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Celastrol, a pentacyclic triterpenoid found in Chinese herb Tripterygium wilfordii, is considered as one of the top-five natural medicinal compounds with high antioxidant property. However, celastrol has poor aqueous solubility and thereby low bioavailability, restricting its clinical application as drug. To overcome this problem, we nanonized celastrol by entrapping it within hydrophilic nanocarrier - calcium phosphate nanoparticle. The synthesized calcium phosphate celastrol nanoparticle (CPCN) had average size of 35 nm, spherical shape, significant stability with (-) 37 mV zeta potential, celastrol entrapment efficiency around 75 % and low celastrol release kinetics spanning over 7 days, as measured by different techniques like FESEM, AFM, DLS, and spectrophotometry. Studies on the antioxidant potency of CPCN by flow cytometry and fluorescence microscopy depicted that the toxicity developed in human neuroblastoma cells SH-SY5Y by treatment with the selective neurotoxin MPP+ iodide (N-Methyl-4-phenylpyridinium iodide) got reduced by pretreatment of the cells with CPCN. Determination of cellular ROS content, depolarization level of mitochondrial membrane potential, cell cycle analysis and nuclear damage in MPP+-exposed cells demonstrated that CPCN had about 65 % more antioxidant efficacy over that of bulk celastrol. Thus, the nanonization process transformed hydrophobic celastrol into hydrophilic CPCN, having high potentiality to be developed as an effective antioxidant drug.

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This study explored the neuroprotective potential of fermented pomegranate (PG-F) against hydrogen peroxide (H2O2)-induced neurotoxicity in human neuroblastoma SH-SY5Y cells and elucidated the underlying molecular mechanisms. The fermentation process, involving probiotics, transforms the hydrolyzable tannins in pomegranate juice into ellagic acid (EA) and gallic acid (GA), which are believed to contribute to its health benefits. Molecular docking simulations confirmed the stable interactions between EA, GA, and proteins associated with the antioxidant and anti-apoptotic pathways. PG-F significantly enhanced the viability of H2O2-treated cells, as evidenced by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays, cell morphology observations, and Hoechst 33342 staining. PG-F mitigated the H2O2-induced intracellular reactive oxygen species (ROS) levels, restored mitochondrial membrane potential, and upregulated antioxidant gene expression. The PG-F treatment also attenuated the H2O2-induced imbalance in the Bax/Bcl-2 ratio and reduced the cleaved caspase-3, caspase-7, and caspase-9 levels, suppressing the apoptotic pathways. Further insights showed that PG-F inhibited the phosphorylation of mitogen-activated protein kinases (MAPKs) and facilitated the nuclear translocation of nuclear factor-erythroid 2-related factor (Nrf2), highlighting its role in modulating the key signaling pathways. A combined treatment with equivalent concentrations of EA and GA, as found in PG-F, induced remarkable cellular protection. Drug combination analysis using the Chou-Talalay method revealed a synergistic effect between EA and GA, emphasizing their combined efficacy. In conclusion, PG-F has significant neuroprotective effects against H2O2-induced neurotoxicity by modulating the antioxidant and anti-apoptotic pathways. The synergistic action of EA and GA suggests the therapeutic potential of PG-F in alleviating oxidative stress-associated neurodegenerative diseases.

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Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain-based on the sequence of the HEP (HEP-Flury) strain-with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

