Gene Expression Endpoints Research Articles

Efficacy of GCN2 inhibition by a novel small molecule AP030 in acute leukemia.

6532 Background: GCN2 is an evolutionarily conserved kinase and a pivotal regulator of the Integrated Stress Response (ISR) that is activated in response to amino acid scarcity. Active GCN2 phosphorylates translation initiation factor eIF2α resulting in the attenuation of global protein synthesis. ISR signaling primarily promotes cell survival but may trigger cell death, dependent on the cellular context. In hematological tumors GCN2 promotes tumor cell survival under conditions of nutrient scarcity. Several GCN2 inhibitors are in clinical development for treatment of both solid and hematological tumors. Here, we describe the preclinical results of a novel, selective ATP-competitive inhibitor of GCN2. Methods: GCN2 inhibition by AP030 was determined using a biochemical Lanthascreen assay and a cell based HTRF assay measuring eIF2a phosphorylation following stimulation with Borrelidin. Kinase selectivity was determined using KinomeScan, and bespoke biochemical and cell-based assays. Interaction of AP030 with GCN2 was determined using X-ray crystallography. AP030 activity in disease-relevant tumor cell lines was determined using cell viability, caspase activation, protein expression and qPCR gene expression endpoints. Inhibition of hematological tumor growth and induction of cell death was investigated in patient samples, and ALL and AML in vivo animal models. Results: Potent inhibition of GCN2 in the Lanthascreen assay was demonstrated with AP030 (Ki of 4.4nM). Following stimulation with Borrelidin, AP030 inhibited eIF2a phosphorylation with an IC50 of 50.8nM and inhibited downstream targets CHAC1 and DDIT3 measured by qPCR. X-ray crystallography confirmed that AP030 binds to the ATP-binding site of GCN2. The KinomeScan confirmed that AP030 was highly selective against the human kinome. AP030 led to partial or complete reduction of AML cell line viability as a single agent and acted synergistically with asparaginase in ALL cell lines. Caspase 3/7 induction was observed in the AML and ALL cell lines evaluated and AP030 increased the proportion of cells undergoing apoptosis in AML patient primary samples. In the CCRF-CEM ALL systemic in vivo tumor model AP030 inhibited tumor growth in combination with asparaginase (TGI 79.33%). Treatment with 0.5-5mg/kg (q.d.) of AP030 resulted in dose-dependent tumor growth inhibition and regression of the MOLM-16 AML in vivo model (TGI 97.37% 5mg/kg). RNA sequencing of residual tumor cells revealed disruption of amino acid and protein metabolism, consistent with GCN2 inhibition. Conclusions: Targeting hematological tumor reliance on the GCN2 arm of the ISR during nutrient scarcity is a novel approach to preventing tumor cell survival. AP030 a novel, potent, selective, ATP-competitive kinase inhibitor has been shown to lead to impressive efficacy in acute leukemia. Based on these preclinical results, a phase 1/2 study has been initiated in AML.

Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/β-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/β-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/β-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

Environmental chemicals change extracellular lipidome of mature human white adipocytes

Certain environmental chemicals affect the body's energy balance and are known as metabolism disrupting chemicals (MDCs). MDCs have been implicated in the development of metabolic diseases, such as obesity and type 2 diabetes. In contrast to their well-known impact on developing adipocytes, MDC effects leading to altered energy balance and development of insulin resistance in mature white adipocytes, constituents of adult adipose tissue, are largely unclear.Here, we investigated the effects of six well-established environmental MDCs (bisphenol A (BPA), perfluorooctanoic acid (PFOA), triclosan (TCS), p,p-dichlorodiphenyl-dichloroethylene (ppDDE), tributyltin chloride (TBT) and triphenyl phosphate (TPP)) on mature human white adipocytes derived from mesenchymal stem cells in vitro. We aimed to identify biomarkers and sensitive endpoints of their metabolism disrupting effects. While most of the tested exposures had no effect on adipocyte glucose consumption, lipid storage and assessed gene expression endpoints, the highest concentration of triclosan affected the total lipid storage and adipocyte size, as well as glucose consumption and mRNA expression of the glucose transporter GLUT1, leptin and adiponectin. Additionally, an increased expression of adiponectin was observed with TPP and the positive control PPARγ agonist rosiglitazone.In contrast, the lipidomic analysis of the cell culture medium after a 3-day exposure was extremely sensitive and revealed concentration-dependent changes in the extracellular lipidome of adipocytes exposed to nearly all studied chemicals. While some of the extracellular lipidome changes were specific for the MDC used, some effects were found common to several tested chemicals and included increases in lysophosphatidylcholines, glycerophospholipids and ceramides and a decrease in fatty acids, with possible implications in inflammation, lipid and glucose uptake.This study points to early signs of metabolic disruption and likely systemic effects of mature adipocyte exposure to environmental chemicals, as well as to the need to include lipidomic endpoints in the assessment of adverse effects of MDCs.

