Case study: Targeted RNA-sequencing of aged formalin-fixed paraffin-embedded samples for understanding chemical mode of action.

Formalin-fixed paraffin-embedded (FFPE) samples are the only remaining biological archive for many toxicological and clinical studies, yet their use in genomics has been limited due to nucleic acid damage from formalin fixation. Older FFPE samples with highly degraded RNA pose a particularly difficult technical challenge. Probe-based targeted sequencing technologies show promise in addressing this issue but have not been directly compared to standard whole-genome RNA-Sequencing (RNA-Seq) methods.

Organocatalyst treatment improves variant calling and mutant detection in archival clinical samples.

Formalin fixation of biological specimens damages nucleic acids and limits their use in genomic analyses. Previously, we showed that RNA isolation with an organocatalyst (2-amino-5-methylphenyl phosphonic acid, used to speed up reversal of formalin-induced adducts) and extended heated incubation (ORGΔ) improved RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples. The primary goal of this study was to evaluate whether ORGΔ treatment improves DNA-sequencing data from clinical FFPE samples.

Genomic comparisons between hepatocarcinogenic and non-hepatocarcinogenic organophosphate insecticides in the mouse liver.

Short-term biomarkers of toxicity have an increasingly important role in the screening and prioritization of new chemicals. In this study, we examined early indicators of liver toxicity for three reference organophosphate (OP) chemicals, which are among the most widely used insecticides in the world. The OP methidathion was previously shown to increase the incidence of liver toxicity, including hepatocellular tumors, in male mice.

Direct formalin fixation induces widespread transcriptomic effects in archival tissue samples.

Sequencing technologies now provide unprecedented access to genomic information in archival formalin-fixed paraffin-embedded (FFPE) tissue samples. However, little is known about artifacts induced during formalin fixation, which could bias results. Here we evaluated global changes in RNA-sequencing profiles between matched frozen and FFPE samples.

Enhanced Quality Metrics for Assessing RNA Derived From Archival Formalin-Fixed Paraffin-Embedded Tissue Samples.

Formalin-fixed paraffin-embedded (FFPE) tissues provide an important resource for toxicogenomic research. However, variability in the integrity or quality of RNA obtained from archival FFPE specimens can lead to unreliable data and wasted resources, and standard protocols for measuring RNA integrity do not adequately assess the suitability of FFPE RNA. The main goal of this study was to identify improved methods for evaluating FFPE RNA quality for whole-genome sequencing.

Authentic Inquiry through Modeling in Biology (AIM-Bio): An Introductory Laboratory Curriculum That Increases Undergraduates' Scientific Agency and Skills.

Providing opportunities for science, technology, engineering, and mathematics undergraduates to engage in authentic scientific practices is likely to influence their view of science and may impact their decision to persist through graduation. Laboratory courses provide a natural place to introduce students to scientific practices, but existing curricula often miss this opportunity by focusing on confirming science content rather than exploring authentic questions. Integrating authentic science within laboratory courses is particularly challenging at high-enrollment institutions and community colleges, where access to research-active faculty may be limiting.

From the Cover: Genomic Effects of Androstenedione and Sex-Specific Liver Cancer Susceptibility in Mice.

Current strategies for predicting carcinogenic mode of action for nongenotoxic chemicals are based on identification of early key events in toxicity pathways. The goal of this study was to evaluate short-term key event indicators resulting from exposure to androstenedione (A4), an androgen receptor agonist and known liver carcinogen in mice. Liver cancer is more prevalent in men compared with women, but androgen-related pathways underlying this sex difference have not been clearly identified.

Metabolic Disruption Early in Life is Associated With Latent Carcinogenic Activity of Dichloroacetic Acid in Mice.

Early-life environmental factors can influence later-life susceptibility to cancer. Recent evidence suggests that metabolic pathways may mediate this type of latency effect. Previously, we reported that short-term exposure to dichloroacetic acid (DCA) increased liver cancer in mice 84 weeks after exposure was stopped.

Dynamics of growth zone patterning in the milkweed bug .

We describe the dynamic process of abdominal segment generation in the milkweed bug We present detailed morphological measurements of the growing germband throughout segmentation. Our data are complemented by cell division profiles and expression patterns of key genes, including and as markers for different stages of segment formation. We describe morphological and mechanistic changes in the growth zone and in nascent segments during the generation of individual segments and throughout segmentation, and examine the relative contribution of newly formed versus existing tissue to segment formation.

