ABSTRACT: Purpose: Cadmium is a nonessential heavy metal and a well known toxic agent. Cadmium is known to alter the gene expression and signaling but the role of miRNAs during Cadmium exposure is not understood. Methods: Mice were treated with 100 mg/ ml cadmium chloride for 120 days and accumulation of cadmium in blood and organs are confirmed by GFAAS. Subsequently, the total RNA was isolated from the whole blood and small RNA sequencing was executed in Illumina HiSeq 1000. Results: The reads were annotated to the mice genome and miRNAs in miRbase using miRDeep* and novel miRNAs were also predicted. The differential expression was studied by DeSeq Conclusions: This is the first report to reveal the cadmium responsive miRNome. These candidate miRNAs can serve as biomarkers for cadmium Whole blood small RNA profiles of control and cadmium exposed mice generated by deep sequencing using Illumina HiSeq 1000
Project description:Purpose: Different studies revealed the importance of microRNAs (miRNAs) in pathological hypertrophy but their role during physiological hypertrophy is largely unexplored. Hence, this study is aimed at revealing the global expression profile of miRNAs during physiological cardiac hypertrophy. Methods: Chronic swimming protocol continuously for eight weeks resulted in induction of physiological hypertrophy in rats and histopathology revealed the absence of tissue damage, apoptosis or fibrosis. Subsequently, the total RNA was isolated and small RNA sequencing was executed in Illumina HiSeq 2000. Results: Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during physiological hypertrophy. The expression profile of the significantly differentially expressed miRNAs were validated by qPCR. Conclusions: This is the first report to reveal the involvement of a large number of apoptotic miRNAs during physiological cardiac hypertrophy including the previously unknown cardiac players like miR-99, miR-100, miR-191, miR-181 and miR-19. Our data indicates that regulation of these apoptotic miRNAs can be one of the major key factor in determining pathological or physiological hypertrophy by controlling apoptosis, fibrosis and cell death mechanisms. Heart small RNA profiles of 16-week old control and physiologically hypertrophied wistar rat were generated by deep sequencing using Illumina HiSeq 2000
Project description:This is a study to explore the transcriptional changes after cadmium treatment in adult rat testes at three time points (control--0 hour, 8 hour and 4 day). Cadmium is an environmental toxicant that is known to affect the male reproductive system. It disrupts the blood-testis barrier irreversibly, and affects the Sertoli-germ cells adhesion, causing germ cell depletion from the seminiferous epithelium. Experiment Overall Design: Adult Sprague-Dawley rats treated with a single dose of cadmium chloride at 3 mg/kg b.w. by i.p. and terminated after 8 hours (n=2) and 20 hours (n=2). Testes from cadmium-treated rats and untreated (control, 0 hour, n=3) rats were harvested and total RNA were prepared. Standard Affymetrix genechip procedures were followed for the subsequent experiments. Data were analysed in MAS 5.0 and GeneSpring 7.2.
Project description:The immortalized human urothelial cell line, UROtsa, was transformed in six parallel cultures with continual passaging in1 µM Cd+2 until the cells were able to attain the ability to form colonies in soft agar and subcutaneous tumors in nude mice. The gene expression profiles between cadmium-transformed and control samples were compared and the differentially expressed genes were identified. Six independent Cadmium-transformed UROtsa cell cultures, three control UROtsa cell cultures
Project description:Purpose: The physiological cardiac hypertrophy is an adaptive condition that does not associate with myocyte cell death while pathological hypertrophy is a maladaptive condition associated with myocyte cell death. Alpha-2 macroglobulin (α-2M) an acute phase protein induces cardiac hypertrophy via the ERK1,2 and PI3K/Akt signaling. This study is aimed at exploring the miRNome of α-2M induced hypertrophied cardiomyocytes and to understand the role of miRNAs in determination of pathological and physiological hypertrophy. Methods: Hypertrophy was induced in H9c2 cardiomyoblasts using alpha-2 macroglobulin. The induction of hypertrophy is confirmed by microscopy and gene expression studies. Subsequently, the total RNA was isolated and small RNA sequencing was executed in Illumina HiSeq 2000. Results: Analysis of small RNA reads revealed the differential expression of a large set of miRNAs during hypertrophy. Among the differentially expressed candidates, miR-99 family (miR-99a, miR-99b and miR-100) showed significant downregulation upon α-2M treatment while isoproterenol treatment (pathological hypertrophy) upregulated their expression. The binding site for Egr1 transcription factor was identified in the promoter region of miR-99 family, and interestingly all miRNAs with Egr1 binding site proven by ChIP-Seq were downregulated during physiological hypertrophy Conclusions: The results proved Egr-1 mediated regulation of miR-99 family determines the uniqueness of pathological and physiological hypertrophy. Upregulated miR-99 expression during pathological hypertrophy suggests that it can be a valuable diagnostic marker and potential therapeutic target for cardiac hypertrophy and heart failure. Small RNA profiles of control and hypertrophied cardiomyocyte H9c2 cells were generated by deep sequencing using Illumina HiSeq 2000
