Human Neuroblastoma Cell Line Research Articles

Isolation and Characterization of A2-EPTX-Nsm1a, a Secretory Phospholipase A2 from Malaysian Spitting Cobra (Naja sumatrana) Venom.

Phospholipase A2 (PLA2) toxins are one of the main toxin families found in snake venom. PLA2 toxins are associated with various detrimental effects, including neurotoxicity, myotoxicity, hemostatic disturbances, nephrotoxicity, edema, and inflammation. Although Naja sumatrana venom contains substantial quantities of PLA2 components, there is limited information on the function and activities of PLA2 toxins from the venom. In this study, a secretory PLA2 from the venom of Malaysian N. sumatrana, subsequently named A2-EPTX-Nsm1a, was isolated, purified, and characterized. A2-EPTX-Nsm1a was purified using a mass spectrometry-guided approach and multiple chromatography steps. Based on LC-MSMS, A2-EPTX-Nsm1a was found to show high sequence similarity with PLA2 from venoms of other Naja species. The PLA2 activity of A2-EPTX-Nsm1 was inhibited by 4-BPB and EDTA. A2-EPTX-Nsm1a was significantly less cytotoxic in a neuroblastoma cell line (SH-SY5Y) compared to crude venom and did not show a concentration-dependent cytotoxic activity. To our knowledge, this is the first study that characterizes and investigates the cytotoxicity of an Asp49 PLA2 isolated from Malaysian N. sumatrana venom in a human neuroblastoma cell line.

Novel chemical inhibitor against SOD1 misfolding and aggregation protects neuron-loss and ameliorates disease symptoms in ALS mouse model

Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disease characterized by selective death of motor neurons. Mutations in Cu, Zn-superoxide dismutase (SOD1) causing the gain of its toxic property are the major culprit of familial ALS (fALS). The abnormal SOD1 aggregation in the motor neurons has been suggested as the major pathological hallmark of ALS patients. However, the development of pharmacological interventions against SOD1 still needs further investigation. In this study, using ELISA-based chemical screening with wild and mutant SOD1 proteins, we screened a new small molecule, PRG-A01, which could block the misfolding/aggregation of SOD1 or TDP-43. The drug rescued the cell death induced by mutant SOD1 in human neuroblastoma cell line. Administration of PRG-A01 into the ALS model mouse resulted in significant improvement of muscle strength, motor neuron viability and mobility with extended lifespan. These results suggest that SOD1 misfolding/aggregation is a potent therapeutic target for SOD1 related ALS.

Mild hyperthermia induced by gold nanorods acts as a dual-edge blade in the fate of SH-SY5Y cells via autophagy

Unraveling unwanted side effects of nanotechnology-based therapies like photothermal therapy (PTT) is vital in translational nanomedicine. Herein, we monitored the relationship between autophagic response at the transcriptional level by using a PCR array and tumor formation ability by colony formation assay in the human neuroblastoma cell line, SH-SY5Y, 48 h after being exposed to two different mild hyperthermia (43 and 48 °C) induced by PTT. In this regard, the promotion of apoptosis and autophagy were evaluated using immunofluorescence imaging and flow cytometry analyses. Protein levels of Ki-67, P62, and LC3 were measured using ELISA. Our results showed that of 86 genes associated with autophagy, the expression of 54 genes was changed in response to PTT. Also, we showed that chaperone-mediated autophagy (CMA) and macroautophagy are stimulated in PTT. Importantly, the results of this study also showed significant changes in genes related to the crosstalk between autophagy, dormancy, and metastatic activity of treated cells. Our findings illustrated that PTT enhances the aggressiveness of cancer cells at 43 °C, in contrast to 48 °C by the regulation of autophagy-dependent manner.

MAPT enhancer containing rs242557 regulates multiple neighboring genes in human microglial cell line.