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Ciguatoxins (CTXs) are neurotoxins responsible for ciguatera poisoning (CP), which affects more than 50,000 people worldwide annually. The development of analytical methods to prevent CP is a pressing global issue, and the N2a assay is one of the most promising methods for detecting CTXs. CTXs are highly toxic, and an action level of 0.01 µg CTX1B equivalent (eq)/kg in fish has been proposed. It is desirable to further increase the detection sensitivity of CTXs in the N2a assay to detect such low concentrations reliably. The opening of voltage-gated sodium channels (NaV channels) and blocking of voltage-gated potassium channels (KV channels) are thought to be involved in the toxicity of CTXs. Therefore, in this study, we developed an assay that could detect CTXs with higher sensitivity than conventional N2a assays, using KV channel inhibitors as sensitizing reagents for N2a cells. The addition of the KV channel inhibitors 4-aminopyridine and tetraethylammonium chloride to N2a cells, in addition to the traditional sensitizing reagents ouabain and veratridine, increased the sensitivity of N2a cells to CTXs by up to approximately 4-fold. This is also the first study to demonstrate the influence of KV channels on the toxicity of CTXs in a cell-based assay.

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Seven undescribed neovibsane-type diterpenoids (1-7) were isolated from the leaves of Viburnum odoratissimum. Their planar structures and relative configurations were elucidated based on a combination of 1D and 2D NMR analysis. The absolute configurations were confirmed by Rh2(OCOCF3)4-induced ECD analysis and comparison of experimental and TDDFT-calculated ECD spectrum. Based on the empirical results of the ECD of in situ formed Rh-complexes, rapid determination of the absolute configuration of C-14 within vibsane-type diterpenoids was proposed. In addition, 3 exhibited a high neuroblastoma cell protective effect of 81.8 % at 50 µM (the control group showed a neuroblastoma cell protective effect of 56.2 % at 50 µM).

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Neurodegenerative disorders of the central nervous system (CNS) such as Alzheimer's and Parkinson's diseases, afflict millions globally, posing a significant public health challenge. Despite extensive research, a critical hurdle in effectively treating neurodegenerative diseases is the lack of neuroprotective drugs that can halt or reverse the underlying disease processes. In this work, we took advantage of the neuroprotective properties of the neuropeptide glycyl-l-prolyl-l-glutamic acid (Glypromate) for the development of new peptidomimetics using l-pipecolic acid as a proline surrogate and exploring their chemical conjugation with relevant active pharmaceutical ingredients (API) via a peptide bond. Together with prolyl-based Glypromate conjugates, a total of 36 conjugates were toxicologically and biologically evaluated. In this series, the results obtained showed that a constrained ring (l-proline) at the central position of the peptide motif accounts for enhanced toxicological profiles and biological effects using undifferentiated and differentiated human neuroblastoma SH-SY5Y cells. Additionally, it was shown that biased biological responses are API-dependent. Conjugation with (R)-1-aminoindane led to a 38-43% reduction of protein aggregation induced by Aß25-35 (10 µM), denoting a 3.2-3.6-fold improvement in comparison with the parent neuropeptide, with no significative difference between functionalization at α and γ-carboxyl ends. On the other hand, the best-performing neuroprotective conjugate against the toxicity elicited by 6-hydroxydopamine (6-OHDA, 125 µM) was obtained by conjugation with memantine at the α-carboxyl end, resulting in a 2.3-fold improvement of the neuroprotection capacity in comparison with Glypromate neuropeptide. Altogether, the chemical strategy explored in this work shows that the neuroprotective capacity of Glypromate can be modified and fine-tuned, opening a new avenue for the development of biased neurotherapeutics for CNS-related disorders.

