Applications of Zebrafish Embryo Models to Predict Developmental Toxicity for Agrochemical Product Development.

The development of safe crop protection products is a complex process that traditionally relies on intensive animal use for hazard identification. Methods that capture toxicity in early stages of agrochemical discovery programs enable a more efficient and sustainable product development pipeline. Here, we explored whether the zebrafish model can be leveraged to identify mammalian-relevant toxicity. We used transgenic zebrafish to assess developmental toxicity following exposures to known mammalian teratogens and captured larval morphological malformations, including bone and vascular perturbations. We further applied toxicogenomics to identify common biomarker signatures of teratogen exposure. The results show that the larval malformation assay predicted teratogenicity with 82.35% accuracy, 87.50% specificity, and 77.78% sensitivity. Similar and slightly lower accuracies were obtained with the vascular and bone assays, respectively. A set of 20 biomarkers were identified that efficiently segregated teratogenic chemicals from nonteratogens. In conclusion, zebrafish are valuable, robust, and cost-effective models for toxicity testing in the early stages of product development.

miRNA family miR-29 inhibits PINK1-PRKN dependent mitophagy via ATG9A.

Loss-of-function mutations in the genes encoding PINK1 and PRKN result in early-onset Parkinson disease (EOPD). Together the encoded enzymes direct a neuroprotective pathway that ensures the elimination of damaged mitochondria via autophagy. We performed a genome-wide high content imaging miRNA screen for inhibitors of the PINK1-PRKN pathway and identified all three members of the miRNA family 29 (miR-29). Using RNAseq we identified target genes and found that siRNA against ATG9A phenocopied the effects of miR-29 and inhibited the initiation of PINK1-PRKN mitophagy. Furthermore, we discovered two rare, potentially deleterious, missense variants (p.R631W and p.S828L) in our EOPD cohort and tested them experimentally in cells. While expression of wild-type ATG9A was able to rescue the effects of miR-29a, the EOPD-associated variants behaved like loss-of-function mutations. Together, our study validates miR-29 and its target gene ATG9A as novel regulators of mitophagy initiation. It further serves as proof-of-concept of finding novel, potentially disease-causing EOPD-linked variants specifically in mitophagy regulating genes. The nomination of genetic variants and biological pathways is important for the stratification and treatment of patients that suffer from devastating diseases, such as EOPD.

High content screening miniaturization and single cell imaging of mature human feeder layer-free iPSC-derived neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are being increasingly used for high content imaging and screening. However, iPSC-derived neuronal differentiation and maturation is time-intensive, often requiring >8 weeks. Unfortunately, the differentiating and maturing iPSC-derived neuronal cultures also tend to migrate and coalesce into ganglion-like clusters making single-cell analysis challenging, especially in miniaturized formats. Using our defined extracellular matrix and low oxygen culturing conditions for the differentiation and maturation of human cortical neurons, we further modified neuronal progenitor cell seeding densities and feeder layer-free culturing conditions in miniaturized formats (i.e., 96 well) to decrease neuronal clustering, enhance single-cell identification and reduce edge effects usually observed after extended neuronal cell culture. Subsequent algorithm development refined capabilities to distinguish and identify single mature neurons, as identified by NeuN expression, from large cellular aggregates, which were excluded from image analysis. Incorporation of astrocyte conditioned medium during differentiation and maturation periods significantly increased the percentage (i.e., ∼10% to ∼30%) of mature neurons (i.e., NeuN+) detected at 4-weeks post-differentiation. Pilot, proof of concept studies using this optimized assay system yielded negligible edge effects and robust Z-factors in population-based as well as image-based neurotoxicity assay formats. Moreover, moxidectin, an FDA-approved drug with documented neurotoxic adverse effects, was identified as a hit using both screening formats. This miniaturized, feeder layer-free format and image analysis algorithm provides a foundational imaging and screening platform, which enables quantitative single-cell analysis of differentiated human neurons.

Accurate gene expression-based biodosimetry using a minimal set of human gene transcripts.

