The majority of human gut microbiome is comprised of obligate anaerobic bacteria that exert essential metabolic functions in the human colon. These anaerobic gut bacteria constantly crosstalk with the colonic epithelium in a mucosal anoxic-oxic interface (AOI). However, in vitro recreation of the metabolically mismatched colonic AOI has been technically challenging. Furthermore, stable co-culture of the obligate anaerobic commensal microbiome and epithelial cells in a mechanically dynamic condition is essential for demonstrating the host-gut microbiome crosstalk. Here, we developed an anoxic-oxic interface-on-a-chip (AOI Chip) by leveraging a modified human gut-on-a-chip to demonstrate a controlled oxygen gradient in the lumen-capillary transepithelial interface by flowing anoxic and oxic culture medium at various physiological milieus. Computational simulation and experimental results revealed that the presence of the epithelial cell layer and the flow-dependent conditioning in the lumen microchannel is necessary and sufficient to create the steady-state vertical oxygen gradient in the AOI Chip. We confirmed that the created AOI does not compromise the viability, barrier function, mucin production, and the expression and localization of tight junction proteins in the 3D intestinal epithelial layer. Two obligate anaerobic commensal gut microbiome, Bifidobacterium adolescentis and Eubacterium hallii, that exert metabolic cross-feeding in vivo, were independently co-cultured with epithelial cells in the AOI Chip for up to a week without compromising any cell viability. Our new protocol for creating an AOI in a microfluidic gut-on-a-chip may enable to demonstrate the key physiological interactions of obligate anaerobic gut microbiome with the host cells associated with intestinal metabolism, homeostasis, and immune regulation.
This paper introduces the use of mass spectrometry to analyze peptide arrays for applications in profiling enzyme specificity. The strategy is illustrated with arrays containing 361 acetylated peptides to profile the activities of several histone deacetylases (HDACs). The arrays reveal distinct substrates that are preferred by members of the HDAC family. This example is particularly relevant because the label-dependent assays now used for these enzymes constrain the range of substrates that can be assayed and can perturb the intrinsic activities of the enzymes. KeywordsEnzymes; Post-translational modifications; SAMDI; Screening The application of peptide arrays for profiling biochemical activities has expanded since Frank and colleagues first reported the SPOT synthesis methodology, wherein peptides are synthesized directly on a cellulose membrane in a cost-efficient manner.[1] These arrays can be treated with enzymes and then analyzed using absorbance, fluorescence, or radio-isotopic assays to rank the activities of the peptides.[2] The requirement for labels can make it challenging to develop assays for certain enzymes and can also lead to false-positive and false-negative results. These limitations have motivated the development of label free formats based on optical methods [3] and mass spectrometry,[4] including our strategy to combine matrix-assisted laser desorption-ionization mass spectrometry with self-assembled monolayer substrates (i.e. the SAMDI method). [5] In this paper we demonstrate the combination of SAMDI-MS with peptide arrays, with an emphasis on the profiling of substrate specificities of several members of the HDAC family, which play a primary role in the regulation of gene expression.The assays now used to measure HDAC activity have been important for mechanistic studies and for identifying hit compounds in high throughput screens, but they have limitations when applied to studies of the substrate specificities. One group of assays identifies active substrates by labeling the deacetylated lysine with haptens that allow the isolation and subsequent sequencing of the peptide substrate. Hence, the original peptide NIH Public Access Author ManuscriptChembiochem. Author manuscript; available in PMC 2010 March 4. substrates must exclude residues susceptible to false positive labeling such as natural lysine, arginine, methionine, and cysteine.[6] A second group of assays conjugates a fluorophore to the carboxy-side of the acetylated lysine. Following HDAC treatment, the chromophore can be proteolytically released only in the deacetylated peptides, providing a convenient assay for HDAC activity compatible with microtiter plates. [7] With this format, the peptide sequence cannot be varied at the carboxy-side of the acetylated lysine and interactions between the fluorophore and enzyme can contribute to activity, making it difficult to understand the intrinsic enzyme specificities. In a clear example of this limitation, the assay revealed that resveratrol activates the SIRT1 deacetylase,[8] ...
This paper reports the development of a class of isoform-selective peptide substrates for measuring endogenous lysine deacetylase (KDAC) activities in cell culture. The peptides were first identified by comparing the substrate specificity profiles of the four KDAC isoforms KDAC2, KDAC3, KDAC8, and sirtuin 1 (SIRT1) on a 361-member hexapeptide array wherein the two C-terminal residues to the acetylated lysine were varied. The arrays were prepared by immobilizing the peptides to a self-assembled monolayer of alkanethiolates on gold and could therefore be analyzed by a mass spectrometry technique termed SAMDI (self-assembled monolayers for matrix assisted laser desorption/ionization time-of-flight mass spectrometry). Arrays presenting the selective substrates were treated with nuclear extracts from HeLa, Jurkat, and smooth muscle cells and analyzed to measure endogenous deacetylase activities. We then use the arrays to profile KDAC activity through the HeLa cell cycle. We find that the activity profile of the KDAC3 selective peptide closely mirrors the changing acetylation state of the H4 histone, suggesting a role for this enzyme in cell cycle regulation. This work is significant because it describes a general route for identifying selective substrates that can be used to understand the differential roles of members of the deacetylase enzyme family in complex biological processes and further because the label-free approach avoids perturbation of enzyme activity that has plagued fluorescence-based assays.
In this paper, we show that PtAu and PdAu random alloy dendrimer-encapsulated nanoparticles with an average size of ∼1.6 nm have different catalytic activity trends for allyl alcohol hydrogenation. Specifically, PtAu nanoparticles exhibit a linear increase in activity with increasing Pt content, whereas PdAu dendrimer-encapsulated nanoparticles show a maximum activity at a Pd content of ∼60%. Both experimental and theoretical results suggest that this contrasting behavior is caused by differences in the strength of H binding on the PtAu and PdAu alloy surfaces. The results have significant implications for predicting the catalytic performance of bimetallic nanoparticles on the basis of density functional theory calculations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.