Ultrasound accelerated γ-aminobutyric acid accumulation in coffee leaves through influencing the microstructure, enzyme activity, and metabolites.

Gamma-aminobutyric acid (GABA) is a non-protein amino acid that possesses various physiological functions. Our previous study has shown that ultrasound increased GABA accumulation in coffee leaves. In this study, we aimed to uncover the GABA enrichment mechanism by investigating the surface microstructure, cellular permeability, enzyme activities, and metabolomics of coffee leaves under ultrasound treatment. The results showed that ultrasound increased the electrical conductivity and the activities of glutamate decarboxylase, γ-aminoaldehyde dehydrogenase, and diamine oxidase by 12.0%, 265.9%, 124.1%, 46.8%, respectively. Environmental scanning electron microscope analysis demonstrated an increased opening of stomata and the rougher surface in the leaves after ultrasound treatment. UPLC-qTOF-MS/MS-based untargeted metabolomics analysis identified 82 differential metabolites involved in various metabolism pathways. Our results indicated that ultrasound changed the surface microstructure of coffee leaves, thereby accelerating the migration of glutamate into the cells; activated related enzymes; regulated C/N metabolism pathways, which led to an increase of GABA.

Caferana seeds (Bunchosia glandulifera) as a new source of nutrients: Evaluation of the proximal composition, solvent extraction, bioactive compounds, and δ-lactam isolation.

The proximal composition, amino acid, carbohydrate, and volatile profiles of caferana (Bunchosia glandulifera) seeds flour were here assessed. Seeds were also subjected to the following extraction processes: one with pressurized ethanol (PLE) and two with ethanol + supercritical CO2 mixture at different temperatures and pressures (SC1 and SC2). Extracts were characterized in terms of caffeine, total phenolic, and δ-lactam. The characterization of caferana seed and its extracts is unprecedented in terms of carbohydrate and volatiles profiles, besides the δ-lactam identification/isolation. SC2 extract exhibited a higher caffeine (9.3 mg/g) and δ-lactam (29.4 mg/g) content, whereas the PLE extract contained a higher total phenolic amount (3.0 mgGAE/g). Caferana is regionally associated to protective effects on mental health. Its byproduct (seed) revealed to be a promising source of bioactive compounds, and a potential raw material of nutritive extracts and flours that can be incorporated into pharmaceutical, nutraceutical, cosmetic, and food products.

Mitochondrial activity as an indicator of fish freshness.

The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨm) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨm and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness.

Metabolic variation in the pulps of two durian cultivars: Unraveling the metabolites that contribute to the flavor.

Durian (Durio zibethinus M.) is a major economic fruit crop in Thailand. In this study, two popular cultivars, namely Chanee and Mon Thong, were collected from three orchards located in eastern Thailand. The pulp metabolome, including 157 annotated metabolites, was explored using capillary electrophoresis-time of flight/mass spectrometry (CE-TOF/MS). Cultivars and harvest years had more impact on metabolite profile separation than cultivation areas. We identified cultivar-dependent metabolite markers related to durian fruit quality traits, such as nutritional value (pyridoxamine), odor (cysteine, leucine), and ripening process (aminocyclopropane carboxylic acid). Interestingly, durian fruit were found to contain high amounts of γ-glutamylcysteine (810.3 ±â€¯257.5 mg/100 g dry weight) and glutathione (158.1 ±â€¯80.4 mg/100 g dry weight), which act as antioxidants and taste enhancers. This metabolite information could be related to consumer preferences and exploited for durian fruit quality improvement.

Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1ß, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage.

Studies on diagnostic biomarkers and therapeutic mechanism of Alzheimer's disease through metabolomics and hippocampal proteomics.

Alzheimer's disease (AD) is the main cause of dementia, but precise diagnosis and treatment are not sufficient so far. The purpose of this study is to develop biomarkers and therapeutic targets for diagnosis and better understanding of AD. As a result, lysophosphatidylcholine and intermediates of sphingolipid metabolism including sphinganine-1-phosphate, sphingosine-1-phosphate, sphingomyelin, and sphingosine in plasma were annotated as potential biomarkers by using UPLC-Q-TOF-MS and UHPLC-Q-Exactive-MS. Besides, glutathione S-transferases (GSTs) including GstA3, Gstm1, Gstm5, Gstm3, Gstk1 and Gstp1 were significantly enhanced in AD hippocampus by using label free nano-LC-MS/MS. Thus, pathogenesis of AD was involved with increasing of choline, decreasing of ACh, enhancement of GSTs and increasing of glutamate which led to oxidative stress and excitotoxity. Effects of donepezil and a natural medicine were evaluated through metabolomics and proteomics. In summary, proteomic and metabolomic analysis on constructed AD rat model were performed through rapid, sensitive and high resolution LC-MS methods to reveal candidate biomarkers. The data suggested that GSTs have great value as therapeutic targets. This study provided valuable information for the diagnosis mechanism and drug discovery of AD.

