Screening of radiation gastrointestinal injury biomarkers in rat plasma by high-coverage targeted lipidomics.

INTRODUCTION: In the event of radiological accidents and cancer radiotherapies in the clinic, the gastrointestinal (GI) system is vulnerable to ionizing radiation and shows GI injury. Accessible biomarkers may provide means to predict, evaluate, and treat GI tissue damage. The current study investigated radiation GI injury biomarkers in rat plasma. MATERIAL AND METHODS: High-coverage targeted lipidomics was employed to profile lipidome perturbations at 72 h after 0, 1, 2, 3, 5, and 8 Gy (60Co γ-rays at 1 Gy/min) total-body irradiation in male rat jejunum. The results were correlated with previous plasma screening outcomes. RESULTS: In total, 93 differential metabolites and 28 linear dose-responsive metabolites were screened in the jejunum. Moreover, 52 lipid species with significant differences both in jejunum and plasma were obtained. Three lipid species with linear dose-response relationship both in jejunum and plasma were put forth, which exhibited good to excellent sensitivity and specificity in triaging different exposure levels. DISCUSSION: The linear dose-effect relationship of lipid metabolites in the jejunum and the triage performance of radiation GI injury biomarkers in plasma were studied for the first time. CONCLUSION: The present study can provide insights into expanded biomarkers of IR-mediated GI injury and minimally invasive assays for evaluation.

Screening of Lipids for Early Triage and Dose Estimation after Acute Radiation Exposure in Rat Plasma Based on Targeted Lipidomics Analysis.

Rapid early triage and dose estimation is vital for limited medical resource allocation and treatment of a large number of the wounded after radiological accidents. Lipidomics has been utilized to delineate biofluid lipid signatures after irradiation. Here, high-coverage targeted lipidomics was employed to screen radiosensitive lipids after 0, 1, 2, 3, 5, and 8 Gy total body irradiation at 4, 24, and 72 h postirradiation in rat plasma. Ultra-performance liquid chromatography-tandem mass spectrometry with a multiple reaction monitoring method was utilized. In total, 416 individual lipids from 18 major classes were quantified and those biomarkers altered in a dose-dependent manner constituted panel A-panel D. Receiver operator characteristic curve analysis using combined lipids showed good to excellent sensitivity and specificity in triaging different radiation exposure levels (area under curve = 0.814-1.000). The equations for dose estimation were established by stepwise regression analysis for three time points. A novel strategy for radiation early triage and dose estimation was first established and validated using panels of lipids. Our study suggests that it is feasible to acquire quantitative lipid biomarker panels using targeted lipidomics platforms for rapid, high-throughput triage, which can provide further insights in developing lipidomics strategies for radiation biodosimetry in humans.

Meta-analysis of Nicorandil effectiveness on myocardial protection after percutaneous coronary intervention.

BACKGROUND: Using the current meta-analysis as well as systematic review, to determine the curative effect of Nicorandil in comparison of no Nicorandil after elective percutaneous coronary intervention(PCI) on patients. METHODS: Published literatures were identified via a computerized literature search of CENTRAL, PubMed, Cochrane, Embase Databases of Systematic Reviews. A set of randomized trials evaluating Nicorandil in comparison of no Nicorandil administered following PCI in patients harboring coronary artery disease were included. Outcomes were revealed based on the following parameters: peak creatine kinase-MB (CK-MB) value, left ventricular ejection fraction (LVEF), peak troponin I (cTnI), and major adverse cardiovascular events (MACEs) per randomized patients. RESULTS: We included a total of 14 RCTs involving 1864 subjects in the present review. According to this meta-analysis, LVEF was significantly improved in Nicorandil group; the peak CK-MB level and the incidence of adverse cardiovascular events were remarkably lower in Nicorandil group. Nicorandil and no Nicorandil administered group appeared to be equivalent with regards to cTnI. CONCLUSIONS: Nicorandil is effective for patients undergoing elective PCI with coronary artery disease in terms of reducing the incidence of adverse cardiovascular events as well as improving heart function. Nicorandil may exert potential role as a valid and adjunctive therapy accompanied with PCI.

