Deamination-triggered exponential signal amplification for chemiluminescent detection of cytosine deaminase at the single-cell level.

We construct a sensitive chemiluminescent biosensor for sensitive detection of cytosine deaminase APOBEC3A based on deamination-triggered exponential signal amplification. This biosensor displays good specificity and high sensitivity, and it can screen APOBEC3A inhibitors and measure endogenous APOBEC3A at the single-cell level, with prospective applications in disease diagnostics and therapy.

[Achene morphology cluster analysis of Taraxacum F. H. Wigg. from northeast China and molecule systematics evidence determined by SRAP].

The achenes morphological and micro-morphological characteristics of six species of genus Taraxacum from northeastern China as well as SRAP cluster analysis were observed for their classification evidences. The achenes were observed by microscope and EPMA. Cluster analysis was given on the basis of the size, shape, cone proportion, color and surface sculpture of achenes. The Taraxacum inter-species achene shape characteristic difference is obvious, particularly spinulose distribution and size, achene color and achene size; with the Taraxacum plant achene shape the cluster method T. antungense Kitag. and the T. urbanum Kitag. should combine for the identical kind; the achene morphology cluster analysis and the SRAP tagged molecule systematics's cluster result retrieves in the table with "the Chinese flora". The class group to divide the result is consistent. Taraxacum plant achene shape characteristic stable conservative, may carry on the inter-species division and the sibship analysis according to the achene shape characteristic combination difference; the achene morphology cluster analysis as well as the SRAP tagged molecule systematics confirmation support dandelion classification result of "the Chinese flora".

[Effects of electromagnetic pulse exposure on the morphological change and excretion function of BV-2 cells and possible mechanism].

OBJECTIVE: To study the effects of electromagnetic pulse (EMP) exposure on the morphological change and excretion functions of mouse microglia (BV-2) cells and possible mechanism. METHODS: BV-2 cells were divided into two groups: the group exposed to EMP at 200 kV/m for 200 pulses and sham exposure group. At 1, 6, 12 and 24 hour after exposure the cells and culture supernatant were collected. Cellular morphological change was observed under invert microscope, the levels of TNF-α, IL-1ß and IL-10 in culture supernatant were determined by enzyme-linked immunosorbent assay (ELISA), nitric oxide (NO) and reactive oxygen species (ROS) were detected by nitrate reductase method and DCFH-DA probe, respectively. The protein and phosphorylation levels of ERK, JNK and p38 were measured by Western Blot method. After the cells pre-treated with the inhibitor of p38 (SB203580) were exposed to EMP, the levels of NO and ROS in culture supernatant were detected. RESULTS: It was found that the large ameboid shape appeared in some microglia cells exposed to EMP for 1, 6 and 12 h. Moreover, the number of microglia cells with ameboid shape increased significantly at 1 h, 6 h and 12 h after EMP exposure compared with sham group (P < 0.05). The levels of cytokines, such as TNF-α, IL-1ß and IL-10, in culture supernatant did not change obviously after EMP exposure. The levels of NO and ROS increased significantly at 1h after EMP exposure, reached the peak at 6 h, began to recover at 12 h and recovered to sham group level at 24 h (P < 0.05). Western blot results showed that the protein and protein phosphorylation levels of ERK and JNK did not change significantly after EMP exposure, however, the protein and protein phosphorylation levels of p38 increased obviously at 1 h and 6 h after EMP exposure, compared with sham group (P < 0.05). In addition, the pretreatment of p38 inhibitor (SB203580) significantly decreased NO and ROS production induced by EMP. CONCLUSION: EMP exposure may activate microglia cells and promote the production of NO and ROS in mouse microglia cells, and p38 pathway is involved in this process.

[Effects of electromagnetic pulse exposure on the permeability of inner blood-retinal barrier model in vitro].