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OBJECTIVE To study the effects of the curcumin derivative bisdemethoxycurcumin （BC） promoting neuronal differentiation of neuroblastoma cells Neuro-2a （N2a） in mice and its mechanism. METHODS The effects of BC （1， 2， 4， 6， 8， 10 μmol/L） on the viability of N2a cells were detected by MTT assay to determine the concentration range of drug treatment. The control group， retinoic acid （RA） group （10 μmol/L） and BC groups （1， 2 and 4 μmol/L） were set up， and the length of neural protrusions of the differentiated cells was measured and the cell differentiation rate was calculated after 48 h and 72 h of culture. Compared with 0 min group， Western blot was used to detect the phosphorylation levels of protein kinase B （Akt）， extracellular- signal regulated kinase 1/2 （ERK1/2）， and p38 mitogen-activated protein kinase （p38） proteins in cells treated by 4 μmol/L BC for 5， 15， 30， 60， 120 min. After intervention with inhibitors LY294002 （LY） and PD98059 （PD）， the effects of BC on Akt and ERK1/2 protein phosphorylation levels and promoting neural differentiation were further validated. RESULTS According to the MTT experiment， the BC concentrations for subsequent induction of cell differentiation were determined to be 1， 2， and 4 μmol/L. After 48 hours of differentiation， compared with the control group， the cell differentiation rate in RA group and BC 1， 2 and 4 μmol/L groups， the length of cellular neural processes wjxhhxx413@163.com in the BC 4 μmol/L group significantly increased （P＜0.05 or P＜0.01）；after inducing differentiation of BC for 72 hours，compared with the control group， the cell differentiation rate and the length of cellular neural processes in the RA group， the cell differentiation rate in the BC 4 μmol/L group， and the length of cellular neural processes in the BC 2 μmol/L group all significantly increased （P＜0.05 or P＜0.01）.Compared with the 0 min group， the phosphorylation levels of Akt， ERK1/2， and p38 proteins in cells of the 5， 15， 30， 60 and 120 min groups increased to varying degrees after treated by 4 μmol/L BC， and some differences were statistically significant （P＜0.05 or P＜0.01）. After adding the inhibitor LY/PD， compared with the BC group， the phosphorylation level of ERK1/2 protein in the PD+BC group cells were significantly reduced （P＜0.01）， and the cell differentiation rates in the LY group， LY+BC group， PD group， and PD+BC group was significantly reduced （P＜0.01）. CONCLUSIONS BC promotes N2a cell differentiation mainly by increasing cell differentiation rate and neural protrusion length. The mechanism may be related to the activation of mitogen-activated protein kinase/ ERK and PI3K/Akt signaling pathways.

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Chronic alcohol consumption may alter mRNA methylation and expression levels of genes related to addiction and reward in the brain, potentially contributing to alcohol tolerance and dependence. Neuron-like (SH-SY5Y) and non-neuronal (SW620) cells were utilized as models to examine chronic intermittent ethanol (CIE) exposure-induced global m6A RNA methylation changes, as well as m6A mRNA methylation changes around the stop codon of three opioid receptor genes (OPRM1, OPRD1, and OPRK1), which are known to regulate pain, reward, and addiction behaviours. CIE exposure for three weeks significantly increased global RNA methylation levels in both SH-SY5Y (t = 3.98, P = 0.007) and SW620 (t = 2.24, P = 0.067) cells. However, a 3-week CIE exposure resulted in hypomethylation around mRNA stop codon regions of OPRM1 and OPRD1 in both cell lines [OPRM1(SH-SY5Y): t = -5.05, P = 0.0005; OPRM1(SW620): t = -3.19, P = 0.013; OPRD1(SH-SY5Y): t = -13.43, P < 0.00001; OPRD1(SW620): t = -4.00, P = 0.003]. Additionally, mRNA expression levels of OPRM1, OPRD1, and OPRK1 were downregulated (corresponding to mRNA hypomethylation) in both SH-SY5Y and SW620 cells after a 3-week CIE exposure. The present study demonstrated that chronic ethanol exposure altered global RNA methylation levels, as well as mRNA methylation and expression levels of opioid receptor genes in both neuron-like and non-neuronal cells. Our findings suggest a potential epitranscriptomic mechanism by which chronic alcohol consumption remodels the expression of reward-related and alcohol responsive genes in the brain, thus increasing the risk of alcohol use disorder development.Abbreviations: OPRM1: the µ-opioid receptor; OPRD1: the δ-opioid receptor; OPRK1: the κ-opioid receptor; CIE: chronic intermittent ethanol exposure; CIE+WD: chronic intermittent ethanol exposure followed by a 24-hr withdrawal; SH-SY5Y: human neuroblastoma cell Line; SW620: human colon carcinoma cell line; RT-qPCR: reverse transcription followed by quantitative polymerase reaction; MazF-RT-qPCR: MazF digestion followed by RT-qPCR.