Alterations in histone acetylation following exposure to 60Co γ-rays and their relationship with chromosome damage in human lymphoblastoid cells.

Chromosome damage is related to DNA damage and erroneous repair. It can cause cell dysfunction and ultimately induce carcinogenesis. Histone acetylation is crucial for regulating chromatin structure and DNA damage repair. Ionizing radiation (IR) can alter histone acetylation. However, variations in histone acetylation in response to IR exposure and the relationship between histone acetylation and IR-induced chromosome damage remains unclear. Hence, this study investigated the variation in the total acetylation levels of H3 and H4 in human lymphocytes exposed to 0-2 Gy 60Co γ-rays. Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor, was added to modify the histone acetylation state of irradiated cells. Then, the total acetylation level, enzyme activity, dicentric plus centric rings (dic + r) frequencies, and micronucleus (MN) frequencies of the treated cells were analyzed. Results indicated that the acetylation levels of H3 and H4 significantly decreased at 1 and 24 h, respectively, after radiation exposure. The acetylation levels of H3 and H4 in irradiated groups treated with SAHA were significantly higher than those in irradiated groups that were not treated with SAHA. SAHA treatment inhibited HDAC activity in cells exposed to 0-1 Gy 60Co γ-rays. SAHA treatment significantly decreased dic + r/cell and MN/cell in cells exposed to 0.5 or 1.0 Gy 60Co γ-rays relative to that in cells that did not receive SAHA treatment. In conclusion, histone acetylation is significantly affected by IR and is involved in chromosome damage induced by 60Co γ-radiation.

Identification and Validation of Candidate Radiation-responsive Genes for Human Biodosimetr.

The aim of the present study is to analyze the global research trend of radiation-responsive genes and identify the highly reproducible radiation-responsive genes. Bibliometric methods were applied to analyze the global research trend of radiation-responsive genes. We found 79 publications on radiation-responsive genes from 2000 to 2017. A total of 35 highly reproducible radiation-responsive genes were identified. Most genes are involved in response to DNA damage, cell proliferation, cell cycle regulation, and DNA repair. The p53 signal pathway was the top enriched pathway. The expression levels of 18 genes in human B lymphoblastoid cell line (AHH-1) cells were significantly up-regulated in a dose-dependent manner at 24 h after exposure to 0-5 Gy 60Co γ-ray irradiation. Our results indicate that developing a gene expression panel with the 35 high reproducibility radiation-responsive genes may be necessary for qualitative and quantitative assessment after exposure.

Dose-effect relationships of nucleoplasmic bridges and complex nuclear anomalies in human peripheral lymphocytes exposed to 60Co γ-rays at a relatively low dose.

The dose effect between nucleoplasmic bridges (NPB) and relatively low doses of ionising radiation remains unknown. Accordingly, this study investigated the NPB frequencies in human peripheral blood lymphocytes exposed to low-dose (60)Co γ-rays. Complex anomalies, including fused nuclei (FUS), horse-shoe nuclei (HS) and circular nuclei (CIR), which possibly originated from multiple NPBs, were also scored. Human peripheral blood samples were collected from three healthy males and irradiated with 0-1 and 0-0.4 Gy (60)Co γ-rays. A cytokinesis-block micronucleus cytome assay was then conducted to analyse NPB, PFHC (NPB plus three complex nuclear anomalies) and micronucleus (MN) in binucleated cells. All dose-response curves followed the linear model for both NPB frequency and PFHC cell frequency. The dose-response curves between NPB frequency and absorbed dose at 0-1 and 0-0.4 Gy were y = 0.0037x + 0.0005 (R (2) = 0.979, P < 0.05) and y = 0.0043x + 0.0004 (R (2) = 0.941, P < 0.05), respectively. The dose-response curves between PFHC cell frequency and absorbed dose at 0-1 and 0-0.4 Gy were y = 0.0044x + 0.0007 (R (2) = 0.982, P < 0.05) and y = 0.0059x + 0.0005 (R (2) = 0.969, P < 0.05), respectively. The statistical significance of differences between the irradiated groups (0-0.4 Gy) and background levels of NPB, PFHC and MN were also analysed. The lowest analysable doses of NPB, PFHC and MN were 0.12, 0.08 and 0.08 Gy, respectively. In conclusion, NPBs and PFHC positively correlated with the absorbed radiation at a relatively low dose.

