Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation.

BACKGROUND: Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD. METHODS: CSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1ß and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay. RESULTS: The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1ß and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1ß gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. CONCLUSION: This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine.

Inhibition of NR2A reduces calcitonin gene-related peptide gene expression induced by cortical spreading depression in rat amygdala.

Despite robust evidence on the role of calcitonin gene-related peptide (CGRP) in migraine via both central and peripheral actions, relatively less is known about how CGRP in the limbic system is involved in migraine progression. This study investigated whether CGRP production machinery exists in the two key limbic regions including hippocampus and amygdala using cortical spreading depression (CSD) as a model of migraine and whether such alteration by CSD is sensitive to N-methyl-d-aspartate (NMDA) receptor regulation in rats. A single or repetitive CSD was induced by topical application of KCl and monitored using electrophysiological methods. The NR2A-containing NMDA receptor antagonist, NVP-AAM077, or its vehicle, was perfused into the contralateral cerebroventricular ventricle of rat. Quantitative PCR was used to measure CGRP mRNA levels in the ipsilateral and contralateral hippocampus and amygdala after CSD events and compared to respective sham treatments. The results showed that neither a single CSD nor repetitive CSD affected CGRP mRNA levels in both the contralateral and ipsilateral hippocampus at 24 h post CSD induction. Differently, significant elevation of CGRP gene expression was observed in the ipsilateral amygdala at 24 h post multiple CSD, but not contralateral side, and not post-single CSD. Further results showed that the CSD-induced CGRP gene expression in the amygdala was markedly reduced by NVP-AAM077 and this reduction corresponded to a reduced cortical susceptibility to CSD in rats. We conclude that repetitive CSD events induce CGRP gene expression in amygdala, which is sensitive to NR2A regulation.

Liquid-Phase Exfoliation and Functionalization of MoS2 Nanosheets for Effective Antibacterial Application.

A lysozyme (Lys)-assisted liquid-phase exfoliation technique was designed to synthesize MoS2 nanosheets (MoS2 -Lys NSs). As a novel nanozyme antibacterial agent with high peroxidase-like catalyst activity, MoS2 -Lys NSs showed good antibacterial efficacy against both Gram-negative ampicillin-resistant Escherichia coli (Ampr E. coli) and Gram-positive Bacillus subtilis. A possible antibacterial mechanism is also proposed. This work provides an effective antibacterial strategy based on the MoS2 -Lys NSs antibacterial agent.

A two-step gas/liquid strategy for the production of N-doped defect-rich transition metal dichalcogenide nanosheets and their antibacterial applications.

Herein, we developed a general two-step gas expansion and exfoliation strategy based on a urea-assisted hydrothermal process combined with sonication exfoliation for the production of nitrogen (N)-doped plus defect-rich transition metal dichalcogenide (TMD) nanosheets (NSs) such as N-MoS2 and N-WS2 NSs. The interlayers of bulk MoS2 (or WS2) were expanded with urea molecules dissolved in distilled water, which were decomposed to NH3 during the hydrothermal process. Simultaneously, sulfur atoms were partly replaced by N atoms to achieve N doping. Subsequently, sonication exfoliation of the urea-treated bulk MoS2 (or WS2) promoted the production of defect-rich NSs. Importantly, the defect-rich N-MoS2 and N-WS2 NSs exhibit enhanced peroxidase-like catalytic activity after being captured by bacteria, and can catalyze hydrogen peroxide (H2O2) to produce more toxic hydroxyl radicals (˙OH) than non-N-doped MoS2 or WS2 NSs. As a result, the N-MoS2 or N-WS2 NSs were capable of effectively killing Gram-negative ampicillin resistant Escherichia coli (AmprE. coli) and Gram-positive endospore-forming Bacillus subtilis (B. subtilis) and promoting bacteria-infected wound healing. This work not only provides a simple, universal exfoliation strategy for producing defect-rich N-doped TMD NSs but also provides a promising catalytic antibacterial option and has potential for many other catalytic applications.

