An electrochemiluminescence based assay for quantitative detection of endogenous and exogenously applied MeCP2 protein variants.

Methyl-CpG-binding protein 2 (MeCP2) is a multifunctional chromosomal protein that plays a key role in the central nervous system. Its levels need to be tightly regulated, as both deficiency and excess of the protein can lead to severe neuronal dysfunction. Loss-of-function mutations affecting MeCP2 are the primary cause of Rett syndrome (RTT), a severe neurological disorder that is thought to result from absence of functional protein in the brain. Several therapeutic strategies for the treatment of RTT are currently being developed. One of them is the use of stable and native TAT-MeCP2 fusion proteins to replenish its levels in neurons after permeation across the blood-brain barrier (BBB). Here we describe the expression and purification of various transactivator of transcription (TAT)-MeCP2 variants and the development of an electrochemiluminescence based assay (ECLIA) that is able to measure endogenous MeCP2 and recombinant TAT-MeCP2 fusion protein levels in a 96-well plate format. The MeCP2 ECLIA produces highly quantitative, accurate and reproducible measurements with low intra- and inter-assay error throughout a wide working range. To underline its broad applicability, this assay was used to analyze brain tissue and study the transport of TAT-MeCP2 variants across an in vitro model of the blood-brain barrier.

Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein.

α-Chlorofatty aldehydes (α-ClFALDs) and α-bromofatty aldehydes (α-BrFALDs) are produced in activated neutrophils and eosinophils. This study investigated the ability of α-BrFALD and α-ClFALD to react with the thiols of GSH and protein cysteinyl residues. Initial studies showed that 2-bromohexadecanal (2-BrHDA) and 2-chlorohexadecanal (2-ClHDA) react with GSH producing the same fatty aldehyde-GSH adduct (FALD-GSH). In both synthetic and cellular reactions, FALD-GSH production was more robust with 2-BrHDA compared with 2-ClHDA as precursor. NaBr-supplemented phorbol myristate acetate (PMA)-activated neutrophils formed more α-BrFALD and FALD-GSH compared with non-NaBr-supplemented neutrophils. Primary human eosinophils, which preferentially produce hypobromous acid and α-BrFALD, accumulated FALD-GSH following PMA stimulation. Mice exposed to Br2 gas had increased levels of both α-BrFALD and FALD-GSH in the lungs, as well as elevated systemic plasma levels of FALD-GSH in comparison to mice exposed to air. Similar relative reactivity of α-ClFALD and α-BrFALD with protein thiols was shown using click analogs of these aldehydes. Collectively, these data demonstrate that GSH and protein adduct formation are much greater as a result of nucleophilic attack of cysteinyl residues on α-BrFALD compared with α-ClFALD, which was observed in both primary leukocytes and in mice exposed to bromine gas.

2-Chlorofatty acids induce Weibel-Palade body mobilization.

Endothelial dysfunction is a hallmark of multiple inflammatory diseases. Leukocyte interactions with the endothelium have significant effects on vascular wall biology and pathophysiology. Myeloperoxidase (MPO)-derived oxidant products released from leukocytes are potential mediators of inflammation and endothelial dysfunction. 2-Chlorofatty acids (2-ClFAs) are produced as a result of MPO-derived HOCl targeting plasmalogen phospholipids. Chlorinated lipids have been shown to be associated with multiple inflammatory diseases, but their impact on surrounding endothelial cells has not been examined. This study tested the biological properties of the 2-ClFA molecular species 2-chlorohexadecanoic acid (2-ClHA) on endothelial cells. A synthetic alkyne analog of 2-ClHA, 2-chlorohexadec-15-ynoic acid (2-ClHyA), was used to examine the subcellular localization of 2-ClFA in human coronary artery endothelial cells. Click chemistry experiments revealed that 2-ClHyA localizes to Weibel-Palade bodies. 2-ClHA and 2-ClHyA promote the release of P-selectin, von Willebrand factor, and angiopoietin-2 from endothelial cells. Functionally, 2-ClHA and 2-ClHyA cause neutrophils to adhere to and platelets to aggregate on the endothelium, as well as increase permeability of the endothelial barrier which has been tied to the release of angiopoietin-2. These findings suggest that 2-ClFAs promote endothelial cell dysfunction, which may lead to broad implications in inflammation, thrombosis, and blood vessel stability.

Therapeutic alliances predict session by session drinking behavior in the treatment of alcohol use disorders.

