Identification of glycolytic proteins as binding partners of Bri2 BRICHOS domain.

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, that can attenuate Aß pathology in the brain. As a result, the identification of novel Bri2 BRICHOS client proteins has been sought to help elucidate signaling pathways and the potential identification of novel therapeutic targets. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, in combination with proteomic analysis on cytosolic and membrane fractions of cortical homogenates from C57BL/6 J WT mouse. We identified 4 proteins from the cytosolic fractions and 44 proteins from the membrane fractions that had significant interactions (p < 0.05) with Bri2 BRICHOS domain, of which 11 proteins were previously identified as proteins that interacted with Bri2 BRICHOS domain. Enrichment analysis of the retained proteins identified glycolysis/gluconeogenesis as the most enriched pathway, with several proteins identified playing roles in carbon metabolism, amino acid synthesis. The data suggested that Bri2 BRICHOS may have a role in cellular energy demands in the brain via glycolysis and mitochondrial oxidative phosphorylation and may play a role in mitochondrial homeostasis.

Active vs passive novelty-related strategies: Sex differences in exploratory behaviour and monoaminergic systems.

Sex differences are apparent in numerous behavioural characteristics. In order to compare and characterise male and female variability of exploratory behaviour, 365 male and 401 female rats were assessed in a task where a bimodal response distribution had previously been established in males. Female rats had significantly higher exploratory activity, and presented normal distribution of the behaviour, very differently from the bimodal distribution of males. No major effect of litter or oestrous cycle was detected. Several differences between male and female rats were found in monoamine metabolism measured ex vivo. Male rats had lower levels of dopamine (DA) in frontal cortex, and higher levels of 3,4-dihydroxyphenylacetic acid (DOPAC) in raphe area; higher levels of serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in dorsal striatum but lower levels of 5-HT and 5-HIAA in locus coeruleus area, 5-HIAA levels were also lower in hippocampus as compared to females. Males had higher noradrenaline (NA) levels in hippocampus and lower normetanephrine (NMN) levels in striatum, in both brain regions male animals had lower NMN/NA ratio. No sex difference was found in accumbens. The only brain region with an interaction between sex and the expression of exploratory activity was raphe: Here 5-HT levels were lower, and DOPAC levels and DOPAC/DA and 5-HIAA/5-HT ratios higher in low exploring male but not female rats. Conclusively, female rats not only display higher levels of exploration but the population distribution of this behaviour is distinct; this may be related to differences in the monoaminergic systems between female and male animals.

Association of Impulsivity With Food, Nutrients, and Fitness in a Longitudinal Birth Cohort Study.

BACKGROUND: Impulsivity is a psychiatric vulnerability factor strongly associated with substance abuse but also with unhealthy diet. Whether these associations extend to specific nutrients is largely unknown. Therefore, we investigated the longitudinal association between diet, cardiorespiratory fitness, and 2 impulsivity dimensions in a representative sample of south Estonian adolescents and young adults. Impulsivity and dietary intake were measured 3 times in 2 birth cohorts at regular intervals in individuals aged 15 to 33 years. METHODS: The sample included 2 birth cohorts of the longitudinal Estonian Children Personality Behaviour and Health Study. The analytic sample size consisted of 2883 observations (56.4% females). The primary outcomes were adaptive and maladaptive impulsivity scores measured by an original 24-item Likert-type questionnaire. Impulsivity scores were predicted from the food diaries data converted into nutrient categories. A linear mixed-effects approach was used to model the time dependence between observations. RESULTS: Lower maladaptive impulsivity was associated with higher cardiorespiratory fitness (ß = -.07; 95% CI = -0.12; -0.03). Higher maladaptive impulsivity was associated with lower dietary intake of zinc (ß = -.10; -0.15; -0.06) and vegetables (ß = -.04; -0.07; -0.01) and higher intake of sodium (ß = .06; 0.02; 0.10). Vitamin B6 was positively associated with adaptive impulsivity (ß = .04; 0.01; 0.07). Additionally, some of the adjusted models showed significant but weak associations with selenium, alcohol, fish, and cereal products. CONCLUSIONS: Food choice may affect the neurochemistry and therefore regulate the manifestations of impulsivity. We identified associations between several (micro)nutrients and maladaptive impulsivity.

Sub-Minute Analysis of Lactate from a Single Blood Drop Using Capillary Electrophoresis with Contactless Conductivity Detection in Monitoring of Athlete Performance.

