Cardiolipin: characterization of distinct oxidized molecular species

Cardiolipin (CL) is a phospholipid predominantly found in the mitochondrial inner membrane and is associated structurally with individual complexes of the electron transport chain (ETC). Because the ETC is the major mitochondrial reactive oxygen species (ROS)-generating site, the proximity to the ETC and bisallylic methylenes of the PUFA chains of CL make it a likely target of ROS in the mitochondrial inner membrane. Oxidized cellular CL products, uniquely derived from ROS-induced autoxidation, could serve as biomarkers for the presence of the ROS and could help in the understanding of the mechanism of oxidative stress. Because major CL species have four unsaturated acyl chains, whereas other phospholipids usually have only one in the sn-2 position, characterization of oxidized CL is highly challenging. In the current study, we exposed CL, under aerobic conditions, to singlet oxygen (¹O₂), the radical initiator 2,2′-azobis(2-methylpropionamidine) dihydrochloride, or room air, and the oxidized CL species were characterized by HPLC-tandem mass spectrometry (MS/MS). Our reverse-phase ion-pair HPLC-MS/MS method can characterize the major and minor oxidized CL species by detecting distinctive fragment ions associated with specific oxidized species. The HPLC-MS/MS results show that monohydroperoxides and bis monohydroperoxides were generated under all three conditions. However, significant amounts of CL dihydroperoxides were produced only by ¹O₂-mediated oxidation. These products were barely detectable from radical oxidation either in a liposome bilayer or in thin film. These observations are only possible due to the chromatographic separation of the different oxidized species.     

Spectral turbulence measuring as feature extraction method from EEG on affective computing

In biomedical and psychological applications dealing with EEG, a suitable selection of the most relevant electrodes is useful for lightening the data acquisition and facilitating the signal processing. Therefore, an efficient method for extracting and selecting features from EEG channels is desirable. Classification methods are more and more applied for obtaining important conclusions from diverse psychological processes, and specifically for emotional processing. In this work, an original and straightforward method, inspired by the spectral turbulence (ST) measure from electrocardiogram and the support vector machine-recursive feature elimination (SVM-RFE) algorithm, is proposed for classifying EEG signals. The goal of this study is to introduce the ST concept in applications of artificial intelligence related to cognitive processes and to determine the best EEG channels for distinguishing between two different experimental conditions. By means of this method, the left temporal region of the brain has revealed to be greatly involved in the affective valence processing elicited by visual stimuli.     

Accumulation of orally administered quercetin in brain tissue and its antioxidative effects in rats

Quercetin is widely distributed in vegetables and herbs and has been suggested to act as a neuroprotective agent. Here, we demonstrate that quercetin can accumulate enough to exert biological activity in rat brain tissues. Homogenates of perfused rat brain without detectable hemoglobin contaminants were treated with β-glucuronidase/sulfatase and the released quercetin and its methylated form were analyzed using high-performance liquid chromatography (HPLC) with three different detection methods. Both quercetin and the methylated form were detected in the brain of quercetin-administered rats using HPLC-UV and HPLC with electrochemical detection and were further identified using HPLC-tandem mass spectrometry. Oral administration of quercetin (50 mg/kg body wt) attenuated the increased oxidative stress in the hippocampus and striatum of rats exposed to chronic forced swimming. The possible transport of quercetin derivatives into the brain tissue was reproduced in vitro by using a rat brain capillary endothelial cell line, a model of the blood-brain barrier. These results show that quercetin could be a potent nutrient that can access the brain and protect it from disorders associated with oxidative stress.     

