A data browsing application for accessing gene and module-level blood transcriptome profiles of healthy pregnant women from high- and low-resource settings.

Transcriptome profiling data, generated via RNA sequencing, are commonly deposited in public repositories. However, these data may not be easily accessible or usable by many researchers. To enhance data reuse, we present well-annotated, partially analyzed data via a user-friendly web application. This project involved transcriptome profiling of blood samples from 15 healthy pregnant women in a low-resource setting, taken at 6 consecutive time points beginning from the first trimester. Additional blood transcriptome profiles were retrieved from the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) public repository, representing a cohort of healthy pregnant women from a high-resource setting. We analyzed these datasets using the fixed BloodGen3 module repertoire. We deployed a web application, accessible at https://thejacksonlaboratory.shinyapps.io/BloodGen3_Pregnancy/which displays the module-level analysis results from both original and public pregnancy blood transcriptome datasets. Users can create custom fingerprint grid and heatmap representations via various navigation options, useful for reports and manuscript preparation. The web application serves as a standalone resource for exploring blood transcript abundance changes during pregnancy. Alternatively, users can integrate it with similar applications developed for earlier publications to analyze transcript abundance changes of a given BloodGen3 signature across a range of disease cohorts. Database URL: https://thejacksonlaboratory.shinyapps.io/BloodGen3_Pregnancy/.

Design of a targeted blood transcriptional panel for monitoring immunological changes accompanying pregnancy.

Background: Immunomodulatory processes exert steering functions throughout pregnancy. Detecting diversions from this physiologic immune clock may help identify pregnant women at risk for pregnancy-associated complications. We present results from a data-driven selection process to develop a targeted panel of mRNAs that may prove effective in detecting pregnancies diverting from the norm. Methods: Based on a de novo dataset from a resource-constrained setting and a dataset from a resource-rich area readily available in the public domain, whole blood gene expression profiles of uneventful pregnancies were captured at multiple time points during pregnancy. BloodGen3, a fixed blood transcriptional module repertoire, was employed to analyze and visualize gene expression patterns in the two datasets. Differentially expressed genes were identified by comparing their abundance to non-pregnant postpartum controls. The selection process for a targeted gene panel considered (i) transcript abundance in whole blood; (ii) degree of correlation with the BloodGen3 module; and (iii) pregnancy biology. Results: We identified 176 transcripts that were complemented with eight housekeeping genes. Changes in transcript abundance were seen in the early stages of pregnancy and similar patterns were observed in both datasets. Functional gene annotation suggested significant changes in the lymphoid, prostaglandin and inflammation-associated compartments, when compared to the postpartum controls. Conclusion: The gene panel presented here holds promise for the development of predictive, targeted, transcriptional profiling assays. Such assays might become useful for monitoring of pregnant women, specifically to detect potential adverse events early. Prospective validation of this targeted assay, in-depth investigation of functional annotations of differentially expressed genes, and assessment of common pregnancy-associated complications with the aim to identify these early in pregnancy to improve pregnancy outcomes are the next steps.

Inherited human c-Rel deficiency disrupts myeloid and lymphoid immunity to multiple infectious agents.

We studied a child with severe viral, bacterial, fungal, and parasitic diseases, who was homozygous for a loss-of-function mutation of REL, encoding c-Rel, which is selectively expressed in lymphoid and myeloid cells. The patient had low frequencies of NK, effector memory cells reexpressing CD45RA (Temra) CD8+ T cells, memory CD4+ T cells, including Th1 and Th1*, Tregs, and memory B cells, whereas the counts and proportions of other leukocyte subsets were normal. Functional deficits of myeloid cells included the abolition of IL-12 and IL-23 production by conventional DC1s (cDC1s) and monocytes, but not cDC2s. c-Rel was also required for induction of CD86 expression on, and thus antigen-presenting cell function of, cDCs. Functional deficits of lymphoid cells included reduced IL-2 production by naive T cells, correlating with low proliferation and survival rates and poor production of Th1, Th2, and Th17 cytokines by memory CD4+ T cells. In naive CD4+ T cells, c-Rel is dispensable for early IL2 induction but contributes to later phases of IL2 expression. The patient's naive B cells displayed impaired MYC and BCL2L1 induction, compromising B cell survival and proliferation and preventing their differentiation into Ig-secreting plasmablasts. Inherited c-Rel deficiency disrupts the development and function of multiple myeloid and lymphoid cells, compromising innate and adaptive immunity to multiple infectious agents.