Microtubule associated protein tau (MAPT) is involved in assembly and stabilization of microtubules. However, mutated or misfolded forms are commonly found to aggregate in Frontotemporal dementia (FTD), Progressive supranuclear palsy (PSP), Corticobasal degeneration (CBD) etc. singly or with another protein as in Alzheimer's disease (AD). The second strongest signal in MAPT locus after H1 haplotype, as identified by multiple GWAS on AD, PD, PSP and CBD, is from MAPT intronic SNP rs242557. It is part of a FANTOM5 enhancer in eye and brain and evolutionarily conserved from humans to armadillo. While it has been hypothesized that rs242557 could exert an effect on MAPT splicing, the results are contradictory. Strong, Independent signals from MAPT neighboring genes KANSL1, NSF LRR37A and WNT3 have also been noted in some of these diseases. In this study we investigate the role of the enhancer region encompassing rs242557 in gene regulation within MAPT locus. Established CRISPR-Cas9 editing protocols were used to delete rs242557 and its flanking sequences in human microglial clone 3 (HMC3) and human neuroblastoma (NBL-S) cell lines. Heterozygous and homozygous clones with deleted rs242557 and its flanking sequences were individually selected for the study. Cells that went through the entire process of genome editing protocol without the presence of any guide RNA were used as wild type controls. cDNA, synthesized from extracted RNA were used to study expression of MAPT isoforms and its neighboring genes by qRT-PCR, while western blots were performed from the corresponding proteins. HMC-3 cells had significant reduction (p=0.0001) of Nsf, Kansl1, Kansl1-AS1, LRR37A, LRR37A2 and LRR37A3 transcripts in both rs242557 homozygous and heterozygous deletion clones. Total MAPT, MAPT isoforms (3R, 4R, 0N, 1N and 2N) and CRHR1 were also low with some having ct values beyond 33. At the protein level reduced expression of NSF and KANSL1 were noted in the homozygotes and some heterozygotes. No significant alteration was observed in the NBL-S clones. Experiments are currently in progress for the remaining neighboring proteins. Our results indicate that the enhancer encompassing rs242557 alters expression of MAPT and several of its neighbors in HMC-3 but not NBL-S cells.

NTRK1/TrkA Activation Overrides the G2/M-Checkpoint upon Irradiation.

Simple SummaryNeuroblastoma (NB) is a solid childhood tumor and needs to be treated with multimodal therapy including radiation in advances stages. TrkA/NTRK1 expression is a hallmark of NB with excellent prognosis, but the impact of TrkA/NTRK1 on radiation response is largely unknown. Here, we report that human neuroblastoma cell lines engineered to express TrkA/NTRK1 in tightly controlled systems fail to activate the G2/M cell cycle checkpoint upon irradiation, which recapitulates the effects of ATM or ATR inhibition. Our findings point to a hitherto unrecognized TrkA/NTRK1-mediated wiring of the radiation response in NB cells.High expression of the receptor tyrosine kinase TrkA/NTRK1 is associated with a favorable outcome in several solid tumors of childhood including neuroblastoma. During development, TrkA/NTRK1 governs migration and differentiation of neuronal precursor cells, while it is associated with mitotic dysfunction and altered DNA damage response, among others, in neuroblastoma. Here, we used human neuroblastoma cell lines with inducible TrkA/NTRK1 expression to mechanistically explore the role of TrkA/NTRK1 signaling in checkpoint activation after DNA damage induced by ionizing radiation (IR). TrkA/NTRK1 activated cells showed increased short-term cell viability upon IR compared to vector control cells. This was accompanied by a deficient G2/M-checkpoint at both low (1 Gy) and high doses (4 Gy) of IR. In a tightly controlled setting, we confirmed that this effect was strictly dependent on activation of TrkA/NTRK1 by its ligand, nerve growth factor (NGF). TrkA/NTRK1-expressing cells displayed impaired ATM and CHK1 phosphorylation, resulting in stabilization of CDC25B. In line with these findings, ATM or ATR inhibition recapitulated the effects of TrkA/NTRK1 activation on the IR-induced G2/M-checkpoint. In conclusion, we here provide first evidence for a previously unrecognized function of NTRK signaling in checkpoint regulation and the response to IR.