A Bi2S3@mSiO2@Ag nanocomposite for enhanced CT visualization and antibacterial response in the gastrointestinal tract.

The non-invasive imaging of the gastrointestinal (GI) tract is highly desired for clinical research due to the various GI tract bacterial infection-induced diseases. To treat GI tract infections, various antibiotics have been used in the clinic. The growing problem of multidrug-resistant bacteria calls for effective antibiotic alternatives. Here, we construct a dual-functional Bi2S3@mSiO2@Ag nanocomposite for simultaneous enhanced X-ray computed tomography (CT) imaging and efficient antibacterial activity in the GI tract. The nanocomposite also has good stability, low cytotoxicity, and negligible hemolysis. Moreover, the investigation of the long-term toxicity and biodistribution of the Bi2S3@mSiO2@Ag nanocomposite after oral administration confirms its safety at the tested dosage. In particular, Ag nanoparticles (NPs) well dispersed on a silica substrate can reduce the antibacterial dosage and enhance the antibacterial activity of the Bi2S3@mSiO2@Ag nanocomposite. Furthermore, we have established bacterially infected enteritis animal models to confirm the antibacterial ability of the nanocomposite. This work opens up a new avenue for the design of a nanotheranostic agent that acts as both a contrast agent for the enhanced visualization of the GI tract and an antibacterial agent as an alternative to antibiotics.

ROS/TRPA1/CGRP signaling mediates cortical spreading depression.

OBJECTIVES: The transient receptor potential ankyrin A 1 (TRPA1) channel and calcitonin gene-related peptide (CGRP) are targets for migraine prophylaxis. This study aimed to understand their mechanisms in migraine by investigating the role of TRPA1 in cortical spreading depression (CSD) in vivo and exploring how reactive oxygen species (ROS)/TRPA1/CGRP interplay in regulating cortical susceptibility to CSD. METHODS: Immunohistochemistry was used for detecting TRPA1 expression. CSD was induced by K+ on the cerebral cortex, monitored using electrophysiology in rats, and intrinsic optical imaging in mouse brain slices, respectively. Drugs were perfused into contralateral ventricle of rats. Lipid peroxidation (malondialdehyde, MDA) analysis was used for indicating ROS level. RESULTS: TRPA1 was expressed in cortical neurons and astrocytes of rats and mice. TRPA1 deactivation by an anti-TRPA1 antibody reduced cortical susceptibility to CSD in rats and decreased ipsilateral MDA level induced by CSD. In mouse brain slices, H2O2 facilitated submaximal CSD induction, which disappeared by the antioxidant, tempol and the TRPA1 antagonist, A-967079; Consistently, TRPA1 activation reversed prolonged CSD latency and reduced magnitude by the antioxidant. Further, blockade of CGRP prolonged CSD latency, which was reversed by H2O2 and the TRPA1 agonist, allyl-isothiocyanate, respectively. CONCLUSIONS: ROS/TRPA1/CGRP signaling plays a critical role in regulating cortical susceptibility to CSD. Inhibition ROS and deactivation of TRPA1 channels may have therapeutic benefits in preventing stress-triggered migraine via CGRP.

Induction of calcitonin gene-related peptide expression in rats by cortical spreading depression.

OBJECTIVE: The neuropeptide calcitonin gene-related peptide (CGRP) has now been established as a key player in migraine. However, the mechanisms underlying the reported elevation of CGRP in the serum and cerebrospinal fluid of some migraineurs are not known. A candidate mechanism is cortical spreading depression (CSD), which is associated with migraine with aura and traumatic brain injury. The aim of this study was to investigate whether CGRP gene expression may be induced by experimental CSD in the rat cerebral cortex. METHODS: CSD was induced by topical application of KCl and monitored using electrophysiological methods. Quantitative PCR and ELISA were used to measure CGRP mRNA and peptide levels in discrete ipsilateral and contralateral cortical regions of the rat brain 24 hours following CSD events and compared with sham treatments. RESULTS: The data show that multiple, but not single, CSD events significantly increase CGRP mRNA levels at 24 hours post-CSD in the ipsilateral rat cerebral cortex. Increased CGRP was observed in the ipsilateral frontal, motor, somatosensory, and visual cortices, but not the cingulate cortex, or contralateral cortices. CSD also induced CGRP peptide expression in the ipsilateral, but not contralateral, cortex. CONCLUSIONS: Repeated CSD provides a mechanism for prolonged elevation of CGRP in the cerebral cortex, which may contribute to migraine and post-traumatic headache.

