BUSCADOR RECOLECTA

Western blot of a protein extract from N. benthamiana leaves expressing PepCPWT. Protein extract aliquots were oxidized with CuCl2 (lanes 1 and 2), reduced with dithiothreitol (lanes 3 and 4), or not pre-treated with any of them (lanes 5–6). Protein extracts loaded in lanes 2, 4 and 6 were incubated with methyl-polyethylene glycol-maleimide (MM(PEG)24) as described by Pant et al. (2021). Reduced cysteines are alkylated with MM(PEG)24 and one conjugation increases the protein mass by 1.24 kDa. A double band was observed in lanes 2 and 4, indicating partial CP oxidation, reduction or alkylation in the controls. The membrane was stripped and re-probed with an anti-Actin antibody as the loading control, Peer reviewed

Western blot of a protein extract from N. benthamiana leaves expressing PepCPWT. Protein extract aliquots were oxidized with CuCl2 (lanes 1 and 2), reduced with dithiothreitol (lanes 3 and 4), or not pre-treated with any of them (lanes 5–6). Protein extracts loaded in lanes 2, 4 and 6 were incubated with methyl-polyethylene glycol-maleimide (MM(PEG)24) as described by Pant et al. (2021). Reduced cysteines are alkylated with MM(PEG)24 and one conjugation increases the protein mass by 1.24 kDa. A double band was observed in lanes 2 and 4, indicating partial CP oxidation, reduction or alkylation in the controls. The membrane was stripped and re-probed with an anti-Actin antibody as the loading control, Peer reviewed

Coomassie-stained SDS-PAGE gel of protein extractions from Nicotiana benthamiana leaves expressing the CP wild type (CP) or the CP mutant CPC127S. EV: pJL-TRBO empty vector; M: Marker. CP and CPC127S are expressed at similar levels, Peer reviewed

[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

Peer reviewed

Supplementary figure S1. Schematic representation of the hypothetical consequences of the THRB variant. In silico prediction of the effect of the spliceogenic variant identified in TRβ1 isoform (NM_001354712.2: c.283+1G>A) showed two scenarios. (A), The skipping of exon 5 disrupts the A/B domain, leaving a protein of 374 amino acids with a secondary structure different from the wild-type protein. The DNAbinding domain (DBD), hinge domain and ligand-binding domain (LBD) are not affected. (B), The skipping of exons 5 and 6 results in a premature codon stop that produces only a small peptide. The 3D models of the proteins were generated using the IntFOLD and PEP-FOLD4 online servers., Inherited retinal dystrophies (IRDs) are a clinically and genetically heterogeneous group of disorders that often severely impair vision. Some patients manifest poor central vision as the first symptom due to cone-dysfunction, which is consistent with cone dystrophy (COD), Stargardt disease (STGD), or macular dystrophy (MD) among others. Here, we aimed to identify the genetic cause of autosomal dominant COD in one family. WGS was performed in 3 affected and 1 unaffected individual using the TruSeq Nano DNA library kit and the NovaSeq 6,000 platform (Illumina). Data analysis identified a novel spliceogenic variant (c.283 + 1G>A) in the thyroid hormone receptor beta gene (THRB) as the candidate disease-associated variant. Further genetic analysis revealed the presence of the same heterozygous variant segregating in two additional unrelated dominant pedigrees including 9 affected individuals with a diagnosis of COD (1), STGD (4), MD (3) and unclear phenotype (1). THRB has been previously reported as a causal gene for autosomal dominant and recessive thyroid hormone resistance syndrome beta (RTHβ); however, none of the IRD patients exhibited RTHβ. Genotype-phenotype correlations showed that RTHβ can be caused by both truncating and missense variants, which are mainly located at the 3′ (C-terminal/ligand-binding) region, which is common to both THRB isoforms (TRβ1 and TRβ2). In contrast, the c.283 + 1G>A variant is predicted to disrupt a splice site in the 5′-region of the gene that encodes the N-terminal domain of the TRβ1 isoform protein, leaving the TRβ2 isoform intact, which would explain the phenotypic variability observed between RTHβ and IRD patients. Interestingly, although monochromacy or cone response alterations have already been described in a few RTHβ patients, herein we report the first genetic association between a pathogenic variant in THRB and non-syndromic IRDs. We thereby expand the phenotype of THRB pathogenic variants including COD, STGD, or MD as the main clinical manifestation, which also reflects the extraordinary complexity of retinal functions mediated by the different THRB isoforms., Peer reviewed