PURPOSE: Rapid and reliable methods for conducting biological dosimetry are a necessity in the event of a large-scale nuclear event. Conventional biodosimetry methods lack the speed, portability, ease of use, and low cost required for triaging numerous victims. Here we address this need by showing that polymerase chain reaction (PCR) on a small number of gene transcripts can provide accurate and rapid dosimetry. The low cost and relative ease of PCR compared with existing dosimetry methods suggest that this approach may be useful in mass-casualty triage situations. METHODS AND MATERIALS: Human peripheral blood from 60 adult donors was acutely exposed to cobalt-60 gamma rays at doses of 0 (control) to 10 Gy. mRNA expression levels of 121 selected genes were obtained 0.5, 1, and 2 days after exposure by reverse-transcriptase real-time PCR. Optimal dosimetry at each time point was obtained by stepwise regression of dose received against individual gene transcript expression levels. RESULTS: Only 3 to 4 different gene transcripts, ASTN2, CDKN1A, GDF15, and ATM, are needed to explain ≥ 0.87 of the variance (R(2)). Receiver-operator characteristics, a measure of sensitivity and specificity, of 0.98 for these statistical models were achieved at each time point. CONCLUSIONS: The actual and predicted radiation doses agree very closely up to 6 Gy. Dosimetry at 8 and 10 Gy shows some effect of saturation, thereby slightly diminishing the ability to quantify higher exposures. Analyses of these gene transcripts may be advantageous for use in a field-portable device designed to assess exposures in mass casualty situations or in clinical radiation emergencies.

Gene expression-based dosimetry by dose and time in mice following acute radiation exposure.

Rapid and reliable methods for performing biological dosimetry are of paramount importance in the event of a large-scale nuclear event. Traditional dosimetry approaches lack the requisite rapid assessment capability, ease of use, portability and low cost, which are factors needed for triaging a large number of victims. Here we describe the results of experiments in which mice were acutely exposed to (60)Co gamma rays at doses of 0 (control) to 10 Gy. Blood was obtained from irradiated mice 0.5, 1, 2, 3, 5, and 7 days after exposure. mRNA expression levels of 106 selected genes were obtained by reverse-transcription real time PCR. Stepwise regression of dose received against individual gene transcript expression levels provided optimal dosimetry at each time point. The results indicate that only 4-7 different gene transcripts are needed to explain ≥ 0.69 of the variance (R(2)), and that receiver-operator characteristics, a measure of sensitivity and specificity, of ≥ 0.93 for these statistical models were achieved at each time point. These models provide an excellent description of the relationship between the actual and predicted doses up to 6 Gy. At doses of 8 and 10 Gy there appears to be saturation of the radiation-response signals with a corresponding diminution of accuracy. These results suggest that similar analyses in humans may be advantageous for use in a field-portable device designed to assess exposures in mass casualty situations.

Gene expression-based detection of radiation exposure in mice after treatment with granulocyte colony-stimulating factor and lipopolysaccharide.

In a large-scale nuclear incident, many thousands of people may be exposed to a wide range of radiation doses. Rapid biological dosimetry will be required on an individualized basis to estimate the exposures and to make treatment decisions. To ameliorate the adverse effects of exposure, victims may be treated with one or more cytokine growth factors, including granulocyte colony-stimulating factor (G-CSF), which has therapeutic efficacy for treating radiation-induced bone marrow ablation by stimulating granulopoiesis. The existence of infections and the administration of G-CSF each may confound the ability to achieve reliable dosimetry by gene expression analysis. In this study, C57BL/6 mice were used to determine the extent to which G-CSF and lipopolysaccharide (LPS, which simulates infection by gram-negative bacteria) alter the expression of genes that are either radiation-responsive or non-responsive, i.e., show potential for use as endogenous controls. Mice were acutely exposed to (60)Co γ rays at either 0 Gy or 6 Gy. Two hours later the animals were injected with either 0.1 mg/kg of G-CSF or 0.3 mg/kg of LPS. Expression levels of 96 different gene targets were evaluated in peripheral blood after an additional 4 or 24 h using real-time quantitative PCR. The results indicate that the expression levels of some genes are altered by LPS, but altered expression after G-CSF treatment was generally not observed. The expression levels of many genes therefore retain utility for biological dosimetry or as endogenous controls. These data suggest that PCR-based quantitative gene expression analyses may have utility in radiation biodosimetry in humans even in the presence of an infection or after treatment with G-CSF.

Developing point of care and high-throughput biological assays for determining absorbed radiation dose.