Functionalized MoS2 Nanovehicle with Near-Infrared Laser-Mediated Nitric Oxide Release and Photothermal Activities for Advanced Bacteria-Infected Wound Therapy.

The rising dangers of bacterial infections have created an urgent need for the development of a new generation of antibacterial nanoagents and therapeutics. A new near-infrared 808 nm laser-mediated nitric oxide (NO)-releasing nanovehicle (MoS2 -BNN6) is reported through the simple assembly of α-cyclodextrin-modified MoS2 nanosheets with a heat-sensitive NO donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine (BNN6) for the rapid and effective treatment of three typical Gram-negative and Gram-positive bacteria (ampicillin-resistant Escherichia coli, heat-resistant Escherichia faecalis, and pathogen Staphylococcus aureus). This MoS2 -BNN6 nanovehicle has good biocompatibility and can be captured by bacteria to increase opportunities of NO diffusion to the bacterial surface. Once stimulated by 808 nm laser irradiation, the MoS2 -BNN6 nanovehicle not only exhibits photothermal therapy (PTT) efficacy but also can precisely control NO release, generating oxidative/nitrosative stress. The temperature-enhanced catalytic function of MoS2 induced by 808 nm laser irradiation simultaneously accelerates the oxidation of glutathione. This acceleration disrupts the balance of antioxidants, ultimately resulting in significant DNA damage to the bacteria. Within 10 min, the MoS2 -BNN6 with enhanced PTT/NO synergetic antibacterial function achieves >97.2% inactivation of bacteria. The safe synergetic therapy strategy can also effectively repair wounds through the formation of collagen fibers and elimination of inflammation during tissue reconstruction.

Synthesis of Surface-Modification-Oriented Nanosized Molybdenum Disulfide with High Peroxidase-Like Catalytic Activity for H2 O2 and Cholesterol Detection.

Abnormal H2 O2 and cholesterol levels are closely related to many diseases. This work reports a facile process for the synthesis of oxidized glutathione (GSSG)-modified MoS2 nanosheets (MoS2 -GSSG NSs). The biocompatible MoS2 -GSSG NSs have good dispersibility and high affinity to the substrate 3,3',5,5'-tetramethylbenzidine (TMB), which is beneficial for improving peroxidase-like catalytic activity of MoS2 . The high peroxidase-like activity of MoS2 -GSSG NSs was applied as a robust nanoplatform for low-cost, rapid, and highly effective colorimetric detection of H2 O2 and total/free cholesterol. Moreover, the peroxidase-like catalytic mechanism was studied by the steady-state kinetics method. The catalytic activity was remarkably high at a wide range of pH (2.4-7.0) and temperature values (25-70 °C). The cholesterol was catalyzed by cholesterol oxidase (ChOx) in the presence of O2 to generate H2 O2 , which oxidized TMB to generate a blue-colored product (oxTMB) under the catalysis of MoS2 -GSSG NSs. The detection limit (DL) of total cholesterol and H2 O2 was as low as 5.36 and 0.51 µm, respectively. The linear ranges for detecting cholesterol and H2 O2 were from 5.36 to 800 µm and from 0.51 to 50 µm, respectively. This method was also successfully applied to the detection of cholesterol in serum. The detection concentration of total cholesterol was consistent with that of the value detected by the blood biochemical method used in the clinic.

The Transient Receptor Potential Ankyrin Type 1 Plays a Critical Role in Cortical Spreading Depression.