Editor's Highlight: Genetic Targets of Acute Toluene Inhalation in Drosophila melanogaster.

Interpretation and use of data from high-throughput assays for chemical toxicity require links between effects at molecular targets and adverse outcomes in whole animals. The well-characterized genome of Drosophila melanogaster provides a potential model system by which phenotypic responses to chemicals can be mapped to genes associated with those responses, which may in turn suggest adverse outcome pathways associated with those genes. To determine the utility of this approach, we used the Drosophila Genetics Reference Panel (DGRP), a collection of ∼200 homozygous lines of fruit flies whose genomes have been sequenced.

Editor's Highlight: Dose-Response Analysis of RNA-Seq Profiles in Archival Formalin-Fixed Paraffin-Embedded Samples.

Use of archival resources has been limited to date by inconsistent methods for genomic profiling of degraded RNA from formalin-fixed paraffin-embedded (FFPE) samples. RNA-sequencing offers a promising way to address this problem. Here, we evaluated transcriptomic dose responses using RNA-sequencing in paired FFPE and frozen (FROZ) samples from 2 archival studies in mice, one <2 years old and the other >20 years old.

Dose and Effect Thresholds for Early Key Events in a PPARα-Mediated Mode of Action.

Current strategies for predicting adverse health outcomes of environmental chemicals are centered on early key events in toxicity pathways. However, quantitative relationships between early molecular changes in a given pathway and later health effects are often poorly defined. The goal of this study was to evaluate short-term key event indicators using qualitative and quantitative methods in an established pathway of mouse liver tumorigenesis mediated by peroxisome proliferator-activated receptor alpha (PPARα).

Mining the Archives: A Cross-Platform Analysis of Gene Expression Profiles in Archival Formalin-Fixed Paraffin-Embedded Tissues.

Formalin-fixed paraffin-embedded (FFPE) tissue samples represent a potentially invaluable resource for transcriptomic research. However, use of FFPE samples in genomic studies has been limited by technical challenges resulting from nucleic acid degradation. Here we evaluated gene expression profiles derived from fresh-frozen (FRO) and FFPE mouse liver tissues preserved in formalin for different amounts of time using 2 DNA microarray protocols and 2 whole-transcriptome sequencing (RNA-seq) library preparation methodologies.

Latent carcinogenicity of early-life exposure to dichloroacetic acid in mice.

Environmental exposures occurring early in life may have an important influence on cancer risk later in life. Here, we investigated carryover effects of dichloroacetic acid (DCA), a small molecule analog of pyruvate with metabolic programming properties, on age-related incidence of liver cancer. The study followed a stop-exposure/promotion design in which 4-week-old male and female B6C3F1 mice received the following treatments: deionized water alone (dH2O, control); dH2O with 0.

Dose-response modeling of early molecular and cellular key events in the CAR-mediated hepatocarcinogenesis pathway.

Low-dose extrapolation and dose-related transitions are paramount in the ongoing debate regarding the quantification of cancer risks for nongenotoxic carcinogens. Phenobarbital (PB) is a prototypical nongenotoxic carcinogen that activates the constitutive androstane receptor (CAR) resulting in rodent liver tumors. In this study, male and female CD-1 mice administered dietary PB at 0, 0.

Concordance of transcriptional and apical benchmark dose levels for conazole-induced liver effects in mice.

The ability to anchor chemical class-based gene expression changes to phenotypic lesions and to describe these changes as a function of dose and time informs mode-of-action determinations and improves quantitative risk assessments. Previous global expression profiling identified a 330-probe cluster differentially expressed and commonly responsive to 3 hepatotumorigenic conazoles (cyproconazole, epoxiconazole, and propiconazole) at 30 days. Extended to 2 more conazoles (triadimefon and myclobutanil), the present assessment encompasses 4 tumorigenic and 1 nontumorigenic conazole.

Transcriptional responses in rat brain associated with sub-chronic toluene inhalation are not predicted by effects of acute toluene inhalation.

A primary public health concern regarding environmental chemicals is the potential for persistent effects from long-term exposure, and approaches to estimate these effects from short-term exposures are needed. Toluene, a ubiquitous air pollutant, exerts well-documented acute and persistent CNS-mediated effects from a variety of exposure scenarios, and so provides a useful case for determining whether its persistent effects can be predicted from its acute effects on the CNS. We recently reported that acute inhalation of toluene produced transcriptional effects in rat brain 18 h following a single, acute 6-h exposure to toluene.