ESTABLISHMENT OF HUMAN NEUROBLASTOMA CELL-LINE (SK-N-SH) AS AN IN VITRO MODEL OF MORPHINE ADDICTION

The usage of morphine for analgesic purposes are widely known, usually for the post-operation procedure and as chronic pain management. However, addiction and overdose liabilities prior to morphine usage are also common. Morphine addiction is observed and studied from various perspectives; tolerance, dependence and withdrawal. With growing and expending research field, researches on addiction were done using in vivo and in vitro model. However, the scientific evidence of morphine addiction using human neuroblastoma cell lines is uncommon. Thus, the present study was designed and conducted to observe the liability of SK-N-SH, as a model for morphine addiction. The cells were administrated with morphine for 24 hours before being treated with methadone. The cytosolic fraction of the cell was collected and used for determining the addiction mechanism. Data showed the involvement of the µ-opioid receptor in expressing the addictive properties of morphine. Exposure to 24-hours morphine had increased the protein level responsible for addiction and reduce the protein levels expressing the endocytic machinery, desensitisation of receptor and cellular adaptation. The altered proteins level was normalised by the treatment of methadone. The study proposed the use of SK-N-SH as an addiction model, as it showed morphine addiction and methadone anti-addiction properties.

Ultrafine black carbon caused mitochondrial oxidative stress, mitochondrial dysfunction and mitophagy in SH-SY5Y cells

Exposure to ambient ultrafine black carbon (uBC, with aerodynamic diameter less than 100 nm) is associated with many neurodegenerative diseases. Oxidative stress is the predominantly reported neurotoxic effects caused by uBC exposure. Mitochondrion is responsible for production of majority of ROS in cells and mitochondrial dysfunction is closely related to adverse nervous outcomes. Mitophagy is an important cellular process to eliminate dysfunctional or damaged mitochondria. However, the mechanisms that modulate mitophagy and mitochondrial dysfunction initiated by uBC remain to be elucidated. The purpose of this study was to investigate how mitochondrial oxidative stress regulated mitochondrial dysfunction and mitophagy in human neuroblastoma cell line (SH-SY5Y) after uBC treatment. RNA interference was further applied to explore the roles of mitophagy in mitochondrial dysfunction. We found uBC triggered cell apoptosis via ROS-mitochondrial apoptotic pathway. The uBC also caused serious mitochondrial damage and respiratory dysfunction, indicated by the abnormalities in mitochondrial division and fusion related proteins, decreased mitochondria number and ATP level. Increased PTEN induced putative kinase 1 (PINK1) and Parkin protein levels and the autolysosome numbers suggested uBC could promote Pink1/Parkin-dependent mitophagy process in SH-SY5Y cells. Mitophagy inhibition could reserve mitochondria number and ATP activity, but not fusion and division related protein levels in SH-SY5Y cells exposed to uBC. Administration of a mitochondria-targeted antioxidant (mitoquinone) significantly eliminated uBC caused apoptosis, mitochondrial dysfunction and mitophagy. Our data suggested mitochondrial oxidative stress regulated uBC induced mitochondrial dysfunction and PINK1/Parkin-dependent mitophagy. PINK1/Parkin-dependent mitophagy probably participated in regulating uBC caused mitochondrial dysfunction but not by controlling mitochondrial fusion and division related proteins. Our results may provide some new insights and evidences to understand the mechanisms of neurotoxicity induced by uBC.

Single-cell reconstruction with spatial context of migrating neural crest cells and their microenvironments during vertebrate head and neck formation.

The dynamics of multipotent neural crest cell differentiation and invasion as cells travel throughout the vertebrate embryo remain unclear. Here, we preserve spatial information to derive the transcriptional states of migrating neural crest cells and the cellular landscape of the first four chick cranial to cardiac branchial arches (BA1-4) using label-free, unsorted single-cell RNA sequencing. The faithful capture of branchial arch-specific genes led to identification of novel markers of migrating neural crest cells and 266 invasion genes common to all BA1-4 streams. Perturbation analysis of a small subset of invasion genes and time-lapse imaging identified their functional role to regulate neural crest cell behaviors. Comparison of the neural crest invasion signature to other cell invasion phenomena revealed a shared set of 45 genes, a subset of which showed direct relevance to human neuroblastoma cell lines analyzed after exposure to the in vivo chick embryonic neural crest microenvironment. Our data define an important spatio-temporal reference resource to address patterning of the vertebrate head and neck, and previously unidentified cell invasion genes with the potential for broad impact.

A Deep Learning Approach with Data Augmentation to Predict Novel Spider Neurotoxic Peptides.