Project description:The study was performed using primary rat hepatocyte in culture from 4 adult male Sprague-Dawley rats to investigate the changes in gene expression under low dose (4μM) and short exposure (3hrs) of cadmium chloride. By comparing the gene expression profiles of control and cadmium-treated cells, the most dramatic and significant changes were for those genes associated with transcriptional regulation, antioxidant response and control of protein integrity. Changes in other genes involved in cellular physiological responses such as inflammation, growth and apoptosis were also observed. Results were further confirmed by quantitative real time polymerase chain reaction (qRT-PCR). Experiment Overall Design: Primary hepatocytes from four rats were isolated using the collagenase-hyaluronidase perfusion and digestion method. The cells of greater than 85% viability were used in this study. The cells were plated at 90% confluency on culture dishes coated with collagen and maintained in Waymouth's medium containing BSA for 4hrs prior to an overnight incubation with BSA free Waymouth's medium. Control samples and cadmium-treated samples from individual rats were hybridized to RAE 230A arrays (n=4 and 8 microarrays); data were generated and analyzed.
Project description:In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity. In this study, primary monocytes were differentiated into macrophages using M-CSF (M-Mac) or with a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in the macrophages, four of which were expressed higher in I-Mac (miRNAs 2.1, 8.1, 9.1 and 14.2) whilst three were detected in both M-Mac and I-Mac (miRNAs 9.3, 13.6 and 15.8). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. screening novel and known miRNAs which may have antiviral properties in 2 different treatments in 2 donors.
Project description:We are investigating the response of human lymphoblastoid cells to low-dose exposure of environmental metals We used microarrays to detail the global programme of gene expression upon response to low-dose metals Keywords: dose TK6 cells were grown to mid log-phase and exposed to low-doses of arsenic and cadmium. RNA was collected 24 hrs after exposure.
Project description:Metals at high concentrations can exert toxic effects on microorganisms. It has been widely reported that lowering environmental pH reduces effects of cadmium toxicity in bacteria. Understanding the effects of pH-mediated cadmium toxicity on bacteria would be useful for minimizing cadmium toxicity in the environment and gaining insight into the interactions between organic and inorganic components of life. Growth curve analysis confirmed that cadmium was less toxic to Escherichia coli at pH 5 than at pH 7 in M9 minimal salts medium. To better understand the cellular mechanisms by which lowering pH decreases cadmium toxicity, we used DNA microarrays to characterize global gene expression patterns in E. coli in response to cadmium exposure at moderately acidic (5) and neutral (7) values of pH. Higher expression of several stress response genes including hdeA, otsA, and yjbJ at pH 5 after only 5 minutes was observed and may suggest that acidic pH more rapidly induces genes that confer cadmium resistance. Genes involved in transport were more highly expressed at pH 7 than at pH 5 in the presence of cadmium. Of the genes that showed an interaction between pH and cadmium effects, 46% encoded hypothetical proteins, which may have novel functions involved in mitigating cadmium toxicity. GROWTH CONDITIONS FOR MICROARRAY EXPERIMENTS: Two overnight cultures of E. coli K-12 were started in M9 medium. A 250 mL Erlenmeyer flask containing 100 mL of M9 medium was inoculated with 0.5 mL for each of the two overnight cultures, each of which was considered a biological replicate. The cultures were grown on a rotary shaker (200 rpm) at 37 °C until the contents of the flask reached an OD600 of 0.3 (mid-log phase of growth). Each culture was divided into four 25 mL aliquots, transferred to 50 mL conical tubes (Corning), and centrifuged at 2540 x g for 12 minutes. The supernatant was