A Novel STK4 Mutation Impairs T Cell Immunity Through Dysregulation of Cytokine-Induced Adhesion and Chemotaxis Genes.

PURPOSE: Human serine/threonine kinase 4 (STK4) deficiency is a rare, autosomal recessive genetic disorder leading to combined immunodeficiency; however, the extent to which immune signaling and host defense are impaired is unclear. We assessed the functional consequences of a novel, homozygous nonsense STK4 mutation (NM_006282.2:c.871C > T, p.Arg291*) identified in a pediatric patient by comparing his innate and adaptive cell-mediated and humoral immune responses with those of three heterozygous relatives and unrelated controls. METHODS: The genetic etiology was verified by whole genome and Sanger sequencing. STK4 gene and protein expression was measured by quantitative RT-PCR and immunoblotting, respectively. Cellular abnormalities were assessed by high-throughput RT-RCR, RNA-Seq, ELISA, and flow cytometry. Antibody responses were assessed by ELISA and phage immunoprecipitation-sequencing. RESULTS: The patient exhibited partial loss of STK4 expression and complete loss of STK4 function combined with recurrent viral and bacterial infections, notably persistent Epstein-Barr virus viremia and pulmonary tuberculosis. Cellular and molecular analyses revealed abnormal fractions of T cell subsets, plasmacytoid dendritic cells, and NK cells. The transcriptional responses of the patient's whole blood and PBMC samples indicated dysregulated interferon signaling, impaired T cell immunity, and increased T cell apoptosis as well as impaired regulation of cytokine-induced adhesion and leukocyte chemotaxis genes. Nonetheless, the patient had detectable vaccine-specific antibodies and IgG responses to various pathogens, consistent with a normal CD19 + B cell fraction, albeit with a distinctive antibody repertoire, largely driven by herpes virus antigens. CONCLUSION: Patients with STK4 deficiency can exhibit broad impairment of immune function extending beyond lymphoid cells.

Humans with inherited T cell CD28 deficiency are susceptible to skin papillomaviruses but are otherwise healthy.

We study a patient with the human papilloma virus (HPV)-2-driven "tree-man" phenotype and two relatives with unusually severe HPV4-driven warts. The giant horns form an HPV-2-driven multifocal benign epithelial tumor overexpressing viral oncogenes in the epidermis basal layer. The patients are unexpectedly homozygous for a private CD28 variant. They have no detectable CD28 on their T cells, with the exception of a small contingent of revertant memory CD4+ T cells. T cell development is barely affected, and T cells respond to CD3 and CD2, but not CD28, costimulation. Although the patients do not display HPV-2- and HPV-4-reactive CD4+ T cells in vitro, they make antibodies specific for both viruses in vivo. CD28-deficient mice are susceptible to cutaneous infections with the mouse papillomavirus MmuPV1. The control of HPV-2 and HPV-4 in keratinocytes is dependent on the T cell CD28 co-activation pathway. Surprisingly, human CD28-dependent T cell responses are largely redundant for protective immunity.

Human STAT3 variants underlie autosomal dominant hyper-IgE syndrome by negative dominance.