As major components of spider venoms, neurotoxic peptides exhibit structural diversity, target specificity, and have great pharmaceutical potential. Deep learning may be an alternative to the laborious and time-consuming methods for identifying these peptides. However, the major hurdle in developing a deep learning model is the limited data on neurotoxic peptides. Here, we present a peptide data augmentation method that improves the recognition of neurotoxic peptides via a convolutional neural network model. The neurotoxic peptides were augmented with the known neurotoxic peptides from UniProt database, and the models were trained using a training set with or without the generated sequences to verify the augmented data. The model trained with the augmented dataset outperformed the one with the unaugmented dataset, achieving accuracy of 0.9953, precision of 0.9922, recall of 0.9984, and F1 score of 0.9953 in simulation dataset. From the set of all RNA transcripts of Callobius koreanus spider, we discovered neurotoxic peptides via the model, resulting in 275 putative peptides of which 252 novel sequences and only 23 sequences showing homology with the known peptides by Basic Local Alignment Search Tool. Among these 275 peptides, four were selected and shown to have neuromodulatory effects on the human neuroblastoma cell line SH-SY5Y. The augmentation method presented here may be applied to the identification of other functional peptides from biological resources with insufficient data.

Clioquinol Decreases Levels of Phosphorylated, Truncated, and Oligomerized Tau Protein.

The neuropathological hallmarks of Alzheimer’s disease (AD) are senile plaques (SPs), which are composed of amyloid β protein (Aβ), and neurofibrillary tangles (NFTs), which consist of highly phosphorylated tau protein. As bio-metal imbalance may be involved in the formation of NFT and SPs, metal regulation may be a direction for AD treatment. Clioquinol (CQ) is a metal-protein attenuating compound with mild chelating effects for Zn2+ and Cu2+, and CQ can not only detach metals from SPs, but also decrease amyloid aggregation in the brain. Previous studies suggested that Cu2+ induces the hyperphosphorylation of tau. However, the effects of CQ on tau were not fully explored. To examine the effects of CQ on tau metabolism, we used a human neuroblastoma cell line, M1C cells, which express wild-type tau protein (4R0N) via tetracycline-off (TetOff) induction. In a morphological study and ATP assay, up to 10 μM CQ had no effect on cell viability; however, 100 μM CQ had cytotoxic effects. CQ decreased accumulation of Cu+ in the M1C cells (39.4% of the control), and both total and phosphorylated tau protein. It also decreased the activity of c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK) (37.3% and 60.7% levels of the control, respectively), which are tau kinases. Of note, activation of protein phosphatase 2A (PP2A), which is a tau phosphatase, was also observed after CQ treatment. Fractionation experiments demonstrated a reduction of oligomeric tau in the tris insoluble, sarkosyl soluble fraction by CQ treatment. CQ also decreased caspase-cleaved tau, which accelerated the aggregation of tau protein. CQ activated autophagy and proteasome pathways, which are considered important for the degradation of tau protein. Although further studies are needed to elucidate the mechanisms responsible for the effects of CQ on tau, CQ may shed light on possible AD therapeutics.

The The Effects of Centella asiatica Extract (CAE) on Methamphetamine-Induced Neurotoxicity via Human Neuroblastoma Cell Line

Methamphetamine (METH) was reported to caused neurotoxicity and cell death, in vitro. Centella asiatica or ‘pegaga’ is a native tropical herb with antioxidant and neuroprotective activities. Although the effects of Centella asiatica against oxidative stress and neuronal cell death have been reported in previous studies, however, the potential effects of Centella asiatica against psychostimulant methamphetamine (METH) are limited. Therefore, this study was aimed to evaluate the effects of Centella asiatica extract (CAE) against METH on all-trans retinoic acid, RA-differentiated human neuroblastoma, SH-SY5Y cells. The RA-differentiated SH-SY5Y cells were used to resemble dopaminergic neuronal-like cells. Cell viability was quantitatively assessed by 3-(4,5-dimethylthiazol-2-yl)-2 tetrazolium bromide, MTS assay. CAE at varying concentrations from 1pg/mL to 1mg/mL significantly decreased the viability of the undifferentiated SH-SY5Y cells in a concentration-dependent manner. At 1mg/mL of CAE, significantly increased the viability of differentiated SH-SY5Y cells. Meanwhile, CAE at 100µg/mL and 1mg/mL significantly reversed the METH-induced neuronal cell death. The results revealed that promising treatment of CAE on METH-induced neurotoxicity is mediated by its high content of asiaticoside, asiatic acid, madecassoside and madecassic acid. Taken together, this study may suggest CAE as a potential therapeutic treatment for METH-induced neurotoxicity, in vitro.

