Netrin-3 Peptide (C-19) is a Chemorepellent and a Growth Inhibitor in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

The netrins are a family of signaling proteins expressed throughout the animal kingdom. Netrins play important roles in developmental processes such as axonal guidance and angiogenesis. Netrin-1, for example, can act as either a chemoattractant or a chemorepellent for axonal growth cones depending upon the concentration of the protein as well as the cell type. Netrin-1 acts as a growth factor in some mammalian cell types and is also expressed by some tumor cells. Netrin-3 appears to share some signaling apparatus with netrin-1, but is less widely expressed, and its physiological roles are much less understood. Netrin-3 is also used as a biomarker for some cancers as well as traumatic kidney injury. Tetrahymena thermophila are free-living, eukaryotic, ciliated protozoas used as a model system for studying chemorepellents and chemoattractants because their swimming behavior is readily observable under a microscope. We have previously found that netrin-1 peptide acts as a chemorepellent in Tetrahymena thermophila at concentrations ranging from micromolar to nanomolar. However, netrin-1 peptide does not affect growth in Tetrahymena at these concentrations. In our current study, we have found that related peptides, netrin-3 peptide (H-19 and C-19; Santa Cruz Biotechnology), act as chemorepellents in Tetrahymena thermophila at concentrations at or below 1 μg/ml. The same concentration of netrin-3 peptide reduces growth of Tetrahymena cultures by approximately 75%. We are currently conducting further studies to determine the mechanism through which these peptides are signaling

Lineage-dependent effects of aryl hydrocarbon receptor agonists contribute to liver tumorigenesis

Rodent cancer bioassays indicate that the aryl hydrocarbon receptor (AHR) agonist, 2,3,7,8-tetracholorodibenzo-p-dioxin (TCDD), causes increases in both hepatocytic and cholangiocytic tumors. Effects of AHR activation have been evaluated on rodent hepatic stem cells (rHpSCs) versus their descendants, hepatoblasts (rHBs), two lineage stages of multipotent, hepatic precursors with overlapping but also distinct phenotypic traits. This was made possible by defining the first successful culture conditions for ex vivo maintenance of rHpScs consisting of a substratum of hyaluronans and Kubota's medium (KM), a serum-free medium designed for endodermal stem/progenitor cells. Supplementation of KM with leukemia inhibitory factor elicited lineage restriction to rHBs. Cultures were treated with various AHR agonists including TCDD, 6-formylindolo-[3,2-b]carbazole (FICZ), and 3-3'-diindolylmethane (DIM) and then analyzed with a combination of immunocytochemistry, gene expression, and high-content image analysis. The AHR agonists increased proliferation of rHpSCs at concentrations producing a persistent AHR activation as indicated by induction of Cyp1a1. By contrast, treatment with TCDD resulted in a rapid loss of viability of rHBs, even though the culture conditions, in the absence of the agonists, were permissive for survival and expansion of rHBs. The effects were not observed with FICZ and at lower concentrations of DIM. Conclusion: Our findings are consistent with a lineage-dependent mode of action for AHR agonists in rodent liver tumorigenesis through selective expansion of rHpSCs in combination with a toxicity-induced loss of viability of rHBs. These lineage-dependent effects correlate with increased frequency of liver tumors. (Hepatology 2015;61:548-560

Identifying genes that mediate anthracyline toxicity in immune cells

The role of the immune system in response to chemotherapeutic agents remains elusive. The interpatient variability observed in immune and chemotherapeutic cytotoxic responses is likely, at least in part, due to complex genetic differences. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at identifying genes underlying these chemotherapeutic cytotoxic effects on immune cells. Using genome-wide association studies (GWAS), we identified four genome-wide significant quantitative trait loci (QTL) that contributed to the sensitivity of doxorubicin and idarubicin in immune cells. Of particular interest, a locus on chromosome 16 was significantly associated with cell viability following idarubicin administration (p = 5.01 × 10−8). Within this QTL lies App, which encodes amyloid beta precursor protein. Comparison of dose-response curves verified that T-cells in App knockout mice were more sensitive to idarubicin than those of C57BL/6J control mice (p < 0.05). In conclusion, the cellular screening approach coupled with GWAS led to the identification and subsequent validation of a gene involved in T-cell viability after idarubicin treatment. Previous studies have suggested a role for App in in vitro and in vivo cytotoxicity to anticancer agents; the overexpression of App enhances resistance, while the knockdown of this gene is deleterious to cell viability. Further investigations should include performing mechanistic studies, validating additional genes from the GWAS, including Ppfia1 and Ppfibp1, and ultimately translating the findings to in vivo and human studies