Most patients with autosomal dominant hyper-IgE syndrome (AD-HIES) carry rare heterozygous STAT3 variants. Only six of the 135 in-frame variants reported have been experimentally shown to be dominant negative (DN), and it has been recently suggested that eight out-of-frame variants operate by haploinsufficiency. We experimentally tested these 143 variants, 7 novel out-of-frame variants found in HIES patients, and other STAT3 variants from the general population. Strikingly, all 15 out-of-frame variants were DN via their encoded (1) truncated proteins, (2) neoproteins generated from a translation reinitiation codon, and (3) isoforms from alternative transcripts or a combination thereof. Moreover, 128 of the 135 in-frame variants (95%) were also DN. The patients carrying the seven non-DN STAT3 in-frame variants have not been studied for other genetic etiologies. Finally, none of the variants from the general population tested, including an out-of-frame variant, were DN. Overall, our findings show that heterozygous STAT3 variants, whether in or out of frame, underlie AD-HIES through negative dominance rather than haploinsufficiency.

Three Copies of Four Interferon Receptor Genes Underlie a Mild Type I Interferonopathy in Down Syndrome.

Down syndrome (DS) is characterized by the occurrence of three copies of human chromosome 21 (HSA21). HSA21 contains a cluster of four interferon receptor (IFN-R) genes: IFNAR1, IFNAR2, IFNGR2, and IL10RB. DS patients often develop mucocutaneous infections and autoimmune diseases, mimicking patients with heterozygous gain-of-function (GOF) STAT1 mutations, which enhance cellular responses to three types of interferon (IFN). A gene dosage effect at these four loci may contribute to the infectious and autoimmune manifestations observed in individuals with DS. We report high levels of IFN-αR1, IFN-αR2, and IFN-γR2 expression on the surface of monocytes and EBV-transformed-B (EBV-B) cells from studying 45 DS patients. Total and phosphorylated STAT1 (STAT1 and pSTAT1) levels were constitutively high in unstimulated and IFN-α- and IFN-γ-stimulated monocytes from DS patients but lower than those in patients with GOF STAT1 mutations. Following stimulation with IFN-α or -γ, but not with IL-6 or IL-21, pSTAT1 and IFN-γ activation factor (GAF) DNA-binding activities were significantly higher in the EBV-B cells of DS patients than in controls. These responses resemble the dysregulated responses observed in patients with STAT1 GOF mutations. Concentrations of plasma type I IFNs were high in 12% of the DS patients tested (1.8% in the healthy controls). Levels of type I IFNs, IFN-Rs, and STAT1 were similar in DS patients with and without recurrent skin infections. We performed a genome-wide transcriptomic analysis based on principal component analysis and interferon modules on circulating monocytes. We found that DS monocytes had levels of both IFN-α- and IFN-γ-inducible ISGs intermediate to those of monocytes from healthy controls and from patients with GOF STAT1 mutations. Unlike patients with GOF STAT1 mutations, patients with DS had normal circulating Th17 counts and a high proportion of terminally differentiated CD8+ T cells with low levels of STAT1 expression. We conclude a mild interferonopathy in Down syndrome leads to an incomplete penetrance at both cellular and clinical level, which is not correlate with recurrent skin bacterial or fungal infections. The constitutive upregulation of type I and type II IFN-R, at least in monocytes of DS patients, may contribute to the autoimmune diseases observed in these individuals.

Chronic mucocutaneous candidiasis and connective tissue disorder in humans with impaired JNK1-dependent responses to IL-17A/F and TGF-ß.