A simple and fast method for the analysis of lactate from a single drop of blood was developed. The finger-prick whole blood sample (10 µL) was diluted (1:20) with a 7% (w/v) solution of [tris(hydroxymethyl)methylamino] propanesulfonic acid and applied to a blood plasma separation device. The device accommodates a membrane sandwich composed of an asymmetric polysulfone membrane and a supporting textile membrane that allows the collection of blood plasma into a narrow glass capillary in less than 20 s. Separated and simultaneously diluted blood plasma was directly injected into a capillary electrophoresis instrument with a contactless conductivity detector (CE-C4D) and analyzed in less than one minute. A separation electrolyte consisted of 10 mmol/L l-histidine, 15 mmol/L dl-glutamic acid, and 30 µmol/L cetyltrimethylammonium bromide. The whole procedure starting from the finger-prick sampling until the CE-C4D analysis was finished, took less than 5 min and was suitable for monitoring lactate increase in blood plasma during incremental cycling exercise. The observed lactate increase during the experiments measured by the developed CE-C4D method correlated well with the results from a hand-held lactate analyzer (R = 0.9882). The advantage of the developed CE method is the speed, significant savings per analysis, and the possibility to analyze other compounds from blood plasma.

The synthesis and characterization of Bri2 BRICHOS coated magnetic particles and their application to protein fishing: Identification of novel binding proteins.

Human integral membrane protein 2B (ITM2B or Bri2) is a member of the BRICHOS family, proteins that efficiently prevent Aß42 aggregation via a unique mechanism. The identification of novel Bri2 BRICHOS client proteins could help elucidate signaling pathways and determine novel targets to prevent or cure amyloid diseases. To identify Bri2 BRICHOS interacting partners, we carried out a 'protein fishing' experiment using recombinant human (rh) Bri2 BRICHOS-coated magnetic particles, which exhibit essentially identical ability to inhibit Aß42 fibril formation as free rh Bri2 BRICHOS, in combination with proteomic analysis on homogenates of SH-SY5Y cells. We identified 70 proteins that had more significant interactions with rh Bri2 BRICHOS relative to the corresponding control particles. Three previously identified Bri2 BRICHOS interacting proteins were also identified in our 'fishing' experiments. The binding affinity of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the top 'hit', was calculated and was identified as a strong interacting partner. Enrichment analysis of the retained proteins identified three biological pathways: Rho GTPase, heat stress response and pyruvate, cysteine and methionine metabolism.

Comprehensive mapping of cytochrome c oxidase activity in the rat brain after sub-chronic ketamine administration.

Ketamine is a noncompetitive antagonist of glutamatergic N-methyl-d-aspartate receptors. Its acute effects on healthy volunteers and schizophrenia patients mimic some acute psychotic, but also cognitive and negative symptoms of schizophrenia, and subchronic treatment with ketamine has been used as an animal model of psychotic disorders. Glutamatergic neurotransmission is tightly coupled to oxidative metabolism in the brain. Quantitative histochemical mapping of cytochrome c oxidase (COX) activity, which reflect long-term energy metabolism, was carried out in rats that received a daily subanaesthetic dose (30 mg/kg) of ketamine for 10 days. In total, COX activity was measured in 190 brain regions to map out metabolic adaptations to the subchronic administration of ketamine. Ketamine treatment was associated with elevated COX activity in nine brain sub-regions in sensory thalamus, basal ganglia, cortical areas, hippocampus and superior colliculi. Changes in pairwise correlations between brain regions were studied with differential correlation analysis. Ketamine treatment was associated with the reduction of positive association between brain regions in 66 % of the significant comparisons. Different layers of the superior colliculi showed the strongest effects. Changes in other visual and auditory brain centres were also of note. The locus coeruleus showed opposite pattern of increased coupling to mainly limbic brain regions in ketamine-treated rats. Our study replicated commonly observed activating effects of ketamine in the hippocampus, cingulate cortex, and basal ganglia. The current study is the first to extensively map the oxidative metabolism in the CNS in the ketamine model of schizophrenia. It shows that ketamine treatment leads to the re-organization of activity in sensory and memory-related brain circuits.

Chronic stress sensitizes amphetamine-elicited 50-kHz calls in the rat: Dependence on positive affective phenotype and effects of long-term fluoxetine pretreatment.