Gene Expression Endpoints Following Subchronic Thiamethoxam Exposure in Adult Male Rabbits

Aim: to investigate the sub chronic toxicity of thiamethoxam on some parameters of reproductive performance in adult male rabbits including gene expression of LDH-C4, FSHβ and LHβ and GnRHR.
 Method: sixteen adult male Chinchilla rabbits were divided into two equal groups. Animals in the first group were treated orally with TMX at dose of 250 mg/kg body weight for 90 days. The second group was served as control.
 Result: Obtained results showed that TMX increased the relative weight of some reproductive organs including testis and prostate. Hormonal analysis revealed that, TMX induced a significant elevation in the serum testosterone level, while the concentrations of FSH and LH hormones did not exhibit any alterations between treated and control groups. In addition, LDH-C4, FSHβ and LHβ and GnRHR genes were down regulated in TMX treated group.
 Conclusion: Administration of thiamethoxam for 90 days in male rabbits induced a noticeable adverse effect on serum testosterone level and down regulated genes related to male rabbit reproductive performance.

Developmental and Hepatic Gene Expression Changes in Chicken Embryos Exposed to p-Tert-Butylphenyl Diphenyl Phosphate and Isopropylphenyl Phosphate via Egg Injection.

Organophosphate flame retardants (OPFRs) are used in a variety of products such as clear coats, resins, and plastics; however, research into their toxicological effects is limited. p-Tert-butylphenyl diphenyl phosphate (BPDP) and isopropylphenyl phosphate (IPPP) are two OPFRs that were prioritized for whole-animal toxicological studies based on observed effects in cultured avian hepatocytes in a previous study. The present study investigates the toxicity of BPDP and IPPP in chicken embryos at different developmental stages by evaluating morphological and gene expression endpoints. Chicken eggs were exposed via air cell injection to 0-250 μg/g (nominal) of either compound and then artificially incubated. At day 11 (midincubation), liver samples were collected for mRNA expression analysis; and at day 20 (1 day prehatch), morphological measurements and liver samples for transcriptomic evaluation were collected. At 250 μg/g, gallbladder size was significantly reduced for both compounds, head/bill length and tarsus length were significantly decreased, and liver somatic index was significantly increased following IPPP exposure only. No effects on mortality were observed up to the highest administered concentration for either chemical. Using a ToxChip polymerase chain reaction array, we report significant differences in hepatic gene expression for both compounds and time points; the most pronounced transcriptomic effects occurred at midincubation. Genes related to xenobiotic metabolism, bile acid/cholesterol regulation, and oxidative stress were significantly dysregulated. Given these changes observed throughout avian embryonic development, further research into the long-term effects of BPDP and IPPP are warranted, especially as they pertain to liver cholestasis. Environ Toxicol Chem 2022;41:739-747. © 2021 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry © 2021 SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Juvenile African Clawed Frogs (Xenopus laevis) Express Growth, Metamorphosis, Mortality, Gene Expression, and Metabolic Changes When Exposed to Thiamethoxam and Clothianidin.

Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57–66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.