Genetic etiologies of chronic mucocutaneous candidiasis (CMC) disrupt human IL-17A/F-dependent immunity at mucosal surfaces, whereas those of connective tissue disorders (CTDs) often impair the TGF-ß-dependent homeostasis of connective tissues. The signaling pathways involved are incompletely understood. We report a three-generation family with an autosomal dominant (AD) combination of CMC and a previously undescribed form of CTD that clinically overlaps with Ehlers-Danlos syndrome (EDS). The patients are heterozygous for a private splice-site variant of MAPK8, the gene encoding c-Jun N-terminal kinase 1 (JNK1), a component of the MAPK signaling pathway. This variant is loss-of-expression and loss-of-function in the patients' fibroblasts, which display AD JNK1 deficiency by haploinsufficiency. These cells have impaired, but not abolished, responses to IL-17A and IL-17F. Moreover, the development of the patients' TH17 cells was impaired ex vivo and in vitro, probably due to the involvement of JNK1 in the TGF-ß-responsive pathway and further accounting for the patients' CMC. Consistently, the patients' fibroblasts displayed impaired JNK1- and c-Jun/ATF-2-dependent induction of key extracellular matrix (ECM) components and regulators, but not of EDS-causing gene products, in response to TGF-ß. Furthermore, they displayed a transcriptional pattern in response to TGF-ß different from that of fibroblasts from patients with Loeys-Dietz syndrome caused by mutations of TGFBR2 or SMAD3, further accounting for the patients' complex and unusual CTD phenotype. This experiment of nature indicates that the integrity of the human JNK1-dependent MAPK signaling pathway is essential for IL-17A- and IL-17F-dependent mucocutaneous immunity to Candida and for the TGF-ß-dependent homeostasis of connective tissues.

A deep intronic splice mutation of STAT3 underlies hyper IgE syndrome by negative dominance.

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

Life-threatening influenza pneumonitis in a child with inherited IRF9 deficiency.

Life-threatening pulmonary influenza can be caused by inborn errors of type I and III IFN immunity. We report a 5-yr-old child with severe pulmonary influenza at 2 yr. She is homozygous for a loss-of-function IRF9 allele. Her cells activate gamma-activated factor (GAF) STAT1 homodimers but not IFN-stimulated gene factor 3 (ISGF3) trimers (STAT1/STAT2/IRF9) in response to IFN-α2b. The transcriptome induced by IFN-α2b in the patient's cells is much narrower than that of control cells; however, induction of a subset of IFN-stimulated gene transcripts remains detectable. In vitro, the patient's cells do not control three respiratory viruses, influenza A virus (IAV), parainfluenza virus (PIV), and respiratory syncytial virus (RSV). These phenotypes are rescued by wild-type IRF9, whereas silencing IRF9 expression in control cells increases viral replication. However, the child has controlled various common viruses in vivo, including respiratory viruses other than IAV. Our findings show that human IRF9- and ISGF3-dependent type I and III IFN responsive pathways are essential for controlling IAV.

A recessive form of hyper-IgE syndrome by disruption of ZNF341-dependent STAT3 transcription and activity.

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

Rapid effects of the aromatase inhibitor fadrozole on steroid production and gene expression in the ovary of female fathead minnows (Pimephales promelas).

Cytochrome P450 aromatase catalyzes conversion of C19 androgens to C18 estrogens and is critical for normal reproduction in female vertebrates. Fadrozole is a model aromatase inhibitor that has been shown to suppress estrogen production in the ovaries of fish. However, little is known about the early impacts of aromatase inhibition on steroid production and gene expression in fish. Adult female fathead minnows (Pimephales promelas) were exposed via water to 0, 5, or 50µg fadrozole/L for a time-course of 0.5, 1, 2, 4, and 6h, or 0 or 50µg fadrozole/L for a time-course of 6, 12, and 24h. We examined ex vivo ovarian 17ß-estradiol (E2) and testosterone (T) production, and plasma E2 concentrations from each study. Expression profiles of genes known or hypothesized to be impacted by fadrozole including aromatase (cytochrome P450 [cyp] 19a1a), steriodogenic acute regulatory protein (star), cytochrome P450 side-chain cleavage (cyp11a), cytochrome P450 17 alpha hydroxylase/17,20 lyase (cyp17), and follicle stimulating hormone receptor (fshr) were measured in the ovaries by quantitative real-time polymerase chain reaction (QPCR). In addition, broader ovarian gene expression was examined using a 15k fathead minnow microarray. The 5µg/L exposure significantly reduced ex vivo E2 production by 6h. In the 50µg/L treatment, ex vivo E2 production was significantly reduced after just 2h of exposure and remained depressed at all time-points examined through 24h. Plasma E2 concentrations were significantly reduced as early as 4h after initiation of exposure to either 5 or 50µg fadrozole/L and remained depressed throughout 24h in the 50µg/L exposure. Ex vivo T concentrations remained unchanged throughout the time-course. Expression of transcripts involved in steroidogenesis increased within the first 24h suggesting rapid induction of a mechanism to compensate for fadrozole inhibition of aromatase. Microarray results also showed fadrozole exposure caused concentration- and time-dependent changes in gene expression profiles in many HPG-axis pathways as early as 4h. This study provides insights into the very rapid effects of aromatase inhibition on steroidogenic processes in fish.

