Xuemei Ma

Oxidative changes and apoptosis induced by 1800-MHz electromagnetic radiation in NIH/3T3 cells

Abstract To investigate the potential adverse effects of mobile phone radiation, we studied reactive oxygen species (ROS), DNA damage and apoptosis in mouse embryonic fibroblasts (NIH/3T3) after intermittent exposure (5u2009min on/10u2009min off, for various durations from 0.5 to 8u2009h) to an 1800-MHz GSM-talk mode electromagnetic radiation (EMR) at an average specific absorption rate of 2u2009W/kg. A 2′,7′-dichlorofluorescin diacetate fluorescence probe was used to detect intracellular ROS levels, immunofluorescence was used to detect γH2AX foci as a marker for DNA damage, and flow cytometry was used to measure apoptosis. Our results showed a significant increase in intracellular ROS levels after EMR exposure and it reached the highest level at an exposure time of 1u2009h (pu2009<u20090.05) followed by a slight decrease when the exposure continued for as long as 8u2009h. No significant effect on the number of γH2AX was detected after EMR exposure. The percentage of late-apoptotic cells in the EMR-exposed group was significantly higher than that in the sham-exposed groups (pu2009<u20090.05). These results indicate that an 1800-MHz EMR enhances ROS formation and promotes apoptosis in NIH/3T3 cells.

Oral intake of hydrogen-rich water ameliorated chlorpyrifos-induced neurotoxicity in rats.

Chronic exposure to low-levels of organophosphate (OP) compounds, such as chlorpyrifos (CPF), induces oxidative stress and could be related to neurological disorders. Hydrogen has been identified as a novel antioxidant which could selectively scavenge hydroxyl radicals. We explore whether intake of hydrogen-rich water (HRW) can protect Wistar rats from CPF-induced neurotoxicity. Rats were gavaged daily with 6.75mg/kg body weight (1/20 LD50) of CPF and given HRW by oral intake. Nissl staining and electron microscopy results indicated that HRW intake had protective effects on the CPF-induced damage of hippocampal neurons and neuronal mitochondria. Immunostaining results showed that the increased glial fibrillary acidic protein (GFAP) expression in astrocytes induced by CPF exposure can be ameliorated by HRW intake. Moreover, HRW intake also attenuated CPF-induced oxidative stress as evidenced by enhanced level of MDA, accompanied by an increase in GSH level and SOD and CAT activity. Acetylcholinesterase (AChE) activity tests showed significant decrease in brain AChE activity after CPF exposure, and this effect can be ameliorated by HRW intake. An in vitro study demonstrated that AChE activity was more intense in HRW than in normal water with or without chlorpyrifos-oxon (CPO), the metabolically-activated form of CPF. These observations suggest that HRW intake can protect rats from CPF-induced neurotoxicity, and the protective effects of hydrogen may be mediated by regulating the oxidant and antioxidant status of rats. Furthermore, this work defines a novel mechanism of biological activity of hydrogen by directly increasing the AChE activity.

Hydrogen-rich saline attenuates skin ischemia/reperfusion induced apoptosis via regulating Bax/Bcl-2 ratio and ASK-1/JNK pathway

INTRODUCTIONnMany pathways have been reported involving the effect of hydrogen-rich saline on protecting skin flap partial necrosis induced by the inflammation of ischemia/reperfusion injury. This study focused on the influence of hydrogen-rich saline treatment on apoptosis pathway of ASK-1/JNK and Bcl-2/Bax radio in I/R injury of skin flaps.nnnMETHODSnAdult male Sprague-Dawley rats were divided into three groups. Group 1 was sham surgery group, Group 2 and 3 were ischemia/reperfusion surgery treated with physiological saline and hydrogen-rich saline respectively. Blood perfusion of flap was measured by Laser doppler flowmeters. Hematoxylin and eosin staining was used to observe morphological changes. Early apoptosis in skin flap was observed through TUNEL staining and presented as the percentage of TUNEL-positive cells of total cells. pASK-1, pJNK, Bcl-2 and Bax were examined by immunodetection. In addition Bcl-2, Bax and caspase-3 were detected by qPCR. Caspase-3 activity was also measured.nnnRESULTSnCompared to the Group 2, tissues from the group 3 were observed with a high expression of Bcl-2 and a low expression of pASK-1, pJNK, and Bax, a larger survival area and a high level of blood perfusion. Hydrogen-rich saline ameliorated inflammatory infiltration and decreased cell apoptosis.nnnCONCLUSIONnThe results indicate that hydrogen-rich saline could ameliorate ischemia/reperfusion injury and improve flap survival rate by inhibiting the apoptosis factor and, at the same time, promoting the expression of anti-apoptosis factor.