Netrin-3 Avoidance and Mitotic Inhibition in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e Involves Intracellular Calcium and Serine/Threonine Kinase Activity

Netrins are a family of signaling proteins ubiquitously expressed throughout the animal kingdom. While netrin-1 has been well characterized, other netrins, such as netrin-3, remain less well understood. In our current study, we characterize the behavior of two netrin-3 peptides, one derived from the N-terminal and one derived from the C-terminal of netrin-3. Both peptides cause avoidance behavior and mitotic inhibition in Tetrahymena thermophila at concentrations as low as 0.5 micrograms (μg) per milliliter. These effects can be reversed by addition of the calcium chelator, EGTA; the intracellular calcium chelator, BAPTA-AM, or the serine/threonine kinase inhibitor, apigenin. The broad spectrum tyrosine kinase inhibitor, genistein, has no effect on netrin-3 signaling, indicating that netrin-3 signaling in this organism uses a different pathway than the previously described netrin-1 pathway. Further studies will allow us to better describe the netrin-3 signaling pathway in this organism

A Qualitative Modeling Approach for Whole Genome Prediction Using High-Throughput Toxicogenomics Data and Pathway-Based Validation

Efficient high-throughput transcriptomics (HTT) tools promise inexpensive, rapid assessment of possible biological consequences of human and environmental exposures to tens of thousands of chemicals in commerce. HTT systems have used relatively small sets of gene expression measurements coupled with mathematical prediction methods to estimate genome-wide gene expression and are often trained and validated using pharmaceutical compounds. It is unclear whether these training sets are suitable for general toxicity testing applications and the more diverse chemical space represented by commercial chemicals and environmental contaminants. In this work, we built predictive computational models that inferred whole genome transcriptional profiles from a smaller sample of surrogate genes. The model was trained and validated using a large scale toxicogenomics database with gene expression data from exposure to heterogeneous chemicals from a wide range of classes (the Open TG-GATEs data base). The method of predictor selection was designed to allow high fidelity gene prediction from any pre-existing gene expression data set, regardless of animal species or data measurement platform. Predictive qualitative models were developed with this TG-GATES data that contained gene expression data of human primary hepatocytes with over 941 samples covering 158 compounds. A sequential forward search-based greedy algorithm, combining different fitting approaches and machine learning techniques, was used to find an optimal set of surrogate genes that predicted differential expression changes of the remaining genome. We then used pathway enrichment of up-regulated and down-regulated genes to assess the ability of a limited gene set to determine relevant patterns of tissue response. In addition, we compared prediction performance using the surrogate genes found from our greedy algorithm (referred to as the SV2000) with the landmark genes provided by existing technologies such as L1000 (Genometry) and S1500 (Tox21), finding better predictive performance for the SV2000. The ability of these predictive algorithms to predict pathway level responses is a positive step toward incorporating mode of action (MOA) analysis into the high throughput prioritization and testing of the large number of chemicals in need of safety evaluation

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways

Health and social impacts of open defecation on women: a systematic review

Background The significance of sanitation to safeguard human health is irrefutable and has important public health dimensions. Access to sanitation has been essential for human dignity, health and well-being. Despite 15 years of conjunctive efforts under the global action plans like Millennium Development Goals (MDGs), 2.3 billion people have no access to improved sanitation facilities (flush latrine or pit latrine) and nearly 892 million of the total world’s population is still practicing open defecation. Methods The study provides a systematic review of the published literature related to implications of open defecation that goes beyond the scope of addressing health outcomes by also investigating social outcomes associated with open defecation. The Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) was used to frame the review, empirical studies focusing upon open defecation in women aged 13–50 in low and middle income countries were included in the review. Research papers included in the review were assessed for quality using appropriate critical appraisal tools. In total 9 articles were included in the review; 5 of these related to health effects and 4 related to social effects of open defecation. Results The review identified 4 overarching themes; Health Impacts of open defecation, Increased risk of sexual exploitation, Threat to women’s privacy and dignity and Psychosocial stressors linked to open defecation, which clearly present a serious situation of poor sanitation in rural communities of Lower-Middle Income Countries (LMICs). The findings of the review identified that open defecation promotes poor health in women with long-term negative effects on their psychosocial well-being, however it is a poorly researched topic. Conclusion The health and social needs of women and girls remain largely unmet and often side-lined in circumstances where toilets in homes are not available. Further research is critically required to comprehend the generalizability of effects of open defecation on girls and women

