Novel histone modifications in microglia derived from a mouse model of chronic pain

As the resident immune cells in the central nervous system, microglia play an important role in the maintenance of its homeostasis. Dysregulation of microglia has been associated with the development and maintenance of chronic pain. However, the relevant molecular pathways remain poorly defined. In this study, we used a mass spectrometry-based proteomic approach to screen potential changes of histone protein modifications in microglia isolated from the brain of control and cisplatin-induced neuropathic pain adult C57BL/6J male mice. We identified several novel microglial histone modifications associated with pain, including statistically significantly decreased histone H3.1 lysine 27 mono-methylation (H3.1K27me1, 54.8% of control) and H3 lysine 56 tri-methylation (7.5% of control), as well as a trend suggesting increased H3 tyrosine 41 nitration. We further investigated the functional role of H3.1K27me1 and found that treatment of cultured microglial cells for 4 consecutive days with 1–10 μM of NCDM-64, a potent and selective inhibitor of lysine demethylase 7A, an enzyme responsible for the demethylation of H3K27me1, dose-dependently elevated its levels with a greater than a two-fold increase observed at 10 μM compared to vehicle-treated control cells. Moreover, pretreatment of mice with NCDM-64 (10 or 25 mg/kg/day, i.p.) prior to cisplatin treatment prevented the development of neuropathic pain in mice. The identification of specific chromatin marks in microglia associated with chronic pain may yield critical insight into the contribution of microglia to the development and maintenance of pain, and opens new avenues for the development of novel nonopioid therapeutics for the effective management of chronic pain.     

Elucidation of the protein composition of mouse seminal vesicle fluid

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.     

Phospholipid transfer protein deficiency ameliorates diet-induced hypercholesterolemia and inflammation in mice

Phospholipid transfer protein (PLTP) facilitates the transfer of phospholipids from triglyceride-rich lipoproteins into HDL. PLTP has been shown to be an important factor in lipoprotein metabolism and atherogenesis. Here, we report that chronic high-fat, high-cholesterol diet feeding markedly increased plasma cholesterol levels in C57BL/6 mice. PLTP deficiency attenuated diet-induced hypercholesterolemia by dramatically reducing apolipoprotein E-rich lipoproteins (-88%) and, to a lesser extent, LDL (-40%) and HDL (-35%). Increased biliary cholesterol secretion, indicated by increased hepatic ABCG5/ABCG8 gene expression, and decreased intestinal cholesterol absorption may contribute to the lower plasma cholesterol in PLTP-deficient mice. The expression of proinflammatory genes (intercellular adhesion molecule-1 and vascular cell adhesion molecule-1) is reduced in aorta of PLTP knockout mice compared with wild-type mice fed either a chow or a high-cholesterol diet. Furthermore, plasma interleukin-6 levels are significantly lower in PLTP-deficient mice, indicating reduced systemic inflammation. These data suggest that PLTP appears to play a proatherogenic role in diet-induced hyperlipidemic mice.     

Matrix metalloproteinases promote motor axon fasciculation in the Drosophila embryo

Matrix metalloproteinases (MMPs) are a large conserved family of extracellular proteases, a number of which are expressed during neuronal development and upregulated in nervous system diseases. Primarily on the basis of studies using pharmaceutical inhibitors, MMPs have been proposed to degrade the extracellular matrix to allow growth cone advance during development and hence play largely permissive roles in axon extension. Here we show that MMPs are not required for axon extension in the Drosophila embryo, but rather are specifically required for the execution of several stereotyped motor axon pathfinding decisions. The Drosophila genome contains only two MMP homologs, Mmp1 and Mmp2. We isolated Mmp1 in a misexpression screen to identify molecules required for motoneuron development. Misexpression of either MMP inhibits the regulated separation/defasciculation of motor axons at defined choice points. Conversely, motor nerves in Mmp1 and Mmp2 single mutants and Mmp1 Mmp2 double mutant embryos are loosely bundled/fasciculated, with ectopic axonal projections. Quantification of these phenotypes reveals that the genetic requirement for Mmp1 and Mmp2 is distinct in different nerve branches, although generally Mmp2 plays the predominant role in pathfinding. Using both an endogenous MMP inhibitor and MMP dominant-negative constructs, we demonstrate that MMP catalytic activity is required for motor axon fasciculation. In support of the model that MMPs promote fasciculation, we find that the defasciculation observed when MMP activity is compromised is suppressed by otherwise elevating interaxonal adhesion -- either by overexpressing Fas2 or by reducing Sema-1a dosage. These data demonstrate that MMP activity is essential for embryonic motor axon fasciculation.     

Custom distinctions in the interaction of G-protein beta subunits with N-type (CaV2.2) versus P/Q-type (CaV2.1) calcium channels

Inhibition of N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gbetagamma complex (Gbetagamma). Far less is known about the important interaction sites on Gbetagamma itself. Here, we developed a novel electrophysiological paradigm, "compound-state willing-reluctant analysis," to describe Gbetagamma interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gbetagamma with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gbeta1, at residues known to interact with Galpha. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gbetagamma for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for "steering" or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel.     

Deeply conserved susceptibility in a multi‐host,multi‐parasite system

Variation in susceptibility is ubiquitous in multi‐host, multi‐parasite assemblages, and can have profound implications for ecology and evolution in these systems. The extent to which susceptibility to parasites is phylogenetically conserved among hosts can be revealed by analysing diverse regional communities. We screened for haemosporidian parasites in 3983 birds representing 40 families and 523 species, spanning ~ 4500 m elevation in the tropical Andes. To quantify the influence of host phylogeny on infection status, we applied Bayesian phylogenetic multilevel models that included a suite of environmental, spatial, temporal, life history and ecological predictors. We found evidence of deeply conserved susceptibility across the avian tree; host phylogeny explained substantial variation in infection status, and results were robust to phylogenetic uncertainty. Our study suggests that susceptibility is governed, in part, by conserved, latent aspects of anti‐parasite defence. This demonstrates the importance of deep phylogeny for understanding present‐day ecological interactions.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Hexasaccharides from the histamine-modified depolymerization of porcine intestinal mucosal heparin

Specific sequences in heparin are responsible for its modulation of the biological activity of proteins. As part of a program to characterize heparin-peptide and heparin-protein binding, we are studying the interaction of chemically discrete heparin-derived oligosaccharides with peptides and proteins. We report here the isolation and characterization, by one- and two-dimensional 1H NMR spectroscopies, of ten hexasaccharides, one pentasaccharide, and one octasaccharide serine that were isolated from depolymerized porcine intestinal mucosal heparin. Hexasaccharides were chosen for study because they fall within the size range, typically tetra- to decasaccharide in length, of heparin sequences that modulate the activity of proteins. The depolymerization reaction was catalyzed by heparinase I (EC 4.2.2.7) in the presence of histamine, which binds site specifically to heparin. Histamine increases both the rate and extent of heparinase I-catalyzed depolymerization of heparin. It is proposed that oligosaccharides produced by heparinase I-catalyzed depolymerization can inhibit the enzyme by binding to the imidazolium group of histidine-203, which together with cysteine-135 forms the catalytic domain of heparinase I. The increased rate and extent of depolymerization are attributed to competitive binding of the oligosaccharides by histamine.