Characteristics of nucleoplasmic bridges induced by 60Co γ-rays in human peripheral blood lymphocytes.

Few studies have shown that the yields of ionising-radiation-induced nucleoplasmic bridges (NPBs) in human cells are dose dependent. However, a dose-response curve between the NPB frequency and the absorbed dose of ionising radiation has not yet been established. This study aimed to investigate NPB frequencies in human peripheral blood lymphocytes induced by cobalt-60 (60Co) γ-rays and to establish a dose-response curve. Human peripheral blood samples were collected from three healthy males, and some of these samples were irradiated with 0-6 Gy 60Co γ-rays. A cytokinesis-block micronucleus cytome assay was then carried out to analyse NPBs and micronuclei (MN) in binucleated cells. The remaining blood samples were irradiated with 0, 2 and 5 Gy of γ-rays, and unstable chromosome aberrations (dicentric chromosome, ring chromosome and acentric chromosome fragment) were analysed. The correlation between NPBs and dicentric plus ring chromosome (dic+r) induced by the same γ-ray dose was also analysed. Results showed that the NPB yields among the three subjects at each dose level were not significantly different. NPBs in binucleated cells at all γ-ray doses conformed to Poisson distribution. The dose-response curve of the γ-ray-induced NPB frequencies followed the linear-quadratic model y = (1.39×10-3)x 2 + (4.94×10-3)x. A positive correlation was observed between the frequencies of NPB and dic+r, as well as between the frequencies of MN and acentric fragments. Therefore, NPB is an important biomarker of early chromosome damage event induced by ionising radiation.

Assessment of retrospective dose estimation, with fluorescence in situ hybridization (FISH), of six victims previously exposed to accidental ionizing radiation.

The present study aims to evaluate the use of the fluorescence in situ hybridization (FISH) translocation assay for retrospective dose estimation of acute accidental exposure to radiation in the past. Reciprocal translocation analysis by FISH with three whole-chromosome probes was performed on normal peripheral blood samples. Samples were irradiated with 0-5Gy (60)Co γ-rays in vitro, and dose-effect curves were established. FISH-based translocation analyses for six accident victims were then performed, and biological doses were estimated retrospectively by comparison with the dose-effect curves. Reconstructed doses by FISH were compared with estimated doses obtained by analysis of di-centrics performed soon after exposure, or with dose estimates from tooth-enamel electron paramagnetic resonance (EPR) data obtained at the same time as the FISH analysis. Follow-up FISH analyses for an adolescent victim were performed. Results showed that dose-effect curves established in the present study follow a linear-quadratic model, regardless of the background translocation frequency. Estimated doses according to two dose-effect curves for all six victims were similar. FISH dose estimations of three adult victims exposed to accidental radiation less than a decade prior to analysis (3, 6, or 7 years ago) were consistent with those estimated with tooth-enamel EPR measurements or analyses of di-centrics. Estimated doses of two other adult victims exposed to radiation over a decade prior to analysis (16 or 33 years ago) were underestimated and two to three times lower than the values obtained from analysis of di-centrics or tooth-enamel EPR. Follow-up analyses of the adolescent victim showed that doses estimated by FISH analysis decrease rapidly over time. Therefore, the accuracy of dose estimates by FISH is acceptable only when analysis is performed less than 7 years after exposure. Measurements carried out more than a decade after exposure through FISH analysis resulted in underestimation of the biological doses compared with values obtained through analysis of di-centrics and tooth-enamel EPR.

Targeted lipidomics-based study of radiation-induced metabolite profiles changes in plasma of total body irradiation cases.