Hemoglobin attenuates the effects of inspired oxygen on plasma isofurans in humans during upper-limb surgery

Reperfusion injury is characterized by significant oxidative stress. F₂-isoprostanes (F₂-IsoP's) and isofurans (IsoF's), the latter preferentially produced during increased oxygen tension, are recognized markers of in vivo oxidative stress. We aimed to determine whether increasing oxygen tension during reperfusion modified levels of plasma total IsoF's and F₂-IsoP's. Forty-five patients undergoing upper-limb surgery were randomized to receive inspired oxygen concentrations of 30, 50, or 80% during the last 15 min of surgery. Venous blood samples were taken before the change in inspired oxygen, after 10 min (before reperfusion), and after 15 min (5 min after reperfusion). IsoF's and F₂-IsoP's were measured by gas chromatography-mass spectrometry. Venous oxygen tension and hemoglobin concentrations were also measured. Plasma IsoF and F₂-IsoP levels in the 50 and 80% O₂ groups were not significantly different from those of the 30% O₂ group. In secondary analyses, using data combining all groups, levels of IsoF's, but not F₂-IsoP's, associated with higher venous oxygen tension (P = 0.038). Hemoglobin negatively modified the influence of oxygen tension on levels of IsoF's (P = 0.014). This study has shown, for the first time, that plasma IsoF levels associate with higher oxygen tension in a human model of reperfusion, and this effect is significantly attenuated by hemoglobin.     

Identification of oxidized phospholipids by electrospray ionization mass spectrometry and LC-MS using a QQLIT instrument

Phospholipids are complex and varied biomolecules that are susceptible to lipid peroxidation after attack by free radicals or electrophilic oxidants and can yield a large number of different oxidation products. There are many available methods for detecting phospholipid oxidation products, but also various limitations and problems. Electrospray ionization mass spectrometry allows the simultaneous but specific analysis of multiple species with good sensitivity and has a further advantage that it can be coupled to liquid chromatography for separation of oxidation products. Here, we explain the principles of oxidized phospholipid analysis by electrospray mass spectrometry and describe fragmentation routines for surveying the structural properties of the analytes, in particular precursor ion and neutral loss scanning. These allow targeted detection of phospholipid headgroups and identification of phospholipids containing hydroperoxides and chlorine, as well as the detection of some individual oxidation products by their specific fragmentation patterns. We describe instrument protocols for carrying out these survey routines on a QTrap5500 mass spectrometer and also for interfacing with reverse-phase liquid chromatography. The article highlights critical aspects of the analysis as well as some limitations of the methodology.     

LC/ESR/MS study of pH-dependent radical generation from 15-LOX-catalyzed DPA peroxidation

Docosapentaenoic acid (DPA) is a unique fatty acid that exists in two isomeric forms (n-3 and n-6), which differ in their physiological behaviors. DPA can undergo free radical-mediated peroxidation via lipoxygenase (LOX). 15-LOX, one of the LOX isomers, has received much attention in cancer research because of its very different expression level in normal tissues compared to tumors and some bioactive fatty acid metabolites modulating the tumorigenic pathways in cancer. However, the mechanism linking 15-LOX, DPA metabolites, and their bioactivities is still unclear, and the free radicals generated in DPA peroxidation have never been characterized. In this study, we have studied radicals formed from both soybean and human cellular (PC3-15LOS cells) 15-LOX-catalyzed peroxidation of DPAs at various pH's using a combination of LC/ESR/MS with the spin trapping technique. We observed a total of three carbon-centered radicals formed in 15-LOX-DPA (n-3) stemming from its 7-, 17-, and 20-hydroperoxides, whereas only one formed from 17-hydroperoxide in DPA (n-6). A change in the reaction pH from 8.5 (15-LOX enzyme optimum) to 7.4 (physiological) and to 6.5 (tumor, acidic) not only decreased the total radical formation but also altered the preferred site of oxygenation. This pH-dependent alteration of radical formation and oxygenation pattern may have significant implications and provide a basis for our ongoing investigations of LOXs as well as fatty acids in cancer biology.     