The transient receptor potential ankyrin type-1 (TRPA1) channels have been proposed as a potential target for migraine therapy. Yet the role of cortical TRPA1 channels in migraine mechanism has not been fully understood. Cortical spreading depression (CSD) is known as an underlying cause of migraine aura. The aim of this study is to investigate if cortical TRPA1 activity is required for CSD genesis and propagation. A mouse brain slice CSD model with intrinsic optical imaging was applied for TRPA1 signaling pharmacology. The results showed that the TRPA1 agonist, umbellulone, facilitated the propagation of submaximal CSD. Correspondingly, an anti-TRPA1 antibody and two selective TRPA1 antagonists, A967079 and HC-030031, prolonged the CSD latency and reduced magnitude, indicating a reduced cortical susceptibility to CSD under TRPA1 deactivation. Furthermore, the TRPA1 agonist, allyl-isothiocyanate (AITC), reversed the suppression of CSD by HC-030031, but not by A967079. Interestingly, the inhibitory action of A967079 on CSD was reversed by exogenous calcitonin-gene-related peptide (CGRP). Consistent to TRPA1 deactivation, the prolonged CSD latency was observed by an anti-CGRP antibody in the mouse brain slice, which was reversed by exogenous CGRP. We conclude that cortical TRPA1 is critical in regulating cortical susceptibility to CSD, which involves CGRP. The data strongly suggest that deactivation of TRPA1 channels and blockade of CGRP would have therapeutic benefits in preventing migraine with aura.

Peroxidase-like activity of MoS2 nanoflakes with different modifications and their application for H2O2 and glucose detection.

MoS2 nanoflakes (MoS2 NFs) with a diameter of ∼390 nm were obtained by a facile one-pot hydrothermal method and the NFs exhibited intrinsic peroxidase-like activity. After being modified by commonly used biocompatible surfactants including polyethyleneimine (PEI), polyacrylic acid (PAA), polyvinylpyrrolidone (PVP), and cysteine (Cys), the peroxidase-like catalytic activities of MoS2 NFs were investigated by using 3,3',5,5'-tetramethylbenzidine (TMB) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS) as chromogenic substrates. Compared to the polymer modified MoS2 NFs, Cys functionalized MoS2 NFs exhibited a high catalytic activity toward H2O2 in the presence of TMB or ABTS. Zeta potential and Michaelis-Menten analyses implied that the electrostatic force induced affinity or repulsion between the MoS2 NFs and substrates, as well as surface modifications of the MoS2 NFs played a key role in the catalytic reactions. Notably, a new peroxidase-like catalytic reaction mechanism was proposed based on the formation of a transient state of Cys-MoS2 NFs containing H2O2 and ABTS, and the catalytic reaction could occur because the Cys on the surface of the MoS2 NFs served as an electron transfer bridge between H2O2 and ABTS. Based on this finding, we also established a platform for colorimetric detection of H2O2 and glucose using Cys-MoS2 NFs as a peroxidase substitution. The limit of detection (LOD) was determined to be 4.103 µmol L-1 for H2O2, and the linear range (LR) was from 0 to 0.3 mmol L-1. The LOD for glucose was 33.51 µmol L-1 and the LR was from 0.05 to 1 mmol L-1, which is suitable for biomedical diagnosis. This work provides a new insight into the catalytic mechanism of peroxidase-like MoS2 NFs, and paves the way for enzyme-like nanomaterials to be used for medical diagnosis.

Sarcoma family kinase activity is required for cortical spreading depression.