Sublethal effects of terbium and praseodymium in juvenile rainbow trout

Abstract The increasing extraction of rare earth elements (REEs) and their subsequent release to freshwater ecosystems are expected to threaten aquatic organisms, yet little is known about the scope of this issue. The purpose of this study was to identify key molecular responses associated with exposure and toxicity of two data-poor REEs, terbium (Tb, heavy REE) and praesodymium (Pr, light REE) in rainbow trout juveniles. Fish were exposed for 96 h to increasing concentrations of Tb and Pr to determine impacts on survival and variation in biomarkers involved in oxidative stress, inflammation, DNA damage and metal homeostasis (metallothioneins). In surviving fish, a suite of 14 gene expression endpoints were also examined as well as genes associated with protein stability, calcium binding, immune response, protein damage, cell division, iron homeostasis and mitochondrial activity. The data showed that Tb was approximately two times more toxic than Pr, with estimated LC50 of 5.8 and 11 mg/L respectively. In addition, Tb and Pr exhibit respectively anti and pro-inflammatory responses relative to controls as demonstrated by the significant variation in the arachidonate cycloocygenase (COX) activity. However only Pr displayed a genotoxic effect due to the increase in the level of DNA strand breaks. Exposure to these two elements lead to the expression of genes involved in DNA repair activity, inflammation, protein denaturation, calcium binding, oxidative stress and divalent metal handling, ATP synthesis and hemoprotein activity. Based on the observed responses to both Tb and Pr, metal homeostasis, protein denaturation and DNA repair activity were commonly affected. The most toxic element (Tb) produced more important changes in the expression of genes involved in oxidative stress, calcium binding, hemoprotein activity and protein folding and turnover. These genes represents potential physiological targets involved in the mode of action for Tb and Pr in fish toxicity.

Histone H3 modifying genes may serve as a predictive marker for metastasis in synovial sarcoma.

11058 Background: Synovial sarcomas (SS) are rare, mesenchymal tumors that exhibit heterogeneous clinical behavior. SS is characterized molecularly by a fusion of the SS18 and one of three SSX genes. While fusion type has been shown to play a role in prognosis, little is known regarding the role of secondary genomic variants in SS. We hypothesized that frequently mutated secondary mutations corresponded to altered molecular pathways associated with accelerated disease progression. Here we present further analysis of the largest study of secondary SS genomic alterations and their potential clinical implications particularly in regard to Histone H3 modifying genes. Methods: Comprehensive Genomic Profiling (CGP) from 203 SS18-SSX fusion positive SS subjects were obtained from Foundation Medicine (FMI). Correlation between gene expression and survival endpoints were assessed in two independent datasets (GSE40018, n = 34; GSE40021, n = 58). A histone H3 score (HH3Score) was calculated through summation of expression values of MLL2, MLL3, SETD2. Altered expression was assessed for genes mutated in > 5% of SS in the FMI dataset. Datasets were also combined with appropriate adjustments for multivariate analysis. Cox proportional hazard, and Chi-squared test were used as appropriate. Results: In CGP profiling of SS, only 4 genes were seen in > 5% of subjects ( MLL3 9%, SETD2 7%, MYO18A 6%, MLL2 6%). 3 of these variants are known to regulate histone H3 activity and are collectively altered in 22% (44 of 203) of the FMI SS subjects. Notably, these H3 targeted gene alterations were found to be mutually exclusive of one another (p < 0.001). In 2 independent SS datasets, subjects with high histone H3 modifying gene RNA expression levels (HH3Score) demonstrated worsened outcomes with earlier tumor metastasis (GSE40018: HR = 0.41, 95%CI 0.15-1.07, p = 0.07; GSE40021: HR = 0.22, 95%CI 0.09-0.52, p < 0.001). Multivariate analysis demonstrated that HH3Score remained significantly correlated to metastasis-free survival (HR = 0.41, 95%CI 0.22-0.77, p < 0.01) when subjects’ age (p < 0.001) and fusion type (p = 0.60) were taken into consideration. Conclusions: Altered expression of histone H3 modifying genes may serve as a key driver for SS progression. Further prospective research is necessary to confirm its prognostic importance.

Subtle morphometric, behavioral and gene expression effects in larval zebrafish exposed to PFHxA, PFHxS and 6:2 FTOH