PURPOSE: Lipidomics is an important tool for triaging exposed individuals, and helps early adoption of prevention and control strategies. The purpose of this study was to screen significantly perturbed lipids between pre- and post-irradiation of human plasma samples after total body irradiation (TBI) and explore potential radiation biomarkers for early radiation classification. METHODS: Plasma samples were collected before and after irradiation from 22 hospitalized cases of acute myeloid leukemia (AML) prepared for bone marrow transplantation. Acute total-body γ irradiation was performed at doses of 0, 4, 8, and 12 Gy. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with multiple reaction monitoring (MRM) method was utilized. Self-paired studies before and after irradiation were performed to screen potential lipid categorization markers and markers of dose-response relationships for radiation perturbation in humans. Based on the screened potential markers, a human TBI dose estimation model was developed. RESULTS: In total, 426 individual lipids from 14 major classes were quantified and 152 potential biomarkers with categorical characteristics were screened. A total of 80 lipids (32 TGs, 29 SMs, 9 FAs, 5 CEs, 5 PIs) were upregulated at 4 Gy, and a total of 91 lipids (39 SMs, 18 TGs, 15 HexCers, 7 CEs, 6 Cers, 3 LacCers, 2 LPEs, 1 PI) were upregulated at 12 Gy. Comparison of the ROC curves between the non-exposed and exposed groups at different doses showed AUC values ranging from 0.807 to 0.876. The metabolic pathways of potential lipid markers are mainly sphingolipid and glycerolipid metabolism, unsaturated fatty acid biosynthesis, fatty acid degradation and biosynthesis. Among the 13 dose-dependent radiosensitive lipids, CE (20:5), CE (18:1) and PI (18:2/18:2) were gradually incorporated into the TBI dose estimation model. CONCLUSION: This study suggested that it was feasible to acquire quantitative lipid biomarker panels using targeted lipidomics platforms for rapid, high-throughput triage. Lipidomics strategies for radiation biodosimetry in humans were established with lipid biomarkers with good dose-response relationship.

Screening radiation-differentially expressed circular RNAs and establishing dose classification models in the human lymphoblastoid cell line AHH-1.

PURPOSE: In the event of a large-scale radiological accident, rapid and high-throughput biodosimetry is the most vital basis in medical resource allocation for the prompt treatment of victims. However, the current biodosimeter is yet to be rapid and high-throughput. Studies have shown that ionizing radiation modulates expressions of circular RNAs (circRNAs) in healthy human cell lines and tumor tissue. circRNA expressions can be quantified rapidly and high-throughput. However, whether circRNAs are suitable for early radiation dose classification remains unclear. METHODS: We employed transcriptome sequencing and bioinformatics analysis to screen for radiation-differentially expressed circRNAs in the human lymphoblastoid cell line AHH-1 at 4 h following exposure to 0, 2, and 5 Gy 60Co γ-rays. The dose-response relationships between differentially expressed circRNA expressions and absorbed doses were investigated using real-time polymerase chain reaction and linear regression analysis at 4 h, 24 h, and 48 h post-exposure to 0, 2, 4, 6, and 8 Gy. Six distinct dose classification models of circRNA panels were established and validated by receiver operating characteristic (ROC) curve analysis. RESULTS: A total of 11 radiation-differentially expressed circRNAs were identified and validated. Based on dose-response effects, those circRNAs changed in a dose-responsive or dose-dependent manner were combined into panels A through F at 4 h, 24 h, and 48 h post-irradiation. ROC curve analysis showed that panels A through C had the potential to effectively classify exposed and non-exposed conditions, which area under the curve (AUC) of these three panels were all 1.000, and the associate p values were .009. Panels D through F excellently distinguished between different dose groups (AUC = 0.963-1.000, p < .05). The validation assay showed that panels A through F demonstrated consistent excellence in sensitivity and specificity in dose classification. CONCLUSIONS: Ionizing radiation can indeed modulate the circRNA expression profile in the human lymphoblastoid cell line AHH-1. The differentially expressed circRNAs exhibit the potential for rapid and high-throughput dose classification.

Biodosimetry Based on Gamma-H2AX Quantification in Human Peripheral Blood Lymphocytes after Partial-body Irradiation.

