Effect of nonylphenol on gene expression in Atlantic salmon liver, gill, hypothalamus, pituitary, and olfactory rosettes
ABSTRACT: Exposure to environmental contaminants like nonylphenol can disrupt smolt development and may be a contributing factor in salmon population declines. We used GRASP 16K cDNA microarrays to identify genes that are differentially expressed in the liver, gill, hypothalamus, pituitary, and olfactory rosettes of Atlantic salmon smolts treated with nonylphenol compared to control smolts. Nonylphenol treatment was confirmed using physiological assays: nonylphenol-treatment significantly decreased gill Na+,K+-ATPase activity and plasma cortisol and T3 levels. Microarray analyses were used to compare expression in nonylphenol-injected fish with expression in vehicle-injected fish: eight arrays each for liver, gill, olfactory rosettes, hypothalamus, and pituitary tissues. Total RNA was isolated from the tissues of eight nonylphenol-injected fish (six males and two females) and eight vehicle-injected fish (two males and six females) and reverse transcribed separately (not pooled); each slide represents a biological replicate. For each tissue, the eight arrays were balanced for dye: nonylphenol-injected fish were labeled with Alexa Fluor 555 and vehicle-injected fish were labeled with Alexa Fluor 647 on four slides, nonylphenol-injected fish were labeled with Alexa Fluor 647 and vehicle-injected fish were labeled with Alexa Fluor 555 on four slides. Liver, gill, hypothalamus, pituitary, and olfactory rosette tissues were analyzed separately.
Project description:To identify genes involved in the developmental process of Atlantic salmon smoltification, gene expression was compared between smolt and parr in tissues involved in osmoregulation (gill), metabolism (liver), imprinting (olfactory rosettes) and neuroendocrine control (hypothalamus and pituitary). Tissue samples were harvested from laboratory-reared parr and smolts on the same date. Smolts were distinguished from parr by size and appearance; developmental status was confirmed by physiological assays. Eight biological replicates (16 fish) balanced for sex and for dye were used in the liver, gill, olfactory rosette, and hypothalamus comparisons. Four male parr were compared to four male smolts and four female parr were compared to four female smolts; smolts were labeled with Alexa Fluor 555 on four arrays and with Alexa Fluor 647 on four arrays. Six biological replicates (12 fish) were used for the pituitary comparison (two female and four male).
Project description:The delta smelt (Hypomesus transpacificus) is a pelagic fish species endemic to the Sacramento-San Joaquin Estuary in Northern California, listed as endangered under both the USA Federal and Californian State Endangered Species Acts and acts as an indicator of ecosystem health in its habitat range. Interrogative tools are required to successfully monitor effects of contaminants upon the delta smelt, and to research potential causes of population decline in this species. We used microarray technology to investigate genome-wide effects in 47-day old larvae after a 7-day exposure to ambient water samples from the Sacramento River at a monitoring field station (Hood) situated 8 miles downstream of the Sacramento regional Wastewater Treatment Plant. Genomic assessments were carried out on surviving organisms and contrasted to laboratory controls. Microarray assessments were conducted on larvae exposed for 7-days to Sacramento River water collected at Hood and pooled laboratory controls. Assessments were carried out in quadruplicate, using 3 fish per treatment. RNA was extracted from frozen whole, individual organisms, using Trizol Reagent (Invitrogen) as per manufacturer's guidelines. Total RNA from 5 fish was pooled per treatment and cDNA was synthesized from a total of 2ug total RNA, amplified using a SuperScripttm Indirect RNA Amplification System (Invitrogen). Resulting aRNA was labeled with Alexa fluor dyes (Invitrogen) as per manufacturer’s instructions. Two color microarray assessments were carried out on quadruplicate treatments, using 5µg of aRNA for pooled controls vs exposed sample, (total 4 samples). Microarray hybridizations were performed manually, and incubated in a waterbath at 42C for 16 hours. Slides were scanned using a GenePix 4000B scanner (Axon Instruments). Data was analyzed using LIMMA GUI (Linear model for microarray analysis graphical user interface) (Smyth, 2005), written in the R-programming language available through Bioconductor http://www.Bioconductor.org. Data was normalized within using print-tip Lowess and between arrays applying average intensity quantile (Aquantile) normalization methods with background correction (Smyth, 2005). A linear model fit was computed using the duplicates on the arrays and the least-squares method, with Benjamin Hochberg false discovery rate adjustment.