Prioritization of Contaminants of Emerging Concern in Wastewater Treatment Plant Discharges Using Chemical:Gene Interactions in Caged Fish.

We examined whether contaminants present in surface waters could be prioritized for further assessment by linking the presence of specific chemicals to gene expression changes in exposed fish. Fathead minnows were deployed in cages for 2, 4, or 8 days at three locations near two different wastewater treatment plant discharge sites in the Saint Louis Bay, Duluth, MN and one upstream reference site. The biological impact of 51 chemicals detected in the surface water of 133 targeted chemicals was determined using biochemical endpoints, exposure activity ratios for biological and estrogenic responses, known chemical:gene interactions from biological pathways and knowledge bases, and analysis of the covariance of ovary gene expression with surface water chemistry. Thirty-two chemicals were significantly linked by covariance with expressed genes. No estrogenic impact on biochemical endpoints was observed in male or female minnows. However, bisphenol A (BPA) was identified by chemical:gene covariation as the most impactful estrogenic chemical across all exposure sites. This was consistent with identification of estrogenic effects on gene expression, high BPA exposure activity ratios across all test sites, and historical analysis of the study area. Gene expression analysis also indicated the presence of nontargeted chemicals including chemotherapeutics consistent with a local hospital waste stream. Overall impacts on gene expression appeared to be related to changes in treatment plant function during rain events. This approach appears useful in examining the impacts of complex mixtures on fish and offers a potential route in linking chemical exposure to adverse outcomes that may reduce population sustainability.

AKT1 has dual actions on the glucocorticoid receptor by cooperating with 14-3-3.

Glucocorticoids are important therapeutic compounds for acute lymphoblastic leukemia (ALL). AKT1 or the protein kinase B is frequently activated in ALL, and contributes to the development of glucocorticoid resistance. We examined impact of AKT1 on glucocorticoid receptor (GR)-induced transcriptional activity in cooperation with phospho-serine/threonine-binding protein 14-3-3. AKT1 has two distinct actions on GR transcriptional activity, one through segregation of GR in the cytoplasm by phosphorylating GR at Ser-134 and subsequent association of 14-3-3, and the other through direct modulation of GR transcriptional activity in the nucleus. For the latter, AKT1 and 14-3-3 are attracted to DNA-bound GR, accompanied by AKT1-dependent p300 phosphorylation, H3S10 phosphorylation and H3K14 acetylation at the DNA site. These two actions of AKT1 regulate distinct sets of glucocorticoid-responsive genes. Our results suggest that specific inhibition of the AKT1/14-3-3 activity on the cytoplasmic retention of GR may be a promising target for treating glucocorticoid resistance observed in ALL.

Ran GTPase promotes cancer progression via Met recepto-rmediated downstream signaling.

It has been shown previously that cancer cells with an activated oncogenic pathway, including Met activation, require Ran for growth and survival.Here, we show that knockdown of Ran leads to a reduction of Met receptor expression in several breast and lung cancer cell lines. This, in turn suppressed HGF expression and the Met-mediated activation of the Akt pathway, as well as cell adhesion, migration, and invasion. In a cell line model where Met amplification has previously been shown to contribute to gefitinib resistance, Ran knockdown sensitized cells to gefitinib-mediated inhibition of Akt and ERK1/2 phosphorylation and consequently reduced cell proliferation. We further demonstrate that Met reduction-mediated by knockdown of Ran, occurs at the post-transcriptional level, probably via a matrix metalloproteinase. Moreover, the level of immunoreactive Ran and Met are positively associated in human breast cancer specimens, suggesting that a high level of Ran may be a pre-requisite for Met overexpression. Interestingly, a high level of immunoreactive Ran dictates the prognostic significance of Met, indicating that the co-overexpression of Met and Ran may be associated with cancer progression and could be used in combination as a prognostic indicator.