ABSTRACT: Quantification of gamma-H2AX foci can estimate exposure to ionizing radiation. Most nuclear and radiation accidents are partial-body irradiation, and the doses estimated using the total-body irradiation dose estimation formula are often lower than the actual dose. To evaluate the dose-response relation of gamma-H2AX foci in human peripheral blood lymphocytes after partial-body irradiation and establish a simple and high throughput model to estimate partial-body irradiation dose, we collected human peripheral blood and irradiated with 0-, 0.5-, 1-, 2-, 3-, 4-, 5-, 6-, and 8-Gy gamma rays to simulate total-body irradiation in vitro. Gamma-H2AX foci were quantitated by flow cytometry at 1 h after irradiation, and a dose-response curve was established for total-body irradiation dose estimation. Then, a partial-body irradiation dose-response calibration curve was established by adding calibration coefficients based on the Dolphin method. To reflect the data distribution of all doses more realistically, the partial-body irradiation dose-response calibration curve was divided into two sections. In addition, partial-body irradiation was simulated in vitro, and the PBI data were substituted into curves to verify the accuracy of the two partial-body irradiation calibration curves. Results showed that the dose estimation variations were all less than 30% except the 25% partial-body irradiation group at 1 Gy, and the partial-body irradiation calibration dose-response curves were YF 1 = - 3.444 x 2 + 18.532 x + 3.109, R 2 = 0.92 (YF ≤ 27.95); YF 2 = - 2.704 x 2 + 37.97 x - 56.45, R 2 = 0.86 (YF > 27.95). Results also suggested that the partial-body irradiation dose-response calibration curve based on the gamma-H2AX foci quantification in human peripheral blood lymphocytes is a simple and high throughput model to assess partial-body irradiation dose.

Multiple exposures to low-dose ionizing radiation induced the initiation and progression of pro-atherosclerotic phenotypes in mice and vascular endothelial cell damage.

OBJECTIVE: This study aimed to investigate the effects of low-dose radiation on the abdominal aorta of mice and vascular endothelial cells. METHODS: Wild-type and tumor-bearing mice were exposed to 15 sessions of low-dose irradiation, resulting in cumulative radiation doses of 187.5, 375, and 750 mGy. The effect on the cardiovascular system was assessed. Immunohistochemistry analyzed protein expressions of PAPP-A, CD62, P65, and COX-2 in the abdominal aorta. Microarray technology, Gene Ontology analysis, and pathway enrichment analysis evaluated gene expression changes in endothelial cells exposed to 375 mGy X-ray. Cell viability was assessed using the Cell Counting Kit 8 assay. Immunofluorescence staining measured γ-H2AX levels, and real-time polymerase chain reaction quantified mRNA levels of interleukin-6 (IL-6), ICAM-1, and Cx43. RESULTS: Hematoxylin and eosin staining revealed thickening of the inner membranes and irregular arrangement of smooth muscle cells in the media membrane at 375 and 750 mGy. Inflammation was observed in the inner membranes at 750 mGy, with a clear inflammatory response in the hearts of tumor-bearing mice. Immunohistochemistry indicated increased levels of PAPP-A, P65, and COX-2 post-irradiation. Microarray analysis showed 425 up-regulated and 235 down-regulated genes, associated with processes like endothelial cell-cell adhesion, IL-6, and NF-κB signaling. Cell Counting Kit 8 assay results indicated inhibited viability at 750 mGy in EA.hy926 cells. Immunofluorescence staining demonstrated a dose-dependent increase in γ-H2AX foci. Reverse transcription quantitative PCR results showed increased expression of IL6, ICAM-1, and Cx43 in EA.hy926 cells post 750 mGy X-ray exposure. CONCLUSION: Repeated low-dose ionizing radiation exposures triggered the development of pro-atherosclerotic phenotypes in mice and damage to vascular endothelial cells.

Cytogenetic analysis in 16-year follow-up study of a mother and fetus exposed in a radiation accident in Xinzhou, China.