High level of positive affectivity acts as a protective factor against adverse effects of stress and decreases vulnerability to mood disorders and drug abuse. Fifty-kHz ultrasonic vocalizations (50-kHz USV) index the level of positive affect in the rat, whereas stable, trait-like inter-individual differences in terms of vocalization activity exist. Previously we have demonstrated that chronic stress can alter the effect of repeated amphetamine administration on 50-kHz vocalizations, and this effect is different in rats with high and low positive affectivity. In the present study it was tested whether the chronic stress effect on amphetamine-induced 50-kHz USV activity is altered by inhibition of serotonin reuptake. Male Wistar high (HC) and low (LC) 50-kHz vocalizing rats were subjected to 43-day chronic variable stress (CVS) regimen. On day 17 of the CVS, the four-week once a day fluoxetine (10 mg/kg) treatment was started. After the CVS and fluoxetine treatment, amphetamine (1 mg/kg) was daily administered for ten days and again nine days after withdrawal. Chronically stressed rats developed cross-sensitization of 50-kHz USV-s with repeated administration of amphetamine except the stressed LC rats that had not received fluoxetine. Amphetamine treatment decreased serotonin turnover in the fluoxetine-treated HC rats, but increased it in fluoxetine-treated LC rats. The effect of amphetamine on levels of amino acids in frontal cortex and hippocampus also depended on previous experience with chronic stress, repeated treatment with fluoxetine, and positive affectivity. Hence, this study provides further evidence the effects of chronic stress, psychostimulants, and a selective serotonin reuptake inhibitor are influenced by the inherent positive affectivity.

The future of metabolomics in ELIXIR.

Metabolomics, the youngest of the major omics technologies, is supported by an active community of researchers and infrastructure developers across Europe. To coordinate and focus efforts around infrastructure building for metabolomics within Europe, a workshop on the "Future of metabolomics in ELIXIR" was organised at Frankfurt Airport in Germany. This one-day strategic workshop involved representatives of ELIXIR Nodes, members of the PhenoMeNal consortium developing an e-infrastructure that supports workflow-based metabolomics analysis pipelines, and experts from the international metabolomics community. The workshop established metabolite identification as the critical area, where a maximal impact of computational metabolomics and data management on other fields could be achieved. In particular, the existing four ELIXIR Use Cases, where the metabolomics community - both industry and academia - would benefit most, and which could be exhaustively mapped onto the current five ELIXIR Platforms were discussed. This opinion article is a call for support for a new ELIXIR metabolomics Use Case, which aligns with and complements the existing and planned ELIXIR Platforms and Use Cases.

Development and characterization of mitochondrial membrane affinity chromatography columns derived from skeletal muscle and platelets for the study of mitochondrial transmembrane proteins.

Mitochondrial membrane fragments from human platelets and monkey skeletal muscles were successfully immobilized onto immobilized artificial membrane chromatographic support for the first time, resulting in mitochondrial membrane affinity chromatography (MMAC) columns. These columns were validated by characterization of translocator protein (TSPO), where multiple concentrations of dipyridamole were run and the binding affinities (Kd) determined. Further, the relative ranking data of TSPO ligands was consistent with previously reported rankings for both, the platelet (MMAC-Platelet) and the skeletal muscle (MMAC-Muscle) column (dipyridamole>PK11195>protoporphyrin IX>rotenone). The functional immobilization of the F-ATPase/ATP synthase was demonstrated on MMAC-Muscle column. Online hydrolysis of ATP to ADP and synthesis of ATP from ADP were both demonstrated on the MMAC-Muscle column. Hydrolysis of ATP to ADP was inhibited by oligomycin A with an IC50 of 40.2±13.5nM (∼60% reduction in ATP hydrolysis, p<0.001), similar to previously reported values. Additionally, the Michaelis-Menten constant (Km) for ADP was found to be 1525±461µM based on the on column dose-dependent increase in ATP production.

Chronic variable stress prevents amphetamine-elicited 50-kHz calls in rats with low positive affectivity.