The cardioprotective effect of sildenafil is mediated by the activation of malate dehydrogenase and an increase in the malate-aspartate shuttle in cardiomyocytes.

Recent evidence has shown the cardioprotective effect of PDE5 inhibition in myocardial ischemia/reperfusion injury, heart failure and cardiac hypertrophy. To investigate the biochemical changes that occur during PDE5 inhibition in cardiac cells, this study assessed the metabolic profile of the HL1 cell line, a murine atrial cell line with adult cardiomyocyte properties. After one hour of treatment with sildenafil, glycolysis was moderately but selectively stimulated, unlike the pentose phosphate pathway and the Krebs cycle. Moreover, malate and a-Ketoglutarate accumulated, paralleled by a decrease in aspartate and glutamate. Interestingly, increased activity of malate dehydrogenase (MDH) was also detected in these cells after sildenafil treatment. Thus, we hypothesized that sildenafil stimulates the malate-aspartate shuttle (MAS) with the final effect of transferring electrons and protons from glycolysis-derived cytosolic NADH into the matrix for use by the electron transport chain, using malate as an electron carrier. Through this metabolic modification, sildenafil may counteract what is often observed in ischemia, i.e. reduced MAS flux as well as a dramatic acceleration of glycolysis, which switches to lactate production. Additionally, the results observed in HL1 cells were also found in isolated mouse hearts. The documented metabolic alteration in cardiomyocytes upon treatment with sildenafil occurred by stimulating cGMP production, which did not activate PKG (cGMP-PKG signaling), since the addition of DT-2, a PKG inhibitor, did not block malate accumulation and increased MDH activity. Conversely, the addition of chelerythrine, a PKC inhibitor, counteracted both malate accumulation and MAS activation, supporting previous evidence that, upon the addition of sildenafil, some PKC isoforms may be implicated in cardioprotection (cGMP-PKC signaling). Interestingly, an increase in cGMP, driven by sildenafil, another cGMP stimulator such as nitroprusside (SNP), or a C-type natriuretic peptide (CNP) which does not inhibit PDE5, led to MAS stimulation and increased MDH activity.

Ginsenosides attenuate d-galactose- and AlCl3-inducedspatial memory impairment by restoring the dysfunction of the neurotransmitter systems in the rat model of Alzheimer's disease.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax ginseng C.A.Mey. is a traditional Chinese herbal medicine, which has been used to treat Alzheimer's disease (AD) for thousands of years. Ginsenoside is one of the major compounds found in P. ginseng. This study aimed to explore the attenuation of spatial memory impairment by ginsenosides and its correlation with restoring the dysfunction of the neurotransmitter systems in AD model rats to understand the mechanism underlying the anti-AD effect of P. ginseng. MATERIALS AND METHODS: In this study, the AD model was established by combining d-galactose (d-gal) with AlCl3 (Al) for 60 days. From day 30, the ginsenosides group was intragastrically administered with ginsenosides for 30 days. The ethology of rats was tested through the Morris water maze test(MWM). Histopathological changes in the hippocampus of rats were observed through hematoxylin and eosin staining. The expressions of amyloid ß peptide (Aß) and phospho-tau (p-tau) in the hippocampus and cortex of rats were detected by immunohistochemistry. A liquid chromatography-mass spectrometry assay was used to measure neurotransmitter concentrations in the hippocampus, cortex, and blood. RESULTS: Ginsenosides could significantly decrease the escape latency time and the average latency time in the place navigation test and increase the times of crossing the platform area, the percentage of residence time, and the distance in the original platform quadrant in the spatial probe test. Ginsenosides could repair the damage of the hippocampus and reduce the expressions of Aß and p-tau. Ginsenosides could also increase γ-aminobutyric acid, acetylcholine, and dopamine levels and decrease glutamate and aspartic acid levels in the hippocampus and cortex and increase glycine and serotonin levels in the blood. CONCLUSIONS: After effectively administrated, ginsenosides attenuate d-gal- and Al-induced spatial memory impairment. The possible mechanism of the beneficial effect is restoring the dysfunction of various neurotransmitters.

A single polycystic kidney disease 2-like 1 channel opening acts as a spike generator in cerebrospinal fluid-contacting neurons of adult mouse brainstem.

