Isolation,cultivation and identification of brain glioma stem cells by magnetic bead sorting

This study describes a detailed process for obtaining brain glioma stem cells from freshly dissected human brain glioma samples using an immunomagnetic bead technique combined with serum-free media pressure screening. Furthermore, the proliferation, differentiation and self-renewal biological features of brain glioma stem cells were identified. Results showed that a small number of CD133 positive tumor cells isolated from brain glioma samples survived as a cell suspension in serum-free media and proliferated. Subcultured CD133 positive cells maintained a potent self-renewal and proliferative ability, and expressed the stem cell-specific markers CD133 and nestin. After incubation with fetal bovine serum, the number of glial fibrillary acidic protein and microtubule associated protein 2 positive cells increased significantly, indicating that the cultured brain glioma stem cells can differentiate into astrocytes and neurons. Western blot analysis showed that tumor suppressor phosphatase and tensin homolog was highly expressed in tumor spheres compared with the differentiated tumor cells. These experimental findings indicate that the immunomagnetic beads technique is a useful method to obtain brain glioma stem cells from human brain tumors.

Reversal Effects of Ivermectin and Moxidectin on Multidrug Resistance in C6/adr Cells in vitro

Multidrug resistance(MDR)is a serious obstacle encountered in cancer treatment.This study was performed to explore the reversal MDR activity of ivermectin(IVM)from avermectin family and moxidectin(MOX)belonging to milbemycin family.The two compounds(5μmol•L-1)showed strong potency to increase adriamycin cytotoxicity toward adriamycin-resistant rat glioma cells C6/adr with fold reversal(FR)of 31.02 and 13.40,respectively.In addition,the mechanisms of them on p-glycoprotein(P-gp)-mediated MDR demonstrated that the two compounds significantly increased the intracellular accumulations of adriamycin and Rh123 via inhibiting P-gp efflux.Based on the analysis of P-gp,MDR1 and MRP1 gene expressions by using immunofluorescence flow cytometry and RT-PCR,the results revealed that the two compounds could down regulate the expression of P-gp,and that MDR1 and MRP1 gene expressions were down regulated.These findings suggested that ivermectin and moxidectin probably represented potent agents for reversing MDR in cancer therapy,and especially ivermectin was a better modulator.

Dantrolene enhances the protective effect of hypothermia on cerebral cortex neurons

Therapeutic hypothermia is the most promising non-pharmacological neuroprotective strategy against ischemic injury. However, shivering is the most common adverse reaction. Many studies have shown that dantrolene is neuroprotective in in vitro and in vivo ischemic injury models. In addition to its neuroprotective effect, dantrolene neutralizes the adverse reaction of hypothermia. Dantrolene may be an effective adjunctive therapy to enhance the neuroprotection of hypothermia in treating ischemic stroke. Cortical neurons isolated from rat fetuses were exposed to 90 minutes of oxygen-glucose deprivation followed by reoxygenation. Neurons were treated with 40 μM dantrolene, hypothermia（at 33°C）, or the combination of both for 12 hours. Results revealed that the combination of dantrolene and hypothermia increased neuronal survival and the mitochondrial membrane potential, and reduced intracellular active oxygen cytoplasmic histone-associated DNA fragmentation, and apoptosis. Furthermore, improvements in cell morphology were observed. The combined treatment enhanced these responses compared with either treatment alone. These findings indicate that dantrolene may be used as an effective adjunctive therapy to enhance the neuroprotective effects of hypothermia in ischemic stroke.

NADPH oxidase 2 does not contribute to early reperfusion-associated reactive oxygen species generation following transient focal cerebral ischemia

Excess production of reactive oxygen species （ROS） critically contributes to occurrence of reperfusion injury, the paradoxical response of ischemic brain tissue to restoration of cerebral blood flow. However, the enzymatic sources of ROS generation remain to be unclear. This study examined Nox2-ontaining NADPH oxidase （Nox2） expression and its activity in ischemic brain tissue following post-ischemic reperfusion to clarify the mechanism of enzymatic reaction of ROS. Male Sprague-Dawley rats were subjected to 90-minute middle cerebral artery occlusion, followed by 3 or 22.5 hours of reperfusion. Quantitative reverse transcriptase PCR and western blot assay were performed to measure mRNA and protein expression of Nox2. Lucigenin fluorescence assays were performed to assess Nox activity. Our data showed that Nox2 mRNA and protein expression levels were significantly increased （3.7-fold for mRNA and 3.6-fold for protein） in ischemic brain tissue at 22.5 hours but not at 3 hours following post-ischemic reperfusion. Similar results were obtained for the changes of NADPH oxidase activity in ischemic cerebral tissue at the two reperfusion time points. Our results suggest that Nox2 may not contribute to the early burst of reperfusion-related ROS generation, but is rather an important source of ROS generation during prolonged reperfusion.