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In recent years, the application of pulsed electric fields with very short durations (nanoseconds) and extremely high amplitudes (MV/m) has been investigated for novel medical purposes. Various electric protocols have been explored for different objectives, including the utilization of fractionated pulse doses to enhance cell electrosensitization to the uptake of different markers or an increase in apoptosis. This study focused on the use of fluorescence imaging to examine molecular calcium fluxes induced by different fractionated protocols of short electric pulses in neuroblastoma (SH-SY5Y) and mesenchymal stem cells (HaMSCs) that were electroporated using nanosecond pulsed electric fields. In our experimental setup, we did not observe cell electrosensitization in terms of an increase in calcium flux following the administration of fractionated doses of nanosecond pulsed electric fields with respect to the non-fractionated dose. However, we observed the targeted activation of calcium-dependent genes (c-FOS, c-JUN, EGR1, NURR-1, ß3-TUBULIN) based on the duration of calcium flux, independent of the instantaneous levels achieved but solely dependent on the final plateau reached. This level of control may have potential applications in various medical and biological treatments that rely on calcium and the delivery of nanosecond pulsed electric fields.

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Amyloidogenic deposition of ß-amyloid (Aß) peptides in human brain involves not only the wild-type Aß (wt-Aß) sequences, but also posttranslationally modified Aß (PTM-Aß) variants. Recent studies hypothesizes that the PTM-Aß variants may trigger the deposition of wt-Aß, which underlies the pathology of Sporadic Alzheimer's disease. Among PTM-Aß variants, the pyroglutamate-3-Aß (pyroE3-Aß) has attracted much attention because of their significant abundances and broad distributions in senile plaques and dispersible and soluble oligomers. pyroE3-specific antibodies are being tested as potential anti-Aß drugs in clinical trials. However, evidence that support the triggering effect of pyroE3-Aß on wt-Aß in cells remain lacking, which diminishes its pathological relevance. We show here that cross-seeding with pyroE3-Aß40 leads to accelerated extracellular and intracellular aggregation of wt-Aß40 in different neuronal cells. Cytotoxicity levels are elevated through the cross-seeded aggregation, comparing with the self-seeded aggregation of wt-Aß40 or the static presence of pyroE3-Aß40 seeds. For the extracellular deposition in mouse neuroblastoma Neuro2a (N2a) cells, the cytotoxicity elevation correlates positively with the seeding efficiency. Besides aggregation rates, cross-seeding with pyroE3-Aß40 also modulates the molecular level structural polymorphisms of the resultant wt-Aß40 fibrils. Using solid-state nuclear magnetic resonance (ssNMR) spectroscopy, we identified key structural differences between the parent pyroE3/ΔE3 and wt-Aß40 fibrils within their fibrillar cores. Structural propagation from seeds to daughter fibrils is demonstrated to be more pronounced in the extracellular seeding in N2a cells by comparing the ssNMR spectra from different seeded wt-Aß40 fibrils, but less significant in the intracellular seeding process in human neuroblastoma SH-SY5Y cells.

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This study aims to screen and identify specific cluster miRNAs of H7N9 virus-infected N2a cells and explore the possible pathogenesis of these miRNAs. The N2a cells are infected with H7N9 and H1N1 influenza viruses, and the cells are collected at 12, 24 and 48 h to extract total RNA. To sequence miRNAs and identify different virus-specific miRNAs, high-throughput sequencing technology is used. Fifteen H7N9 virus-specific cluster miRNAs are screened, and eight of them are included in the miRBase database. These cluster-specific miRNAs regulate many signaling pathways, such as the PI3K-Akt signaling pathway, the RAS signaling pathway, the cAMP signaling pathway, actin cytoskeleton regulation and cancer-related genes. The study provides a scientific basis for the pathogenesis of H7N9 avian influenza, which is regulated by miRNAs.