Cerebrospinal fluid contacting neurons (CSF-cNs) are found around the central canal of all vertebrates. They present a typical morphology, with a single dendrite that projects into the cavity and ends in the CSF with a protuberance. These anatomical features have led to the suggestion that CSF-cNs might have sensory functions, either by sensing CSF movement or composition, but the physiological mechanisms for any such role are unknown. This hypothesis was recently supported by the demonstration that in several vertebrate species medullo-spinal CSF-cNs selectively express Polycystic Kidney Disease 2-Like 1 proteins (PKD2L1). PKD2L1 are members of the 'transient receptor potential (TRP)' superfamily, form non-selective cationic channels of high conductance, are regulated by various stimuli including protons and are therefore suggested to act as sensory receptors. Using patch-clamp whole-cell recordings of CSF-cNs in brainstem slices obtained from wild type and mutant PKD2L1 mice, we demonstrate that spontaneously active unitary currents in CSF-cNs are due to PKD2L1 channels that are capable, with a single opening, of triggering action potentials. Thus PKD2L1 might contribute to the setting of CSF-cN spiking activity. We also reveal that CSF-cNs have the capacity of discriminating between alkalinization and acidification following activation of specific conductances (PKD2L1 vs. ASIC) generating specific responses. Altogether, this study reinforces the idea that CSF-cNs represent sensory neurons intrinsic to the central nervous system and suggests a role for PKD2L1 channels as spike generators.

Two cellular hypotheses explaining the initiation of ketamine's antidepressant actions: Direct inhibition and disinhibition.

A single, low dose of ketamine evokes antidepressant actions in depressed patients and in patients with treatment-resistant depression (TRD). Unlike classic antidepressants, which regulate monoamine neurotransmitter systems, ketamine is an antagonist of the N-methyl-D-aspartate (NMDA) family of glutamate receptors. The effectiveness of NMDAR antagonists in TRD unveils a new set of targets for therapeutic intervention in major depressive disorder (MDD) and TRD. However, a better understanding of the cellular mechanisms underlying these effects is required for guiding future therapeutic strategies, in order to minimize side effects and prolong duration of efficacy. Here we review the evidence for and against two hypotheses that have been proposed to explain how NMDAR antagonism initiates protein synthesis and increases excitatory synaptic drive in corticolimbic brain regions, either through selective antagonism of inhibitory interneurons and cortical disinhibition, or by direct inhibition of cortical pyramidal neurons. This article is part of the Special Issue entitled 'Synaptopathy--from Biology to Therapy'.

(1)H NMR metabolomic profiling of the blue crab (Callinectes sapidus) from the Adriatic Sea (SE Italy): A comparison with warty crab (Eriphia verrucosa), and edible crab (Cancer pagurus).

The metabolomic profile of blue crab (Callinectes sapidus) captured in the Acquatina lagoon (SE Italy) was compared to an autochthonous (Eriphia verrucosa) and to a commercial crab species (Cancer pagurus). Both lipid and aqueous extracts of raw claw muscle were analyzed by (1)H NMR spectroscopy and MVA (multivariate data analysis). Aqueous extracts were characterized by a higher inter-specific discriminating power compared to lipid fractions. Specifically, higher levels of glutamate, alanine and glycine characterized the aqueous extract of C. sapidus, while homarine, lactate, betaine and taurine characterized E. verrucosa and C. pagurus. On the other hand, only the signals of monounsaturated fatty acids distinguished the lipid profiles of the three crab species. These results support the commercial exploitation and the integration of the blue crab in human diet of European countries as an healthy and valuable seafood.

C/EBPß and C/EBPδ transcription factors: Basic biology and roles in the CNS.

CCAAT/enhancer binding protein (C/EBP) ß and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPß mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPß and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPß and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPß and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPß and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPß and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.

Gabapentin inhibits the activity of the rat excitatory glutamate transporter 3 expressed in Xenopus oocytes.

Gabapentin, a derivative of γ-aminobutyric acid (GABA), is used to treat epilepsy and neuropathic pain. The pharmacological mechanisms for gabapentin effects are not completely elucidated. We investigated the effect of gabapentin on the activity of excitatory amino acid transporter 3 (EAAT3) that can regulate extracellular glutamate concentrations. EAAT3 was expressed in Xenopus oocytes. Membrane currents were recorded after application of l-glutamate in the presence or absence of different concentrations of gabapentin (1-300µM) by using a two-electrode voltage clamp. To determine the effect of gabapentin on Vmax and Km of EAAT3 for l-glutamate, l-glutamate at 3-300µM was used. To study the effects of protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI3K) on gabapentin-induced changes in EAAT3 activity, oocytes were incubated with the PKC activator (Phorbol 12-myristate 13-acetate, PMA), the PKC inhibitors (chelerythrine or staurosporine), and the PI3K inhibitor wortmannin. Gabapentin decreased EAAT3 activity in a concentration-dependent manner and EAAT3 activity was significantly inhibited by 10-300µM gabapentin. Gabapentin significantly decreased Vmax without affecting Km. PMA increased EAAT3 activity; however, gabapentin attenuated the PMA-induced increase in EAAT3 activity. Pre-incubation of oocytes with chelerythrine, staurosporine, or wortmannin decreased basal EAAT3 activity, which was further reduced by gabapentin. We conclude that gabapentin decreases EAAT3 activity at clinically relevant and higher concentrations, in which PKC and PI3K may not be involved. The results suggest that EAAT3 might not be a target for the anticonvulsant action of gabapentin.

