Modification of polyelectrolyte multilayer coatings using nanoparticles to optimize adhesion and proliferation of different cell types

Adapting characteristics of biomaterials specifically for in vitro and in vivo applications is becoming increasingly important in order to control interactions between material and biological systems. These complex interactions are influenced by surface properties like chemical composition, charge, mechanical and topographic attributes. In many cases it is not useful or even not possible to alter the base material but changing surface, to improve biocompatibility or to make surfaces bioactive, may be achieved by thin coatings. An already established method is the coating with polyelectrolyte multilayers (PEM). To adjust adhesion, proliferation and improve vitality of certain cell types, we modified the roughness of PEM coatings. We included different types nanoparticles (NP’s) in different concentrations into PEM coatings for controlling surface roughness. Surface properties were characterized and the reaction of 3 different cell types on these coatings was tested

Additional file 4: Figure S2. of SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

Melting peak analysis for the rRT-PCR assay performance in field-caught ZIKV-infected mosquitoes. The evaluation is based on the primer combination F2 + R3. The melting peak for ZIKV infected mosquitoes (1–7) and ZIKV non-infected mosquito species (8–14) falls within the same range as the positive control (ATCC® VR-84). (TIFF 679 kb

Additional file 3: Figure S1. of SYBR green-based one step quantitative real-time polymerase chain reaction assay for the detection of Zika virus in field-caught mosquitoes

Melting curve analysis demonstrating the non-specific amplification by primer combinations F + R3, F + R4 and F3 + R4 in the presence of mosquito-derived RNA and specificity of F + R3 in a panel of flavivirus and alphavirus RNA. a. Melting peak analysis for the F + R4 primer pair b. Melting peak analysis for the F3 + R4 primer pair c. Melting peak analysis for the F + R3 primer pair. d. Melting peak analysis for the F + R3 primer pairs within a panel of flavivirus and alphavirus RNA. Abbreviation: NTC, Negative control. (TIFF 2225 kb

The fine-specificity of an Env183/A2 mAb and soluble TCR tetramers.

<p>(A) The Env_183–191 peptide modeled into the Tax/HLA-A2 crystal structure, inset: lateral view of the peptide in HLA-A*02:01. The Arg187 side chain protrudes from the binding pocket, potentially interacting with the TCR or antibody. (B) Nine different Env_183–191 peptides with single position alanine substitutions were used to probe the fine specificity. (C and D) The alanine variant pMHCs were loaded onto beads and stained with Env183/A2 mAb or TCR tetramer (1 µg/mL).</p

Recognition of pMHC presented on the surface of cells.

<p>(A) Untreated (−) and T2 cells treated (+) with IFN-γ were pulsed with 1 or 10 µM of Env_183–191 or 10 µM of Core_18–27 peptide control and stained with 1 µg/mL TCR tetramer. (B) Identically treated T2 cells stained with 1 µg/mL Env183/A2 mAb demonstrates that both treatment with IFN-γ and pulsing with higher peptide concentrations results in a higher surface expression of Env183/A2 complexes. (C) HepG2.2.15 and EBO-PreS1 cells, transfected with the full and PreS1 fragment of the HBV genome, respectively, endogenously process and present Env183/A2 pMHC complexes on their cell surface. The mAb (dashed) shows improved detection compared to the TCR tetramers (solid). Peptide pulsed HepG2.2.15 and EBO-PreS1 further increased surface levels of Env183/A2 (filled histograms). Treatment of cells with IFN-γ demonstrably up-regulated HLA-A2 expression (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051397#pone.0051397.s005" target="_blank">Fig. S5B</a>).</p

Expression and purfication of the Env183/A2 soluble T cell receptor.

<p>(A) The gene encoding for TCR α chain residues 1–202 and TCR β chain residues 1–243 were separately cloned into pET28a vectors. A BirA recognition sequence was added to the C-terminus of the β chain. Cα Thr48Cys and Cβ Ser57Cys mutations were introduced to facilitate an inter-chain disulfide bond (red bar). (B) SDS PAGE analysis of two distinct fractions obtained after chromatographic purification of refolded TCR. (C) Gel shift analysis of the purified TCR shows >90% biotinylation. (D) Surface plasmon resonance analysis of refolded TCR monomers demonstrated functional binding to immobilized Env183/A2 with a K_D of 0.6 µM, as determined with a steady-state model.</p

pMHC-coated beads as an artificial antigen presenting surface.

<p>(A) Schematic of the fluorescent bead assay. Streptavidin-coated beads were loaded with biotinylated pMHC monomer thus providing a homogenous binding surface for the Env183/A2 mAb or TCR tetramers. (B) Beads were stained with 10 µg/mL of monomeric TCRs or (C) 1 µg/mL APC-conjugated TCR tetramers. Beads presenting Core_18–27 HLA-A*02:01 were used as a control. (D) Beads were loaded with HLA-A*02:01 presenting 6 different peptides and stained with a mouse anti-β2m followed by detection with an APC-conjugated goat anti-mouse antibody. (E) Staining shows equal levels of pMHC on beads. (F) The beads were furthermore stained with 1 µg/mL APC-conjugated TCR tetramers, showing that they only bound Genotype A/C/D and Genotype B variants of Env_183–191 peptides presented by HLA-A*02:01.</p