OBJECTIVE: To establish the inner blood-retinal barrier (BRB) model in vitro by co-culturing RF/6A cells and C6 cells and to investigate the effects of EMP (200 kV/m, 200 pulses) exposure on the permeability of the inner BRB model in vitro. METHODS: RF/6A cells and C6 cells were co-cultured on transwell, and the characteristic of the inner BRB model was assessed by detecting transendothelial electrical resistance (TEER) and the permeability of horseradish peroxidase (HRP). The co-cultured model was exposed or sham exposed to the EMP (200 kV/m 200 pulses) for 0.5, 3, 6, 12, 24 h in vitro, then TEER and the permeability of HRP were measured for studying the effects of EMP on the permeability of inner BRB model in vitro. RESULTS: TEER value (145 Ωcm(2)) of the co-culturing inner BRB model significantly increased, as compared to that of RF/6A cells alone model (P < 0.05) on the 6th day after inoculation. There was significant difference of permeability of HRP between the co-culturing inner BRB model and RF/6A cells alone model (P < 0.05). The ability of inhibiting large molecular materials in the co-culturing inner BRB model enhanced. The TEER value decreased and the permeability of HRP increased as compared to the sham group at 0.5, 3, 6 h after the exposure. CONCLUSION: The inner BRB model by co-culturing RF/6A cells and C6 cells in vitro is efficient and suitable to study the alterations of the restricted permeability function of the inner BRB. EMP (200 kV/m for 200 pulses) could induce the enhanced permeability of the inner BRB model in vitro.

Assay of dried blood spot from finger prick for sodium valproate via ink auxiliary headspace gas chromatography mass spectrometry.

Sodium valproate is the most commonly used antiepileptic drug that patients need to keep taking over a long period of time or on a permanent basis. Its blood concentration should be accurately detected to avoid toxicity or side-effects, especially for children and the aged. Dried blood spot (DBS) sampling from finger prick is a minimally invasive and patient-friendly procedure for blood collection. However, there are few studies about rapid detection of sodium valproate in DBS samples in current literatures. In this work, we developed an ink auxiliary headspace gas chromatography mass spectrometry (GC-MS) strategy for direct detection of sodium valproate in DBS from epilepsy patients, which does not need extra solvent extraction or elution. It was discovered that carbon black ink could provide better capacity of heat absorption and dissociation, and higher quality of headspace sampling. The detection sensitivity has been improved with reported headspace GC-MS methods, and the limit of quantitation could reach to 200 ng/mL. Finally, this strategy was practically applied to quantify sodium valproate in DBS samples from 29 epilepsy patients. The result showed higher accuracy with lower relative errors by comparing with the clinical immunoassay results. In conclusion, we developed a direct detection method for DBS samples that is suitable for high-throughput clinical test with great potential for clinical application.

Microwave radiation leading to shrinkage of dendritic spines in hippocampal neurons mediated by SNK-SPAR pathway.

The popularization of microwave raised concerns about its influence on health including cognitive function which is associated greatly with dendritic spines plasticity. SNK-SPAR is a molecular pathway for neuronal homeostatic plasticity during chronically elevated activity. In this study, Wistar rats were exposed to microwaves (30 mW/cm2 for 6 min, 3 times/week for 6 weeks). Spatial learning and memory function, distribution of dendritic spines, ultrastructure of the neurons and their dendritic spines in hippocampus as well as the related critical molecules of SNK-SPAR pathway were examined at different time points after microwave exposure. There was deficiency in spatial learning and memory in rats, loss of spines in granule cells and shrinkage of mature spines in pyramidal cells, accompanied with alteration of ultrastructure of hippocampus neurons. After exposure to 30 mW/cm2 microwave radiation, the up-regulated SNK induced decrease of SPAR and PSD-95, which was thought to cause the changes mentioned above. In conclusion, the microwave radiation led to shrinkage and even loss of dendritic spines in hippocampus by SNK-SPAR pathway, resulting in the cognitive impairments.

Electromagnetic pulse activated brain microglia via the p38 MAPK pathway.

Previously, we found that electromagnetic pulses (EMP) induced an increase in blood brain barrier permeability and the leakage of albumin from blood into brain tissue. Albumin is known to activate microglia cells. Thus, we hypothesised that microglia activation could occur in the brain after EMP exposure. To test this hypothesis, the morphology and secretory function of microglia cells, including the expression of OX-42 (a marker of microglia activation), and levels of TNF-α, IL-10, IL-1ß, and NO were determined in the rat cerebral cortex after EMP exposure. In addition, to examine the signalling pathway of EMP-induced microglia activation, protein and phosphorylated protein levels of p38, JNK and ERK were determined. It was found that the expression of OX-42increased significantly at 1, 6 and 12h (p<0.05) and recovered to the sham group level at 24h after EMP exposure. Levels of NO, TNF-α and IL-10 also changed significantly in vivo and in vitro after EMP exposure. The protein level of p38 and phosphorylated p38 increased significantly after EMP exposure (p<0.05) and recovered to sham levels at 12 and 24h, respectively. Protein and phosphorylated protein levels of ERK and JNK did not change. SB203580 (p38 inhibitor) partly prevented the change in NO, IL-10, IL-1ß, TNF-α levels induced by EMP exposure. Taken together, these results suggested that EMP exposure (200kV/m, 200 pulses) could activate microglia in rat brain and affect its secretory function both in vivo and in vitro, and the p38 pathway is involved in this process.