Recent studies of perfluoroalkylated substances (PFASs) have focused on the toxicity of long chain PFASs, such as PFOS or PFOA, which have been demonstrated to cause an array of developmental and behavioral effects. However, less is known about low molecular weight PFASs and alternatives. This study examined the morphometric and behavioral effects in zebrafish following developmental exposures of C6 PFASs: perfluorohexanoic acid, PFHxA, perfluorohexane sulfonate, PFHxS, and 6:2 fluorotelomer alcohol, 6:2 FTOH. Embryos were exposed to 0.02–20 μM concentrations of these compounds from the high stage (˜3 h post fertilization, hpf) until 120 hpf. Morphometric and gene expression endpoints were examined at 120 hpf. Genes selected for analysis were previously shown to be altered in zebrafish developmentally exposed to PFOS and PFOA. Additionally, exposed larvae were transferred to clean water and reared until 14 days post fertilization, dpf, when behavioral assays were completed and morphometric endpoints examined. While PFHxA was found to be the most acutely toxic at 120 hpf, few morphometric effects were observed. Gene expression was the most sensitive endpoint with significant increased tgfb1a, bdnf, and ap1s1 expression observed with PFHxA exposure. PFHxS exposure produced morphometric effects in the larvae, specifically increased length and yolk sac area at 2 and 20 μM. This phenotype persisted to the 14 dpf time point, where these larvae additionally displayed decreased distance traveled and crosses through the center of the arena of the behavioral assay. Exposure to 6:2 FTOH caused no morphometric effects at 120 hpf, and this compound was the least acutely toxic. However, expression of both tgfb1a and bdnf were increased by greater than 2 fold change at this time point. Effects also persisted to 14 dpf where a significant increase in distance traveled and velocity were observed in the behavioral assay. This study demonstrates effects on behavioral, morphometric and gene expression endpoints with developmental PFHxA, PFHxS, and 6:2 FTOH exposures in zebrafish.

Skeletal muscle PGC-1β signaling is sufficient to drive an endurance exercise phenotype and to counteract components of detraining in mice.

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α and -1β serve as master transcriptional regulators of muscle mitochondrial functional capacity and are capable of enhancing muscle endurance when overexpressed in mice. We sought to determine whether muscle-specific transgenic overexpression of PGC-1β affects the detraining response following endurance training. First, we established and validated a mouse exercise-training-detraining protocol. Second, using multiple physiological and gene expression end points, we found that PGC-1β overexpression in skeletal muscle of sedentary mice fully recapitulated the training response. Lastly, PGC-1β overexpression during the detraining period resulted in partial prevention of the detraining response. Specifically, an increase in the plateau at which O2 uptake (V̇o2) did not change from baseline with increasing treadmill speed [peak V̇o2 (ΔV̇o2max)] was maintained in trained mice with PGC-1β overexpression in muscle 6 wk after cessation of training. However, other detraining responses, including changes in running performance and in situ half relaxation time (a measure of contractility), were not affected by PGC-1β overexpression. We conclude that while activation of muscle PGC-1β is sufficient to drive the complete endurance phenotype in sedentary mice, it only partially prevents the detraining response following exercise training, suggesting that the process of endurance detraining involves mechanisms beyond the reversal of muscle autonomous mechanisms involved in endurance fitness. In addition, the protocol described here should be useful for assessing early-stage proof-of-concept interventions in preclinical models of muscle disuse atrophy.

Linking the response of endocrine regulated genes to adverse effects on sex differentiation improves comprehension of aromatase inhibition in a Fish Sexual Development Test

The Fish Sexual Development Test (FSDT) is a non-reproductive test to assess adverse effects of endocrine disrupting chemicals. With the present study it was intended to evaluate whether gene expression endpoints would serve as predictive markers of endocrine disruption in a FSDT. For proof-of-concept, a FSDT according to the OECD TG 234 was conducted with the non-steroidal aromatase inhibitor fadrozole (test concentrations: 10μg/L, 32μg/L, 100μg/L) using zebrafish (Danio rerio). Gene expression analyses using quantitative RT-PCR were included at 48h, 96h, 28days and 63days post fertilization (hpf, dpf). The selection of genes aimed at finding molecular endpoints which could be directly linked to the adverse apical effects of aromatase inhibition. The most prominent effects of fadrozole exposure on the sexual development of zebrafish were a complete sex ratio shift towards males and an acceleration of gonad maturation already at low fadrozole concentrations (10μg/L). Due to the specific inhibition of the aromatase enzyme (Cyp19) by fadrozole and thus, the conversion of C19-androgens to C18-estrogens, the steroid hormone balance controlling the sex ratio of zebrafish was altered. The resulting key event is the regulation of directly estrogen-responsive genes. Subsequently, gene expression of vitellogenin 1 (vtg1) and of the aromatase cyp19a1b isoform (cyp19a1b), were down-regulated upon fadrozole treatment compared to controls. For example, mRNA levels of vtg1 were down-regulated compared to the controls as early as 48 hpf and 96 hpf. Further regulated genes cumulated in pathways suggested to be controlled by endocrine mechanisms, like the steroid and terpenoid synthesis pathway (e.g. mevalonate (diphospho) decarboxylase (mvd), lanosterol synthase (2,3-oxidosqualene-lanosterol cyclase; lss), methylsterol monooxygenase 1 (sc4mol)) and in lipid transport/metabolic processes (steroidogenic acute regulatory protein (star), apolipoprotein Eb (apoEb)). Taken together, this study demonstrated that the existing Adverse Outcome Pathway (AOP) for aromatase inhibition in fish can be translated to the life-stage of sexual differentiation. We were further able to identify MoA-specific marker gene expression which can be instrumental in defining new measurable key events (KE) of existing or new AOPs related to endocrine disruption.