Dual neuronal response to tumor necrosis factor-alpha following spinal cord injury

BACKGROUND： Numerous studies have shown that tumor necrosis factor α （TNF-α） is closely correlated with spinal cord injury （SCI）, but the mechanisms of TNF-α and therapeutic treatments for SCI are still poorly understood. OBJECTIVE： To determine the role of TNF-α in the pathogenesis of SCI. DESIGN, TIME AND SETTING： An in vivo experiment based on genetically engineered animals was performed at the Medical University of South Carolina, Charleston, South Carolina, USA, between June 2007 and October 2008. MATERIALS： TNF-α transgenic rats （Xenogen Biosciences in Cranbury, New Jersey, USA） were utilized in this study. METHODS： TNF-α transgenic （tg） and wild-type （WT） rats underwent a complete single-level laminectomy at the 10^th thoracic vertebra （T10）. MAIN OUTCOME MEASURES： Motor function of rat hindlimb was assessed using the Basso, Beattie, and Bresnahan hindlimb locomotor rating scale. Histological evaluation of spinal cord tissue loss was conducted. Immunohistochemistry for astrocytes, microglia/macrophages, and TNF receptors （TNFRs） was performed on spinal cord tissue sections. TNF-α mRNA expression was detected by real-time polymerase chain reaction. The concentrations of nerve growth factor （NGF） and brain-derived neurotrophic factor （BDNF） in the supernatant were determined using an enzyme-linked immunosorbent assay kit for rat NGF or BDNF, respectively. The rats were injected subcutaneously with etanercept to verify that TNF-α was the direct effect of the modulation of behavioral and neurodegenerative outcomes in the TNF-α tg rats. RESULTS： TNF-α tg rats showed higher expression of TNF-α mRNA in the spinal cord prior to SCI. TNF-α tg rats showed worse motor deficits than WT rats in the acute period （〈 3 days） after SCI （P 〈 0.01）, while in the chronic period, TNF-α tg rats exhibited persistent elevated baseline levels of TNF-α mRNA and improved recovery in motor function and tissue healing compared to WT rats （P 〈 0.01 ）. Following SCI, the number of microglia/macrophages in TNF-α tg rat was always greater than in WT rat （P 〈 0.01）. There were no significant differences in NGF and BDNF levels in the supernatant of spinal cord homogenates. TNFR1 expression was significantly greater in the TNF-α tg rats compared to the WT rats （P 〈 0.01）. However, TNFR2 expression did not reveal a significant increase in the TNF-α tg rats compared to the WT rats. Finally, treatment with etanercept reduced injury acutely, but exacerbated the injury chronically. CONCLUSION： Overexpression of TNF-α is deleterious in the acute phase, but beneficial in the chronic phase in the response to SCI. The role of TNF-α post-injury may depend on TNF-α expression in the spinal cord and its differential binding to TNFRI. Our observations may have clinical relevance that antagonists or inhibitors of TNF-α could be administered within the early time window post-injury, and appropriate amounts of TNF-α could be administered during the chronic stage, in order to improve the final neurological recovery in patients with SCI.

Tumor Suppressor miR-637 Is Associated with Cellular Migration, Invasion, and Glioma Diagnosis

Objective: Abnormal miRNA expression is observed in several human tumors;moreover, normal cell regulation can be disrupted by tumor-suppressive or oncogenic miRNAs. We aimed to investigate the role of miR-637 in gliomas. Methods: We assessed miR-637 expression in 98 and 16 gliomas and non-tumoral brain tissues, respectively, using in situ hybridization. We calculated receiver operating characteristic curves to determine the specificity and sensitivity of miR-637 biomarkers. Next, the effects of miR-637 on glioma cell migration and invasion were determined by using the transwell assay. Candidate target genes were identified through Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. Results: There was significant miR-637 downregulation in glioma tissues (P < 0.001). Further, it showed potential as a diagnostic biomarker for gliomas. In addition, miR-637 suppressed glioma cell migration and invasion. Conclusions: Our findings suggest that miR-637 inhibits glioma invasion and migration and could be a potential diagnostic marker for gliomas. Future studies should examine the potential mechanisms underlying miR-637 as a diagnostic marker and therapeutic target for gliomas.