Effect of Baicalin-loaded PEGylated cationic solid lipid nanoparticles modified by OX26 antibody on regulating the levels of baicalin and amino acids during cerebral ischemia-reperfusion in rats.

Baicalin has many pharmacological activities, including neuroprotective function against ischemia and neurodegeneration. In our previous study, we found that Baicalin-loaded PEGylated cationic solid lipid nanoparticles modified by OX26 antibody (OX26-PEG-CSLN) might be a promising carrier to deliver drugs across the blood-brain barrier for the treatment of brain diseases. So, the aim of this present study was to further elucidate the mechanisms of OX26-PEG-CSLN cerebral ischemia protection by monitoring the changes of extracellular amino acids. In addition, we investigated the effect of OX26-PEG-CSLN on the excitotoxic neuronal injury as well as the pharmacokinetic profiles of baicalin in cerebrospinal fluid during ischemia-reperfusion period. The cerebrospinal fluid was collected by a microdialysis technique and divided into two parts - one part for pharmacokinetic study of baicalin using LC-MS/MS method and the other for pharmacodynamic study which was done by pre- column derivatization of the amino acids and analysis using a high-performance liquid chromatography with fluorescence detector (HPLC-FLD). The pharmacokinetic study showed that the AUC value of OX26-PEG-CSLN was 5.69-fold higher than that of the baicalin solution (Sol) (P<0.05) and the Cmax value of OX26-PEG-CSLN was 6.84-fold higher than that of the Sol (P<0.05). Moreover, the extracellular levels of glutamate (Glu), aspartic acid (Asp), glycine (Gly), taurine (Tau) and γ-aminobutyric acid (GABA) were measured for monitoring the imbalance of amino acids caused by ischemia and reperfusion. The excitotoxic index (EI) was also calculated for evaluating the imbalance between excitatory amino acid and inhibitory amino acid. The pharmacodynamic study showed that OX26-PEG-CSLN had stronger effect than Sol in reducing the content of aspartic, glutamic acid and increasing the concentrations of glycine, taurine and γ-aminobutyric acid during ischemia-reperfusion period. In conclusion, OX26-PEG-CSLN improved uptake of baicalin across the BBB into the brain, and elevated bioavailability of baicalin in cerebral spinal fluid of rats under the cerebral ischemia-reperfusion injury. OX26-PEG-CSLN had much higher protection effect against cerebral ischemia injury than Sol by relieving the excitotoxic neuronal injury via regulating amino acid levels in cerebral spinal fluid.

Riluzole attenuates the effects of chemoconvulsants acting on glutamatergic and GABAergic neurotransmission in the planarian Dugesia tigrina.

Planarians, the non-parasitic flatworms, display dose-dependent, distinct (C-like and corkscrew-like) hyperkinesias upon exposure to 0.001-10 mM aqueous solutions of glutamatergic agonists (L-glutamate and N-methyl-D-aspartate (NMDA)) and 0.001-5 mM concentrations of the glutamate decarboxylase (GAD) inhibitor (semicarbazide). In the planarian seizure-like activity (PSLA) experiments the three chemoconvulsants displayed the following order of potency (EC50): L-glutamate (0.6mM)>NMDA (1.4 mM)>semicarbazide (4.5mM). Planarian hyperkinesias behavior counting experiments also revealed that riluzole (0.001 to 1mM), an anti-convulsive agent, displayed no significant behavioral activity by itself, but attenuated hyperkinesias elicited by the three chemoconvulsants targeting either glutamatergic or GABAergic neurotransmission with the following order of potency (IC50): NMDA (44.7 µM)>semicarbazide (88.3 µM)>L-glutamate (160 µM). Further, (+)-MK-801, a specific NMDA antagonist, alleviated 3mM NMDA (47%) or 3mM L-glutamate (27%) induced planarian hyperkinesias. The results provide pharmacological evidence for the presence of glutamatergic receptor-like and semicarbazide sensitive functional GAD enzyme-like proteins in planaria in addition to demonstrating, for the first time, the anti-convulsive effects of riluzole in an invertebrate model. High performance liquid chromatography coupled with fluorescence detection (HPLC-F) analysis performed on planarian extracts post no drug treatment (control) or treatment with 3mM semicarbazide, combination of 3mM semicarbazide and 0.1 mM riluzole, or 0.1 mM riluzole revealed that 3 mM semicarbazide induced 35% decrease in the GABA levels and a combination of 3mM semicarbazide and 0.1 mM riluzole induced 42% decrease in glutamate levels with respect to the control group.