ABSTRACT
Sugar-Will-Eventually-be-Exported-Transporters (SWEETs) are an important family of sugar transporters that appear to be ubiquitous in all organisms. Recent research has determined the structure of SWEETs in higher plants, identified specific residues required for monosaccharide or disaccharide transport, and begun to understand the specific functions of individual plant SWEET proteins. However, in green algae (Chlorophyta) these transporters are poorly characterised. This study identified SWEET proteins from across representative Chlorophyta with the aim to characterise their phylogenetic relationships and perform protein structure modelling in order to inform functional prediction. The algal genomes analysed encoded between one and six SWEET proteins, which is much less than a typical higher plant. Phylogenetic analysis identified distinct clusters of over 70 SWEET protein sequences, taken from almost 30 algal genomes. These clusters remain separate from representative higher or non-vascular plant SWEETs, but are close to fungi SWEETs. Subcellular localisation predictions and analysis of conserved amino acid residues revealed variation between SWEET proteins of different clusters, suggesting different functionality. These findings also showed conservation of key residues at the substrate-binding site, indicating a similar mechanism of substrate selectivity and transport to previously characterised higher plant monosaccharide-transporting SWEET proteins. Future work is now required to confirm the predicted sugar transport specificity and determine the functional role of these algal SWEET proteins.
ABSTRACT
Repairing articular cartilage by combining microfracture and various scaffolds has been extensively performed in in vivo animal models. We previously described a novel extracellular matrix (ECM) scaffold for cartilage tissue engineering. The aim of this study was to investigate the effect of a bone marrow-derived mesenchymal stem cells-derived ECM (BMSC-dECM) scaffold on the chondrogenic differentiation of marrow clots following microfracture in vitro. In this study, we manufactured the BMSC-dECM scaffold using a freeze-drying method. To obtain the marrow clots, a full-thickness cartilage defect was established and microholes were created in the trochlear groove of New Zealand white rabbits. The samples were divided and cultured in vitro for 1, 2, 4, and 8 weeks. The samples included a culture of the marrow clot alone (Group 1), a culture of the marrow clot with transforming growth factor-beta 3 (TGF-ß3) (Group 2), a culture of the composite of the BMSC-dECM scaffold and the marrow clot alone (Group 3), and a culture of the composite with TGF-ß3 (Group 4). A smooth and glossy surface was observed in Group 2 and Group 4 over time, but the surface for Group 4 was larger from week 1 onward. Compressive strength gradually increased in Groups 2 and 4, and greater increases were observed in Group 4 during the 8-week culture period. Enhanced cartilage-like matrix deposition of glycosaminoglycan (GAG) and type II collagen were confirmed by Safranin O and immunohistochemistry staining, respectively, in Groups 2 and 4. The GAG and collagen contents also gradually increased over time in Groups 2 and 4; the increase was greater in Group 4. In addition, real-time-polymerase chain reaction demonstrated that the expression of chondrogenic genes, such as COL2, ACAN, and SOX9, was gradually upregulated in Groups 2 and 4. However, greater increases in the expression of these cartilage-like genes were observed in Group 4 from week 4 onward. Our results suggest that the BMSC-dECM scaffold may favor the chondrogenesis of marrow clots following microfracture in vitro. In conclusion, these tissue engineering-like constructs could be potential candidates for cartilage repair.