel, PhosTag SDS PAGE analysis of Cut7-3xHA in asynchronous and mitotically blocked nda3-KM311 cells in the presence or absence of Ppe1. Bottom panel, the same samples submitted to SDS PAGE analysis using an anti-tubulin antibody. E: SDS PAGE analysis of Pkl1-13xMyc levels on wild type or ppe1∆ cells. The upper panel corresponds to the blot incubated with an anti-Myc antibody, while the bottom panel corresponds to the blot incubated with an anti-tubulin antibody. F: Plot showing the total Pkl1-13xMyc levels relative to the corresponding tubulin levels in wild type and ppe1∆ cells. n for wild type: 3; n for ppe1∆: 3. a.u.: arbitrary units. Statistical significance was determined using a T-test (p = 0.121). G: Upper panel, PhosTag SDS PAGE analysis of Pkl1-13xMyc in asynchronous and mitotically blocked nda3-KM311 cells in the presence or absence of Ppe1. Bottom panel, the same samples submitted to SDS PAGE analysis using an anti-tubulin antibody., Peer reviewed

A: Serial dilution assay of the wild type, elp3∆, cut7–24, cut7–24 elp3∆, cut7–22 and cut7–22 elp3∆ strains incubated for 3 days at the indicated temperatures. B: Selected kymographs showing interphasic MT dynamics in wild type, ppe1∆, elp3∆ and elp3-YY527AA cells expressing GFP-Atb2, showing the inverted green channel in a grey scale. Scale bar: 5 μm. C: Plot showing MT growth rate of the indicated strains expressing GFP-Atb2 (n for wild type: 74; n for ppe1∆: 87; n for elp3∆: 79; n for elp3-YY527AA: 79). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.409; wt vs. elp3∆, p = 0.374; wt vs. elp3-YY527AA, p = 0.001). D: Plot showing MT catastrophe rate of the indicated strains expressing GFP-Atb2 (n for wild type: 95; n for ppe1∆: 73; n for elp3∆: 82; n for elp3-YY527AA: 88). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.054; wt vs. elp3∆, p = 0.003; wt vs. elp3-YY527AA, p = 0.294). E: Plot showing MT dwelling time at the cell end of the indicated strains expressing GFP-Atb2 (n for wild type: 63; n for ppe1∆: 67; n for elp3∆: 67; n for elp3-YY527AA: 65). Statistical differences were determined using a T-test (wt vs. ppe1∆, p = 0.931; wt vs. elp3∆, p = 0.500; wt vs. elp3-YY527AA, p = 0.667). F: Maximum intensity projections from confocal time lapse images of wild type cells expressing mCherry-Atb2 and Elp3 or Elp4 fused to GFP. The upper series shows the magenta (mCherry-Atb2) and green (Elp3-GFP or Elp4-GFP) merged channels. The bottom series shows the inverted green channel in a grey scale. Time 0 corresponds to mitotic entry. The dashed line represents the cell contour. Scale bar: 2 μm. G: Western-blot analysis of SDS PAGE of Elp3-GFP and Elp4-GFP from the strains shown in C., Peer reviewed

A: Serial dilution assay of the wild type, ppe1∆, klp2∆, ppe1∆ klp2∆, cut7–22, cut7–22 ppe1∆, cut7–22 klp2∆ and cut7–22 ppe1∆ klp2∆ strains incubated for 3 days at the indicated temperatures. B: Serial dilution assay of the wild type, ppe1∆, alp7∆, ppe1∆ alp7∆, cut7–22, cut7–22 ppe1∆, cut7–22 alp7∆ and cut7–22 ppe1∆ alp7∆ strains incubated for 3 days at the indicated temperatures. C: Serial dilution assay of the wild type, ppe1∆, ase1∆, ppe1∆ ase1∆, cut7–22, cut7–22 ppe1∆, cut7–22 ase1∆ and cut7–22 ppe1∆ ase1∆ strains incubated for 3 days at the indicated temperatures., Peer reviewed