Molecular analysis of SMN1, SMN2, NAIP, GTF2H2, and H4F5 genes in 157 Chinese patients with spinal muscular atrophy

Spinal muscular atrophy (SMA) is a common and lethal autosomal recessive neurodegenerative disorder, which is caused by mutations of the survival motor neuron 1 (SMN1) gene. Additionally, the phenotype is modified by several genes nearby SMN1 in the 5q13 region. In this study, we analyzed mutations in SMN1 and quantified the modifying genes, including SMN2, NAIP, GTF2H2, and H4F5 by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP), multiplex ligation-dependent probe amplification (MLPA), TA cloning, allele-specific long-range PCR, and Sanger sequencing in 157 SMA patients. Most SMA patients (94.90%) possessed a homozygous SMN1 deletion, while 10 patients demonstrated only the absence of exon 7, but the presence of exon 8. Two missense mutations (c.689 C > T and c.844 C > T) were identified in 2 patients who both carried a single copy of SMN1. We found inverse correlations between SMN2, the NAIP copy number, and the clinical severity of the disease. Furthermore, 7 severe type I patients possessed large-scale deletions, including SMN1, NAIP, and GTF2H2. We conclude that SMN1 gene conversion, SMN1 subtle mutations, SMN2 copy number, and the extent of deletion in the 5q13 region should all be considered in the genotype–phenotype analysis of SMA.     

Evaluation of 18F-labeled exendin(9-39) derivatives targeting glucagon-like peptide-1 receptor for pancreatic β-cell imaging

β-cell mass (BCM) is known to be decreased in subjects with type-2 diabetes (T2D). Quantitative analysis for BCM would be useful for understanding how T2D progresses and how BCM affects treatment efficacy and for earlier diagnosis of T2D and development of new therapeutic strategies. However, a noninvasive method to measure BCM has not yet been developed.We developed four ¹⁸F-labeled exendin(9-39) derivatives for β-cell imaging by PET: [¹⁸F]FB9-Ex(9-39), [¹⁸F]FB12-Ex(9-39), [¹⁸F]FB27-Ex(9-39), and [¹⁸F]FB40-Ex(9-39). Affinity to the glucagon-like peptide-1 receptor (GLP-1R) was evaluated with dispersed islet cells of ddY mice. Uptake of exendin(9-39) derivatives in the pancreas as well as in other organs was evaluated by a biodistribution study. Small-animal PET study was performed after injecting [¹⁸F]FB40-Ex(9-39).FB40-Ex(9-39) showed moderate affinity to the GLP-1R. Among all of the derivatives, [¹⁸F]FB40-Ex(9-39) resulted in the highest uptake of radioactivity in the pancreas 30?min after injection. Moreover, it showed significantly less radioactivity accumulated in the liver and kidney, resulting in an overall increase in the pancreas-to-organ ratio. In the PET imaging study, pancreas was visualized at 30?min after injection of [¹⁸F]FB40-Ex(9-39).[¹⁸F]FB40-Ex(9-39) met the basic requirements for an imaging probe for GLP-1R in pancreatic β-cells. Further enhancement of pancreatic uptake and specific binding to GLP-1R will lead to a clear visualization of pancreatic β-cells.     

Lifting Industrial Ecology Modeling to a New Level of Quality and Transparency: A Call for More Transparent Publications and a Collaborative Open Source Software Framework

Industrial ecology (IE) is a maturing scientific discipline. The field is becoming more data and computation intensive, which requires IE researchers to develop scientific software to tackle novel research questions. We review the current state of software programming and use in our field and find challenges regarding transparency, reproducibility, reusability, and ease of collaboration. Our response to that problem is fourfold: First, we propose how existing general principles for the development of good scientific software could be implemented in IE and related fields. Second, we argue that collaborating on open source software could make IE research more productive and increase its quality, and we present guidelines for the development and distribution of such software. Third, we call for stricter requirements regarding general access to the source code used to produce research results and scientific claims published in the IE literature. Fourth, we describe a set of open source modules for standard IE modeling tasks that represent our first attempt at turning our recommendations into practice. We introduce a Python toolbox for IE that includes the life cycle assessment (LCA) framework Brightway2, the ecospold2matrix module that parses unallocated data in ecospold format, the pySUT and pymrio modules for building and analyzing multiregion input‐output models and supply and use tables, and the dynamic_stock_model class for dynamic stock modeling. Widespread use of open access software can, at the same time, increase quality, transparency, and reproducibility of IE research.