The increasing expression of GPX7 related to the malignant clinical features leading to poor prognosis of glioma patients

Background:Glioma is the most common malignant brain tumor in adults.The standard treatment scheme of glioma is surgical resection combined alternative radio-and chemotherapy.However,the outcome of glioma patients was unsatisfied.Here,we aimed to explore the molecular and biological function characteristics of GPX7 in glioma.Methods:The multidimensional data of glioma samples were downloaded from Chinese Glioma Genome Atlas(CGGA).RT-qPCR method was used to identify the expression status of GPX7.Kaplan–Meier curves and Cox regression analysis were used to explore the prognostic value of GPX7.Gene Set Enrichment Analysis(GSEA)was applied to investigate the GPX7-related functions in glioma.Results:The results indicated that the expression of GPX7 in glioma was higher compared to that in normal brain tissue.Univariate and multivariate Cox regression analyses confirmed that the expression value of GPX7 was an independent prognostic factor in glioma.The GSEA analysis showed that GPX7 was significantly enriched in the cell cycle pathway,ECM pathway,focal adhesion pathway,and toll-like receptor pathway.Conclusions:The GPX7 was recommended as an independent risk factor for patients diagnosed with glioma for the first time and GPX7 could be potentially used as the therapy target in future.Furthermore,we attempted to explore a potential biomarker for improving the diagnosis and prognosis of patients with glioma.

Exploration and functionalization of M1-macrophage extracellular vesicles for effective accumulation in glioblastoma and strong synergistic therapeutic effects

Glioblastoma multiforme(GBM)is a highly aggressive brain tumor with an extremely low survival rate.New and effective approaches for treatment are therefore urgently needed.Here,we successfully developed M1-like macrophage-derived extracellular vesicles(M1EVs)that overcome multiple challenges via guidance from two macrophage-related observations in clinical specimens from GBM patients:enrichment of M2 macrophages in GBM;and origination of a majority of infiltrating macrophage from peripheral blood.To maximize the synergistic effect,we further functionalized the membranes of M1EVs with two hydrophobic agents(the chemical excitation source CPPO(C)and the photosensitizer Ce6(C))and loaded the hydrophilic hypoxiaactivated prodrug AQ4N(A)into the inner core of the M1EVs.After intravenous injection,the inherent nature of M1-derived extracellular vesicles CCA-M1EVs allowed for blood-brain barrier penetration,and modulated the immunosuppressive tumor microenvironment via M2-to-M1 polarization,which increased hydrogen peroxide(H_(2)O_(2))levels.Furthermore,the reaction between H_(2)O_(2) and CPPO produced chemical energy,which could be used for Ce6 activation to generate large amounts of reactive oxygen species to achieve chemiexcited photodynamic therapy(CDT).As this reaction consumed oxygen,the aggravation of tumor hypoxia also led to the conversion of non-toxic AQ4N into toxic AQ4 for chemotherapy.Therefore,CCA-M1EVs achieved synergistic immunomodulation,CDT,and hypoxia-activated chemotherapy in GBM to exert a potent therapeutic effect.Finally,we demonstrated the excellent effect of CCA-M1EVs against GBM in cell-derived xenograft and patient-derived xenograft models,underscoring the strong potential of our highly flexible M1EVs system to support multi-modal therapies for difficult-to-treat GBM.

Urine biomarkers discovery by metabolomics and machine learning for Parkinson’s disease diagnoses

Parkinson’s disease(PD)is a complex neurological disorder that typically worsens with age.A wide range of pathologies makes PD a very heterogeneous condition,and there are currently no reliable diagnostic tests for this disease.The application of metabolomics to the study of PD has the potential to identify disease biomarkers through the systematic evaluation of metabolites.In this study,urine metabolic profiles of 215 urine samples from 104 PD patients and 111 healthy individuals were assessed based on liquid chromatography-mass spectrometry.The urine metabolic profile was first evaluated with partial leastsquares discriminant analysis,and then we integrated the metabolomic data with ensemble machine learning techniques using the voting strategy to achieve better predictive performance.A combination of 8-metabolite predictive panel performed well with an accuracy of over 90.7%.Compared to control subjects,PD patients had higher levels of 3-methoxytyramine,N-acetyl-l-tyrosine,orotic acid,uric acid,vanillic acid,and xanthine,and lower levels of 3,3-dimethylglutaric acid and imidazolelactic acid in their urine.The multi-metabolite prediction model developed in this study can serve as an initial point for future clinical studies.