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HYPOTHESIS: Colloidal gold nanoparticles (AuNPs) functionalised with hydrophilic thiols can be used as drug delivery probes, thanks to their small size and hydrophilic character. AuNPs possess unique properties for their use in nanomedicine, especially in cancer treatment, as diagnostics and therapeutic tools. EXPERIMENTS: Thiol functionalised AuNPs were synthesised and loaded with methotrexate (MTX). Spectroscopic and morphostructural characterisations evidenced the stability of the colloids upon interaction with MTX. Solid state (GISAXS, GIWAXS, FESEM, TEM, FTIR-ATR, XPS) and dispersed phase (UV-Vis, DLS, ζ-potential, NMR, SAXS) experiments allowed to understand structure-properties correlations. The nanoconjugate was tested in vitro (MTT assays) against two neuroblastoma cell lines: SNJKP and IMR5 with overexpressed n-Myc. FINDINGS: Molar drug encapsulation efficiency was optimised to be >70%. A non-covalent interaction between the π system and the carboxylate moiety belonging to MTX and the charged aminic group of one of the thiols was found. The MTX loading slightly decreased the structural order of the system and increased the distance between the AuNPs. Free AuNPs showed no cytotoxicity whereas the AuNPs-MTX nanoconjugate had a more potent effect when compared to free MTX. The active role of AuNPs was evidenced by permeation studies: an improvement on penetration of the drug inside cells was evidenced.

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Human-relevant three-dimensional (3D) models of cerebral tissue can be invaluable tools to boost our understanding of the cellular mechanisms underlying brain pathophysiology. Nowadays, the accessibility, isolation and harvesting of human neural cells represents a bottleneck for obtaining reproducible and accurate models and gaining insights in the fields of oncology, neurodegenerative diseases and toxicology. In this scenario, given their low cost, ease of culture and reproducibility, neural cell lines constitute a key tool for developing usable and reliable models of the human brain. Here, we review the most recent advances in 3D constructs laden with neural cell lines, highlighting their advantages and limitations and their possible future applications.

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Ciguatera fish poisoning (CFP) is a foodborne illness affecting > 50,000 people worldwide annually. It is caused by eating marine invertebrates and fish that have accumulated ciguatoxins (CTXs). Recently, the risk of CFP to human health, the local economy, and fishery resources have increased; therefore, detection methods are urgently needed. Functional assays for detecting ciguatoxins in fish include receptor binding (RBA) and neuroblastoma cell-based assay (N2a assay), which can detect all CTX congeners. In this study, we made these assays easier to use. For RBA, an assay was developed using a novel near-infrared fluorescent ligand, PREX710-BTX, to save valuable CTXs. In the N2a assay, a 1-day assay was developed with the same detection performance as the conventional 2-day assay. Additionally, in these assays, we used calibrated CTX standards from the Pacific determined by quantitative NMR for the first time to compare the relative potency of congeners, which differed significantly among previous studies. In the RBA, there was almost no difference in the binding affinity among congeners, showing that the differences in side chains, stereochemistry, and backbone structure of CTXs did not affect the binding affinity. However, this result did not correlate with the toxic equivalency factors (TEFs) based on acute toxicity in mice. In contrast, the N2a assay showed a good correlation with TEFs based on acute toxicity in mice, except for CTX3C. These findings, obtained with calibrated toxin standards, provide important insights into evaluating the total toxicity of CTXs using functional assays.