KDM6B histone demethylase is an epigenetic regulator of estrogen receptor ß expression in human pleural mesothelioma.

AIM: To assess the correlation between KDM6B and estrogen receptor ß (ERß) expression in malignant pleural mesothelioma (MPM). MATERIALS & METHODS: We evaluated gene expression by in silico analysis of microarray data, real-time PCR and western blot in MPM tumors and cell lines. RESULTS & CONCLUSION: We report a strong positive correlation between the expression of KDM6B and ERß in MPM tumors and cell lines. We describe that, in hypoxia, the HIF2α-KDM6B axis induces an epithelioid morphology and ERß expression in biphasic MPM cells with estrogen receptor-negative phenotype. Reduced histone H3K27 tri-methylation confirms KDM6B activity under hypoxic conditions. Importantly, cells treated during reoxygenation with the selective ERß agonist, KB9520, maintain ERß expression and the less aggressive phenotype acquired in hypoxia.

Coral-zooxanthellae meta-transcriptomics reveals integrated response to pollutant stress.

BACKGROUND: Corals represent symbiotic meta-organisms that require harmonization among the coral animal, photosynthetic zooxanthellae and associated microbes to survive environmental stresses. We investigated integrated-responses among coral and zooxanthellae in the scleractinian coral Acropora formosa in response to an emerging marine pollutant, the munitions constituent, 1,3,5-trinitro-1,3,5 triazine (RDX; 5 day exposures to 0 (control), 0.5, 0.9, 1.8, 3.7, and 7.2 mg/L, measured in seawater). RESULTS: RDX accumulated readily in coral soft tissues with bioconcentration factors ranging from 1.1 to 1.5. Next-generation sequencing of a normalized meta-transcriptomic library developed for the eukaryotic components of the A. formosa coral holobiont was leveraged to conduct microarray-based global transcript expression analysis of integrated coral/zooxanthellae responses to the RDX exposure. Total differentially expressed transcripts (DET) increased with increasing RDX exposure concentrations as did the proportion of zooxanthellae DET relative to the coral animal. Transcriptional responses in the coral demonstrated higher sensitivity to RDX compared to zooxanthellae where increased expression of gene transcripts coding xenobiotic detoxification mechanisms (i.e. cytochrome P450 and UDP glucuronosyltransferase 2 family) were initiated at the lowest exposure concentration. Increased expression of these detoxification mechanisms was sustained at higher RDX concentrations as well as production of a physical barrier to exposure through a 40% increase in mucocyte density at the maximum RDX exposure. At and above the 1.8 mg/L exposure concentration, DET coding for genes involved in central energy metabolism, including photosynthesis, glycolysis and electron-transport functions, were decreased in zooxanthellae although preliminary data indicated that zooxanthellae densities were not affected. In contrast, significantly increased transcript expression for genes involved in cellular energy production including glycolysis and electron-transport pathways was observed in the coral animal. CONCLUSIONS: Transcriptional network analysis for central energy metabolism demonstrated highly correlated responses to RDX among the coral animal and zooxanthellae indicative of potential compensatory responses to lost photosynthetic potential within the holobiont. These observations underscore the potential for complex integrated responses to RDX exposure among species comprising the coral holobiont and highlight the need to understand holobiont-species interactions to accurately assess pollutant impacts.

Differential effects and potential adverse outcomes of ionic silver and silver nanoparticles in vivo and in vitro.