The relationship between stress response and positive affective states is thought to be bidirectional: whilst stress can lead to a blunted hedonic response, positive affect reduces the negative effects of stress. We have previously shown that persistently high positive affectivity as measured by 50-kHz ultrasonic vocalizations (USVs) is protective against chronic variable stress (CVS). The present study examined the effect of CVS on 50-kHz USVs elicited by amphetamine administration, simultaneously considering the stable inter-individual differences in positive affectivity. Forty juvenile male Wistar rats were categorised as of high (HC) or low (LC) positive affectivity based on their 50-kHz USV response to imitation of rough-and-tumble play ('tickling'). As adults, the rats were subjected to four weeks of CVS, after which D-amphetamine was administered in five daily doses followed by a challenge dose (all 1mg/kg IP) nine days later. CVS reduced sucrose preference in LC-rats only. After CVS, amphetamine-elicited 50-kHz USVs were significantly reduced in LC-rats, the effect of stress in HC-rats being smaller and less consistent. In previously stressed and amphetamine-treated LC-rats, locomotor response to amphetamine was attenuated. In stressed LC-rats, DOPAC levels and dopamine turnover were increased in striatum after amphetamine treatment, and dopamine D1 receptor levels were upregulated in nucleus accumbens. LC-rats had lower isoleucine levels in frontal cortex. These results show that stress-related changes in response to amphetamine are dependent on inter-individual differences in positive affectivity both at neurochemical and behavioural levels, and further support the notion of higher vulnerability of animals with low positive affect.

Amyloid ß25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aß) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aß through ROS and metals are mainly studied in neuronal cells using full-length Aß1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aß25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aß25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aß25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aß25-35 induced ROS.

Toxicity of 11 Metal Oxide Nanoparticles to Three Mammalian Cell Types In Vitro.

The knowledge on potential harmful effects of metallic nanomaterials lags behind their increased use in consumer products and therefore, the safety data on various nanomaterials applicable for risk assessment are urgently needed. In this study, 11 metal oxide nanoparticles (MeOx NPs) prepared using flame pyrolysis method were analyzed for their toxicity against human alveolar epithelial cells A549, human epithelial colorectal cells Caco2 and murine fibroblast cell line Balb/c 3T3. The cell lines were exposed for 24 h to suspensions of 3-100 µg/mL MeOx NPs and cellular viability was evaluated using. Neutral Red Uptake (NRU) assay. In parallel to NPs, toxicity of soluble salts of respective metals was analyzed, to reveal the possible cellular effects of metal ions shedding from the NPs. The potency of MeOx to produce reactive oxygen species was evaluated in the cell-free assay. The used three cell lines showed comparable toxicity responses to NPs and their metal ion counterparts in the current test setting. Six MeOx NPs (Al2O3, Fe3O4, MgO, SiO2, TiO2, WO3) did not show toxic effects below 100 µg/mL. For five MeOx NPs, the averaged 24 h IC50 values for the three mammalian cell lines were 16.4 µg/mL for CuO, 22.4 µg/mL for ZnO, 57.3 µg/mL for Sb2O3, 132.3 µg/mL for Mn3O4 and 129 µg/mL for Co3O4. Comparison of the dissolution level of MeOx and the toxicity of soluble salts allowed to conclude that the toxicity of CuO, ZnO and Sb2O3 NPs was driven by release of metal ions. The toxic effects of Mn3O4 and Co3O4 could be attributed to the ROS-inducing ability of these NPs. All the NPs were internalized by the cells according to light microscopy studies but also proven by TEM, and internalization of Co3O4 NPs seemed to be most prominent in this aspect. In conclusion, this work provides valuable toxicological data for a library of 11 MeOx NPs. Combining the knowledge on toxic or non-toxic nature of nanomaterials may be used for safe-by-design approach.

Potential of polyE-323 coated capillaries for capillary electrophoresis of lipids.

In this note the feasibility of a polyamine-based capillary coating, polyE-323, for capillary electrophoresis (CE) of lipids is explored. PolyE-323 has previously been demonstrated to be suitable to suppress analyte-wall interaction of proteins in CE. However, the full applicability range of polyE-323 has not been exploited yet and it might be useful in the analysis of hydrophobic analytes, such as lipids. In this study, the stability of polyE-323 when using highly organic background electrolytes (BGEs), which are needed to solubilize the lipid analytes, was studied. For this, we used three different lipid samples: sphingomyelin, cardiolipin and a lipid extract from a cell culture. The highly organic BGEs that were used in this study consisted of 94.5% of organic solvents and 5.5% of an aqueous buffer. First, the influence of pure acetonitrile, methanol, propylene carbonate, isopropanol and chloroform on the polyE-323 coating was investigated. Then BGEs were developed and tested, using sphingomyelin and cardiolipin as test analytes in CE-UV experiments. After establishing the best BGEs (in terms of analysis time and repeatability) by CE-UV, sphingomyelin was used as a test analyte to demonstrate that method was also suitable for CE with mass-spectrometry detection (CE-MS). The LOD of sphingomyelin was estimated to be 100 nM and its migration time repeatability was 1.3%. The CE-MS analysis was further applied on a lipid extract obtained from human glioblastoma cells, which resulted in the separation and detection of a multitude of putative lipids. The results of our feasibility study indicate that CE systems based on polyE-323 coated capillaries and highly organic BGEs are promising for fast electromigration-based analysis of lipids.