Model Identifies Genetic Predisposition of Alzheimer's Disease as Key Decider in Cell Susceptibility to Stress.

Accumulation of unfolded/misfolded proteins in neuronal cells perturbs endoplasmic reticulum homeostasis, triggering a stress cascade called unfolded protein response (UPR), markers of which are upregulated in Alzheimer’s disease (AD) brain specimens. We measured the UPR dynamic response in three human neuroblastoma cell lines overexpressing the wild-type and two familial AD (FAD)-associated mutant forms of amyloid precursor protein (APP), the Swedish and Swedish-Indiana mutations, using gene expression analysis. The results reveal a differential response to subsequent environmental stress depending on the genetic background, with cells overexpressing the Swedish variant of APP exhibiting the highest global response. We further developed a dynamic mathematical model of the UPR that describes the activation of the three branches of this stress response in response to unfolded protein accumulation. Model-based analysis of the experimental data suggests that the mutant cell lines experienced a higher protein load and subsequent magnitude of transcriptional activation compared to the cells overexpressing wild-type APP, pointing to higher susceptibility of mutation-carrying cells to stress. The model was then used to understand the effect of therapeutic agents salubrinal, lithium, and valproate on signalling through different UPR branches. This study proposes a novel integrated platform to support the development of therapeutics for AD.

Reactive Oxygen Species (ROS) Are Not a Key Determinant for Zika Virus-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells.

Introduction: ZIKV is a highly neurotropic virus that can cause the death of infected neuroprogenitor cells through mitochondrial damage and intrinsic apoptotic signaling. In this context, the role of reactive oxygen species (ROS) in neuronal cell death caused by ZIKV still remains elusive. Objective: We aimed at evaluating the role of these cellular components in the death of human undifferentiated neuroblastoma cell line infected with ZIKV. Results: ZIKV infection resulted in the extensive death of SH-SY5Y cells with the upregulation of several genes involved in survival and apoptotic responses as well as the colocalization of mitochondrial staining with ZIKV Envelope (E) protein. Notably, levels of intracellular reactive oxygen species (ROS) were not altered during ZIKV infection in undifferentiated SH-SY5Y cells, and consistent with these results, the treatment of infected cells with the widely studied ROS scavenger N-acetylcysteine (NAC) did not prevent cell death in these cells. Conclusion: Altogether, our results suggest that excessive ROS production is not the main trigger of SH-SY5Y cells death in ZIKV infection.

Novel Hydroxytyrosol-Donepezil Hybrids as Potential Antioxidant and Neuroprotective Agents.

It is well-accepted that the endogenous antioxidant protection system progressively decays in elderly people, and that the oxidative stress contributes to different neurodegenerative disorders such as Alzheimer’s Diseases (AD). The lower incidence of AD in countries which feature the Mediterranean Diet was associated to the high consumption of extra virgin olive oil and its polyphenolic fraction, in particular hydroxytyrosol. The protective role of these bio-phenols against oxidative stress, suggested that we combine their antioxidant/free radical scavenging activity with donepezil, an active ingredient which has just been approved for the treatment of AD. Different synthetic strategies were tested to conjugate the two different synthons in good yields. Additionally, a nitro-hydroxytyrosol derivative was synthesized to extend the application to other neurodegeneration inflammatory models. Then, their bioactivity was measured in different chemical and biological tests on a human neuroblastoma cell line (SHSY-5Y). Remarkable results on cell viability and the regulation of the redox state of cells were obtained. All hybrids showed negligible cell death under 1 μM and are stable and non toxic. Reactive oxygen species (ROS) measurements showed that the nitro-hybrid was the more effective one at reducing the ROS amount to physiological values. Then, in light of the bio-metal hypothesis of diverse neurodegenerative disorders, we tested these new compounds on the chelation properties of redox-active metals. The nitro-hybrid was able to chelate all of the tested metal cations, suggesting that we propose it as potential lead compound for a new class of neuroprotective antioxidant agents.