Nanoparticles are of concern because of widespread use, but it is unclear if metal nanoparticles cause effects directly or indirectly. We explored whether polyvinylpyrrolidone-coated silver nanoparticles (PVP-AgNPs) cause effects through intact nanoparticles or dissolved silver. Females of the model species fathead minnow (Pimephales promelas) were exposed to either 4.8 µg/L of AgNO3 or 61.4 µg/L of PVP-AgNPs for 96h. Microarray analyses were used to identify impacted receptors and toxicity pathways in liver and brain tissues that were confirmed using in vitro mammalian assays. AgNO3 and PVP-AgNP exposed fish had common and distinct effects consistent with both intact nanoparticles and dissolved silver causing effects. PVP-AgNPs and AgNO3 both affected pathways involved in Na(+), K(+), and H(+) homeostasis and oxidative stress but different neurotoxicity pathways. In vivo effects were supported by PVP-AgNP activation of five in vitro nuclear receptor assays and inhibition of ligand binding to the dopamine receptor. AgNO3 inhibited ligand binding to adrenergic receptors α1 and α2 and cannabinoid receptor CB1, but had no effect in nuclear receptor assays. PVP-AgNPs have the potential to cause effects both through intact nanoparticles and metal ions, each interacting with different initiating events. Since the in vitro and in vivo assays examined here are commonly used in human and ecological hazard screening, this work suggests that environmental health assessments should consider effects of intact nanoparticles in addition to dissolved metals.

Integrated approach to explore the mechanisms of aromatase inhibition and recovery in fathead minnows (Pimephales promelas).

Aromatase, a member of the cytochrome P450 superfamily, is a key enzyme in estradiol synthesis that catalyzes the aromatization of androgens into estrogens in ovaries. Here, we used an integrated approach to assess the mechanistic basis of the direct effects of aromatase inhibition, as well as adaptation and recovery processes in fish. We exposed female fathead minnows (Pimephales promelas) via the water to 30 µg/L of a model aromatase inhibitor, fadrozole, during 8 days (exposure phase). Fish were then held in clean water for 8 more days (recovery phase). Samples were collected at 1, 2, 4, and 8 days of both the exposure and the recovery phases. Transcriptomics, metabolomics, and network inference were used to understand changes and infer connections at the transcript and metabolite level in the ovary. Apical endpoints directly indicative of endocrine function, such as plasma estradiol, testosterone, and vitellogenin levels were also measured. An integrated analysis of the data revealed changes in gene expression consistent with increased testosterone in fadrozole-exposed ovaries. Metabolites such as glycogen and taurine were strongly correlated with increased testosterone levels. Comparison of in vivo and ex vivo steroidogenesis data suggested the accumulation of steroidogenic enzymes, including aromatase, as a mechanism to compensate for aromatase inhibition.

The good, the bad, and the toxic: approaching hormesis in Daphnia magna exposed to an energetic compound.

A hormetic response is characterized by an opposite effect in small and large doses of chemical exposure, often resulting in seemingly beneficial effects at low doses. Here, we examined the potential mechanisms underlying the hormetic response of Daphnia magna to the energetic trinitrotoluene (TNT). Daphnia magna were exposed to TNT for 21 days, and a significant increase in adult length and number of neonates was identified at low concentrations (0.002-0.22 mg/L TNT), while toxic effects were identified at high concentrations (0.97 mg/L TNT and above). Microarray analysis of D. magna exposed to 0.004, 0.12, and 1.85 mg/L TNT identified effects on lipid metabolism as a potential mechanism underlying hormetic effects. Lipidomic analysis of exposed D. magna supported the hypothesis that TNT exposure affected lipid and fatty acid metabolism, showing that hormetic effects could be related to changes in polyunsaturated fatty acids known to be involved in Daphnia growth and reproduction. Our results show that Daphnia exposed to low levels of TNT presented hormetic growth and reproduction enhancement, while higher TNT concentrations had an opposite effect. Our results also show how a systems approach can help elucidate potential mechanisms of action and adverse outcomes.