Development of novel biodosimetry assays and medical countermeasures is needed to obtain a level of radiation preparedness in the event of malicious or accidental mass exposures to ionizing radiation ...(IR). For biodosimetry, metabolic profiling with mass spectrometry (MS) platforms has identified several small molecules in easily accessible biofluids that are promising for dose reconstruction. As our microbiome has profound effects on biofluid metabolite composition, it is of interest how variation in the host microbiome may affect metabolomics based biodosimetry. Here, we 'knocked out' the microbiome of male and female C57BL/6 mice (Abx mice) using antibiotics and then irradiated (0, 3, or 8 Gy) them to determine the role of the host microbiome on biofluid radiation signatures (1 and 3 d urine, 3 d serum). Biofluid metabolite levels were compared to a sham and irradiated group of mice with a normal microbiome (Abx-con mice). To compare post-irradiation effects in urine, we calculated the Spearman's correlation coefficients of metabolite levels with radiation dose. For selected metabolites of interest, we performed more detailed analyses using linear mixed effect models to determine the effects of radiation dose, time, and microbiome depletion. Serum metabolite levels were compared using an ANOVA. Several metabolites were affected after antibiotic administration in the tryptophan and amino acid pathways, sterol hormone, xenobiotic and bile acid pathways (urine) and lipid metabolism (serum), with a post-irradiation attenuative effect observed for Abx mice. In urine, dose×time interactions were supported for a defined radiation metabolite panel (carnitine, hexosamine-valine-isoleucine Hex-V-I, creatine, citric acid, and Nε,Nε,Nε-trimethyllysine TML) and dose for N1-acetylspermidine, which also provided excellent (AUROC ≥ 0.90) to good (AUROC ≥ 0.80) sensitivity and specificity according to the area under the receiver operator characteristic curve (AUROC) analysis. In serum, a panel consisting of carnitine, citric acid, lysophosphatidylcholine (LysoPC) (14:0), LysoPC (20:3), and LysoPC (22:5) also gave excellent to good sensitivity and specificity for identifying post-irradiated individuals at 3 d. Although the microbiome affected the basal levels and/or post-irradiation levels of these metabolites, their utility in dose reconstruction irrespective of microbiome status is encouraging for the use of metabolomics as a novel biodosimetry assay.
The development of in vitro molecular biomarkers to accurately predict toxicological effects has become a priority to advance testing strategies for human health risk assessment. The application of ...in vitro transcriptomic biomarkers promises increased throughput as well as a reduction in animal use. However, the existing protocols for predictive transcriptional signatures do not establish appropriate guidelines for dose selection or account for the fact that toxic agents may have pleiotropic effects. Therefore, comparison of transcriptome profiles across agents and studies has been difficult. Here we present a dataset of transcriptional profiles for TK6 cells exposed to a battery of well-characterized genotoxic and nongenotoxic chemicals. The experimental conditions applied a new dose optimization protocol that was based on evaluating expression changes in several well-characterized stress-response genes using quantitative real-time PCR in preliminary dose-finding studies. The subsequent microarray-based transcriptomic analyses at the optimized dose revealed responses to the test chemicals that were typically complex, often exhibiting substantial overlap in the transcriptional responses between a variety of the agents making analysis challenging. Using the nearest shrunken centroids method we identified a panel of 65 genes that could accurately classify toxicants as genotoxic or nongenotoxic. To validate the 65-gene panel as a genomic biomarker of genotoxicity, the gene expression profiles of an additional three well-characterized model agents were analyzed and a case study demonstrating the practical application of this genomic biomarker-based approach in risk assessment was performed to demonstrate its utility in genotoxicity risk assessment.