Abatacept Treatment Does Not Preserve Renal Function in the Streptozocin-Induced Model of Diabetic Nephropathy.

Diabetic nephropathy (DN) is one of the most severe complications of diabetes and remains the largest cause of end-stage renal disease in the Western world. Treatment options are limited and novel therapies that effectively slow disease progression are warranted. Previous work suggested that treatment with CTLA4-Ig (abatacept), a molecule that binds and blocks B7-1 and is licensed for the treatment of rheumatoid arthritis, could ameliorate DN. This study was designed to assess whether B7-1 signalling constitutes a promising therapeutic pathway for DN. Mice injected with streptozotocin (STZ) were treated with abatacept and glycemia and renal function were assessed. No differences were found in diabetes progression, albumin excretion rates or albumin/creatine ratios, while mesangial expansion was unaltered at endpoint. Except for increased renal CCL5, treatment did not affect a panel of gene expression endpoints reflecting early fibrotic changes, inflammation and kidney injury. Finally, abatacept treatment effectively reduced the accumulation of activated CD4+ T cells in the kidney, suggesting that renal T cell inflammation is not a driving factor in the pathology of the STZ model. In conjunction with the recent data discounting the expression of B7-1 on podocytes, our present data do not support a role for abatacept in DN treatment.

PFOS, PFNA, and PFOA sub-lethal exposure to embryonic zebrafish have different toxicity profiles in terms of morphometrics, behavior and gene expression

Polyfluorinated compounds (PFC) are a class of anthropogenic, persistent and toxic chemicals. PFCs are detected worldwide and consist of fluorinated carbon chains of varying length, terminal groups, and industrial uses. Previous zebrafish studies in the literature as well as our own studies have shown that exposure to these chemicals at a low range of concentrations (0.02–2.0μM; 20–2000ppb) resulted in chemical specific developmental defects and reduced post hatch survival. It was hypothesized that sub-lethal embryonic exposure to perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), or perfluorooctanoic acid (PFOA) would result in different responses with regard to morphometric, behavior, and gene expression in both yolk sac fry and larval zebrafish. Zebrafish were exposed to PFOS, PFOA, and PFNA (0.02, 0.2, 2.0μM) for the first five days post fertilization (dpf) and analyzed for morphometrics (5 dpf, 14 dpf), targeted gene expression (5 dpf, 14 dpf), and locomotive behavior (14 dpf). All three PFCs commonly resulted in a decrease in total body length, increased tfc3a (muscle development) expression and decreased ap1s (protein transport) expression at 5dpf, and hyperactive locomotor activity 14 dpf. All other endpoints measured at both life-stage time points varied between each of the PFCs. PFOS, PFNA, and PFOA exposure resulted in significantly altered responses in terms of morphometric, locomotion, and gene expression endpoints, which could be manifested in field exposed teleosts.

A Hormone-responsive 3D Culture Model of the Human Mammary Gland Epithelium.

The process of mammary epithelial morphogenesis is influenced by hormones. The study of hormone action on the breast epithelium using 2D cultures is limited to cell proliferation and gene expression endpoints. However, in the organism, mammary morphogenesis occurs in a 3D environment. 3D culture systems help bridge the gap between monolayer cell culture (2D) and the complexity of the organism. Herein, we describe a 3D culture model of the human breast epithelium that is suitable to study hormone action. It uses the commercially available hormone-responsive human breast epithelial cell line, T47D, and rat tail collagen type 1 as a matrix. This 3D culture model responds to the main mammotropic hormones: estradiol, progestins and prolactin. The influence of these hormones on epithelial morphogenesis can be observed after 1- or 2-week treatment according to the endpoint. The 3D cultures can be harvested for analysis of epithelial morphogenesis, cell proliferation and gene expression.