A multi-cell, multi-scale model of vertebrate segmentation and somite formation.

Somitogenesis, the formation of the body's primary segmental structure common to all vertebrate development, requires coordination between biological mechanisms at several scales. Explaining how these mechanisms interact across scales and how events are coordinated in space and time is necessary for a complete understanding of somitogenesis and its evolutionary flexibility. So far, mechanisms of somitogenesis have been studied independently.

Acute toluene exposure alters expression of genes in the central nervous system associated with synaptic structure and function.

Toluene is a volatile organic compound (VOC) and a ubiquitous air pollutant of interest to EPA regulatory programs. Whereas its acute functional effects are well described, several modes of action in the CNS have been proposed. Therefore, we sought to identify potential pathways mediating direct or indirect effects of VOCs by investigating the genomic response of the rat CNS to acutely-inhaled toluene.

Transcriptional profile of diuron-induced toxicity on the urinary bladder of male Wistar rats to inform mode of action.

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide that induces rat urinary bladder urothelial tumors at high dietary levels (2500 ppm). The specific mode of action and molecular alterations triggered by diuron, however, have not been clarified. The present study evaluated the dose-dependent effects of mucosal alterations and transcriptional changes in the urinary bladder of rats exposed to diuron.

Gene expression of normal human epidermal keratinocytes modulated by trivalent arsenicals.

Chronic exposure to inorganic arsenic (iAs) is associated with the development of benign and malignant human skin lesions including nonmelanoma skin cancers. The precise arsenical form(s) responsible for this carcinogenic effect are unknown, although trivalent inorganic arsenic (iAs(III)) and two of its toxic metabolites, monomethylarsonous acid (MMA(III)) and methylarsinous acid (DMA(III)), are attractive candidates. In an effort to better understand and compare their toxic effects in the skin, we compared the global gene expression profiles of normal human epidermal keratinocytes (NHEKs) exposed to varying noncytotoxic/slightly cytotoxic concentrations of iAs(III), MMA(III), and DMA(III) for 24 h.

Induction of abasic sites by the drinking-water mutagen MX in Salmonella TA100.

Mutagen X (MX) is a chlorinated furanone that accounts for more of the mutagenic activity of drinking water than any other disinfection by-product. It is one of the most potent base-substitution mutagens in the Salmonella (Ames) mutagenicity assay, producing primarily GC to TA mutations in TA100. MX does not produce stable DNA adducts in cellular or acellular DNA.

Comparison of comparative genomic hybridization technologies across microarray platforms.

In the 2007 Association of Biomolecular Resource Facilities Microarray Research Group project, we analyzed HL-60 DNA with five platforms: Agilent, Affymetrix 500K, Affymetrix U133 Plus 2.0, Illumina, and RPCI 19K BAC arrays. Copy number variation was analyzed using circular binary segmentation (CBS) analysis of log ratio scores from four independently assessed hybridizations of each platform.

Multicell simulations of development and disease using the CompuCell3D simulation environment.

Mathematical modeling and computer simulation have become crucial to biological fields from genomics to ecology. However, multicell, tissue-level simulations of development and disease have lagged behind other areas because they are mathematically more complex and lack easy-to-use software tools that allow building and running in silico experiments without requiring in-depth knowledge of programming. This tutorial introduces Glazier-Graner-Hogeweg (GGH) multicell simulations and CompuCell3D, a simulation framework that allows users to build, test, and run GGH simulations.

Discrimination of tumorigenic triazole conazoles from phenobarbital by transcriptional analyses of mouse liver gene expression.

Conazoles are fungicides used to control fungal growth in environmental settings and to treat humans with fungal infections. Mouse hepatotumorigenic conazoles display many of the same hepatic toxicologic responses as the mouse liver carcinogen phenobarbital (PB): constitutive androstane receptor (CAR) activation, hypertrophy, Cyp2b induction, and increased cell proliferation. The goal of this study was to apply transcriptional analyses to hepatic tissues from mice exposed to PB, propiconazole (Pro) or triadimefon (Tri) at tumorigenic exposure levels to reveal similarities and differences in response among these treatments.