Interpretation of positive genotoxicity findings using the current in vitro testing battery is a major challenge to industry and regulatory agencies. These tests, especially mammalian cell assays, ...have high sensitivity but suffer from low specificity, leading to high rates of irrelevant positive findings (i.e., positive results in vitro that are not relevant to human cancer hazard). We developed an in vitro transcriptomic biomarker-based approach that provides biological relevance to positive genotoxicity assay data, particularly for in vitro chromosome damage assays, and propose its application for assessing the relevance of the in vitro positive results to carcinogenic hazard. The transcriptomic biomarker TGx-DDI (previously known as TGx-28.65) readily distinguishes DNA damage-inducing (DDI) agents from non-DDI agents. In this study, we demonstrated the ability of the biomarker to classify 45 test agents across a broad set of chemical classes as DDI or non-DDI. Furthermore, we assessed the biomarker’s utility in derisking known irrelevant positive agents and evaluated its performance across analytical platforms. We correctly classified 90% (9 of 10) of chemicals with irrelevant positive findings in in vitro chromosome damage assays as negative. We developed a standardized experimental and analytical protocol for our transcriptomics biomarker, as well as an enhanced application of TGx-DDI for high-throughput cell-based genotoxicity testing using nCounter technology. This biomarker can be integrated in genetic hazard assessment as a follow-up to positive chromosome damage findings. In addition, we propose how it might be used in chemical screening and assessment. This approach offers an opportunity to significantly improve risk assessment and reduce cost.
Transcriptomic biomarkers can be used to inform molecular initiating and key events involved in a toxicant’s mode of action. To address the limited approaches available for identifying ...epigenotoxicants, we developed and assessed a transcriptomic biomarker of histone deacetylase inhibition (HDACi). First, we assembled a set of ten prototypical HDACi and ten non-HDACi reference compounds. Concentration–response experiments were performed for each chemical to collect TK6 human lymphoblastoid cell samples after 4 h of exposure and to assess cell viability following a 20-h recovery period in fresh media. One concentration was selected for each chemical for whole transcriptome profiling and transcriptomic signature derivation, based on cell viability at the 24-h time point and on maximal induction of HDACi-response genes (
RGL1
,
NEU1
,
GPR183
) or cellular stress-response genes (
ATF3
,
CDKN1A
,
GADD45A
) analyzed by TaqMan qPCR assays after 4 h of exposure. Whole transcriptomes were profiled after 4 h exposures by Templated Oligo-Sequencing (TempO-Seq). By applying the nearest shrunken centroid (NSC) method to the whole transcriptome profiles of the reference compounds, we derived an 81-gene toxicogenomic (TGx) signature, referred to as TGx-HDACi, that classified all 20 reference compounds correctly using NSC classification and the Running Fisher test. An additional 4 HDACi and 7 non-HDACi were profiled and analyzed using TGx-HDACi to further assess classification performance; the biomarker accurately classified all 11 compounds, including 3 non-HDACi epigenotoxicants, suggesting a promising specificity toward HDACi. The availability of TGx-HDACi increases the diversity of tools that can facilitate mode of action analysis of toxicants using gene expression profiling.
The diagnosis of Behçet's disease (BD) remains challenging due to the lack of diagnostic biomarkers. This study aims to identify potential serum metabolites associated with BD and its disease ...activity.
Medical records and serum samples of 24 pretreated BD patients, 12 post-treated BD patients, and age-matched healthy controls (HC) were collected for metabolomics and lipidomics profiling using UPLC-QTOF-MS and UPLC-QTOF-MS
approaches. Additionally, serum samples from an independent cohort of BD patients, disease controls including rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Takayasu's arteritis (TA), Crohn's disease (CD) patients, and HC were collected for further validation of two potential biomarkers using UPLC-QTOFMS analysis.
Unsupervised principal component analysis (PCA) showed a clear separation of metabolomics profiles of BD patients from HC. Statistical analysis of the data revealed differential metabolites between BD patients and HC. The serum levels of some phosphatidylcholines (PCs) were found to be significantly lower in BD patients, while the levels of several polyunsaturated fatty acids (PUFAs) were increased markedly in the BD group compared with HC. Furthermore, the serum level of two omega-6 PUFAs, linoleic acid (LA) and arachidonic acid (AA), were dramatically decreased in patients with remission. A validation cohort confirmed that the serum LA and AA levels in BD patients were significantly higher than those in HC and patients with RA, SLE, TA, and CD. In addition, receiver operating characteristic (ROC) analysis indicated good sensitivity and specificity.