BACKGROUND AND PURPOSE: Systems are being developed to assess radiation exposure based on leukocyte mRNA levels obtained by finger-stick sampling. The goal is to provide accurate detection of dose exposures up to 10 Gy for up to 1 week following exposure. We previously showed that specific mRNA sequences increase expression within an hour of exposure, and some genes continue to show elevated expression for at least 24 h. Full duration and dose-dependence of this persistence remain to be determined. In the present study, real-time quantitative PCR (qPCR) was used to determine changes in gene expression. qPCR can rapidly analyze small blood samples and could be adopted into a field-portable instrument that provides a radiation dose readout within 30 min. MATERIALS AND METHODS: From previous microarray analysis of 21,000 genes expressed in human lymphoblastoid cells 4 h post-irradiation (0-4 Gy), 118 genes were selected for evaluation by qPCR of gene expression in the leukocytes of human blood irradiated in vitro with doses of 0-10 Gy from a Co-60 gamma source at a dose rate of 30 cGy/min. RESULTS: Blood from 20 normal healthy human donors yielded many mRNA sequences that could be used for radiation dosimetry. We observed four genes with large and persistent responses following exposure: ASTN2, CDKN1A, GADD45A, and GDF15. Five genes were identified as reliably non-responsive and were suitable for use as endogenous controls: DPM1, ITFG1, MAP4, PGK1, and SLC25A36; of these, ITFG1 was used for the analyses presented here. A significant dose-responsive increase in expression occurred for CDKN1A that was >16-fold at 10 Gy and 3-fold at 0.5 Gy compared to pre-irradiation values. CONCLUSIONS: These data show large, selective increases in mRNA transcript levels that persist for at least 48 h after single exposures between 0.5 and 10 Gy. Stable, non-responsive mRNA sequences for use as endogenous controls were also identified. These results indicate that following further study to establish the most reproducible gene and dose-response models under a wide range of conditions in vivo, rapid real-time qPCR on blood samples could potentially be used to establish biologically-effective dosimetry from either accidental irradiation or clinical radiotherapy.

Ethanol increases fetal human neurosphere size and alters adhesion molecule gene expression.

BACKGROUND: Ethanol (ETOH) consumption by pregnant women can result in Fetal Alcohol Spectrum Disorder (FASD). To date, the cellular targets and mechanisms responsible for FASD are not fully characterized. Our aim was to determine if ETOH can affect fetal human brain-derived neural progenitor cells (NPC). METHODS: Neural progenitor cells were isolated by positive selection from normal second trimester fetal human brains (n = 4) and cultured, for up to 72 hours, in mitogenic media containing 0, 1, 10, or 100 mM ETOH. From 48 to 72 hours in culture, neurospheres generated in these conditions were filmed using time-lapse video microscopy. At the end of 72 hours, neurosphere diameter and roundness were measured using videographic software. Mitotic phase analysis of cell-cycle activity and apoptotic cell count were also performed at this time, by flow cytometry using propidium iodide (PI) staining. Real-time PCR was used to estimate expression of genes associated with cell adhesion pathways. RESULTS: Neurosphere diameter correlated positively (r = 0.87) with increasing ETOH concentrations. There was no significant difference in cell-cycle activity and no significant increase in apoptosis with increasing ETOH concentrations. Time-lapse video microscopy showed that ETOH (100 mM) reduced the time for neurosphere coalescence. Real-time PCR analysis showed that ETOH significantly altered the expression of genes involved in cell adhesion. There was an increase in the expression of alpha and beta Laminins 1, beta Integrins 3 and 5, Secreted phosphoprotein1 and Sarcoglycan epsilon. No change in the expression of beta Actin was observed while the expression of beta Integrin 2 was significantly suppressed. CONCLUSIONS: ETOH had no effect on NPC apoptosis but, resulted in more rapid coalescence and increased volume of neurospheres. Additionally, the expression of genes associated with cell adhesion was significantly altered. ETOH induced changes in NPC surface adhesion interactions may underlie aspects of neurodevelopmental abnormalities in FASD.

Evaluation of polymer and self-assembled monolayer-coated silicone surfaces to reduce neural cell growth.

The development of silicone catheters has improved the treatment of hydrocephalus. Unfortunately, the functionality of the catheters used for the treatment of hydrocephalus is compromised by cell obstruction. In this study silicone surfaces coated with biopolymers (heparin and hyaluronan) and self-assembled monolayers (SAM) (octadecyltrichlorosilane-OTS and fluoroalkylsilane-FAS) were employed to investigate the effect of these coatings on astrocyte and choroid plexus cell growth in vitro. Compared to unmodified silicone, FAS surfaces significantly reduced (p<0.05) astrocyte proliferation, heparin (p<0.001) and hyaluronan (p<0.001) surfaces significantly increased astrocyte growth, while no significant difference was observed on OTS surfaces. A similar trend was observed for choroid plexus cell growth on heparin (p<0.05) and hyaluronan (p<0.05) coatings, however, no significant reduction in cell growth was observed on FAS- or OTS-coated surfaces compared to silicone. Low cell growth may be attributed to hydrophobicity of the surfaces (FAS 112.2+/-2.6 degrees , OTS 102.2+/-1.3 degrees). Contact angle measurements confirmed the stability of the hydrophobic and hydrophilic properties of all the coatings on the silicone surfaces for 30 days. Surface roughness did not play an important role on cell growth. Silicone shunts coated with SAMs may be suitable for future clinical applications to improve the treatment of hydrocephalus.