Chemically mediated plant-herbivore coevolution: influence of plant cyanogenic compounds on feeding and growth of the Eastern Tent Caterpillar, Malacosoma americanum.

We investigated the evolutionary stance of Malacosoma americanum in the stepwise coevolutionary process between herbivores and plants, in field and laboratory experiments. We tested the benefits of M. americanum's specialization on P. serotina. Our hypothesis states that the cyanogenic glycoside found in P. serotina is used as a feeding stimulant by M. americanum. Our statistical data shows a higher relative growth rate in P. serotina in comparison to some of our tested plant species. However, we also concluded that cyanogenic glycosides are not significantly used as a feeding stimulant.http://deepblue.lib.umich.edu/bitstream/2027.42/54497/1/2935.pdfDescription of 2935.pdf : Access restricted to on-site users at the U-M Biological Station

Characterization of a Netrin-1-like Protein Secreted by \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of pleiotropic signaling proteins, expressed throughout the animal kingdom, that have guidance functions in the development of the nervous system and other branched tissues, Netrins often serve a chemotactic role, acting as chemoattractants or chemorepellents depending upon the type of receptors expressed within the tissue. Chemorepellent transduction usually involves the UNC-5 family of receptors along with the tyrosine kinase, src-1. The best-characterized netrin in the family, netrin-1, has previously been shown to be a chemorepellent in the ciliated protozoan Tetrahymena thermophila, and the tyrosine kinase inhibitor genistein, blocked netrin-1 signaling in this organism. T. thermophila secrete a protein that is immunologically similar to netrin-1, suggesting that this netrin-1-like protein may play a role in intercellular communication. In this study, we find that the netrin-1-like protein of Tetrahymena is a basic protein, approximately 52 kD, which is found in whole cell extract but enriched in secreted protein. In addition, our data indicate that T. thermophila have proteins that are immunologically similar to src-1 and UNC-5, suggesting that parts of the netrin signaling pathway conserved throughout the animal kingdom may also be present in Kingdom Protista. Further characterization will be necessary to learn more about these signaling proteins and their physiological role in this organism

Netrin-3-peptides Are Chemorepellents and Mitotic Inhibitors in \u3cem\u3eTetrahymena thermophila\u3c/em\u3e

Netrins are a family of guidance proteins involved in developmental signaling as well as maintenance of homeostasis within the adult organism. Netrin-1 is the best characterized of all the netrins and has been linked to cancer, apoptosis, angiogenesis, and immune signaling. However, much less is known about netrin-3, especially its roles outside of development. In this study, we performed behavioral assays that demonstrated the chemorepellent effect of netrin-3-peptides on Tetrahymena thermophila. Our pharmacological inhibition assays showed that signaling is dependent on calcium and serine/threonine kinases. We also observed that exposing cells to netrin-3-peptides for two days caused a significant decrease in the mitotic rate. Inhibition of mitosis was rescued by adding calcium chelators or a serine/threonine kinase inhibitor to the culture media alongside the netrin-3-peptides. We also used immunofluorescence and enzyme-linked immunosorbent assay (ELISA) assays to determine that Tetrahymena secrete a netrin-3-like protein, suggesting a physiological role for a netrin-3-like signal in keeping cell populations from depleting their resources too rapidly. Finally, we contrasted signaling data from our studies with netrin-3-peptides to our data from our previous studies with netrin-1-peptide, showing that the two peptides use different signaling pathways, resulting in dissimilar physiological consequences for the organism