Project description:a salmonid microarray was used to characterize environmentally-regulated shifts in gene expression between ocean and river habitats in gill and liver tissues of wild migrating adult Pacific sockeye salmon (Oncorhynchus nerka). To correlate gene expression with survival, non-lethal biopsy sampling of gill tissue and microarray-based profiling was combined with biotelemetry and genetic stock identification so that transcriptomic profiles could be compared between fish reaching spawning grounds and presumed mortalities. Fish were captured fish at two marine sampling sites, one within Johnstone Strait (JS), BC. Canada and one within Juan De Fuca Strait (JDFS), BC Canada. Ocean sites were contrasted to fish sampled within the Fraser River at Whonnock (W), BC, Canada. Gill and liver tissues were dissected at each of these sites. Non-lethal biopsy sampling was performed on migrating sockeye salmon intercepted within the Fraser River at Mission, BC, Canada and genetically-based stock ID was used to determine the stock-specific spawning grounds for each fish, giving an intended end-point of migration for each of the stocks investigated in this study.Gene expression levels were determined by comparing the amount of mRNA transcript in the experimental samples relative to a reference sample. A total of 123 microarrays were used to generate the dataset, corresponding to individual hybridizations of both gill and liver samples collected from JS (gill n=14; liver n=15), JDFS (gill n=15; liver n=13), W (gill n=11; liver n=10), and biopsy sampled gill tissue collected at Mission (n=45).Total RNA was amplified (1 round) with MessageAmpTMII-96 kit (Ambion, TX, USA), and reverse transcribed to cDNA before labelling with ALEXA dyes using the Invitrogen Indirect Labelling Kit. The reference contained the combined aRNA of all individuals used in the experiment, excluding bioposy sampled fish. Individual samples were labelled with Alexa 555 and the reference control with Alexa 647, and no dye swaps were perfromed.
Project description:Transcriptional response of Bacillus subtilis to daptomycin in wild-type and in a daptomycin resistant mutant. Bacillus subtilis 168, WT (-DAP) vs. DapR1 (-DAP), WT (+DAP) vs. DapR1 (+DAP), DapR1 (+DAP) vs. DapR1 (-DAP). Each experiment was conducted at least twice using two independent total RNA preparations. For daptomycin untreated comparison between 168 WT and DapR1 mutant, DapR1 was labeled with Alexa Fluor 647 and WT was labeled with Alexa Fluor 555. For daptomycin treated experiments between WT and DapR1, DapR1 was labeled with Alexa Fluor 647 and WT with Alexa Fluor 555. For treated vs. untreated DapR1, the DAP treated samples were labeled with Alexa Fluor 647 and the untreated with Alexa Fluor 555. For dye swap, untreated DapR1 was labeled with Alexa Fluor 647 and DAP treated with Alexa Fluor 555.
Project description:Although studies have established that exogenous growth hormone (GH) treatment stimulates growth in fish, its effects on target tissue gene expression are not well characterized. We assessed the effects Posilac® (Monsanto Co., St. Louis, MO), a recombinant bovine somatotropin, on tissue transcript levels. Transcript abundance was measure in liver and muscle using the GRASP 16 K cDNA microarray. A selection of the genes identified as altered with the microarray, and also transcripts for insulin-like growth factors, growth hormone receptors (GHR) and myostatins were measured by realtime PCR in the liver, muscle, brain, kidney, intestine, stomach, gill and heart. In general, transcripts identified as differentially regulated in the muscle on the microarray showed similar direction of expression in the other non-hepatic tissues. Rainbow trout were selected from two high growth rate and two low growth rate families. A total of 113 and 67 transcripts were identified by microarray as differentially expressed with GH treatment across growth rate for muscle and liver respectively. The largest proportion of the transcripts represented novel transcripts, followed by immune and metabolism related genes. The immune related genes were primarily modulated in the liver and indicate activation of a non-specific immune response. The metabolic genes include lipid metabolism, oxidative phosphorylation and one carbon metabolism pathway transcripts. Most notable among the growth axis genes measured by realtime PCR were increases in GHR1 and-2 transcript in liver and muscle. Our results indicate that short-term GH treatment activates the immune system, shifts the metabolic sectors and modulates growth regulating genes. Keywords: Growth Hormone Injection Muscle and Liver Gene Expression Rainbow trout (hatched March 2005) selected for extreme growth rate were obtained from NCCCWA brood stock. Families were selected based on body weight at 7 months of age and thermal growth coefficient for the final month of growth. The two high growth families used in the study were in the top 2% in terms of growth rate, and the low growth families were in the lowest 10% for growth rate. Fish acclimated to the new tanks for two weeks prior to initiation of the treatments. Fish from each family were randomly selected to receive one of three treatments: 1) Posilac® injection (120 mg/kg BW, n = 4 per family); 2) vehicle injection (n = 4 per family); or 3) untouched controls (n = 2 per family). We had determined there was no effect to growth or the GH/IGF-I axis in the vehicle treated fish, and therefore, all of the microarray hybridizations were made between the GH and vehicle injected groups. This study included a total of 16 two-channel arrays designed for the direct comparison of GH treatment levels. That is, for each of the four groups, 1) High Growth Rate Liver; 2) Low Growth Rate Liver; 3) High Growth Rate Muscle; and 4) Low Growth Rate Muscle, four slide were hybridizes using individual RNA samples from individual fish. RNA isolation from each tissue/organ sample was handled separately (without pooling) with the purpose of using biological replications. We hybridized two slides with the GH cDNA labeled with Alexa 555 and vehicle cDNA labeled with Alexa 647; and two slides, using unique RNA samples, for the with GH cDNA labeled with Alexa 647 and tissue from the vehicle injected group labeled with Alexa 647 within each tissue and growth rate. Sixteen slides were used in the current study representing 32 individual tissue samples, meaning a total of four biological replicates for each treatment group.
