18 research outputs found
Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice
BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Global Analysis of Secreted Proteins and Glycoproteins in <i>Saccharomyces cerevisiae</i>
Protein secretion
is essential for numerous cellular activities,
and secreted proteins in bodily fluids are a promising and noninvasive
source of biomarkers for disease detection. Systematic analysis of
secreted proteins and glycoproteins will provide insight into protein
function and cellular activities. Yeast (<i>Saccharomyces cerevisiae</i>) is an excellent model system for eukaryotic cells, but global analysis
of secreted proteins and glycoproteins in yeast is challenging due
to the low abundances of secreted proteins and contamination from
high-abundance intracellular proteins. Here, by using mild separation
of secreted proteins from cells, we comprehensively identified and
quantified secreted proteins and glycoproteins through inhibition
of glycosylation and mass spectrometry-based proteomics. In biological
triplicate experiments, 245 secreted proteins were identified, and
comparison with previous experimental and computational results demonstrated
that many identified proteins were located in the extracellular space.
Most quantified secreted proteins were down-regulated from cells treated
with an N-glycosylation inhibitor (tunicamycin). The quantitative
results strongly suggest that the secretion of these down-regulated
proteins was regulated by glycosylation, while the secretion of proteins
with minimal abundance changes was contrarily irrelevant to protein
glycosylation, likely being secreted through nonclassical pathways.
Glycoproteins in the yeast secretome were globally analyzed for the
first time. A total of 27 proteins were quantified in at least two
protein and glycosylation triplicate experiments, and all except one
were down-regulated under N-glycosylation inhibition, which is solid
experimental evidence to further demonstrate that the secretion of
these proteins is regulated by their glycosylation. These results
provide valuable insight into protein secretion, which will further
advance protein secretion and disease studies
Comprehensive Analysis of Protein N‑Glycosylation Sites by Combining Chemical Deglycosylation with LC–MS
Glycosylation
is one of the most important protein modifications
in biological systems. It plays a critical role in protein folding,
trafficking, and stability as well as cellular events such as immune
response and cell-to-cell communication. Aberrant protein glycosylation
is correlated with several diseases including diabetes, cancer, and
infectious diseases. The heterogeneity of glycans makes comprehensive
identification of protein glycosylation sites very difficult by MS
because it is challenging to match mass spectra to peptides that contain
different types of unknown glycans. We combined a chemical deglycosylation
method with LC–MS-based proteomics techniques to comprehensively
identify protein N-glycosylation sites in yeast. On the basis of the
differences in chemical properties between the amide bond of the N-linkage
and the glycosidic bond of the O-linkage of sugars, O-linked sugars
were removed and only the innermost N-linked GlcNAc remained, which
served as a mass tag for MS analysis. This chemical deglycosylation
method allowed for the identification of 555 protein N-glycosylation
sites in yeast by LC–MS, which is 46% more than those obtained
from the parallel experiments using the Endo H cleavage method. A
total of 250 glycoproteins were identified, including 184 membrane
proteins. This method can be extensively used for other biological
samples
Systematic Investigation of Cellular Response and Pleiotropic Effects in Atorvastatin-Treated Liver Cells by MS-Based Proteomics
For
decades, statins have been widely used to lower cholesterol
levels by inhibiting the enzyme HMG Co-A reductase (HMGCR). It is
well-known that statins have pleiotropic effects including improving
endothelial function and inhibiting vascular inflammation and oxidation.
However, the cellular responses to statins and corresponding pleiotropic
effects are largely unknown at the proteome level. Emerging mass spectrometry-based
proteomics provides a unique opportunity to systemically investigate
protein and phosphoprotein abundance changes as a result of statin
treatment. Many lipid-related protein abundances were increased in
HepG2 cells treated by atorvastatin, including HMGCR, FDFT, SQLE,
and LDLR, while the abundances of proteins involved in cellular response
to stress and apoptosis were decreased. Comprehensive analysis of
protein phosphorylation demonstrated that several basic motifs were
enriched among down-regulated phosphorylation sites, which indicates
that kinases with preference for these motifs, such as protein kinase
A and protein kinase C, have attenuated activities. Phosphopeptides
on a group of G-protein modulators were up-regulated, which strongly
suggests that cell signal rewiring was a result of the effect of protein
lipidation by the statin. This work provides a global view of liver
cell responses to atorvastatin at the proteome and phosphoproteome
levels, which provides insight into the pleiotropic effects of statins
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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration.
Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery
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Competitive Protein Binding Influences Heparin-Based Modulation of Spatial Growth Factor Delivery for Bone Regeneration
Tissue engineering strategies involving the in vivo delivery of recombinant growth factors are often limited by the inability of biomaterials to spatially control diffusion of the delivered protein within the site of interest. The poor spatiotemporal control provided by porous collagen sponges, which are used for the clinical delivery of bone morphogenetic protein-2 (BMP-2) for bone regeneration, has necessitated the use of supraphysiological protein doses, leading to inflammation and heterotopic ossification. This study describes a novel tissue engineering strategy to spatially control rapid BMP-2 diffusion from collagen sponges in vivo by creating a high-affinity BMP-2 sink around the collagen sponge. We designed an electrospun poly-ɛ-caprolactone nanofiber mesh containing physically entrapped heparin microparticles, which have been previously demonstrated to bind and retain large amounts of BMP-2. Nanofiber meshes containing 0.05 and 0.10 mg of microparticles/cm2 demonstrated increased BMP-2 binding and decreased BMP-2 release in vitro compared with meshes without microparticles. However, when microparticle-containing meshes were used in vivo to limit the diffusion of BMP-2 delivered by using collagen sponges in a rat femoral defect, no differences in heterotopic ossification or biomechanical properties were observed. Further investigation revealed that, although BMP-2 binding to heparin microparticles was rapid, the presence of serum components attenuated microparticle-BMP-2 binding and increased BMP-2 release in vitro. These observations provide a plausible explanation for the results observed in vivo and suggest that competitive protein binding in vivo may hinder the ability of affinity-based biomaterials to modulate growth factor delivery
Image1_Peanut butter feeding induces oral tolerance in genetically diverse collaborative cross mice.tif
BackgroundEarly dietary introduction of peanut has shown efficacy in clinical trials and driven pediatric recommendations for early introduction of peanut to children with heightened allergy risk worldwide. Unfortunately, tolerance is not induced in every case, and a subset of patients are allergic prior to introduction. Here we assess peanut allergic sensitization and oral tolerance in genetically diverse mouse strains.ObjectiveWe aimed to determine whether environmental adjuvant-driven airway sensitization and oral tolerance to peanut could be induced in various genetically diverse mouse strains.MethodsC57BL/6J and 12 Collaborative Cross (CC) mouse strains were fed regular chow or ad libitum peanut butter to induce tolerance. Tolerance was tested by attempting to sensitize mice via intratracheal exposure to peanut and lipopolysaccharide (LPS), followed by intraperitoneal peanut challenge. Peanut-specific immunoglobulins and peanut-induced anaphylaxis were assessed.ResultsWithout oral peanut feeding, most CC strains (11/12) and C57BL/6J induced peanut-specific IgE and IgG1 following airway exposure to peanut and LPS. With oral peanut feeding none of the CC strains nor C57BL/6J mice became sensitized to peanut or experienced anaphylaxis following peanut challenge.ConclusionAllergic sensitization and oral tolerance to peanut can be achieved across a range of genetically diverse mice. Notably, the same strains that became allergic via airway sensitization were tolerized by feeding high doses of peanut butter before sensitization, suggesting that the order and route of peanut exposure are critical for determining the allergic fate.</p