Polydopamine as an adhesive coating for open tubular capillary electrochromatography.

Polydopamine (PolyD) coating was used as an adhesive layer in the preparation of biological stationary phases for open tubular capillary electrochromatography (OT-CEC). The influence of coating solution freshness, coating time, temperature and dopamine hydrochloride concentration on the PolyD layer formation was studied. The performance of the polyD coating was monitored by measuring the electro-osmotic flow in coated capillaries. Following polyD coating of the capillary, secondary layer material (e.g. cell membrane solutions, phospholipid mixtures or mitochondria) was inserted into the capillary for at least 1 h. The performance of these double-coated capillaries (a polyD layer+a biological material layer) was compared with capillaries containing the respective biological material directly attached to the capillary wall. The study reveals that the presence of polyD layer in fused silica capillaries improves the performance of lipid and membrane fragment coatings in capillaries. At the same time, the thickness of the polyD layer does not have marked impact on the secondary coatings. Analysis with test analytes demonstrated that double-coated capillaries can be applied to study membrane-drug interactions.

New capillary coatings in open tubular CEC as models for biological membranes.

Novel stationary phases in open tubular CEC were investigated. The coating procedure was fast and simple. The coating material contained membrane suspension of different neuronal cell lines. The performance and stability of three cell lines: human neuroblastoma SH-SY5Y, murine microglia Bv-2 and human glioma U87-MG cells were studied. The coating solution was expected to contain both membrane proteins and membrane lipids. The presence of membrane proteins was tested by Western blotting and the presence of phospholipids by the analysis of phosphorus content. The stability of the coating was estimated by monitoring the mobility of EOF over successive runs. The effects of pH, storage time and temperature on the coating stability were also studied. The results showed that the cell membrane-based coating was stable over pH range of 6.5-8.5. Coatings derived from different cells yielded similar stability and EOF mobility. Capillary coated with a membrane solution was stable over 3-day period. The same coating solution could be used for 3 weeks.

Liposomes in capillary electromigration techniques.

The use of phospholipid vesicles (liposomes) in EKC and CEC, as well as the use of CE for studying liposomes and lipid-analyte aggregates, are briefly reviewed. The works are presented in the order of appearance. The review describes the most common liposome dispersions used for achieving separation of neutral and charged compounds in liposome EKC. The CEC part is divided into two parts, discriminating between liposomes immobilized on capillaries for hindering analytes from interacting with the fused-silica wall and liposomes immobilized on capillaries for achieving chromatographic phases for separation of compounds. Finally, the use of CE for studying liposomes and lipid-analyte aggregates as analytes is discussed in brief.

Capillary electrophoresis of diuretics and probenecid in methanol.

Actual mobilities and dissociation constants of six diuretics (benzthiazide, bumetanide, ethacrynic acid, furosemide, hydrochlorothiazide and chlorothiazide) and probenecid were investigated in methanol by capillary zone electrophoresis (CZE). The actual mobilities were derived from the dependence of the effective mobilities of the analytes on the pH(*) of the methanolic background electrolyte (BGE) solution. The measurement of effective mobilities was carried out mainly by a pressure-mediated capillary electrophoresis (CE) method. For comparison, parallel measurements were carried for two of the analytes by the conventional capillary electrophoresis approach, without pressure. The pK(a)(*) values in methanol were calculated by non-linear curve fitting to the measured mobility values. The difference between the pK(a) values in water and methanol was about 5 units for the loop diuretics (furosemide, bumetanide, ethacrynic acid) and probenecid and around 3-4 units for the thiazide diuretics. Knowledge of the ionisation behaviour of compounds in methanol paves the way for the wider use of methanol as background electrolyte solvent in capillary electrophoresis. Moreover, as is demonstrated in the current study, the calculation of actual mobilities and pK(a) values facilitates the optimisation of pH conditions for the separation, thereby applications can be expanded, and the combination of capillary electrophoresis with electrospray ionisation mass spectrometry becomes easier.