Gene expression during normal and FSHD myogenesis

<p>Abstract</p> <p>Background</p> <p>Facioscapulohumeral muscular dystrophy (FSHD) is a dominant disease linked to contraction of an array of tandem 3.3-kb repeats (D4Z4) at 4q35. Within each repeat unit is a gene, <it>DUX4</it>, that can encode a protein containing two homeodomains. A <it>DUX4 </it>transcript derived from the last repeat unit in a contracted array is associated with pathogenesis but it is unclear how.</p> <p>Methods</p> <p>Using exon-based microarrays, the expression profiles of myogenic precursor cells were determined. Both undifferentiated myoblasts and myoblasts differentiated to myotubes derived from FSHD patients and controls were studied after immunocytochemical verification of the quality of the cultures. To further our understanding of FSHD and normal myogenesis, the expression profiles obtained were compared to those of 19 non-muscle cell types analyzed by identical methods.</p> <p>Results</p> <p>Many of the ~17,000 examined genes were differentially expressed (> 2-fold, <it>p </it>< 0.01) in control myoblasts or myotubes vs. non-muscle cells (2185 and 3006, respectively) or in FSHD vs. control myoblasts or myotubes (295 and 797, respectively). Surprisingly, despite the morphologically normal differentiation of FSHD myoblasts to myotubes, most of the disease-related dysregulation was seen as dampening of normal myogenesis-specific expression changes, including in genes for muscle structure, mitochondrial function, stress responses, and signal transduction. Other classes of genes, including those encoding extracellular matrix or pro-inflammatory proteins, were upregulated in FSHD myogenic cells independent of an inverse myogenesis association. Importantly, the disease-linked <it>DUX4 </it>RNA isoform was detected by RT-PCR in FSHD myoblast and myotube preparations only at extremely low levels. Unique insights into myogenesis-specific gene expression were also obtained. For example, all four Argonaute genes involved in RNA-silencing were significantly upregulated during normal (but not FSHD) myogenesis relative to non-muscle cell types.</p> <p>Conclusions</p> <p><it>DUX4</it>'s pathogenic effect in FSHD may occur transiently at or before the stage of myoblast formation to establish a cascade of gene dysregulation. This contrasts with the current emphasis on toxic effects of experimentally upregulated <it>DUX4 </it>expression at the myoblast or myotube stages. Our model could explain why <it>DUX4</it>'s inappropriate expression was barely detectable in myoblasts and myotubes but nonetheless linked to FSHD.</p

Crystal structure of the neurotrophin-3 and p75 NTR

Neurotrophins (NTs) are important regulators for the survival, differentiation and maintenance of different peripheral and central neurons. NTs bind to two distinct classes of glycosylated receptor: the p75 neurotrophin receptor (p75(NTR)) and tyrosine kinase receptors (Trks). Whereas p75(NTR) binds to all NTs, the Trk subtypes are specific for each NT. The question of whether NTs stimulate p75(NTR) by inducing receptor homodimerization is still under debate. Here we report the 2.6-A resolution crystal structure of neurotrophin-3 (NT-3) complexed to the ectodomain of glycosylated p75(NTR). In contrast to the previously reported asymmetric complex structure, which contains a dimer of nerve growth factor (NGF) bound to a single ectodomain of deglycosylated p75(NTR) (ref. 3), we show that NT-3 forms a central homodimer around which two glycosylated p75(NTR) molecules bind symmetrically. Symmetrical binding occurs along the NT-3 interfaces, resulting in a 2:2 ligand-receptor cluster. A comparison of the symmetrical and asymmetric structures reveals significant differences in ligand-receptor interactions and p75(NTR) conformations. Biochemical experiments indicate that both NT-3 and NGF bind to p75(NTR) with 2:2 stoichiometry in solution, whereas the 2:1 complexes are the result of artificial deglycosylation. We therefore propose that the symmetrical 2:2 complex reflects a native state of p75(NTR) activation at the cell surface. These results provide a model for NTs-p75(NTR) recognition and signal generation, as well as insights into coordination between p75(NTR) and Trks

Genome Modification Approaches to Improve Performance, Quality, and Stress Tolerance of Important Mediterranean Fruit Species (Olea europaea L., Vitis vinifera L., and Quercus suber L.).

In the last decades the interest on traditional Mediterranean fruits highly increased, not only due to the constant demand of consumers for new crop alternatives, but also due to the identification in such species of molecules with important properties for human health (e.g. resveratrol from grapes and oleuropein from olives). Efforts to improve the production capacity and fruit quality in such fruit species, as well as the resistance to biotic and abiotic stress, was achieved by plant breeders using mainly classical breeding approaches (e.g. selection, hybridization and mutagenesis), nevertheless, breeding support by plant tissue culture techniques, marker assisted selection, as well as by genome modification, was also used. Here we will present the state of the art related with the production of transgenic plants in three Mediterranean fruit species with important impact on the economy, olive, grapevine and cork. The achievements, problems and future perspectives will be discussed.This work was financially supported by national funds through FCT (Foundation for Science and Technology) under the Project UID/AGR/00115/2013, PTDC/BIA-BQM/28539/2017, by the Project OLEAVALOR (ALT20-03-0145-FEDER000014) funded by FEDER funds through the Program Alentejo 2020; Hélia Cardoso and Susana Serrazina were supported by FCT through post-doc fellowship SFRH/BPD/109849/2015 and SFRH/BPD/108653/2015, respectively; Andreia Figueiredo was also supported by the investigator FCT program IF/00819/2015