3 research outputs found
Smaller CpG-Conjugated Gold Nanoconstructs Achieve Higher Targeting Specificity of Immune Activation
This
study describes a side-by-side comparison of the in vitro immunostimulatory
activity of cytosine–phosphate–guanine (CpG)-conjugated
gold nanoparticles. Three different gold nanoparticle cores (13 nm
spheres, 50 nm spheres, and 40 nm stars) with the same CpG surface
density were investigated for toll-like receptor 9 activation. For
this parameter set, 13 nm spheres displayed significantly higher specificity
for targeting immune receptors and larger nanoparticles (50 nm spheres
and 40 nm stars) showed higher cellular uptake and higher immune activation
because of off-target effects. Changes in nanoparticle size and presentation
of activating ligands affect construct-induced immune responses at
different levels, and care must be taken when considering practical
and global design rules for CpG delivery
Characterization of Molecules Binding to the 70K N‑Terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry
Fibronectin (Fn) is a large glycoprotein
present in plasma and
extracellular matrix and is important for many processes. Within Fn
the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated
Fn assembly, a process that contributes to embryogenesis, development,
and platelet thrombus formation. In addition, major human pathogens
including <i>Staphlycoccus aureus</i> and <i>Streptococcus
pyogenes</i> bind the 70k-Fn region by a novel form of protein–protein
interaction called β-zipper formation, facilitating bacterial
spread and colonization. Knowledge of blood plasma and platelet proteins
that interact with 70k-Fn by β-zipper formation is incomplete.
In the current study, we aimed to characterize these proteins through
affinity purification. For this affinity purification, we used a novel
purification technique termed immiscible filtration assisted by surface
tension (IFAST). The foundation of this technology is immiscible phase
filtration, using a magnet to draw paramagnetic particle (PMP)-bound
analyte through an immiscible barrier (oil or organic solvent) that
separates an aqueous sample from an aqueous eluting buffer. The immiscible
barrier functions to remove unbound proteins via exclusion rather
than dilutive washing used in traditional isolation methods. We identified
31 interactors from plasma, of which only seven were previously known
to interact with Fn. Furthermore, five proteins were identified to
interact with 70k-Fn from platelet lysate, of which one was previously
known. These results demonstrate that IFAST offers advantages for
proteomic studies of interacting molecules in that the technique requires
small sample volumes, can be done with high enough throughput to sample
multiple interaction conditions, and is amenable to exploratory mass
spectrometric and confirmatory immuno-blotting read-outs
Characterization of Molecules Binding to the 70K N‑Terminal Region of Fibronectin by IFAST Purification Coupled with Mass Spectrometry
Fibronectin (Fn) is a large glycoprotein
present in plasma and
extracellular matrix and is important for many processes. Within Fn
the 70 kDa N-terminal region (70k-Fn) is involved in cell-mediated
Fn assembly, a process that contributes to embryogenesis, development,
and platelet thrombus formation. In addition, major human pathogens
including <i>Staphlycoccus aureus</i> and <i>Streptococcus
pyogenes</i> bind the 70k-Fn region by a novel form of protein–protein
interaction called β-zipper formation, facilitating bacterial
spread and colonization. Knowledge of blood plasma and platelet proteins
that interact with 70k-Fn by β-zipper formation is incomplete.
In the current study, we aimed to characterize these proteins through
affinity purification. For this affinity purification, we used a novel
purification technique termed immiscible filtration assisted by surface
tension (IFAST). The foundation of this technology is immiscible phase
filtration, using a magnet to draw paramagnetic particle (PMP)-bound
analyte through an immiscible barrier (oil or organic solvent) that
separates an aqueous sample from an aqueous eluting buffer. The immiscible
barrier functions to remove unbound proteins via exclusion rather
than dilutive washing used in traditional isolation methods. We identified
31 interactors from plasma, of which only seven were previously known
to interact with Fn. Furthermore, five proteins were identified to
interact with 70k-Fn from platelet lysate, of which one was previously
known. These results demonstrate that IFAST offers advantages for
proteomic studies of interacting molecules in that the technique requires
small sample volumes, can be done with high enough throughput to sample
multiple interaction conditions, and is amenable to exploratory mass
spectrometric and confirmatory immuno-blotting read-outs