Structural adaptations of octaheme nitrite reductases from haloalkaliphilic <i>Thioalkalivibrio</i> bacteria to alkaline pH and high salinity - Fig 4

<p>Intersubunit contacts in GsNiR (the trimer contact (A, B, E, F,) and the dimer contact (I, J,)) and TvNiR (the trimer contact (C, D, G, H) and the dimer contact (K, L)); hydrophobic regions are highlighted in red, hydrophilic are blue. The inset in the center of the figure shows localization of contacts in the hexamer. The intersubunit contacts in GsNiR and TvNiR are shown with relative electrostatic surface potentials on left (A, E, I) and right (D, H, L) respectively.</p

Shape of ONR from <i>Tv</i>. <i>nitratireducens</i> [5] (multiscale models of macromolecular assembly were generated with USCF Chimera).

<p>The figure shows the trimer (top view and view at the base) and the hexamer (side view). Subunits in the hexamer are designated by letters. Central axis is shown as a dotted line. Intersubunit contacts (trimers and dimers) and solvent-accessible protein surface are shown in yellow, hemes of the C subunit are numbered. Secondary structure of the B-subunit (cartoon representation) is shown in blue.</p

Complementarity of hydrophobic/hydrophilic properties in ONRs (Sll–hydrophobic-hydrophobic buried surface area in ONR intersubunit contacts, Shh–hydrophilic-hydrophilic buried surface area between subunits, Sburied–total area of contact between subunits (buried surface area), Stotal–total surface area, Sasa–surface accessible area).

<p>Complementarity of hydrophobic/hydrophilic properties in ONRs (Sll–hydrophobic-hydrophobic buried surface area in ONR intersubunit contacts, Shh–hydrophilic-hydrophilic buried surface area between subunits, Sburied–total area of contact between subunits (buried surface area), Stotal–total surface area, Sasa–surface accessible area).</p

ONR structural analysis (hexameric state).

<p>ONR structural analysis (hexameric state).</p

Scaffolds cytotoxicity, seeding efficiency and cell proliferation: a) analysis of scaffold cytotoxicity via LDH assay 24 hours post seeding; b) qualitative demonstration of scaffold population with hASCs 30 min and 4 hours after seeding (scale bars = 100 μm); analysis of proliferation of (c) hASCs and (d) hBMSCs.

<p>Scaffolds cytotoxicity, seeding efficiency and cell proliferation: a) analysis of scaffold cytotoxicity via LDH assay 24 hours post seeding; b) qualitative demonstration of scaffold population with hASCs 30 min and 4 hours after seeding (scale bars = 100 μm); analysis of proliferation of (c) hASCs and (d) hBMSCs.</p

Mechanical properties of the non aged and aged scaffolds (a); Phase contrast (upper row) and Alizarin Red S staining (lower row) of cellular calcium phosphate deposits on non aged and aged Zr-Si scaffolds seeded with hASCs and cultured for 26 days in osteogenic media (b): Arrows indicate calcium phosphate deposits. Scale bars = 50 μm.

<p>Mechanical properties of the non aged and aged scaffolds (a); Phase contrast (upper row) and Alizarin Red S staining (lower row) of cellular calcium phosphate deposits on non aged and aged Zr-Si scaffolds seeded with hASCs and cultured for 26 days in osteogenic media (b): Arrows indicate calcium phosphate deposits. Scale bars = 50 μm.</p

EDX spectrum and SEM demonstration of 2PP fabricated Zr-Si inorganic-organic hybrid scaffold with a pore size of 250 μm.

<p>Scale bars: 500 μm, 100 μm and 50 μm for the left, middle and right SEM images, respectively.</p

Light microscopy images demonstrating staining on bone matrix specific marker protein osteocalcin.

<p>hASCs (a) and hBMSCs (c) cultured on TCP in control media; hASCs (b) and hBMSCs (d) cutured on TCP in osteogenic media; hASCs cultured in scaffolds with 150, 200 and 250 μm pore sizes in control (e,i,m) and in osteogenic media (f,j,n); hBMSCs cultured in scaffolds with 150, 200 and 250 μm pore sizes in control (g,k,o) and in osteogenic media (h,l,p). Scale bars = 100 μm.</p

Quantification of calcium deposited by hASCs and hBMSCs in Zr-Si scaffolds during 21 days in culture.

<p>Student’s- t-test: * indicate significant difference of p<0.01; ** indicate significant difference of p<0.05, *** indicate significant difference of p <0.5.</p

Cells on scaffolds after 21 days in osteogenic and control cultures: (a and c) hASCs and hBMSCs from osteogenic culture; (e and g) hASCs and hBMSCs from control culture.

<p>High magnification images showing calcium phosphate deposits of hASCs and hBMSCs cultured on Zr-Si scaffolds in osteogenic (b and d) and control (f and h) conditions; (i-l) EDX mapping confirming the presence of calcium and phosphor in the accumulations. Scale bars: (a, c, e, g) 60 μm, (b, d, f, h) 5 μm, (i) 50 μm and (j-l) 10 μm.</p