A novel in vitro assay model developed to measure both extracellular and intracellular acetylcholine levels for screening cholinergic agents.

Cholinergic neurons utilize choline (Ch) to synthetize acetylcholine (ACh) and contain a high-affinity Ch transporter, Ch acetyltransferase (ChAT), ACh receptors, and acetylcholinesterase (AChE). As the depletion or malfunction of each component of the cholinergic system has been reported in patients with dementia, many studies have sought to evaluate whether treatment candidates affect each of the cholinergic components. The associated changes in the cholinergic components may be reflected by intra- or extra-cellular ACh levels, with an increase in extracellular ACh levels occurring following AChE inhibition. We hypothesized that increases in intracellular ACh levels can be more sensitively detected than those in extracellular ACh levels, thereby capturing subtle effects in the cholinergic components other than AChE. The objective of this study was to test this hypothesis. We developed an in vitro model to measure both extracellular and intracellular ACh levels using the human cholinergic neuroblastoma cell line, LA-N-2, which have been reported to express Ch transporter, ChAT, muscarinic ACh receptor (mAChR), and AChE. With this model, we evaluated several drug compounds and food constituents reported to improve cholinergic function through various mechanisms. In addition, we conducted western blotting to identify the subtype of mAChR that is expressed on the cell line. Our cell-based assay system was capable of detecting increases in extracellular ACh levels induced by an AChE inhibitor at relatively high doses, as well as increases in intracellular ACh levels following the administration of lower AChE-inhibitor doses and an mAChR agonist. Moreover, increases in intracellular ACh levels were observed even after treatment with food constituents that have different mechanisms of action, such as Ch provision and ChAT activation. In addition, we revealed that LA-N-2 cells expressed mAChR M2. The findings support our hypothesis and indicate that the developed assay model can broadly screen compounds from drugs to food ingredients, with varying strengths and mechanisms of action, to develop treatments for ACh-relevant phenomena, including dementia and aging-related cognitive decline.

Development of an in vitro neuroblastoma 3D model and its application for sterigmatocystin-induced cytotoxicity testing

Given the increasing importance of establishing better risk assessments for mycotoxins, novel in vitro tools for the evaluation of their toxicity are mandatory. In this study, an in vitro 3D spheroid model from SH-SY5Y cells, a human neuroblastoma cell line, was developed, optimized and characterized to test the cytotoxic effects caused by the mycotoxin sterigmatocystin (STE). STE induced a concentration- and time-dependent cell viability decrease in spheroids. Spheroids displayed cell disaggregation after STE exposure, increasing in a dose-dependent manner and over time. STE also induced apoptosis as confirmed by immunofluorescence staining and Western blot. Following the decreased proliferation and increased apoptosis, STE cytostasis effects were observed by migration assays both in 2D and 3D cell culture. Increased ROS generation, as well as DNA damage were also observed. Taken together, these data highlight the cytotoxic properties of STE and suggest that cell culture models play a pivotal role in the toxicological risk assessment of mycotoxins. The evaluation of cytotoxicity in spheroids (3D) rather than monolayer cultures (2D) is expected to more accurately reflect in vivo-like cell behaviour.

Protective Effect of Butylphthalide on Neuronal Apoptosis in Parkinson Rats and Its Effect on miR-146a-5p Expression and Phosphatidylinositide 3-Kinases/Protein Kinase B Pathway

80 male Wistar rats were stochastically assigned to Sham + Vehicle group, Sham + BUT group, PD + Vehicle group and PD + BUT group. Rotenone PD model rats were prepared by subcutaneous injection of rotenone sunflower oil emulsion 2 mg/(kg · d) for 5 consecutive weeks. Butylphthalide 80 mg/(kg · d) were given to the rats in Sham + BUT group and PD + BUT group by gavage from the first day of rotenone injection for 5 weeks. Subsequently, the motor retardation ability and the morphological changes of the substantia nigra (SN) of each group were evaluated. Meanwhile, the levels of neuronal injury, apoptosis, inflammation and oxidative stress in each group of rats were assayed. The impact of BUT treatment on miR-146a-5p expression and PI3K/AKT signal pathway in rat brain tissue was assayed. Finally, by constructing a PD cell model of the neurotoxin 6-hydroxydopamine (6-OHDA)-treated human neuroblastoma cell line SH-SY5Y, the in vitro anti-PD pharmacological effect of BUT was further verified.