Quantification of Epstein-Barr Virus DNA in Patients with Idiopathic Orbital Inflammatory Pseudotumor

Inflammatory pseudotumors (IPT) are soft tissue tumors that include a diverse group of lesions characterized by inflammatory cell infiltration and variable fibrotic responses. Idiopathic orbital inflammatory pseudotumors (IOIP) are IPTs of unknown etiology that develop in the orbit. Due to the lack of well-defined pathogenic mechanisms, diagnosis and treatment of this disease remain a significant challenge. Epstein-Barr virus (EBV) infection, which causes significant lymphocyte infiltration, has been proposed to be involved in IOIP. This study tries to validate the relationship between EBV infection and the development of IOIP. Sixteen IOIP tissue samples were obtained from patients during surgical resection of the lesion. One Graves ophthalmopathy tissue sample and 20 normal donors plasma serves as controls. The plasma level of five EBV antibodies, including VCA-IgG, VCA-IgA, VCA-IgM, EA-IgG and EBNA1-IgG were examined. All plasma samples were EB-VCA-IgG positive and EB-VCA-IgM negative, suggesting that all people tested had been infected with EBV but not in the acute infection stage. EBV-DNA was detected in 15/16 (94%) of IOIP tissue samples despite different levels of lymphocyte infiltration and 5/16 plasma samples (31%) were detected EBV DNA positive which is higher than the normal controls (10%). Percent of positive plus suspected positive samples with one or more of the three important risk markers (VCA-IgA, EA-IgG, EBV-DNA) is 50% of the patients (8/16) which is much higher compare with the normal controls (20%). The results further reveal the relationship between IOIP and EBV infection.

Hyperbaric oxygen preconditioning inhibits skin flap apoptosis in a rat ischemia-reperfusion model.

BACKGROUNDnHyperbaric oxygen (HBO) improves skin flap function and inhibits partial necrosis induced by ischemia-reperfusion (I/R) injury. Our study aimed to evaluate the mechanism underlying HBO regulation of the antiapoptosis factors associated with I/R injury of skin flaps.nnnMETHODSnThe rats were divided into sham surgery, I/R, and HBO groups. Rats from the HBO group received HBO preconditioning followed by I/R surgery. Blood perfusion of the skin flaps was measured with laser Doppler flowmeters. Tissue morphology and apoptosis were subsequently assessed based on hematoxylin-eosinhe and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining. Protein expression of phosphorylated apoptosis signal-regulating kinase 1 (pASK-1), phosphorylated c-Jun N-terminal kinase (pJNK), B-cell lymphoma-2 (Bcl-2), and Bcl2-associated X protein (Bax) was examined by immunodetection, and Bcl-2 messenger RNA expression was detected by quantitative polymerase chain reaction. In addition, caspase-3 activity was also measured.nnnRESULTSnThe result of microcirculation analysis showed that the survival and blood perfusion rates significantly increased in the skin flap after HBO exposure. Terminal deoxynucleotidyl transferase dUTP nick-end labeling staining revealed that cell apoptosis was significantly attenuated in the HBO group. Furthermore, HBO preconditioning increased the expression of Bcl-2 and inhibited pASK-1, pJNK, and Bax expression as determined by both immunohistochemistry and Western blot. Caspase-3 activity and the Bax/Bcl-2 ratio declined in the HBO group.nnnCONCLUSIONSnHBO preconditioning effectively ameliorates I/R injury by regulating the apoptosis signal-regulating kinase 1 and/or c-Jun N-terminal kinase pathway and anti- and proapoptosis factors.

Chlorpyrifos Induces the Expression of the Epstein-Barr Virus Lytic Cycle Activator BZLF-1 via Reactive Oxygen Species

Organophosphate pesticides (OPs) are among the most widely used synthetic chemicals for the control of a wide variety of pests, and reactive oxygen species (ROS) caused by OPs may be involved in the toxicity of various pesticides. Previous studies have demonstrated that a reactivation of latent Epstein-Barr virus (EBV) could be induced by oxidative stress. In this study, we investigated whether OPs could reactivate EBV through ROS accumulation. The Raji cells were treated with chlorpyrifos (CPF), one of the most commonly used OPs. Oxidative stress indicators and the expression of the EBV immediate-early gene BZLF-1 were determined after CPF treatment. Our results show that CPF induces oxidative stress as evidenced by decreased malondialdehyde (MDA) level, accompanied by an increase in ROS production, DNA damage, glutathione (GSH) level, and superoxide dismutase (SOD) and catalase (CAT) activity. Moreover, CPF treatment significantly enhances the expression of BZLF-1, and the increased BZLF-1 expression was ameliorated by N-acetylcysteine (NAC) incubation. These results suggest that OPs could contribute to the reactivation of the EBV lytic cycle through ROS induction, a process that may play an important role in the development of EBV-associated diseases.

Combining Gold Nanoparticles with Real-Time Immuno-PCR for Analysis of HIV p24 Antigens

We have improved real-time immuno-PCR (IPCR) with nanoparticle based amplification. Gold magnetic particles were functionalized with antibodies against the HIV capsid protein p24 and gold nanoparticles were functionalized with both oligonucleotide barcodes and antibodies against a non- overlapping region of the cognate p24. In the presence of p24, the gold magnetic particles and the gold nanoparticles form sandwiched structures that are magnetically separated from mixture. After dehybridization of the barcode DNA on the gold nanoparticle, the released barcodes are quickly identified by realtime PCR. The limit of detection was 100 copies of p24 antigens. Results show that the real-time IPCR through nanoparticle based barcode amplification offers an disruptive approach to p24 detection and quantification, thus may potentially shorten the window of HIV-1 diagnosis, and might be valuable to monitor the response to anti-retroviral treatment when RNA copy number is very below.