Project description:Antibodies against cell surface antigens may be internalized through their specific interactions with these proteins and in some cases may induce or perturb antigen internalization. The anti-cancer efficacy of antibody-drug conjugates is thought to rely on their uptake by cancer cells expressing the surface antigen. Numerous techniques, including microscopy and flow cytometry, have been used to identify antibodies with desired cellular uptake rates. To enable quantitative measurements of internalization of labeled antibodies, an assay based on internalized and quenched fluorescence was developed. For this approach, we generated novel anti-Alexa Fluor monoclonal antibodies (mAbs) that effectively and specifically quench cell surface-bound Alexa Fluor 488 or Alexa Fluor 594 fluorescence. Utilizing Alexa Fluor-labeled mAbs against the EphA2 receptor tyrosine kinase, we showed that the anti-Alexa Fluor reagents could be used to monitor internalization quantitatively over time. The anti-Alexa Fluor mAbs were also validated in a proof of concept dual-label internalization assay with simultaneous exposure of cells to two different mAbs. Importantly, the unique anti-Alexa Fluor mAbs described here may also enable other single- and dual-label experiments, including label detection and signal enhancement in macromolecules, trafficking of proteins and microorganisms, and cell migration and morphology.
Project description:We demonstrate that Alexa Fluor 633 hydrazide (Alexa Fluor 633) selectively labels neocortical arteries and arterioles by binding to elastin fibers. We measured sensory stimulus-evoked arteriole dilation dynamics in mouse, rat and cat visual cortex using Alexa Fluor 633 together with neuronal activity using calcium indicators or blood flow using fluorescein dextran. Arteriole dilation decreased fluorescence recorded from immediately underlying neurons, representing a potential artifact during neuronal functional imaging experiments.
Project description:Transcriptional response of Bacillus subtilis to moenomycin in wild-type 168. Bacillus subtilis 168, WT (-MOE) vs. WT (+MOE). The experiment was conducted in triplicate using three independent total RNA preparations. Untreated samples were labeled with Alexa Fluor 555 and moenomycin treated samples were labeled with Alexa Fluor 647.
Project description:The long-term viability of Pacific salmon stocks and the fisheries they support are threatened if large numbers die prematurely en-route to spawning grounds. Physiological profiles that were correlated with the fate of wild sockeye salmon during river migration were discovered using functional genomics studies on biopsied tissues. Three independent biotelemetry studies tracked the biopsied fish after tagging in the marine environment over 200 km from the Fraser River, in the lower river 69 km from the river mouth and at the spawning grounds. Salmon carrying the poor performance (unhealthy) profile in the ocean exhibited a 4-times lower probability of arriving to spawning grounds than those with a healthy genomic signature, although generally migrated into the river and to the spawning grounds faster. A related unhealthy signature observed in the river was associated with a 30% reduction in survival to spawning grounds in one of the three stocks tested. At spawning grounds, the same poor performance signature was associated with twice the pre-spawning mortality compared with healthy fish. Functional analysis revealed that the unhealthy signature, which intensified during migration to spawning grounds, was consistent with an intracellular pathogenic infection, likely a virus. These results are the first to suggest a pathogen present in salmon in the marine environment could be a major source of mortality during migration and spawning in the river. This series are gill expression profiles from the study of fish sampled and tagged in the lower river and tracked as they swam towards the spawning grounds. Fish were caught in seine nets, gastrically implanted with radio transmitters, and biopsy sampled for blood, gill, muscle, and fin. Individual fish were tracked by receivers placed throughout the Fraser River watershed to identify and fate (i.e. the location of the receiver that last detected the fish). Targeted stocks of interest were genetically identified. Gene expression was profiled in gill tissue, a critical respiratory and ionoregulatory organ that is highly responsive to stress, chemical exposure and disease. Gene expression was assayed on the GRASP salmonid 16K cDNA microarray.
Project description:We collected sockeye salmon from the Fraser River, British Columbia, and held them at ecologically relevant temperatures (14C and 19C) determine the effect of elevated water temperature on cellular processes in non-lethally sampled gill tissue and blood plasma over a period of seven days that represents a significant portion of their upstream migration. Time-matched fish that died prematurely over the course of the holding study were also sampled for gill tissue and the transcriptomic responses in moribund fish were compared with surviving fish. This is the first study to experimentally examine transcriptomic responses to high water temperature and premature mortality in wild-caught Pacific salmon and the results will help in understanding some of the cellular mechanisms involved in large-scale migration mortality in Pacific salmon during warm water periods and for predicting or understanding causes of mortality in naturally senescing adult Pacific salmon. Forty samples were analyzed on forty two-channel microarrays, using a common reference design, with multiple biological replicates for each temperature condition. Fish were further classified into survivor and moribund based on their status after 7 days in captivity.