Correlating behaviour and gene expression endpoints in the dopaminergic system after modafinil administration in mouse

The mechanisms of action of modafinil continue to be poorly characterised and its potential for abuse in preclinical models remains controverted. The aim of this study was to further elucidate the mechanism of action of modafinil, through a potential behavioural and molecular association in the mouse. A conditioned place preference (CPP) paradigm was implemented to investigate the rewarding properties of modafinil. Whole genome expression and qRT-PCR analysis were performed on the ventral tegmental area (VTA), nucleus accumbens (NAC) and prefrontal cortex (PFC) of modafinil-treated and control animals. Modafinil administration (65mg/kg) induced an increase in locomotor activity, an increase in the change of preference for the drug paired side after a conditioning period as well as changes to gene expression profiles in the VTA (120 genes), NAC (23 genes) and PFC (19 genes). A molecular signature consisting of twelve up-regulated genes was identified as common to the three brain regions. Multiple linear correlation analysis showed a strong correlation (R2>0.70) between the behavioural and molecular endpoints in the three brain regions. We show that modafinil had a concomitant effect on CPP, locomotor activity, and up-regulation of interferon-γ (IFN-γ) regulated genes (Gbp2, Gbp3, Gbp10, Cd274, Igtp), while correlating the latter set of genes with behaviour changes evaluated through the CPP. A potential association can be proposed based on the dysregulation of p47 family genes and Gbp family of IFN-γ induced GTPases. In conclusion, these findings suggest a link between the behavioural and molecular events in the context of modafinil administration.

104 EMBRYO MANIPULATION TECHNIQUES ALTER PRE-IMPLANTATION DEVELOPMENT AND GENE EXPRESSION IN MOUSE EMBRYOS

Micromanipulation techniques are commonly employed with human embryos; however, the long-term effects on growth and development of embryos are unknown. Subjective techniques, such as embryo grading, may not be sensitive enough to show differences in developmental potential and more objective assessments are needed. The objective of this experiment was to evaluate whether manipulation techniques and cryopreservation alter morphologic and gene expression endpoints of embryos. Two-cell mouse embryos were cultured to blastocyst stage and divided into 6 treatment groups: nonmanipulated control (UNMAN; n = 252), laser-assisted hatching (LAH; n = 124), LAH with slow cooling cryopreservation (LAHcryo; n = 78), simulated biopsy (BIOP; n = 90) performed by laser disruption of 1/8 of the embryo’s cellular volume, BIOP with slow cooling cryopreservation (BIOPcryo; n = 91), and BIOP with vitrification (BIOPvit; n = 66). After manipulation or thaw, embryos were cultured 28 h to blastocyst stage. Fully hatched and hatching blastocysts were collected for cell counts and quantitative PCR to assay absolute transcript abundance for Plac8, Glut1, Oct4, and Cox2, 18s rRNA, and Ppia using standard curves generated from serial dilutions of digested plasmids. Data were analysed using ANOVA with Duncan’s post hoc test and significance was defined as P < 0.05. Manipulation groups differed in developmental stage at 28 h postmanipulation (P = 0.03), with a larger percentage of embryos completely hatched in LAH (46%) and BIOP (45%) groups compared to UNMAN (11%). Cryopreservation reduced percentages of completely hatched embryos (LAHcryo = 26%; BIOPcryo = 31%; BIOPvit = 19%). The number of cells per embryo also varied (P < 0.001); UNMAN and LAH groups had similar numbers of blastomeres (UNMAN = 71; LAH = 74), whereas BIOP groups had reduced cell counts (BIOP = 53; BIOPcryo = 56; BIOPvit = 48). Patterns of gene expression varied between groups; the BIOP group had more cDNA per cell (UNMAN = 125 ng cell–1; BIOP = 202 ng cell–1; P = 0.004), whereas BIOPcryo group did not show this effect (BIOPcryo = 111 ng cell–1). Transcript abundance of Cox2, Oct4, and Glut1 per embryo was significantly decreased (P < 0.05) in biopsied embryos that were cryopreserved, either by slow-cooling or vitrification (Cox2: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268; Oct4: UNMAN = 266, BIOP = 181, BIOPcryo = 95, BIOPvit = 97; Glut1: UNMAN = 643, BIOP = 490, BIOPcryo = 259, BIOPvit = 268). The data suggests that micromanipulation alters developmental timelines and gene expression of embryos. While manipulations differ in degree and nature of effect, our investigation implies all manipulations have some effect on the transcriptome of the developing embryo. Differences in cDNA quantity in the BIOP group may indicate a stress response or recovery attempt that becomes blunted in biopsied embryos which are subsequently cryopreserved. Thus, our study suggests that cryopreservation of biopsied embryos may come at a price and offers scientific support for only judiciously selected and medically indicated embryo biopsies.

