GPR18 drives FAAH inhibition-induced neuroprotection against HIV-1 Tat-induced neurodegeneration

Human immunodeficiency virus type 1 (HIV-1) is known to provoke microglial immune responses which likely play a paramount role in the development of chronic neuroinflammatory conditions and neuronal damage related to HIV-1 associated neurocognitive disorders (HAND). In particular, HIV-1 Tat protein is a proinflammatory neurotoxin which predisposes neurons to synaptodendritic injury. Drugs targeting the degradative enzymes of endogenous cannabinoids have shown promise in reducing inflammation with minimal side effects in rodent models. Considering that markers of neuroinflammation can predict the extent of neuronal injury in HAND patients, we evaluated the neurotoxic effect of HIV-1 Tat-exposed microglia following blockade of fatty acid amid hydrolyze (FAAH), a catabolic enzyme responsible for degradation of endocannabinoids, e.g. anandamide (AEA). In the present study, cultured murine microglia were incubated with Tat and/or a FAAH inhibitor (PF3845). After 24 h, cells were imaged for morphological analysis and microglial conditioned media (MCM) was collected. Frontal cortex neuron cultures (DIV 7–11) were then exposed to MCM, and neurotoxicity was assessed via live cell calcium imaging and staining of actin positive dendritic structures. Results demonstrate a strong attenuation of microglial responses to Tat by PF3845 pretreatment, which is indicated by 1) microglial changes in morphology to a less proinflammatory phenotype using fractal analysis, 2) a decrease in release of neurotoxic cytokines/chemokines (MCP-1/CCL2) and matrix metalloproteinases (MMPs; MMP-9) using ELISA/multiplex assays, and 3) enhanced production of endocannabinoids (AEA) using LC/MS/MS. Additionally, PF3845\u27s effects on Tat-induced microglial-mediated neurotoxicity, decreased dysregulation of neuronal intracellular calcium and prevented the loss of actin-positive staining and punctate structure in frontal cortex neuron cultures. Interestingly, these observed neuroprotective effects appeared to be independent of cannabinoid receptor activity (CB1R & CB2R). We found that a purported GPR18 antagonist, CID-85469571, blocked the neuroprotective effects of PF3845 in all experiments. Collectively, these experiments increase understanding of the role of FAAH inhibition and Tat in mediating microglial neurotoxicity in the HAND condition

Verification of Protein Biomarker Specificity for the Identification of Biological Stains by Quadrupole Time-of-flight Mass Spectrometry

Advances in proteomics technology over the past decade offer forensic serologists a greatly improved opportunity to accurately characterize the tissue source from which a DNA profile has been developed. Such information can provide critical context to evidence and can help to prioritize downstream DNA analyses. Previous proteome studies compiled panels of “candidate biomarkers” specific to each of five body fluids (i.e., peripheral blood, vaginal/menstrual fluid, seminal fluid, urine, and saliva). Here, a multiplex quadrupole time‐of‐flight mass spectrometry assay has been developed in order to verify the tissue/body fluid specificity the 23 protein biomarkers that comprise these panels and the consistency with which they can be detected across a sample population of 50 humans. Single‐source samples of these human body fluids were accurately identified by the detection of one or more high‐specificity biomarkers. Recovery of body fluid samples from a variety of substrates did not impede accurate characterization and, of the potential inhibitors assayed, only chewing tobacco juice appeared to preclude the identification of a target body fluid. Using a series of 2‐component mixtures of human body fluids, the multiplex assay accurately identified both components in a single‐pass. Only in the case of saliva and peripheral blood did matrix effects appear to impede the detection of salivary proteins

A Novel, In-Solution Separation of Endogenous Cardiac Sarcomeric Proteins and Identification of Distinct Charged Variants of Regulatory Light Chain

The molecular conformation of the cardiac myosin motor is modulated by inter-molecular interactions among the heavy chain, the light chains, myosin binding protein-C (MyBP-C) and titin, and is governed by post-translational modifications (PTMs). In-gel digestion followed by liquid chromatography mass spectrometry (LC/MS/MS) has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based workflow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Sub-cellular fractionation followed by OFFGEL electrophoresis (OGE) resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain (MHC), and MyBPC of the thick filament. Further purification of RLC using reverse phase (RP) -HPLC separation and UV detection enriched for RLC PTMs at the intact protein level, and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OGE fractions were un-phosphorylated (78.8%), singly- (18.1%) and doubly-phosphorylated (3.1%) RLC. The novel aspects of this study are: 1) milligram amounts of endogenous cardiac sarcomeric sub-proteome were focused with resolution comparable to 2DE, 2) separation and quantification of post-translationally modified variants was achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in-solution, 4) endogenous charge variants of RLC were separated; a novel doublyphosphorylated form was identified in mouse, and singly-phosphorylated, singly-deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method