In November 1992, a radiation accident occurred in Xinzhou, due to the collection by a farmer of an unused (60)Co source; 37 individuals were exposed to ionizing radiation. Three individuals died and the farmer's 19-weeks-pregnant wife suffered acute radiation symptoms. Conventional chromosome analysis, cytokinesis-block micronuclei (CBMN) assay and fluorescence in situ hybridization (FISH) painting with three pairs of whole chromosome probes were used to analyze chromosomal aberrations for the pregnant female and her baby during the 16 years following the accident. The yields of dicentrics and rings (dic+r) continually declined between 41 days and 16 years after the accident. The frequency of binucleated MN also decreased over time for both mother and daughter. Sixteen years after exposure, the yields of dic+r and binucleated MN decreased to normal levels, but the reciprocal translocation frequencies remained elevated, for both mother and daughter. FISH results showed a decreasing yield of translocations with time. Based on the changes in maternal translocation frequency, the daughter's dose at the time of exposure was estimated as 1.82 (1.35-2.54)Gy. This was consistent with the clinical manifestations of severe mental retardation and low IQ score. FISH-based translocation analysis can be used for follow-up studies on accidental exposure and, after correction, for retrospective dose estimation for individuals prenatally exposed to radiation.

[Hyperspectral remote sensing image classification based on SVM optimized by clonal selection].

Model selection for support vector machine (SVM) involving kernel and the margin parameter values selection is usually time-consuming, impacts training efficiency of SVM model and final classification accuracies of SVM hyperspectral remote sensing image classifier greatly. Firstly, based on combinatorial optimization theory and cross-validation method, artificial immune clonal selection algorithm is introduced to the optimal selection of SVM (CSSVM) kernel parameter a and margin parameter C to improve the training efficiency of SVM model. Then an experiment of classifying AVIRIS in India Pine site of USA was performed for testing the novel CSSVM, as well as a traditional SVM classifier with general Grid Searching cross-validation method (GSSVM) for comparison. And then, evaluation indexes including SVM model training time, classification overall accuracy (OA) and Kappa index of both CSSVM and GSSVM were all analyzed quantitatively. It is demonstrated that OA of CSSVM on test samples and whole image are 85.1% and 81.58, the differences from that of GSSVM are both within 0.08% respectively; And Kappa indexes reach 0.8213 and 0.7728, the differences from that of GSSVM are both within 0.001; While the ratio of model training time of CSSVM and GSSVM is between 1/6 and 1/10. Therefore, CSSVM is fast and accurate algorithm for hyperspectral image classification and is superior to GSSVM.

[A method of hyperspectral quantificational identification of minerals based on infrared spectral artificial immune calculation].

Rapid identification of minerals based on near infrared (NIR) and shortwave infrared (SWIR) hyperspectra is vital to remote sensing mine exploration, remote sensing minerals mapping and field geological documentation of drill core, and have leaded to many identification methods including spectral angle mapping (SAM), spectral distance mapping (SDM), spectral feature fitting(SFF), linear spectral mixture model (LSMM), mathematical combination feature spectral linear inversion model(CFSLIM) etc. However, limitations of these methods affect their actual applications. The present paper firstly gives a unified minerals components spectral inversion (MCSI) model based on target sample spectrum and standard endmember spectral library evaluated by spectral similarity indexes. Then taking LSMM and SAM evaluation index for example, a specific formulation of unified MCSI model is presented in the form of a kind of combinatorial optimization. And then, an artificial immune colonial selection algorithm is used for solving minerals feature spectral linear inversion model optimization problem, which is named ICSFSLIM. Finally, an experiment was performed to use ICSFSLIM and CFSLIM to identify the contained minerals of 22 rock samples selected in Baogutu in Xinjiang China. The mean value of correctness and validness identification of ICSFSLIM are 34.22% and 54.08% respectively, which is better than that of CFSLIM 31.97% and 37.38%; the correctness and validness variance of ICSFSLIM are 0.11 and 0.13 smaller than that of CFSLIM, 0.15 and 0.25, indicating better identification stability.

Effects of cyclophosphamide and mitomycin C on radiation-induced transcriptional biomarkers in human lymphoblastoid cells.