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Thymoquinone (TQ), an active compound from Nigella sativa seeds, is often described as a pharmacologically relevant compound with antioxidative properties, while the synthesis of TQ in the plant via oxidations makes it inapplicable for scavenging radicals. Therefore, the present study was designed to reassess the radical scavenging properties of TQ and explore a potential mode of action. The effects of TQ were studied in models with mitochondrial impairment and oxidative stress induced by rotenone in N18TG2 neuroblastoma cells and rotenone/MPP+ in primary mesencephalic cells. Tyrosine hydroxylase staining revealed that TQ significantly protected dopaminergic neurons and preserved their morphology under oxidative stress conditions. Quantification of the formation of superoxide radicals via electron paramagnetic resonance showed an initial increase in the level of superoxide radicals in the cell by TQ. Measurements in both cell culture systems revealed that the mitochondrial membrane potential was tendentially lowered, while ATP production was mostly unaffected. Additionally, the total ROS levels were unaltered. In mesencephalic cell culture under oxidative stress conditions, caspase-3 activity was decreased when TQ was administered. On the contrary, TQ itself tremendously increased the caspase-3 activity in the neuroblastoma cell line. Evaluation of the glutathione level revealed an increased level of total glutathione in both cell culture systems. Therefore, the enhanced resistance against oxidative stress in primary cell culture might be a consequence of a lowered caspase-3 activity combined with an increased pool of reduced glutathione. The described anti-cancer ability of TQ might be a result of the pro-apoptotic condition in neuroblastoma cells. Our study provides evidence that TQ has no direct scavenging effect on superoxide radicals.

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The pathological accumulation of quinolinic acid (QA) is often associated with neuritis and neuronal cell death in several neurodegenerative diseases, through the overproduction of free radicals. Urolithin B and auraptene have been reported to exert potent antioxidant effects - however, little is known about the protective effects of these compounds against QA-induced neurotoxicity. Therefore, this study aimed to explore the in vitro protective effects of urolithin B and auraptene against QA-induced neurotoxicity in the SH-SY5Y neuroblastoma cell line. The MTT assay was used to evaluate cell viability, and flow cytometry was carried out to evaluate effects on the cell cycle and apoptosis. The intracellular levels of reactive oxygen species (ROS) were also determined. Our findings showed that auraptene at non-toxic concentrations had no protective effect on QA-induced toxicity. However, urolithin B at concentrations of 0.6 µM and 2.5 µM enhanced the viability of cells treated with QA. Moreover, while the percentage of apoptotic cells (i.e. in the sub-G1 phase) was shown to significantly increase after QA treatment, pre-treatment with urolithin B reduced the number of these apoptotic cells. Furthermore, urolithin B, as an antioxidant, also significantly reduced QA-induced ROS production. Our findings suggest that urolithin B may possess potent antioxidant and neuroprotective effects against QA-induced neurotoxicity that merit further investigation.

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Human neuronal cells are a more appropriate cell model for neurological disease studies such as Alzheimer and Parkinson's disease. SH-SY5Y neuroblastoma cells have been widely used for differentiation into a mature neuronal cell phenotype. The cellular differentiation process begins with retinoic acid incubation, followed by incubation with brain-derived neurotrophic factor (BDNF), a recombinant protein produced in E. coli cells. Endotoxin or lipopolysaccharide (LPS) is the major component of the outer membrane of bacterial cells that triggers the activation of pro-inflammatory cytokines and ultimately cell death. Consequently, any endotoxin contamination of the recombinant BDNF used for cell culture experiments would impact on data interpretation. Therefore, in this study, we expressed the BDNF recombinant protein in bacterial endotoxin-free cells that were engineered to modify the oligosaccharide chain of LPS rendering the LPS unable to trigger the immune response of human cells. The expression of DCX and MAP-2 in differentiated cells indicate that in-house and commercial BDNF are equally effective in inducing differentiation. This suggests that our in-house BDNF protein can be used to differentiate SH-SY5Y neuroblastoma cells without the need for an endotoxin removal step.