Ribosome profiling to investigate in vivo molecular mechanisms of immune function (TECH2P.906)

Abstract Genomic analyses of gene expression of immune cells have so far focused on their transcriptional program by quantifying mRNA abundance, measured by microarray or more recently by RNA-seq. The mRNA abundance is often presumed as a proxy of the gene expression, but the correlation between mRNA abundance and protein level, which is the true end point of gene expression, frequently shows lack of sufficient correlation due to the apparent regulation of gene expression at the layer of translation. Here, we present the applications of an emerging technique, Ribosome profiling (Ribo-seq), on primary immune cells derived from various genetic mutant mice developing immune diseases. This directly captured genome-wide maps of protein synthesis (translatome) by allowing quantification of ribosome footprint of each mRNA with high resolution and depth. Side-by-side comparison of transcriptome and translatome in the disease models revealed the signaling pathways that are predominantly regulated in translation levels are prevalent, highlighting the critical roles of translational control in immune function.

Ribosome profiling: new views of translation, from single codons to genome scale

Genome-wide analyses of gene expression have so far focused on the abundance of mRNA species as measured either by microarray or, more recently, by RNA sequencing. However, neither approach provides information on protein synthesis, which is the true end point of gene expression. Ribosome profiling is an emerging technique that uses deep sequencing to monitor in vivo translation. Studies using ribosome profiling have already provided new insights into the identity and the amount of proteins that are produced by cells, as well as detailed views into the mechanism of protein synthesis itself.

Streamlining gene expression analysis: integration of co-culture and mRNA purification

Co-culture of multiple cell types within a single device enables the study of paracrine signaling events. However, extracting gene expression endpoints from co-culture experiments is laborious, due in part to pre-PCR processing of the sample (i.e., post-culture cell sorting and nucleic acid purification). Also, a significant loss of nucleic acid may occur during these steps, especially with microfluidic cell culture where lysate volumes are small and difficult to access. Here, we describe an integrated platform for performing microfluidic cell culture and extraction of mRNA for gene expression analysis. This platform was able to recover 30-fold more mRNA than a similar, non-integrated system. Additionally, using a breast cancer/bone marrow stroma co-culture, we recapitulated stromal-dependent, estrogen-independent growth of the breast cancer cells, coincident with transcriptional changes. We anticipate that this platform will be used for streamlined analysis of paracrine signaling events as well as for screening potential drugs and/or patient samples.

Characterization of Gene Expression Endpoints During Postembryonic Development of the Northern Green Frog (Rana clamitans melanota)

Postembryonic development of a larval tadpole into a juvenile frog involves the coordinated action of thyroid hormone (TH) across a diversity of tissues. Changes in the frog transcriptome represent a highly sensitive endpoint in the detection of developmental progression, and for the identification of environmental chemical contaminants that possess endocrine disruptive properties. Unfortunately, in contrast with their vital role as sentinels of environmental change, few gene expression tools currently exist for the majority of native North American frog species. We have isolated seven expressed gene sequences from the Northern green frog (Rana clamitans melanota) that encode proteins associated with TH-mediated postembryonic development and global stress response, and established a quantitative real-time polymerase chain reaction (qPCR) assay. We also obtained three additional species-specific gene sequences that functioned in the normalization of the expression data. Alterations in mRNA abundance profiles were identified in up to eight tissues during R. clamitans postembryonic development, and following exogenous administration of TH to premetamorphic tadpoles. Our results characterize tissue distribution and sensitivity to TH of select mRNA of a common North American frog species and support the potential use of this qPCR assay in identification of the presence of chemical agents in aquatic environments that modulate TH action.