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Secondary human lymphoid tissue immune reactions take place in a highly coordinated environment with compartmentalization representing a fundamental feature of this organization.In situprofiling methodologies are indispensable for the understanding of this compartmentalization. Here, we propose a complementary experimental approach aiming to reveal different aspects of this process. The analysis of human tonsils, using a combination of single cell phenotypic analysis based on flow cytometry and multiplex imaging and mass spectrometry-based methodologies, revealed a compartmentalized organization at cellular and molecular level. More specifically, the skewed distribution of highly specialized immune cell subsets and relevant soluble mediators was accompanied by a compartmentalized localization of several lipids across different anatomical areas of the tonsillar tissue. The performance of such combinatorial experimental approaches could lead to the identification of novelin situinteractions and molecular targets for thein vivomanipulation of lymphoid organ, particularly the germinal center, immune reactions.

International audience; Mass spectrometric investigations of noncovalent binding between low molecular weight compounds revealed the existence of gas-phase (GP) noncovalent complex (NCC) ions involving zwitterionic structures. ESI MS is used to prove the formation of stable sodiated NCC anions between fructose (F6P) and arginine (R) moieties. Theoretical calculations indicate a folded solvated salt (i.e., sodiated carboxylate interacting with phosphate) rather than a charge-solvated form. Under standard CID conditions, [(F6P+R-H+Na)-H]− competitively forms two major product ions (PIs) through partner splitting [(R-H+Na) loss] and charge-induced cross-ring cleavage while preserving the noncovalent interactions (noncovalent product ions (NCPIs)). MS/MS experiments combined with in-solution proton/deuteron exchanges (HDXs) demonstrated an unexpected labeling of PIs, i.e., a correlated D-enrichment/D-depletion. An increase in activation time up to 3000 ms favors such processes when limited to two H/D exchanges. These results are rationalized by interpartner hydride/deuteride exchanges (⟨HDX⟩) through stepwise isomerization/dissociation of sodiated NCC-d11 anions. In addition, the D-enrichment/D-depletion discrepancy is further explained by back HDX with residual water in LTQ (selective for the isotopologue NCPIs as shown by PI relaxation experiments). Each isotopologue leads to only one back HDX unlike multiple HDXs generally observed in GP. This behavior shows that NCPIs are zwitterions with charges solvated by a single water molecule, thus generating a back HDX through a relay mechanism, which quenches the charges and prevents further back HDX. By estimating back HDX impact on D-depletion, the interpartner ⟨HDX⟩ complementarity was thus illustrated. This is the first description of interpartner ⟨HDX⟩ and selective back HDX validating salt-solvated structures.

We describe an innovative use for the recently reported fast lipid analysis technique (FLAT) that allows for the generation of MALDI tandem mass spectrometry data suitable for lipid A structure analysis directly from a single Gram-negative bacterial colony. We refer to this tandem MS version of FLAT as FLATn. Neither technique requires sophisticated sample preparation beyond the selection of a single bacterial colony, which significantly reduces overall analysis time (∼1 h), as compared to conventional methods. Moreover, the tandem mass spectra generated by FLATn provides comprehensive information on fragments of lipid A, for example, ester bonded acyl chain dissociations, cross-ring cleavages, and glycosidic bond dissociations, all of which allow the facile determination of novel lipid A structures or confirmation of expected structures. In addition to generating tandem mass spectra directly from single colonies, we also show that FLATn can be used to analyze lipid A structures taken directly from a complex biological clinical sample without the need for ex vivo growth. From a urine sample from a patient with an E. coli infection, FLATn identified the organism and demonstrated that this clinical isolate carried the mobile colistin resistance-1 gene (mcr-1) that results in the addition of a phosphoethanolamine moiety and subsequently resistance to the antimicrobial, colistin (polymyxin E). Moreover, FLATn allowed for the determination of the existence of a structural isomer in E. coli lipid A that had either a 1- or 4'-phosphate group modification by phosphoethanolamine generated by a change of bacterial culture conditions.

We developed a method to directly detect and map the Gram-negative bacterial virulence factor lipid A derived from lipopolysaccharide (LPS) by coupling acid hydrolysis with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). As the structure of lipid A (endotoxin) determines the innate immune outcome during infection, the ability to map its location within an infected organ or animal is needed to understand localized inflammatory responses that results during host-pathogen interactions. We previously demonstrated detec- tion of free lipid A from infected tissue; however detection of lipid A derived from intact (smooth) LPS from host-pathogen MSI studies, proved elusive. Here, we detected LPS-derived lipid A from the Gram-negative pathogens, Escherichia coli (Ec, m/z 1797) and Pseudomonas aeruginosa (Pa, m/z 1446) using on-tissue acid hydrolysis to cleave the glycosidic linkage between the polysaccharide (core and 0-antigen) and lipid A moieties of LPS. Using accurate mass methods, the ion corresponding to the major Ec and Pa lipid A species (m/z 1797 and 1446, respectively) were unambiguously discriminated from complex tissue substrates. Further, we evaluated potential delocalization and signal loss of other tissue lipids and found no evidence for either, making this LPS-to-Lipid A-MSI (LLA-MSI) method, compatible with simultaneous host-pathogen lipid imaging following acid hydrolysis. This spatially sensitive technique is the first step in mapping host-influenced de novo lipid A modifications, such as those associated with antimicrobial resistance phenotypes, during Gram-negative bacterial infection and will advance our understanding of the host-pathogen interface.