PURPOSE: Ionizing radiation (IR)-induced transcriptional changes are considered a potential biodosimetry for dose evaluation and health risk monitoring of acute or chronic radiation exposure. It is crucial to understand the impact of confounding factors on the radiation-responsive gene expressions for accurate and reproducible dose assessment. This study aims to explore the potential influence of exposures to chemotherapeutic agents such as cyclophosphamide (CP) and mitomycin C (MMC) on IR-induced transcriptional biomarkers. METHODS: The human B lymphoblastoid cells (AHH-1) were exposed to 0, 20, 50, 100, 200 and 500 µg/ml CP or 0, 0.025, 0.05, 0.1 and 1 µg/ml MMC, respectively. The appropriate concentrations of CP and MMC were added for 1 h before irradiation with 0, 2, 4 and 6 Gy of 60Co γ-rays at a dose rate of 1 Gy/min. Cell viability was evaluated by CCK-8 assay. The gene expression responses of 18 radiation-induced transcriptional biomarkers were examined at 24 h after exposures to CP and MMC, respectively. The expression levels of five crucial DNA interstrand crosslinks (ICLs) repair genes were also evaluated. The biodosimetry models were established based on the specific radiation-responsive gene combinations. RESULTS: The baseline transcriptional levels of the 18 selected genes were slightly affected by CP treatment in the absence of IR, while the transcript responses to IR could be inhibited as the concentration of CP up to 50 µg/ml. MMC treatment up-regulated the background levels in most radiation-responsive gene expressions. Of 18 genes, only the relative mRNA expression levels of CDKN1A and BBC3 were repressed after treatment with IR and MMC in combination. The relative mRNA level of RAD51 was significantly up-regulated after exposure to CP, while the expression of FANCD2, RAD51 and BLM showed an overall increase in response to MMC treatment. After irradiation, the relative mRNA expression levels of FANCD2, BRCA2 and RAD51 exhibited dose-dependent increases in IR alone and MMC treatment groups. In addition, the biodosimetry models were established using 2-4 radiation-responsive genes based on different radiation exposure scenarios. CONCLUSION: Our findings suggested that IR-induced gene expression changes were slightly affected after exposure to a relatively low concentration of CP and MMC. Gene expression combinations might improve the broad applicability of transcriptional biodosimetry across diverse radiation exposures.

Genome-Wide Identification and Evolutionary Analyses of SrfA Operon Genes in Bacillus.

A variety of secondary metabolites contributing to plant growth are synthesized by bacterial nonribosomal peptide synthases (NRPSs). Among them, the NRPS biosynthesis of surfactin is regulated by the SrfA operon. To explore the molecular mechanism for the diversity of surfactins produced by bacteria within the genus Bacillus, we performed a genome-wide identification study focused on three critical genes of the SrfA operon-SrfAA, SrfAB and SrfAC-from 999 Bacillus genomes (belonging to 47 species). Gene family clustering indicated the three genes can be divided into 66 orthologous groups (gene families), of which a majority comprised members of multiple genes (e.g., OG0000009 had members of all three SrfAA, SrfAB and SrfAC genes), indicating high sequence similarity among the three genes. Phylogenetic analyses also found that none of the three genes formed monophyletic groups, but were usually arranged in a mixed manner, suggesting the close evolutionary relationship among the three genes. Considering the module structure of the three genes, we propose that self-duplication, especially tandem duplications, might have contributed to the initial establishment of the entire SrfA operon, and further gene fusion and recombination as well as accumulated mutations might have continuously shaped the different functional roles of SrfAA, SrfAB and SrfAC. Overall, this study provides novel insight into metabolic gene clusters and operon evolution in bacteria.

Low-dose ionizing radiation: Effects on the proliferation and migration of lens epithelial cells via activation of the Wnt/ß-catenin pathway.

Eye lens opacification (cataract) induced by ionizing radiation is an important concern for radiation protection. Human lens epithelial cells (HLE-B3) were irradiated with γ-rays and radiation effects, including cell proliferation, cell migration, cell cycle distribution, and other changes related to the ß-catenin pathway, were determined after 8-72 h and 7 d. In an in vivo model, mice were irradiated; DNA damage (γH2AX foci) in the cell nucleus of the anterior capsule of the lens was detected within 1 h, and radiation effects on the anterior and posterior lens capsules were observed after 3 months. Low-dose ionizing radiation promoted cell proliferation and migration. The expression levels of ß-catenin, cyclin D1, and c-Myc were significantly increased in HLE-B3 cells after irradiation and ß-catenin was translocated into the cell nucleus (activation of the Wnt/ß-catenin pathway). In C57BL/6 J mouse lens, even a very low irradiation dose (0.05 Gy) induced the formation of γH2AX foci, 1 h after irradiation. At 3 months, migratory cells were found in the posterior capsule; expression of ß-catenin was increased and it was clustered at the nucleus in the epithelial cells of the lens anterior capsule. The Wnt/ß-catenin signaling pathway may an important role in promoting abnormal proliferation and migration of lens epithelial cells after low-dose irradiation.