The serum metabolomics profiles in BD patients are altered. Serum LA and AA are promising diagnostic biomarkers for BD.
Common recommendations for cell line authentication, annotation and quality control fall short addressing genetic heterogeneity. Within the Human Toxome Project, we demonstrate that there can be ...marked cellular and phenotypic heterogeneity in a single batch of the human breast adenocarcinoma cell line MCF-7 obtained directly from a cell bank that are invisible with the usual cell authentication by short tandem repeat (STR) markers. STR profiling just fulfills the purpose of authentication testing, which is to detect significant cross-contamination and cell line misidentification. Heterogeneity needs to be examined using additional methods. This heterogeneity can have serious consequences for reproducibility of experiments as shown by morphology, estrogenic growth dose-response, whole genome gene expression and untargeted mass-spectroscopy metabolomics for MCF-7 cells. Using Comparative Genomic Hybridization (CGH), differences were traced back to genetic heterogeneity already in the cells from the original frozen vials from the same ATCC lot, however, STR markers did not differ from ATCC reference for any sample. These findings underscore the need for additional quality assurance in Good Cell Culture Practice and cell characterization, especially using other methods such as CGH to reveal possible genomic heterogeneity and genetic drifts within cell lines.
The hybrid plasmonic surface lattice resonance (SLR) laser constructed by a MAPbBr3 perovskite thin film on the Ag nanoparticle array is unambiguously demonstrated in this research. The relatively ...high refractive index of the perovskite thin film provides an excellent coupling between the photonic mode and SLR, leading to a high spontaneous emission coupling factor and a low threshold lasing. Furthermore, a novel spin‐coating process applied to grow the MAPbBr3 perovskite thin film can leave air gaps under the perovskite that provides a large index difference in the periodic array, making the hybrid plasmonic SLR exhibits a high density of state, which is beneficial for laser operation. Via theoretical design and experimental verification, the lasing behaviors of the hybrid plasmonic SLR perovskite laser show excellent characteristics with a fixed polarization. This demonstration facilitates an enhanced lasing performance and realization of the low‐cost and low‐energy‐consumption laser application.
The hybrid plasmonic surface lattice resonance (SLR) laser constructed by a perovskite thin film on the Ag nanoparticle array is unambiguously demonstrated in this research. By coupling between the photonic mode and SLR, the lasing behaviors show excellent characteristics with a fixed polarization. This demonstration facilitates an enhanced lasing performance and realization of the low‐cost and low‐energy‐consumption laser application.
Protein phosphatase 2A (PP2A) has been implicated to exert its tumor suppressive function via a small subset of regulatory subunits. In this study, we reported that the specific B regulatory subunits ...of PP2A B56γ1 and B56γ3 mediate dephosphorylation of p53 at Thr55. Ablation of the B56γ protein by RNAi, which abolishes the Thr55 dephosphorylation in response to DNA damage, reduces p53 stabilization, Bax expression and cell apoptosis. To investigate the molecular mechanisms, we have shown that the endogenous B56γ protein level and association with p53 increase after DNA damage. Finally, we demonstrate that Thr55 dephosphorylation is required for B56γ3‐mediated inhibition of cell proliferation and cell transformation. These results suggest a molecular mechanism for B56γ‐mediated tumor suppression and provide a potential route for regulation of B56γ‐specific PP2A complex function.
One of the most cited limitations of capillary and microchip electrophoresis is the poor sensitivity. This review continues to update this series of biannual reviews, first published in ...Electrophoresis in 2007, on developments in the field of online/in‐line concentration methods in capillaries and microchips, covering the period July 2016–June 2018. It includes developments in the field of stacking, covering all methods from field‐amplified sample stacking and large‐volume sample stacking, through to isotachophoresis, dynamic pH junction, and sweeping. Attention is also given to online or in‐line extraction methods that have been used for electrophoresis.
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