Cardiolipins (CLs) are an important, regulated lipid class both in prokaryotic and eukaryotic cells, yet they remain largely unexplored by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) in tissues. To date, no in-depth optimization studies of label-free visualization of CLs in complex biological samples have been reported. Here we report a streamlined modification to our previously reported MALDI-MSI method for detection of endogenous CLs in prokaryotic and eukaryotic cells based on preparation with norharmane (NRM) matrix. Notably, the use of NRM matrix permitted sensitive detection (4.7 pg/mm(2)) of spotted CL synthetic standards. By contrast, four other MALDI matrices commonly used for lipid analysis failed to generate CL ions. Using this NRM-based method, endogenous CLs were detected from two types of complex biological samples: dried bacterial arrays and mouse tissue sections. In both cases, using NRM resulted in a better signal/noise for CL ions than the other matrices. Furthermore, inclusion of a washing step improved CL detection from tissue and this combined tissue preparation method (washing and NRM matrix) was used to profile normal mouse lung. Mouse lung yielded 26 unique CLs that were mapped and identified. Consistent with previous findings, CLs containing polyunsaturated fatty acids (PUFAs) were found in abundance in the airway and vascular features of the lung. This work represents a comprehensive investigation of detection conditions for CL using MALDI-MSI in complex biological samples that resulted in a streamlined method that enables future studies of the biological role(s) of CL in tissue.

Purpose This conceptual paper aims to explore the possibility of human beings reaching a virtual form of immortality. Design/methodology/approach The paper is an investigation of the path from an early example of human knowledge to the birth of artificial intelligence (AI) and robots. A critical analysis of different point of views, from philosophers to scientists, is presented. Findings From ancient rock art paintings to the moon landing, human knowledge has made a huge progress to the point of creating robots resembling human features. While these humanoid robots can successfully undertake risky tasks, they also generate ethical issues for the society they interact with. Research limitations/implications The paper is conceptual, and it does attempt to provide one theory by which human beings can achieve the dream of immortality. It is part of a work in progress on the use of AI and the issues related to the creation/use of humanoid robots in society. Originality/value This paper provides an overview of some of the key issues and themes impacting our modern society. Its originality resides in the linking of human knowledge to collective knowledge and then of collective mind to the hyper-collective mind. The idea of humans reaching immortality is burdened by the imperative need to define ethical guidelines for the field of AI and its uses.

International audience; We investigated the product ion spectra of [M + Na](+) from diterpene diester species and low molecular mass metabolites analyzed by electrospray ionization (ESI). Mainly, the formation of protonated salt structures was proposed to explain the observed neutral losses of carboxylic acids. It also facilitates understanding sodium retention on product ions or on neutral losses. In addition, the occurrence of consecutive carboxylic acid losses is rather unexpected under resonant excitation conditions. Quantum calculation demonstrated that the exothermic character of such neutral losses can represent a relevant explanation. There is no doubt that the formation and role of the protonated salt structures will be helpful for a better understanding and software-assisted interpretation of tandem mass spectra from small molecules, especially in the ever-growing metabolomics field.

In this paper, drug-drug chemical interactions between two different aromatic compounds were studied by mass spectrometry. Specifically, we examined non-covalent complexes (NCX) between paclitaxel, a chemotherapeutic compound, and medications widely used in palliative care for depression, psychosis, and anxiety. It is unknown whether psychotropic medications directly interact with paclitaxel. Here, we use a simple and rapid electrospray ionization mass spectrometry in vitro assay, which has been predictive in the case of neuropeptides, to measure the relative strength of non-covalent interactions. This chemical interaction is most likely due to the overlap of aromatic rings of π-orbitals between paclitaxel and five commonly used medications: diazepam, clonozepam, sertraline, fluoxetine, and haloperidol. Molecular modeling illustrates that differences in the stability of the NCXs are likely due to the distance between the aromatic rings present in both the paclitaxel and antidepressant medications. Graphical Abstract.