Drug Repurposing Patent Applications April–June 2021

Drug Repurposing Patent Applications April–June 2021

Acetonitrile adduct analysis of underivatised amino acids offers improved sensitivity for hydrophilic interaction liquid chromatography tandem mass-spectrometry

LC-MS/MS method development for native amino acid detection can be problematic due to low ionisation efficiencies, in source fragmentation, potential for cluster ion formation and incorrect application of chromatography techniques. This has led to the majority of the scientific community derivatising amino acids for more sensitive analysis. Derivatisation has several benefits including reduced signal-to-noise ratios, more efficient ionisation, and a change in polarity, allowing the use of reverse phase chromatography. However, derivatisation of amino acids can be expensive, requires additional sample preparation steps, is more time consuming and increases sample instability, due to the most derivatised amino acids only be stable for finite amount of time. While showing initial promise, development of reliable hydrophilic interaction liquid chromatography (HILIC) separation methods has presented difficulties for the analyst including irreproducible separation and poor sensitivity. This study aimed to find a means to improve the detection sensitivity of the 20 protein amino acids by HILIC-MS/MS. We describe the use of previously undescribed amino acid-acetonitrile (ACN) adducts to improve detection of 16 out of the 20 amino acids. While all amino acids examined did form an ACN adduct, 4 had low intensity adduct formation compared to their protonated state, 3 of which are classified as basic amino acids. For 15 of the 20 amino acids tested, we used the ACN adduct for both quantification and qualification ions and demonstrated a significant enhancement in signal-to-noise ratio, ranging from 23 to 1762% improvement. Lower LODs, LOQs and lower ranges of linearity were also achieved for these amino acids. The optimised method was applied to a human neuroblastoma cell line (SH-SY5Y) with the potential to be applied to other complex sample types. The improved sensitivity this method offers simplifies sample preparation and reduces the costs of amino acid analysis compared to those methods that rely on derivatisation for sensitivity.

Chemical constituents isolated from the aerial parts of Swertia pseudochinensis and their potential neuroprotective effects

Abstract Objective: Swertia pseudochinensis, an annual herb of the genus Swertia in the family Gentianaceae. Some constituents and extracts from the Swertia genus have been recently reported to possess neuroprotective effects, suggesting their potential utility in the prevention and treatment of Parkinson disease (PD). The aim of this work is to identify the chemical constituents and evaluate the potential biological activists of Swertia pseudochinensis. Methods: The phytochemicals from the aerial parts of S. pseudochinensis were isolated and purified by silica gel, Sephadex LH-20 gel, semi-preparative high-performance liquid chromatography, and identified by the spectroscopic methods. All compounds were evaluated for their potential neuroprotective effects against 1-methyl-4-phenylpyridinium-induced apoptosis in the SH-SY5Y human neuroblastoma cell line using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. Then, we performed an enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery and investigated the mechanisms by which bellidifolin regulates neurodegenerative disease. Results: Two new anthraquinone, 1,5,6-trimethoxy-2-hydroxy-3-methy-anthraquinone (1) and 1,5,6,8-tetramethoxy-2-hydroxy-3-methyanthraquinone (2), together with nine known including 7-O-β-d-glucopyranosyl-1,8-dihydroxy-3-methoxyxanthone (3), gentisin (4), swertianolin (5), bellidifolin (6), gentiacaulein (7), norswertianolin (8), 5-O-β-d-glucopyranosyl-1,3,8-trihydroxyapatone (9), 1-hydroxy-3,5,8-trimethoxyxanthone (10), and aurantio-obtusin (11), were isolated and compounds 6–8 and 10 exhibited neuroprotective effects at a concentration of 50 μmol/L. Among them, bellidifolin showed significant protective activity, and might have potential as a neuroprotective agent for the treatment of PD, possibly by acting on oxidative damage and reactive oxygen species. Conclusions: These findings indicate that further research on the genus Swertia and its bioactive constituents toward neurodegenerative disorders could be extremely rewarding.