Chemokine-Like Factor 1 (CKLF-1) is Overexpressed in Keloid Patients: A Potential Indicating Factor for Keloid-Predisposed Individuals.

AbstractChemokine-like factor 1 (CKLF-1) is a novel cytokine which have a crucial role in immune and inflammatory responses. In this study, the expression level of CKLF-1 was measured to assess the difference between keloid patients and people without keloid.Fifty samples were taken from 30 patients: 10 keloid patients; 10 scar patients; and 10 patients without obvious scarring. Patients were randomly selected from the hospitalized patients of Peking Union Medical College Hospital from September 2013 to July 2015. Five groups of samples were established: keloid samples from keloid patients (K); normal skin samples from keloid patients (KS); scar samples from scar patients (C); normal skin samples from scar patients (CS); and normal skin samples from patients without obvious scarring (S). Hematoxylin and eosin (H&E) staining was used to observe morphological changes. CKLF-1, IL-6, IL-8, IL-18, and TGF-&bgr; were detected by immunohistochemical and western blot technology. The expression of CKLF-1s mRNA was also measured by the real-time quantitative polymerase chain reaction (RT-qPCR).Compared to the K group, the other 4 groups presented significantly less inflammatory infiltration and lower expression levels of CKLF-1, IL-6, IL-8, IL-18, and TGF-&bgr;. Among the 3 normal skin groups, the expression level of CKLF-1 was significantly higher in the KS group than in the CS or S group. The mRNA expression was also obvious in the K and KS groups.CKLF-1 and other inflammatory factors were overexpressed in the samples from keloid patients, indicating that the formation of keloid may be related to inflammation and that CKLF-1 may play an important role in this process.

Detection of Epstein-Barr Virus in Idiopathic Orbital Inflammatory Pseudotumor

Purpose: The etiology of idiopathic orbital inflammatory pseudotumors (IOIP) is unknown. Epstein-Barr virus (EBV) infection, which causes significant lymphocyte infiltration, has been proposed to be involved in IOIP. This study tries to validate the relationship between EBV infection and the development of IOIP. Methods: Eleven surgical samples from IOIP patients and four non-IOIP control samples from surgery removal of eye due to trauma were analyzed by microarray. The data were confirmed by real time RT-PCR. The existence of EBV-DNA segments in IOIP samples was determined by PCR. Results: Hierarchical clustering of microarray data successfully distinguished the IOIP group and the non-IOIP control group. The gene EBI2, usually induced by EBV infection, was significantly up-regulated in the IOIP group (max = 11.7-fold over control). EBV-DNA was detected in 9/10 (90%) of the IOIP samples and in 0/4 of the control samples. Up-regulation of EB12, the immunoglobulin kappa light chain (IGKC) and the immunoglobulin heavy chain gene 1(IGHA1) were detected in the IOIP group compared to the control group. Conclusion: Collectively, these findings suggest that IOIP may be related to EBV infection and IGKC and IGHA1 activation. Furthermore, we hypothesize that the expression of the EBI2 gene may serve as a molecular marker for IOIP. More IOIP patient samples and detection of EBV related genes ( EBER1, EBNA2 and LMP1) expression need to be investigated.

Suspension Array for Multiplex Detection of Eight Fungicide-Resistance Related Alleles in Botrytis cinerea

A simple and high-throughput assay to detect fungicide resistance is required for large-scale monitoring of the emergence of resistant strains of Botrytis cinerea. Using suspension array technology performed on a Bio-Plex 200 System, we developed a single-tube allele-specific primer extension assay that can simultaneously detect eight alleles in one reaction. These eight alleles include E198 and 198A of the β-Tubulin gene (BenA), H272 and 272Y of the Succinate dehydrogenase iron–sulfur subunit gene (SdhB), I365 and 365S of the putative osmosensor histidine kinase gene (BcOS1), and F412 and 412S of the 3-ketoreductase gene (erg27). This assay was first established and optimized with eight plasmid templates containing the DNA sequence variants BenA-E198, BenA-198A, SdhB-H272, SdhB-272Y, BcOS1-I365, BcOS1-365S, erg27-F412, and erg27-412S. Results indicated that none of the probes showed cross-reactivity with one another. The minimum limit of detection for these genotypes was one copy per test. Four mutant plasmids were mixed with 10 ng/μL wild-type genomic DNA in different ratios. Detection sensitivity of mutant loci was 0.45% for BenA-E198A, BcOS1-I365S, and erg27-F412S, and was 4.5% for SdhB-H272Y. A minimum quantity of 0.1 ng of genomic DNA was necessary to obtain reliable results. This is the first reported assay that can simultaneously detect mutations in BenA, SdhB, BcOS1, and erg27.