While it is known that opioid receptors (ORs) are densely expressed in both the brain and periphery, it is widely accepted that hypoxic effects of opioids result solely from their direct action in the CNS. To examine the role of peripheral ORs in triggering brain hypoxia, we used oxygen sensors in freely moving rats to examine how naloxone-HCl and naloxone-methiodide, the latter which is commonly believed to be peripherally restricted, affect brain oxygen responses induced by intravenous heroin at low, human-relevant doses. Similar to naloxone-HCl, naloxone-methiodide at a relatively low dose (2 mg/kg) fully blocked heroin-induced decreases in brain oxygen levels. As measured by mass spectrometry, naloxone-methiodide was found to be ~40-fold less permeable than naloxone-HCl across the blood-brain barrier, thus acting as a selective blocker of peripheral ORs. Despite this selectivity, a low but detectable amount of naloxone was found in brain tissue after naloxone-methiodide administration, potentially influencing our results. Therefore, we examined the effects of naloxone-methiodide at a very low dose (0.2 mg/kg; at which naloxone was undetectable in brain tissue) and found that this drug still powerfully attenuates heroin-induced brain oxygen responses. These data demonstrate the role of peripheral ORs in triggering heroin-induced respiratory depression and subsequent brain hypoxia.

Supplemental Digital Content is available in the text., Objective— Cells use various mechanisms to maintain cellular cholesterol homeostasis including efflux of cholesterol from the cellular plasma membrane to cholesterol acceptors such as HDLs (high-density lipoproteins). Little is known about the transfer of cholesterol from cells into the extracellular matrix. Using a unique monoclonal antibody that detects ordered cholesterol arrays (ie, cholesterol micro[or nano]-domains), we previously identified that particles containing these cholesterol domains accumulate in the extracellular matrix during cholesterol enrichment of human monocyte-derived macrophages and are found in atherosclerotic lesions. In this study, we further investigate these deposited particles containing cholesterol microdomains and discover their unexpected morphology. Approach and Results— Although appearing spherical at the resolution of the conventional fluorescence microscope, super-resolution immunofluorescence and atomic force microscopy of in situ cholesterol microdomains, and immunoelectron microscopy of isolated cholesterol microdomains revealed that the microdomains are not vesicles or 3-dimensional crystals but rather appear as branching irregularly shaped deposits of varying size. These cholesterol microdomain-containing deposits are shed from the plasma membrane into the extracellular matrix. Conclusions— To date, research on cellular excretion of excess cholesterol has demonstrated cellular cholesterol efflux in the form of membranous vesicles and discoidal HDL particles released into the fluid-phase medium. Shedding of plasma membrane cholesterol microdomains provides an additional mechanism for cells such as macrophages to maintain plasma membrane cholesterol homeostasis. Furthermore, recognition that macrophages shed cholesterol microdomains into the extracellular matrix is important to our understanding of extracellular buildup of cholesterol in atherosclerosis.

Traumatic brain injury (TBI) is a serious public health problem and the leading cause of death in children and young adults. It also contributes to a substantial number of cases of permanent disability. As lipids make up over 50% of the brain mass and play a key role in both membrane structure and cell signaling, their profile is of particular interest. In this study, we show that advanced mass spectrometry imaging (MSI) has sufficient technical accuracy and reproducibility to demonstrate the anatomical distribution of 50 μm diameter microdomains that show changes in brain ceramide levels in a rat model of controlled cortical impact (CCI) 3 days post injury with and without treatment. Adult male Sprague-Dawley rats received one strike and were euthanized 3 days post trauma. Brain MS images showed increase in ceramides in CCI animals compared to control as well as significant reduction in ceramides in CCI treated animals, demonstrating therapeutic effect of a peptide agonist. The data also suggests the presence of diffuse changes outside of the injured area. These results shed light on the extent of biochemical and structural changes in the brain after traumatic brain injury and could help to evaluate the efficacy of treatments.

Mass spectrometry imaging (MSI) of tissue implanted with silver nanoparticulate (AgNP) matrix generates reproducible imaging of lipids in rodent models of disease and injury. Gas-phase production and acceleration of size-selected 8 nm AgNP is followed by controlled ion beam rastering and soft landing implantation of 500 eV AgNP into tissue. Focused 337 nm laser desorption produces high quality images for most lipid classes in rat brain tissue (in positive mode: galactoceramides, diacylglycerols, ceramides, phosphatidylcholines, cholesteryl ester, and cholesterol, and in negative ion mode: phosphatidylethanolamides, sulfatides, phosphatidylinositol, and sphingomyelins). Image reproducibility in serial sections of brain tissue is achieved within10% tolerance by selecting argentated instead of alkali cationized ions. The imaging of brain tissues spotted with pure standards was used to demonstrate that Ag cationized ceramide and diacylglycerol ions are from intact, endogenous species. In contrast, almost all Ag cationized fatty acid ions are a result of fragmentations of numerous lipid types having the fatty acid as a subunit. Almost no argentated intact fatty acid ions come from the pure fatty acid standard on tissue. Graphical Abstract ᅟ.

