Substrate recognition and proton coupling by a bacterial member of solute carrier family 17.

The solute carrier 17 family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl-. Other solute carrier 17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied Escherichia coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+:galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

Structures suggest a mechanism for energy coupling by a family of organic anion transporters.

Members of the solute carrier 17 (SLC17) family use divergent mechanisms to concentrate organic anions. Membrane potential drives uptake of the principal excitatory neurotransmitter glutamate into synaptic vesicles, whereas closely related proteins use proton cotransport to drive efflux from the lysosome. To delineate the divergent features of ionic coupling by the SLC17 family, we determined the structure of Escherichia coli D-galactonate/H+ symporter D-galactonate transporter (DgoT) in 2 states: one open to the cytoplasmic side and the other open to the periplasmic side with substrate bound. The structures suggest a mechanism that couples H+ flux to substrate recognition. A transition in the role of H+ from flux coupling to allostery may confer regulation by trafficking to and from the plasma membrane.

Hydrogel biophysical properties instruct coculture-mediated osteogenic potential.

Cell-based approaches for bone formation require instructional cues from the surrounding environment. As an alternative to pharmacological strategies or transplanting single cell populations, one approach is to coimplant populations that can establish a new vasculature and differentiate to bone-forming osteoblasts. Mesenchymal stem/stromal cells (MSCs) possess osteogenic potential and produce numerous angiogenic growth factors. Endothelial colony-forming cells (ECFCs) are a subpopulation of endothelial progenitor cells capable of vasculogenesis in vivo and may provide endogenous cues to support MSC function. We investigated the contribution of the carrier biophysical properties to instruct entrapped human MSCs and ECFCs to simultaneously promote their osteogenic and proangiogenic potential. Compared with gels containing MSCs alone, fibrin gels engineered with increased compressive stiffness simultaneously increased the osteogenic and proangiogenic potential of entrapped cocultured cells. ECFCs produced bone morphogenetic protein-2 (BMP-2), a potent osteoinductive molecule, and increases in BMP-2 secretion correlated with gel stiffness. Coculture of MSCs with ECFCs transduced to knockdown BMP-2 production abrogated the osteogenic response to levels observed with MSCs alone. These results demonstrate that physical properties of engineered hydrogels modulate the function of cocultured cells in the absence of inductive cues, thus increasing the translational potential of coimplantation to speed bone formation and repair.

Effect of calcium phosphate particle shape and size on their antibacterial and osteogenic activity in the delivery of antibiotics in vitro.

Powders composed of four morphologically different calcium phosphate particles were prepared by precipitation from aqueous solutions: flaky, brick-like, elongated orthogonal, and spherical. The particles were then loaded with either clindamycin phosphate as the antibiotic of choice or fluorescein, a model molecule used to assess the drug release properties. A comparison was carried out of the effect of such antibiotic-releasing materials on: sustained drug release profiles; Staphylococcus aureus growth inhibition; and osteogenic propensities in vitro. Raman spectroscopic analysis indicated the presence of various calcium phosphate phases, including monetite (flaky and elongated orthogonal particles), octacalcium phosphate (brick-shaped particles), and hydroxyapatite (spherical particles). Testing the antibiotic-loaded calcium phosphate powders for bacterial growth inhibition demonstrated satisfying antibacterial properties both in broths and on agar plates. All four calcium-phosphate-fluorescein powders exhibited sustained drug release over 21 days. The calcium phosphate sample with the highest specific surface area and the smallest, spherical particle size was the most effective in both drug loading and release, consequently having the highest antibacterial efficiency. Moreover, the highest cell viability, the largest gene expression upregulation of three different osteogenic markers--osteocalcin, osteopontin, and Runx2--as well as the least disrupted cell cytoskeleton and cell morphologies were also noticed for the calcium phosphate powder composed of the smallest, spherical nanosized particles. Still, all four powders exerted a viable effect on osteoblastic MC3T3-E1 cells in vitro, as evidenced by both morphological assessments on fluorescently stained cells and measurements of their mitochondrial activity. The obtained results suggest that the nanoscale particle size and the corresponding coarseness of the surface of particle conglomerates as the cell attachment points may present a favorable starting point for the development of calcium-phosphate-based osteogenic drug delivery devices.