Objectives Sarcoma family kinase activity is associated with multiple diseases including ischemia and cancer; however, its role in the mechanism of migraine aura has been less well characterised. This study aims to investigate whether sarcoma family kinase is required for cortical spreading depression. Methods Cortical spreading depression was induced by topical application of K+ to the cerebral cortex and was monitored using electrophysiology in rats, and intrinsic optical signal in mouse brain slices. Drugs were perfused into the contralateral cerebral ventricle for pharmacological manipulations in rats. Western blot analysis was used for detecting the level of phosphorylated, and total, sarcoma family kinase in the ipsilateral cortex of rats. Key results The data demonstrate that a single cortical spreading depression in rats induced ipsilateral cortical sarcoma family kinase phosphorylation at the Y416 site. Deactivation of sarcoma family kinase by its inhibitor (3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1 H-pyrazolo[3,4- dpyrimidin-4-amine) suppressed the elevated enzyme activity and cortical susceptibility to cortical spreading depression. Interestingly, the inhibitory effect of the N-methyl-D-aspartate receptor antagonist NVP-AAM077 on cortical spreading depression was reversed by the sarcoma family kinase activator pYEEI (EPQY(PO3H2)EEEIPIYL), suggesting a link between this enzyme and N-methyl-D-aspartate receptors. Similarly, after deactivation of sarcoma family kinase, a reduction of sarcoma family kinase phosphorylation and cortical susceptibility to cortical spreading depression was observed with NVP-AAM077. Conclusions We conclude that activation of sarcoma family kinase is required for cortical spreading depression, and this process is regulated by recruiting N-methyl-D-aspartate receptors. This study provides novel insight for sarcoma family kinase function in the mechanism of migraine aura.

Proteomic analysis of immature rat pups brain in response to hypoxia and ischemia challenge.

Hypoxia and ischemia significantly affects perinatal brain development, even worse in preterm infants. However, the details of the mechanism leading to permanent brain damage after hypoxia-ischemia attack have not been fully elucidated. Proteomics could provide insight into the potential mechanism and help to promote the clinical treatment. In this study, quantitative analysis was performed 24 hours after hypoxia-ischemia using liquid-chromatography mass spectrometry coupled to label-free analysis. Compared to control, 193 proteins were present only in hypoxic-ischemic group. In addition, 34 proteins were more than 2 folds up-regulated and 14 proteins were more than 2 folds down-regulated in hypoxia-ischemia group. Gene Ontology database showed that the majority of differentially expressed proteins comprised mitochondrial proteins et al. Molecular function analysis revealed that the majority of proteins were involved in ion binding et al. Biological process analysis showed that the majority of proteins were involved in response to organic substance et al. STRING 9.0 software analysis were used to explore the complex interactions existed among the proteins. Western blot were used to verify the fold changes of some proteins-microtubule-associated protein 2 and microtubule-associated protein tau. This novel study performed a full-scale screening of the proteomics research in hypoxic-ischemic brain damage of immature rat.

In vivo induction of apoptosis by fucoxanthin, a marine carotenoid, associated with down-regulating STAT3/EGFR signaling in sarcoma 180 (S180) xenografts-bearing mice.

Previous in vitro researches have showed that fucoxanthin, a natural carotenoid isolated from sargassum, can inhibit proliferation or induce apoptosis in human neuroblastoma, hepatoma, leukemia, colon carcinoma, prostate cancer or urinary bladder cancer cells. But the precise mechanism by which fucoxanthin exerts anticarcinogenic effects is not yet fully understood. In this study, we performed an in vivo study to investigate the anti-tumor effect and mechanisms of fucoxanthin on xenografted sarcoma 180 (S180) in mice. Results revealed that fucoxanthin significantly inhibited the growth of sarcoma at the dose of 50 or 100 mg/kg. TUNEL analysis showed that the number of positive cells in the fucoxanthin-treated group was higher than that in the control group. Western blotting analysis also revealed the suppressed expression of bcl-2 and enhanced expression of cleaved caspase-3 by fucoxanthin. In addition, immunohistochemistry analysis and Western blotting analysis showed that fucoxanthin significantly decreased the expressions of survivin and vascular endothelial growth factor (VEGF). Most importantly, fucoxanthin inhibited the expressions of the epidermal growth factor receptor (EGFR) and STAT3 and phosphorylated STAT3 proteins. These results indicated that in vivo induction of apoptosis by fucoxanthin is associated with down-regulating STAT3/EGFR signaling in S180 xenografts-bearing mice.