Humans are increasingly aware that their fate will depend on the wisdom they apply in interacting with the ecosystem. Its health is defined as the condition in which the ecosystem can deliver and continuously renew its fundamental services. A healthy ecosystem allows optimal interactions between humans and the other biotic/abiotic components, and only in a healthy ecosystem can humans survive and efficiently reproduce. Thus, both the human and ecosystem health should be considered together in view of their interdependence. The present article suggests that this relationship could be considered starting from the Hippocrates (460 BC&ndash, 370 BC) work &ldquo, On Airs, Waters, and Places&rdquo, to derive useful medical and philosophical implications for medicine which is indeed a topic that involves scientific as well as philosophical concepts that implicate a background broader than the human body. The brain-body-ecosystem medicine is proposed as a new more complete approach to safeguarding human health. Epidemiological data demonstrate that exploitation of the environment resulting in ecosystem damage affects human health and in several instances these diseases can be detected by modifications in the heart-brain interactions that can be diagnosed through the analysis of changes in heart rate variability.

Electrostatic interactions are one of the main factors influencing biomolecular conformation. The formation of noncovalent complexes by electrostatic interactions is governed by certain amino acid residues and post-translational modifications. It has been demonstrated that adjacent arginine forms noncovalent complex with phosphate; however, histidine noncovalent complexes have rarely been investigated. In the present work, we compare the interaction between basic epitopes (NLRRITRVN, SHHGLHSTPD) and diverse acidic and aromatic-rich peptides using both MALDI and ESI Mass spectrometry. We show that adjacent histidines can also form stable noncovalent bonds and that those bonds are probably formed by a salt bridge between the phosphate or the acid residues and the histidines. However, noncovalent complexes with the arginine epitopes form more readily and are stronger than those with histidine-containing epitopes.

Background Mild traumatic brain injury (TBI) is a common public health issue that may contribute to chronic degenerative disorders. Membrane lipids play a key role in tissue responses to injury, both as cell signals and as components of membrane structure and cell signaling. This study demonstrates the ability of high resolution mass spectrometry imaging (MSI) to assess sequences of responses of lipid species in a rat controlled cortical impact model for concussion. New method A matrix of implanted silver nanoparticles was implanted superficially in brain sections for matrix-assisted laser desorption (MALDI) imaging of 50 μm diameter microdomains across unfixed cryostat sections of rat brain. Ion-mobility time-of-flight MS was used to analyze and map changes over time in brain lipid composition in a rats after Controlled Cortical Impact (CCI) TBI. Results Brain MS images showed changes in sphingolipids near the CCI site, including increased ceramides and decreased sphingomyelins, accompanied by changes in glycerophospholipids and cholesterol derivatives. The kinetics differed for each lipid class; for example ceramides increased as early as 1 day after the injury whereas other lipids changes occurred between 3 and 7 days post injury. Comparison with existing method(s) Silver nanoparticles MALDI matrix is a sensitive new tool for revealing previously undetectable cellular injury response and remodeling in neural, glial and vascular structure of the brain. Conclusions Lipid biochemical and structural changes after TBI could help highlighting molecules that can be used to determine the severity of such injuries as well as to evaluate the efficacy of potential treatments.

Alcohol abuse is a chronic disease characterized by the consumption of alcohol at a level that interferes with physical and mental health and causes serious and persistent changes in the brain. Lipid metabolism is of particular interest due to its high concentration in the brain. Lipids are the main component of cell membranes, are involved in cell signaling, signal transduction, and energy storage. In this study, we analyzed lipid composition of chronically ethanol exposed mouse brains. Juvenile (JUV) and adult (ADU) mice were placed on a daily limited-access ethanol intake model for 52 days. After euthanasia, brains were harvested, and total lipids were extracted from brain homogenates. Samples were analyzed using high resolution mass spectrometry and processed by multivariate and univariate statistical analysis. Significant lipid changes were observed in different classes including sphingolipids, fatty acids, lysophosphatidylcholines, and other glycerophospholipids.

Schnyder corneal dystrophy (SCD) is a rare autosomal dominant disease in humans, characterized by abnormal deposition of cholesterol and phospholipids in cornea caused by mutations in the UbiA prenyltransferase domain containing 1 (UBIAD1) gene. In this study, we generated a mouse line carrying Ubiad1 N100S point mutation using the CRISPR/Cas9 technique to investigate the pathogenesis of SCD. In vivo confocal microscopy revealed hyper-reflective dot-like deposits in the anterior cornea in heterozygotes and homozygotes. No significant change was found in corneal epithelial barrier function or wound healing. Electron microscopy revealed abnormal mitochondrial morphology in corneal epithelial, stromal, and endothelial cells. Mitochondrial DNA copy number assay showed 1.27 ± 0.07 fold change in homozygotes versus 0.98 ± 0.05 variation in wild type mice (P Ubiad1N100S mouse provides a promising animal model of SCD revealing that mitochondrial dysfunction is a prominent component of the disease. The different phenotype in human and mouse may due to difference in cholesterol metabolism between species.

Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) is widely used as a unique tool to record the distribution of a large range of biomolecules in tissues. 2,6-Dihydroxyacetophenone (DHA) matrix has been shown to provide efficient ionization of lipids, especially gangliosides. The major drawback for DHA as it applies to MS imaging is that it sublimes under vacuum (low pressure) at the extended time necessary to complete both high spatial and mass resolution MSI studies of whole organs. To overcome the problem of sublimation, we used an atmospheric pressure (AP)-MALDI source to obtain high spatial resolution images of lipids in the brain using a high mass resolution mass spectrometer. Additionally, the advantages of atmospheric pressure and DHA for imaging gangliosides are highlighted. The imaging of [M-H]- and [M-H2O-H]- mass peaks for GD1 gangliosides showed different distribution, most likely reflecting the different spatial distribution of GD1a and GD1b species in the brain. Graphical Abstract ᅟ.

Investigations of brain complex integrative actions should consider beside neural networks, glial, extracellular molecular, and fluid channels networks. The present paper proposes that all these networks are assembled into the brain hyper-network that has as fundamental components, the tetra-partite synapses, formed by neural, glial, and extracellular molecular networks. Furthermore, peri-synaptic astrocytic processes by modulating the perviousness of extracellular fluid channels control the signals impinging on the tetra-partite synapses. It has also been surmised that global signalling via astrocytes networks and highly pervasive signals, such as electromagnetic fields (EMFs), allow the appropriate integration of the various networks especially at crucial nodes level, the tetra-partite synapses. As a matter of fact, it has been shown that astrocytes can form gap-junction-coupled syncytia allowing intercellular communication characterised by a rapid and possibly long-distance transfer of signals. As far as the EMFs are concerned, the concept of broadcasted neuroconnectomics (BNC) has been introduced to describe highly pervasive signals involved in resetting the information handling of brain networks at various miniaturisation levels. In other words, BNC creates, thanks to the EMFs, generated especially by neurons, different assemblages among the various networks forming the brain hyper-network. Thus, it is surmised that neuronal networks are the "core components" of the brain hyper-network that has as special "nodes" the multi-facet tetra-partite synapses. Furthermore, it is suggested that investigations on the functional plasticity of multi-partite synapses in response to BNC can be the background for a new understanding and perhaps a new modelling of brain morpho-functional organisation and integrative actions.

Long-chain PUFAs (LC-PUFAs; C20–C22; e.g., DHA and arachidonic acid) are highly enriched in vertebrate retina, where they are elongated to very-long-chain PUFAs (VLC-PUFAs; C ≥28) by the elongation of very-long-chain fatty acids-4 (ELOVL4) enzyme. These fatty acids play essential roles in modulating neuronal function and health. The relevance of different lipid requirements in rods and cones to disease processes, such as age-related macular degeneration, however, remains unclear. To better understand the role of LC-PUFAs and VLC-PUFAs in the retina, we investigated the lipid compositions of whole retinas or photoreceptor outer segment (OS) membranes in rodents with rod- or cone-dominant retinas. We analyzed fatty acid methyl esters and the molecular species of glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine) by GC-MS/GC-flame ionization detection and ESI-MS/MS, respectively. We found that whole retinas and OS membranes in rod-dominant animals compared with cone-dominant animals had higher amounts of LC-PUFAs and VLC-PUFAs. Compared with those of rod-dominant animals, retinas and OS membranes from cone-dominant animals also had about 2-fold lower levels of di-DHA (22:6/22:6) molecular species of glycerophospholipids. Because PUFAs are necessary for optimal G protein-coupled receptor signaling in rods, these findings suggest that cones may not have the same lipid requirements as rods.

The well-characterized cellular and structural components of the kidney show distinct regional compositions and distribution of lipids. In order to more fully analyze the renal lipidome we developed a matrix-assisted laser desorption/ionization mass spectrometry approach for imaging that may be used to pinpoint sites of changes from normal in pathological conditions. This was accomplished by implanting sagittal cryostat rat kidney sections with a stable, quantifiable and reproducible uniform layer of silver using a magnetron sputtering source to form silver nanoparticles. Thirty-eight lipid species including seven ceramides, eight diacylglycerols, 22 triacylglycerols, and cholesterol were detected and imaged in positive ion mode. Thirty-six lipid species consisting of seven sphingomyelins, 10 phosphatidylethanolamines, one phosphatidylglycerol, seven phosphatidylinositols, and 11 sulfatides were imaged in negative ion mode for a total of seventy-four high-resolution lipidome maps of the normal kidney. Thus, our approach is a powerful tool not only for studying structural changes in animal models of disease, but also for diagnosing and tracking stages of disease in human kidney tissue biopsies.

Ceramides (CER) are involved in alcohol-induced neuroinflammation. In a mouse model of chronic alcohol exposure, 16 CER and 18 sphingomyelin (SM) concentrations from whole brain lipid extracts were measured using electrospray mass spectrometry. All 18 CER concentrations in alcohol exposed adults increased significantly (range: 25–607%); in juveniles, 6 CER decreased (range: −9 to −37%). In contrast, only three SM decreased in adult and one increased significantly in juvenile. Next, regional identification at 50 μm spatial resolution from coronal sections was obtained with matrix implanted laser desorption/ionization mass spectrometry imaging (MILDI-MSI) by implanting silver nanoparticulate matrices followed by focused laser desorption. Most of the CER and SM quantified in whole brain extracts were detected in MILDI images. Coronal sections from three brain levels show qualitative regional changes in CER-SM ion intensities, as a function of group and brain region, in cortex, striatum, accumbens, habenula, and hippocampus. Highly correlated changes in certain white matter CER-SM pairs occur in regions across all groups, including the hippocampus and the lateral (but not medial) cerebellar cortex of adult mice. Our data provide the first microscale MS evidence of regional lipid intensity variations induced by alcohol.

