Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease

Granulocytes as models for human protein marker identification following nicotine exposure

Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian CNS, in the peripheral nervous system, and in skeletal muscle. Neuronal-type nAChRs are also found in several non-neuronal cell types, including leukocytes. Granulocytes are a subtype of leukocytes that include basophils, eosinophils, and neutrophils. Granulocytes, also known as polymorphonuclear leukocytes, are characterized by their ability to produce, store, and release compounds from intracellular granules. Granulocytes are the most abundant type of leukocyte circulating in the peripheral blood. Granulocyte abundance, nAChR expression, and nAChR upregulation following chronic nicotine administration makes granulocytes interesting models for identifying protein markers of nicotine exposure. Nicotinic receptor subunits and several non-nAChR proteins have been identified as protein markers of granulocyte nicotine exposure. We review methods to isolate granulocytes from human tissue, summarize present data about the expression of nAChRs in the three granulocyte cell types (basophils, eosinophils, and neutrophils), describe current knowledge of the effects of nicotine exposure on human granulocyte protein expression, and highlight areas of interest for future investigation

1999-2000 Young Musician Competition - Winds and Brass

https://spiral.lynn.edu/conservatory_other-competitions/1000/thumbnail.jp

The effects of collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering.

In this study, we examined the effects of varying collagen concentration and crosslink density on the biological, structural and mechanical properties of collagen-GAG scaffolds for bone tissue engineering. Three different collagen contents (0.25%, 0.5% and 1% collagen) and two different dehydrothermal (DHT) crosslinking processes [1] 105 degrees C for 24 h and [2] 150 degrees C for 48 h were investigated. These scaffolds were assessed for (1) pore size, (2) permeability (3) compressive strength and (4) cell viability. The largest pore size, permeability rate, compressive modulus, cell number and cell metabolic activity was all found to occur on the 1% collagen scaffold due to its increased collagen composition and the DHT treatment at 150 degrees C was found to significantly improve the mechanical properties and not to affect cellular number or metabolic activity. These results indicate that doubling the collagen content to 1% and dehydrothermally crosslinking the scaffold at 150 degrees C for 48 h has enhanced mechanical and biological properties of the scaffold making it highly attractive for use in bone tissue engineering

Early Onset of Franciscan Subduction

The Franciscan subduction complex of California is considered a type example of a subduction-accretion system, yet the age of subduction initiation and relationship to the tectonic history of western North America remain controversial. Estimates for the timing of Franciscan subduction initiation are largely based either indirectly on regional tectonic arguments or from the ages of high-grade blocks within mélange. Many of the high-grade blocks record counterclockwise pressure-temperature paths with early amphibolite overprinted by later eclogite and blueschist; however, their origin and significance with respect to subduction initiation have been debated. In contrast, some high-grade blocks show evidence for clockwise pressure-temperature paths and an early eclogite assemblage overprinted by later amphibolite Zircon U-Pb ages from inclusions in garnet and Lu-Hf estimates of initial garnet growth ages from these samples record early eclogite metamorphism at ~176 Ma. Matrix zircon U-Pb ages and Lu-Hf estimates of final garnet growth ages record a barroisite-amphibolite assemblage overprint of eclogite at ~160 Ma. Combined with petrologic data and existing geochronology, the data suggest that (1) Franciscan subduction was underway by no later than 180 Ma, (2) continuous subduction metamorphism occurred for at least 100 Ma, and (3) Franciscan subduction initiation predated the formation of the overlying Coast Range Ophiolite, supporting models that form the ophiolite above an east dipping Franciscan subduction zone

Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease

Brain Region-Specific nAChR and Associated Protein Abundance Alterations Following Chronic Nicotine and/or Menthol Exposure

The identification of biomarkers that are altered following nicotine/tobacco exposure can facilitate the investigation of tobacco-related diseases. Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian central and peripheral nervous systems and the neuromuscular junction. Neuronal nAChR subunits (11) have been identified in mammals (α2-7, α9-10, β2-4). We examined changes in β2 nAChR subunit protein levels after chronic nicotine, (±)-menthol, or nicotine co-administered with (±)-menthol in nine murine brain regions. Our investigation of β2 nAChR subunit level changes identified the hypothalamus as a novel region of interest for menthol exposure that demonstrated increased β2 nAChR levels after (±)-menthol plus nicotine exposure compared to nicotine exposure alone. Using mass spectrometry, we further characterized changes in membrane protein abundance profiles in the hypothalamus to identify potential biomarkers of (±)-menthol plus nicotine exposure and proteins that may contribute to the elevated β2 nAChR subunit levels. In the hypothalamus, 272 membrane proteins were identified with altered abundances after chronic nicotine plus menthol exposure with respect to chronic nicotine exposure without menthol. A comprehensive investigation of changes in nAChR and non-nAChR protein expression resulting from (±)-menthol plus nicotine in the brain may establish biomarkers to better understand the effects of these drugs on addiction and addiction-related diseases

Brain Region-Specific nAChR and Associated Protein Abundance Alterations Following Chronic Nicotine and/or Menthol Exposure

The identification of biomarkers that are altered following nicotine/tobacco exposure can facilitate the investigation of tobacco-related diseases. Nicotinic acetylcholine receptors (nAChRs) are pentameric cation channels expressed in the mammalian central and peripheral nervous systems and the neuromuscular junction. Neuronal nAChR subunits (11) have been identified in mammals (α2-7, α9-10, β2-4). We examined changes in β2 nAChR subunit protein levels after chronic nicotine, (±)-menthol, or nicotine co-administered with (±)-menthol in nine murine brain regions. Our investigation of β2 nAChR subunit level changes identified the hypothalamus as a novel region of interest for menthol exposure that demonstrated increased β2 nAChR levels after (±)-menthol plus nicotine exposure compared to nicotine exposure alone. Using mass spectrometry, we further characterized changes in membrane protein abundance profiles in the hypothalamus to identify potential biomarkers of (±)-menthol plus nicotine exposure and proteins that may contribute to the elevated β2 nAChR subunit levels. In the hypothalamus, 272 membrane proteins were identified with altered abundances after chronic nicotine plus menthol exposure with respect to chronic nicotine exposure without menthol. A comprehensive investigation of changes in nAChR and non-nAChR protein expression resulting from (±)-menthol plus nicotine in the brain may establish biomarkers to better understand the effects of these drugs on addiction and addiction-related diseases

Implementation of U.K. Earth system models for CMIP6

We describe the scientific and technical implementation of two models for a core set of experiments contributing to the sixth phase of the Coupled Model Intercomparison Project (CMIP6). The models used are the physical atmosphere-land-ocean-sea ice model HadGEM3-GC3.1 and the Earth system model UKESM1 which adds a carbon-nitrogen cycle and atmospheric chemistry to HadGEM3-GC3.1. The model results are constrained by the external boundary conditions (forcing data) and initial conditions.We outline the scientific rationale and assumptions made in specifying these. Notable details of the implementation include an ozone redistribution scheme for prescribed ozone simulations (HadGEM3-GC3.1) to avoid inconsistencies with the model's thermal tropopause, and land use change in dynamic vegetation simulations (UKESM1) whose influence will be subject to potential biases in the simulation of background natural vegetation.We discuss the implications of these decisions for interpretation of the simulation results. These simulations are expensive in terms of human and CPU resources and will underpin many further experiments; we describe some of the technical steps taken to ensure their scientific robustness and reproducibility