OBJECTIVE: The therapeutic alliance is recognized as an important contributor to treatment outcomes. In this study, the session-to-session interplay of the alliance (as perceived by the patient) and alcohol involvement (drinking days and heavy drinking days between successive treatment sessions) was examined. The analyses also tested the extent to which pretreatment changes in drinking altered these interrelationships. METHOD: Participants (N = 63) seeking treatment for an alcohol use disorder received 12 weeks of CBT for alcohol dependence and completed weekly assessments of the alliance. RESULTS: Higher session alliance scores at a given session significantly predicted lower alcohol involvement (both drinking days and heavy drinking days) in the period until the next treatment session, controlling for previous alcohol involvement. This relationship was further moderated by pretreatment change (changes in drinking before the first treatment session). Among those who demonstrated low pretreatment change, alliances continued to predict alcohol involvement. In contrast, alliances were not associated with alcohol involvement among those who significantly reduced their drinking before the first treatment session (high pretreatment changers). Finally, alcohol involvement during the period preceding a treatment session did not significantly predict alliance ratings. CONCLUSIONS: These data demonstrate that more positive patient ratings of the alliance at any given treatment session are associated with less alcohol involvement during the period until the next treatment session, most particularly among patients who have not initiated reductions in their drinking before the first treatment session. For such patients, efforts to maximize therapeutic alliances may be warranted and productive. (PsycINFO Database Record

Formation of chlorinated lipids post-chlorine gas exposure.

Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.

Intestinal Phospholipid Remodeling Is Required for Dietary-Lipid Uptake and Survival on a High-Fat Diet.

Phospholipids are important determinants of membrane biophysical properties, but the impact of membrane acyl chain composition on dietary-lipid absorption is unknown. Here we demonstrate that the LXR-responsive phospholipid-remodeling enzyme Lpcat3 modulates intestinal fatty acid and cholesterol absorption and is required for survival on a high-fat diet. Mice lacking Lpcat3 in the intestine thrive on carbohydrate-based chow but lose body weight rapidly and become moribund on a triglyceride-rich diet. Lpcat3-dependent incorporation of polyunsaturated fatty acids into phospholipids is required for the efficient transport of dietary lipids into enterocytes. Furthermore, loss of Lpcat3 amplifies the production of gut hormones, including GLP-1 and oleoylethanolamide, in response to high-fat feeding, contributing to the paradoxical cessation of food intake in the setting of starvation. These results reveal that membrane phospholipid composition is a gating factor in passive lipid absorption and implicate LXR-Lpcat3 signaling in a gut-brain feedback loop that couples absorption to food intake.

Identification of glutathione adducts of α-chlorofatty aldehydes produced in activated neutrophils.

α-Chlorofatty aldehydes (α-ClFALDs) are produced by hypochlorous acid targeting plasmalogens during neutrophil activation. This study investigated the reaction of the α-chlorinated carbon of α-ClFALD with the nucleophile, GSH. Utilizing ESI/MS/MS, the reaction product of GSH and the 16-carbon α-ClFALD, 2-chlorohexadecanal (2-ClHDA), was characterized. The resulting conjugate of 2-ClHDA and GSH (HDA-GSH) has an intact free aldehyde, and the chlorine at the α-carbon is ejected. Stable isotope-labeled [d4]HDA-GSH was synthesized, which further confirmed the structure, and was used to quantify natural α-ClFALD conjugates of GSH (FALD-GSH) using reverse-phase LC with detection by ESI/MS/MS using selected reaction monitoring. HDA-GSH is elevated in RAW 264.7 cells treated with physiologically relevant concentrations of exogenous 2-ClHDA. Furthermore, PMA-treated primary human neutrophils have elevated levels of HDA-GSH and the conjugate of 2-chlorooctadecanal (2-ClODA) and GSH (ODA-GSH), as well as elevated levels of 2-ClHDA and 2-ClODA. Production of both conjugates in PMA-stimulated neutrophils was reduced by 3-aminotriazole pretreatment, which also blocks endogenous α-ClFALD production. Additionally, plasma FALD-GSH levels were elevated in the K/BxN mouse arthritis model. Taken together, these studies demonstrate novel peptidoaldehydes derived from GSH and α-ClFALD in activated human neutrophils and in vivo in K/BxN mice.

LXRs regulate ER stress and inflammation through dynamic modulation of membrane phospholipid composition.

The fatty acyl composition of phospholipids determines the biophysical character of membranes and impacts the function of membrane proteins. Here, we define a nuclear receptor pathway for the dynamic modulation of membrane composition in response to changes in cellular lipid metabolism. Ligand activation of liver X receptors (LXRs) preferentially drives the incorporation of polyunsaturated fatty acids into phospholipids through induction of the remodeling enzyme Lpcat3. Promotion of Lpcat3 activity ameliorates endoplasmic reticulum (ER) stress induced by saturated free fatty acids in vitro or by hepatic lipid accumulation in vivo. Conversely, Lpcat3 knockdown in liver exacerbates ER stress and inflammation. Mechanistically, Lpcat3 modulates inflammation both by regulating inflammatory kinase activation through changes in membrane composition and by affecting substrate availability for inflammatory mediator production. These results outline an endogenous mechanism for the preservation of membrane homeostasis during lipid stress and identify Lpcat3 as an important mediator of LXR effects on metabolism.