The development of bivalent ligands has attracted interest as a way to potentially improve the selectivity and/or affinity for a specific receptor subtype. The ability to bind two distinct receptor binding sites simultaneously can allow the selective activation of specific G-protein dependent or β-arrestin-mediated cascade pathways. Herein, we developed an extended SAR study using sumanirole (1) as the primary pharmacophore. We found that substitutions in the N-1- and/or N-5-positions, physiochemical properties of those substituents, and secondary aromatic pharmacophores can enhance agonist efficacy for the cAMP inhibition mediated by G(i/o)-proteins, while reducing or suppressing potency and efficacy toward β-arrestin recruitment. Compound 19 was identified as a new lead for its selective D(2) G-protein biased agonism with an EC(50) in the subnanomolar range. Structure—activity correlations were observed between substitutions in positions N-1 and/or N-5 of 1 and the capacity of the new bivalent compounds to selectively activate G-proteins versus β-arrestin recruitment in D(2)R-BRET functional assays.

Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes’ participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. This article is protected by copyright. All rights reserved.

Complex molecular and cellular mechanisms regulate G protein-coupled receptors (GPCRs). It is suggested that proteins intrinsically disordered regions (IDRs) are to play a role in GPCR’s intra and extracellular regions plasticity, due to their potential for post-translational modification and interaction with other proteins. These regions are defined as lacking a stable three-dimensional (3D) structure. They are rich in hydrophilic and charged, amino acids and are capable to assume different conformations which allow them to interact with multiple partners. In this study we analyzed 75 GPCR involved in synaptic transmission using computational tools for sequence-based prediction of IDRs within a protein. We also evaluated putative ligand-binding motifs using receptor sequences. The disorder analysis indicated that neurotransmitter GPCRs have a significant amount of disorder in their N-terminus, third intracellular loop (3IL) and C-terminus. About 31%, 39% and 53% of human GPCR involved in synaptic transmission are disordered in these regions. Thirty-three percent of receptors show at least one predicted PEST motif, this being statistically greater than the estimate for the rest of human GPCRs. About 90% of the receptors had at least one putative site for dimerization in their 3IL or C-terminus. ELM instances sampled in these domains were 14-3-3, SH3, SH2 and PDZ motifs. In conclusion, the increased flexibility observed in GPCRs, added to the enrichment of linear motifs, PEST and heteromerization sites, may be critical for the nervous system’s functional plasticity.

Certain amino acid residues and posttranslational modifications play an important role in the formation of noncovalent complexes (NCXs) by electrostatic interactions. Electrospray ionization mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs, due to its softer ionization process and compatibility with the solution phase of NCX mixtures. In order to locate the site where interactions are forming in NCXs involving phosphopeptides and adjacent arginines, tandem mass spectrometry studies using collision-induced dissociation (CID) and electron transfer dissociation (ETD) were performed on NCXs at different charge states. CID fragmentation revealed two dissociation pathways: one in which the electrostatic interaction is disrupted and another in which the covalent bond attaching the phosphate group to the amino acid residue is cleaved, while the electrostatic interaction is maintained. ETD and sequential ETD/ETD, and CID/ETD allow the determination of the NCX interaction site. These results confirmed the involvement of the phosphorylated amino acid and at least two adjacent arginines as the binding site.

Profiling and imaging MALDI mass spectrometry (MS) allows detection and localization of biomolecules in tissue, of which lipids are a major component. However, due to the in situ nature of this technique, complexity of tissue and need for a chemical matrix, the recorded signal is complex and can be difficult to assign. Ion mobility adds a dimension that provides coarse shape information, separating isobaric lipids, peptides, and oligonucleotides along distinct familial trend lines before mass analysis. Previous work using MALDI-ion mobility mass spectrometry to analyze and image lipids has been conducted mainly in positive ion mode, although several lipid classes ionize preferentially in negative ion mode. This work highlights recent data acquired in negative ion mode to detect glycerophosphoethanolamines (PEs), glycerophosphoserines (PSs), glycerophosphoglycerols (PGs), glycerolphosphoinositols (PIs), glycerophosphates (PAs), sulfatides (STs), and gangliosides from standard tissue extracts and directly from mouse brain tissue. In particular, this study focused on changes in ion mobility based upon lipid head groups, composition of radyl chain (# of carbons and double bonds), diacyl versus plasmalogen species, and hydroxylation of species. Finally, a MALDI-ion mobility imaging run was conducted in negative ion mode, resulting in the successful ion mapping of several lipid species.