Melatonin inhibits senescence-associated melanin pigmentation through the p53-TYR pathway in human primary melanocytes and the skin of C57BL/6 J mice after UVB irradiation.

UVB exposure accelerates skin aging and pigmentation. Melatonin effectively regulates tyrosinase (TYR) activity and aging. The purpose of this study was to determine the association between premature senescence and pigmentation, and the mechanism of melanin synthesis effected by melatonin. Primary melanocytes were extracted and identified from the male foreskin. To inhibit TYR expression, primary melanocytes were transduced with the lentivirus pLKD-CMV-EGFP-2A-Puro-U6-TYR. The wild-type TYR(+/+) and TYR(-/-) or TYR(+/-) knockout C57BL/6 J mice were used to determine the role of TYR on melanin synthesis in vivo. Results showed that UVB-induced melanin synthesis is dependent on TYR in primary melanocytes and mice. Furthermore, in primary melanocytes pretreated with Nutlin-3 or PFT-α to up or downregulate p53, results showed that premature senescence and melanin synthesis increased in primary melanocytes after UVB irradiation at 80 mJ/cm2, and further increased after being treated with Nutlin-3, while significantly decreased with PFT-α. In addition, melatonin inhibited UVB-induced premature senescence associated with inactivation of p53 and phosphorylation of p53 on Ser15 (ser-15), a decrease of melanin synthesis accompanied by reduced TYR expression. Moreover, skin erythema and pigmentation induced by UVB were reduced in the dorsal and ear skin of mice topically pretreated with 2.5% melatonin. These indicate that melatonin inhibits UVB-induced senescence-associated pigmentation via the p53-TYR pathway in primary melanocytes and prevents pigmentation obviously in the dorsal and ear skin of C57BL/6 J mice after UVB irradiation. KEY MESSAGES: P53 links UVB irradiation-induced senescence and senescence-associated pigmentation and regulates TYR in primary melanocytes after UVB irradiation. Melatonin inhibits senescence-associated pigmentation through the p53-TYR pathway in primary melanocytes. Melatonin prevents skin erythema and melanin pigmentation induced by UVB irradiation in the dorsal and ear skin of C57BL/6J mice.

Identification of two novel mitochondrial DNA deletions induced by ionizing radiation.

OBJECTIVE: We identify ionizing radiation-induced mitochondrial DNA (mtDNA) deletions in human lymphocytes and their distribution in normal populations. METHODS: Long-range polymerase chain reactions (PCR) using two pairs of primers specific for the human mitochondrial genome were used to analyze the lymphoblastoid cell line following exposure to 10 Gy (60)Co γ-rays. Limited-condition PCR, cloning and sequencing techniques were applied to verify the mtDNA deletions detected with long-range PCR. Human peripheral blood samples were irradiated with 0, 2 and 6 Gy (60)Co γ-rays, and real-time PCR analysis was performed to validate the mtDNA deletions. In order to know the distribution of mtDNA deletions in normal population, 222 healthy Chinese adults were also investigated. RESULTS: Two mtDNA deletions, a 7455-bp deletion (nt475-nt7929 in heavy strand) and a 9225-bp deletion (nt7714 -nt369 in heavy strand), occurring between two 8-bp direct repeats, were identified in lymphoblastoid cells using long-range PCR, limited-condition PCR and sequencing. These results were also observed for (60)Co γ-rays irradiated human peripheral blood cells. CONCLUSION: Two novel mtDNA deletions, a 7455-bp deletion and a 9225-bp deletion, were induced by ionizing radiation. The rate of the mtDNA deletions within a normal population was related to the donors' age, but was independent of gender.