Affinity Constants of Bovine Serum Albumin for 5 nm Gold Nanoparticles (AuNPs) with ω-Functionalized Thiol Monolayers Determined by Fluorescence Spectroscopy.

A detailed understanding of the binding of serum proteins to small (dcore <10 nm) nanoparticles (NPs) is essential for the mediation of protein corona formation in next generation nanotherapeutics. While a number of studies have investigated the details of protein adsorption on large functionalized NPs, small NPs (with a particle surface area comparable in size to the protein) have not received extensive study. This study determined the affinity constant (Ka) of BSA when binding to three different functionalized 5 nm gold nanoparticles (AuNPs). AuNPs were synthesized using three ω-functionalized thiols (mercaptoethoxy-ethoxy-ethanol (MEEE), mercaptohexanoic acid (MHA), and mercaptopentyltrimethylammonium chloride (MPTMA)), giving rise to particles with three different surface charges. The binding affinity of bovine serum albumin (BSA) to the different AuNP surfaces was investigated using UV-visible absorbance spectroscopy, dynamic light scattering (DLS), and fluorescence quenching titrations. Fluorescence titrations indicated that the affinity of BSA was actually highest for small AuNPs with a negative surface charge (MHA-AuNPs). Interestingly, the positively charged MPTMA-AuNPs showed the lowest Ka for BSA, indicating that electrostatic interactions are likely not the primary driving force in binding of BSA to these small AuNPs. Ka values at 25 °C for MHA, MEEE, and MPTMA-AuNPs were 5.2 ± 0.2 × 107, 3.7 ± 0.2 × 107, and 3.3 ± 0.16 × 107 M-1 in water, respectively. Fluorescence quenching titrations performed in 100 mM NaCl resulted in lower Ka values for the charged AuNPs, while the Ka value for the MEEE-AuNPs remained unchanged. Measurement of the hydrodynamic diameter (Dh) by dynamic light scattering (DLS) suggests that adsorption of 1-2 BSA molecules is sufficient to saturate the AuNP surface. DLS and negative-stain TEM images indicate that, despite the lower observed Ka values, the binding of MPTMA-AuNPs to BSA likely induces significant protein misfolding and may lead to extensive BSA aggregation at specific BSA:AuNP molar ratios.

The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFß ligand and the Gfrl co-receptor.

The RET receptor tyrosine kinase is crucial for the development of the enteric nervous system (ENS), acting as a receptor for Glial cell line-derived neurotrophic factor (GDNF) via GFR co-receptors. Drosophila has a well-conserved RET homolog (Ret) that has been proposed to function independently of the Gfr-like co-receptor (Gfrl). We find that Ret is required for development of the stomatogastric (enteric) nervous system in both embryos and larvae, and its loss results in feeding defects. Live imaging analysis suggests that peristaltic waves are initiated but not propagated in mutant midguts. Examination of axons innervating the midgut reveals increased branching but the area covered by the branches is decreased. This phenotype can be rescued by Ret expression. Additionally, Gfrl shares the same ENS and feeding defects, suggesting that Ret and Gfrl might function together via a common ligand. We identified the TGFß family member Maverick (Mav) as a ligand for Gfrl and a Mav chromosomal deficiency displayed similar embryonic ENS defects. Our results suggest that the Ret and Gfrl families co-evolved before the separation of invertebrate and vertebrate lineages.

Genetic Tools for the Analysis of Drosophila Stomatogastric Nervous System Development.

The Drosophila stomatogastric nervous system (SNS) is a compact collection of neurons that arises from the migration of neural precursors. Here we describe genetic tools allowing functional analysis of the SNS during the migratory phase of development. We constructed GAL4 lines driven by fragments of the Ret promoter, which yielded expression in a subset of migrating neural SNS precursors and also included a distinct set of midgut associated cells. Screening of additional GAL4 lines driven by fragments of the Gfrl/Munin, forkhead, twist and goosecoid (Gsc) promoters identified a Gsc fragment with expression from initial selection of SNS precursors until the end of embryogenesis. Inhibition of EGFR signaling using three identified lines disrupted the correct patterning of the frontal and recurrent nerves. To manipulate the environment traveled by SNS precursors, a FasII-GAL4 line with strong expression throughout the entire intestinal tract was identified. The transgenic lines described offer the ability to specifically manipulate the migration of SNS precursors and will allow the modeling and in-depth analysis of neuronal migration in ENS disorders such as Hirschsprung's disease.