Noncovalent interactions govern how molecules communicate. Mass spectrometry is an important and versatile tool for the analysis of noncovalent complexes (NCX). Electrospray mass spectrometry (ESI-MS) is the most widely used MS technique for the study of NCXs because of its softer ionization and easy compatibility with the solution phase of NCX mixtures. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has also been used to study NCXs. However, successful analysis depends upon several experimental factors, such as matrix selection, solution pH, and instrumental parameters. In this study, we employ MALDI imaging mass spectrometry to investigate the location and formation of NCXs, involving both peptides and proteins, in a MALDI sample spot.

Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced "mild" traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5-5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology.

Matrix-assisted laser/desorption ionization (MALDI) time-of-flight mass spectrometry (TOF) has been investigated for use in the field of metabolomics; however, difficulties, mainly due to chemical interferences, are typically encountered. By coupling MALDI with ion mobility time-of-flight mass spectrometry (IMMS), isomers and isobars are resolved in mobility space reducing the chemical interference from matrix/background ions. MALDI-IMMS offers the advantages of high sensitivity, high throughput and low sample consumption. For this study, MALDI-IMMS was evaluated by monitoring metabolic changes in lymphatic fluid collected from fasting and fed rats. The number of metabolite features detected in the samples ranged between 1200 and 3400 depending on the duration between the feeding time and lymph sample collection. There were 747 metabolite features that were statistically analyzed by principal component analysis (PCA). From the 3-D score plots of PC1, PC2 and PC3 65 % of the original variation of the system was explained and the differences between the samples were demonstrated.

This work shows how a chemical inkjet printer was used for this purpose resulting in the imaging of phosphatidylcholines and sulfatides. The intricacies involved in effective matrix deposition are discussed.

Gangliosides are amphiphilic molecules found in the outer layer of plasma membranes of all vertebrate cells. They play a major role in cell recognition and signaling and are involved in diseases affecting the central nervous system (CNS). We are reporting the differential distribution of ganglioside species in the rat brain's cerebrum, based on their ceramide associated core, and for the first time the presence of acetylation detected by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, which was used to map and image gangliosides with detailed structural information and histological accuracy. In the hippocampus, localization of the major species GM1, GD1, O-acetylGD1, GT1, and O-acetylGT1 depends on the sphingoïd base (d18:1 sphingosine or d20:1 eïcosasphingosine) in the molecular layer of the dentate gyrus (ML), which is made up of three distinct layers, the inner molecular layer (IML), which contains sphingosine exclusively, and the middle molecular layer (MML) and the outer molecular layer (OML) where eïcosasphingosine is the only sphingoïd base. These results demonstrate that there is a different distribution of gangliosides in neuronal axons and dendrites depending on the ceramide core of each layer. GM3, GM2, GD3, and GD2 contain sphingosine predominantly and are mainly present in body cell layers, which are made up of the pyramidal cell layer (Py) and the granular layer of the dentate gyrus (GL), in contrast with GQ1 and the O-acetylated forms of GD1, GT1, and GQ1 gangliosides, which contain both sphingoïd bases. However their distribution is based on the sialylated and acetylated oligosaccharide chains in the neuronal cell bodies.

The combination of ion mobility with matrix-assisted laser desorption/ionization allows for the rapid separation and analysis of biomolecules in complex mixtures (such as tissue sections and cellular extracts), as isobaric lipid, peptide, and oligonucleotide molecular ions are pre-separated in the mobility cell before mass analysis. In this study, MALDI-IM MS is used to analyze gangliosides, a class of complex glycosphingolipids that has different degrees of sialylation. Both GD1a and GD1b, structural isomers, were studied to see the effects on gas-phase structure depending upon the localization of the sialic acids. A total ganglioside extract from mouse brain was also analyzed to measure the effectiveness of ion mobility to separate out the different ganglioside species in a complex mixture.

Dopamine D(2) and D(4) receptors partially codistribute in the dorsal striatum and appear to play a fundamental role in complex behaviors and motor function. The discovery of D(2)R-D(4.)(x)R (D(4.2)R, D(4.4)R or D(4.7)R) heteromers has been made in cellular models using co-immunoprecipitation, in situ Proximity Ligation Assays and BRET(1) techniques with the D(2)R and D(4.7)R receptors being the least effective in forming heteromers. Allosteric receptor-receptor interactions in D(2)R-D(4.2)R and D(2)R-D(4.4) R heteromers were observed using the MAPK assays indicating the existence of an enhancing allosteric receptor-receptor interaction in the corresponding heteromers between the two orthosteric binding sites. The bioinformatic predictions suggest the existence of a basic set of common triplets (ALQ and LRA) in the two participating receptors that may contribute to the receptor-receptor interaction interfaces.