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OBJECTIVE: The objective of this study was to investigate the potential for erucic acid (EA), an omega-9 monounsaturated fatty acid, to act as a neuroprotective agent. MATERIALS AND METHODS: In this study, EA was investigated against N2a cell lines and a rotenone (ROT)-induced model of Parkinson's disease for its neuroprotective potential. The N2a cell line was incubated with fetal bovine serum, penicillin, and streptomycin supplemented with Dulbecco's Modified Eagle's Medium, and the following assays were carried out: (i) MTT, (ii) biocompatibility, (iii) DCFDA, and (iv) diphenylamine. A cell morphology study was also performed. Further, ROT 1 mg/kg s.c. and EA 3 and 10 mg/kg p.o. were given to rats on a daily basis for 21 days, and the following parameters were assessed: (i) neurobehavioral studies, (ii) oxidative stress markers, (iii) neuroinflammatory markers, (iv) neurotransmitters, and (v) histopathological study. RESULTS: The cell viability assay revealed that EA showed protection against ROT-induced toxicity in N2a cells, which was confirmed by a cell morphology study. EA decreased oxidative stress and % DNA fragmentation significantly. EA also prevented ROT-induced motor impairment and altered levels of oxidative stress markers, neurotransmitters, and neuroinflammatory markers significantly. When compared to the ROT group, a histological investigation of the EA group showed partial neuronal loss with the existence of intact neurons in between the vacuolated gaps. CONCLUSION: This study revealed that EA possesses profound neuroprotective properties in in vitro and in vivo studies. Additional research can be carried out to study the mechanism of EA with respect to its neuroprotective potential.

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Background and Objectives: Variants of GABRA2 have been repeatedly associated with alcohol dependence risk. However, no study investigated potential epigenetic alterations in the GABRA2 gene in alcohol-dependent (AD) subjects during alcohol withdrawal. We investigated DNA methylation pattern in the regulatory region of GABRA2 gene in peripheral leukocytes of AD patients and controls. Further, GABRA2 methylation patterns were analysed in neuroblastoma cells under ethanol exposure and withdrawal. Materials and Methods: In the present study, blood samples were obtained from 41 AD subjects on the day of inpatient admission, after the first and second week of inpatient treatment. The comparison group included 47 healthy controls. GABRA2 methylation of 4 CpG sites in the CpG island was compared to neuroblastoma cells which were exposed to 100 mM of ethanol for 2, 5 and 9 days, followed by a withdrawal interval of 4 days. Results: no significant differences in GABRA2 methylation patterns were found in AD subjects over time and vs. controls, after controlling for age. Further, no influence of withdrawal severity, alcohol consumption before admission and other alcohol dependence characteristics were found. Conclusions: The results indicate that GABRA2 methylation in AD individuals and in a cell model is unaffected by alcohol exposition and withdrawal. Influences of GABRA2 on characteristics of alcohol dependence may be exerted by mechanisms other than epigenetic alterations related to alcohol intoxication or withdrawal.

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The potent environmental herbicide and weedicide paraquat is linked to neuromotor defects and Parkinson's disease (PD). We have evaluated the neuroprotective role of citric acid-sourced carbon quantum dots (Cit-CQDs) on paraquat-insulted human neuroblastoma-derived SH-SY5Y cell lines and on a paraquat-exposed nematode (Caenorhabditis elegans). Our data reveal that Cit-CQDs are able to scavenge free radicals in test tube assays and mitigate paraquat-elevated reactive oxygen species (ROS) levels in SH-SY5Y cells. Furthermore, Cit-CQDs protect the cell line from paraquat, which otherwise elicits cell death. Cit-CQDs-challenged nematodes demonstrate enhanced survival rates 72 h post-paraquat exposure compared to controls. Paraquat ablates dopamine (DA) neurons, which results in compromised locomotor function in nematodes. However, the neurons remained intact when the nematodes were incubated with Cit-CQDs prior to neurotoxicant exposure. The collective data suggest Cit-CQDs offer neuroprotection for cell lines and organisms from xenotoxicant-associated neuronal injury and death. The study suggests Cit-CQDs as a potentially viable green chemistry-synthesized, biobased nanomaterial for intervention in neurodegenerative disorders.

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