Identification of a Novel Rat NR2B Subunit Gene Promoter Region Variant and Its Association with Microwave-Induced Neuron Impairment.

Microwave radiation has been implicated in cognitive dysfunction and neuronal injury in animal models and in human investigations; however, the mechanism of these effects is unclear. In this study, single nucleotide polymorphism (SNP) sites in the rat GRIN2B promoter region were screened. The associations of these SNPs with microwave-induced rat brain dysfunction and with rat pheochromocytoma-12 (PC12) cell function were investigated. Wistar rats (n = 160) were exposed to microwave radiation (30 mW/cm(2) for 5 min/day, 5 days/week, over a period of 2 months). Screening of the GRIN2B promoter region revealed a stable C-to-T variant at nucleotide position -217 that was not induced by microwave exposure. The learning and memory ability, amino acid contents in the hippocampus and cerebrospinal fluid, and NR2B expression were then investigated in the different genotypes. Following microwave exposure, NR2B protein expression decreased, while the Glu contents in the hippocampus and CSF increased, and memory impairment was observed in the TT genotype but not the CC and CT genotypes. In PC12 cells, the effects of the T allele were more pronounced than those of the C allele on transcription factor binding ability, transcriptional activity, NR2B mRNA, and protein expression. These effects may be related to the detrimental role of the T allele and the protective role of the C allele in rat brain function and PC12 cells exposed to microwave radiation.

Alterations of cognitive function and 5-HT system in rats after long term microwave exposure.

The increased use of microwaves raises concerns about its impact on health including cognitive function in which neurotransmitter system plays an important role. In this study, we focused on the serotonin system and evaluated the long term effects of chronic microwave radiation on cognition and correlated items. Wistar rats were exposed or sham exposed to 2.856GHz microwaves with the average power density of 5, 10, 20 or 30mW/cm(2) respectively for 6min three times a week up to 6weeks. At different time points after the last exposure, spatial learning and memory function, morphology structure of the hippocampus, electroencephalogram (EEG) and neurotransmitter content (amino acid and monoamine) of rats were tested. Above results raised our interest in serotonin system. Tryptophan hydroxylase 1 (TPH1) and monoamine oxidase (MAO), two important rate-limiting enzymes in serotonin synthesis and metabolic process respectively, were detected. Expressions of serotonin receptors including 5-HT1A, 2A, 2C receptors were measured. We demonstrated that chronic exposure to microwave (2.856GHz, with the average power density of 5, 10, 20 and 30mW/cm(2)) could induce dose-dependent deficit of spatial learning and memory in rats accompanied with inhibition of brain electrical activity, the degeneration of hippocampus neurons, and the disturbance of neurotransmitters, among which the increase of 5-HT occurred as the main long-term change that the decrease of its metabolism partly contributed to. Besides, the variations of 5-HT1AR and 5-HT2CR expressions were also indicated. The results suggested that in the long-term way, chronic microwave exposure could induce cognitive deficit and 5-HT system may be involved in it.

Electromagnetic-pulse-induced activation of p38 MAPK pathway and disruption of blood-retinal barrier.

The blood-retinal barrier (BRB) is critical for maintaining retina homeostasis and low permeability. In this study, we evaluated the effects of electromagnetic pulse (EMP) exposure on the permeability of BRB, alterations of tight junction (TJ) proteins of BRB and if any, involvement of mitogen-activated protein kinase (MAPK) pathway. Male Sprague-Dawley (SD) rats and RF/6A cells which were pretreated with or without MAPKs inhibitors were sham exposed or exposed to EMP at 200kV/m for 200 pulses. The alteration of BRB permeability was examined through fluorescence microscope and quantitatively assessed using Evans blue (EB) and endogenous albumin as tracers. The expressions of TJ proteins and some signaling molecules of MAPK pathway were measured by Western blots. The observations were that EMP exposure resulted in increased BRB permeability concurrent with the decreased expressions of occludin and claudin-5, which were correlated with the increased expressions of phospho-p38, phospho-JNK and phospho-ERK and could be blocked when pretreated with p38 MAPK inhibitor. Thus, the results suggested that the alterations of occludin and claudin-5 may play an important role in the disruption of TJs, which may lead to the transient breakdown of BRB after EMP exposure with the involvement of p38 MAPK pathway through phosphorylation of signaling molecules.