Comprehensive metabolome analysis using mass spectrometry (MS) often results in a complex mass spectrum and difficult data analysis resulting from the signals of numerous small molecules in the metabolome. In addition, MS alone has difficulty measuring isobars and chiral, conformational and structural isomers. When a matrix-assisted laser desorption ionization (MALDI) source is added, the difficulty and complexity are further increased. Signal interference between analyte signals and matrix ion signals produced by MALDI in the low mass region (

The adenosine A(2A)-dopamine D(2) receptor heteromer is one of the most studied receptor heteromers. It has important implications for basal ganglia function and pathology. Recent studies using Bioluminescence and Sequential Resonance Energy Transfer techniques shed light on the role of Ca(2+) in the modulation of the quaternary structure of the A(2A)-D(2) receptor heteromer, which was found to depend on the binding of calmodulin (CaM) to the carboxy-terminus of the A(2A) receptor in the A(2A)-D(2) receptor heteromer. Importantly, the changes in quaternary structure correlate with changes in function. A Ca(2+)/CaM-dependent modulation of MAPK signaling upon agonist treatment could be observed in cells expressing A(2A)-D(2) receptor heteromers. These studies provide a first example of a Ca(2+)-mediated modulation of the quaternary structure and function of a receptor heteromer.

The concept of Receptor Mosaic (RM) is discussed; hence the integrative functions of the assemblage of G-protein coupled receptors physically interacting in the plane of the plasma membrane. The main focus is on a hetero-trimer of G-protein coupled receptors, namely the A2A-D2-CB1 receptor trimer. A bioinformatics analysis was carried out on the amino acid sequence of these receptors to indicate domains possibly involved in the receptor-receptor interactions. Such a bioinformatic analysis was also carried out on the RM formed by mGLU R5, D2 and A2A. The importance of topology, i.e., of the reciprocal localisation of the three interacting receptors in the plan of the membrane for the RM integrative functions is underlined. However, it is also pointed out that this fundamental aspect still waits techniques capable of an appropriate investigation. Finally, it is discussed how RM topology can give hints for a structural definition of the concept of hub receptor. Thus, just as in any network, the receptor operating as a hub is the one that in the molecular network formed by the receptors has the highest number of inputs.

Advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) have allowed for the direct analysis of biological molecules from tissue. Although most of the early studies of direct tissue profiling by MALDI-TOFMS have focused on proteins and peptides, analysis of lipids has increased dramatically in recent years. This review gives an overview of the factors to consider when analyzing lipids directly from tissue and some recent examples of the use of MALDI-TOFMS for the direct profiling of lipids in tissue.

The concept of intramembrane receptor-receptor interactions and evidence for their existence was introduced by Agnati and Fuxe in 1980/81 suggesting the existence of heteromerization of receptors. In 1982, they proposed the existence of aggregates of multiple receptors in the plasma membrane and coined the term receptor mosaics (RM). In this way, cell signaling becomes a branched process beginning at the level of receptor recognition at the plasma membrane where receptors can directly modify the ligand recognition and signaling capacity of the receptors within a RM. Receptor-receptor interactions in RM are classified as operating either with classical cooperativity, when consisting of homomers or heteromers of similar receptor subtypes having the same transmitter, or non-classical cooperativity, when consisting of heteromers. It has been shown that information processing within a RM depends not only on its receptor composition, but also on the topology and the order of receptor activation determined by the concentrations of the ligands and the receptor properties. The general function of RM has also been demonstrated to depend on allosteric regulators (e.g., homocysteine) of the receptor subtypes present. RM as integrative nodes for receptor-receptor interactions in conjunction with membrane associated proteins may form horizontal molecular networks in the plasma membrane coordinating the activity of multiple effector systems modulating the excitability and gene expression of the cells. The key role of electrostatic epitope-epitope interactions will be discussed for the formation of the RM. These interactions probably represent a general molecular mechanism for receptor-receptor interactions and, without a doubt, indicate a role for phosphorylation-dephosphorylation events in these interactions. The novel therapeutic aspects given by the RMs will be discussed in the frame of molecular neurology and psychiatry and combined drug therapy appears as the future way to go.

Receptor heteromers constitute a new area of research that is reshaping our thinking about biochemistry, cell biology, pharmacology and drug discovery. In this commentary, we recommend clear definitions that should facilitate both information exchange and research on this growing class of transmembrane signal transduction units and their complex properties. We also consider research questions underlying the proposed nomenclature, with recommendations for receptor heteromer identification in native tissues and their use as targets for drug development.

Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60-min middle cerebral artery occlusion (MCAo). ATX was present in the infarction area at 70-75 min after onset of MCAo. Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 d after MCAo. To evaluate the protective mechanisms of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis, and excitoxicity. ATX antagonized ischemia-mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome c, and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, our data suggest that ATX can reduce ischemia-related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and antiapoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events.—Shen, H., Kuo, C.-C., Chou, J., Delvolve, A., Jackson, S. N., Post, J., Woods, A. S., Hoffer, B. J., Wang, Y., Harvey, B. K. Astaxanthin reduces ischemic brain injury in adult rats.