decanted, and the cells were resuspended in 25 mL of M9 medium at pH 7 or pH 5 in the presence or absence (two cultures of each) of 5.4 µM (1 µg/mL) total cadmium, added as CdCl2. The cultures were incubated at 25 °C for either 5 or 15 minutes with manual rotation of the flasks once per minute. After the appropriate amount of time, 15 mL of RNAProtect Bacteria Reagent (Qiagen) was added to each culture to immediately halt all metabolic processes. The solutions were vortexed, incubated at 25 °C for 5 minutes, and centrifuged for 12 minutes at 3750 x g. RNA was extracted from the cell pellets immediately following centrifugation. RNA EXTRACTION AND HYBRIDIZATION PROCEDURES: RNA was extracted and purified using a Masterpure RNA purification kit (Epicentre Technologies). The quantity and quality of the RNA samples were determined spectrophotometrically. Preparation of the cDNA, labeling with Cy3 and Cy5, and successive hybridizations were accomplished using a 3DNA Array 900MPX kit following the manufacturer’s protocols (Genisphere) with the following modifications. 3DNA reverse transcriptase enzyme (Genisphere # RT300320) rather than SuperScript II was added to 1 µg of RNA and 2 µL of a random primer (1 µg/µL). The final cDNA hybridization mix contained 29 µL 2X enhanced cDNA hybridization buffer rather than 2X SDS-based hybridization buffer or 2X formamide-based hybridization buffer. The cDNA mix was hybridized to a cDNA microarray printed by the Microarray and Proteomics Facility at the University of Alberta (Operon version 1.0 oligonucleotides). The arrays were scanned with a Versarray ChipReader (BioRad) with laser power at 75%, photomultiplier tube (PMT) sensitivity at 800 V, and detector gain at 1. DATA ANALYSIS: Each array directly compared transcription at pH 5 and pH 7 for a given cadmium treatment (0 or 5.4 µM cadmium) and exposure time. Dye swaps were performed for each biological replicate for each of the following treatments (8 total arrays): 5 minutes with cadmium exposure, 5 minutes without cadmium exposure, 15 minutes with cadmium exposure, and 15 minutes without cadmium exposure. The 0-minute exposure to cadmium treatment was obtained from the 5-minute microarray without cadmium exposure. Spot intensities and locations were determined using TIGR Spotfinder, Version 3.1.1. All subsequent analyses were performed using the ma-anova package in the open-source statistical software package, R (www.r-project.org), Version 2.4.1. The data were normalized using the regional lowess method. Following normalization the median expression values of genes represented in triplicate on each array were determined for each gene. A mixed model two-way ANOVA for the main fixed effects of pH, cadmium, and their interaction (array and spot were the random effects) were performed (using Type III F-tests) separately for each time point to identify genes for which pH and cadmium interacted to significantly affect expression (FDR-adjusted p ≤ 0.05).
Project description:While numerous examples of male reproductive disorders have been reported in vertebrates, invertebrate’s organisms have been considerably less studied, despite their ecological importance. The aim of this study is to investigate male infertility in the amphipod Gammarus fossarum, a sentinel species in freshwater risk assessment. Thus in laboratory, we exposed male gammarids to different concentrations of three different xenobiotics: cadmium, and two potent arthropods endocrine-disruptor chemicals, methoxyfenozide and pyriproxyfen. Afterward, we investigated alterations of reproductive health by sperm quality markers and proteomes dynamics on the male reproductive tissue by nanoLC-MS/MS for evidencing proteins modulated by toxic exposure.
Project description:Ecotoxicological tests may be biased by the use of laboratory strains that usually contain very limited genetic diversity. It is therefore essential to study how genetic variation influences stress tolerance relevant for toxicity outcomes. To that end we studied sensitivity to cadmium in two distinct genotypes of the parthogenetic soil ecotoxicological model organism Folsomia candida. Clonal lines of both genotypes (TO1 and TO2) showed divergent fitness responses to cadmium exposure; TO2 reproduction was 20% less affected by cadmium. Statistical analyses revealed significant differences between the cadmium-affected transcriptomes; i) the number of genes affected by cadmium in TO2 was only minor (~22%) compared to TO1; ii) 97 genes showed a genotype × cadmium interaction and their response to cadmium showed globally larger fold changes in TO1 when compared to TO2; iii) the interaction genes showed a concerted manner of expression in TO1 while a less coordinated pattern was observed in TO2. We conclude that (1) there is genetic variation in parthenogenetic populations of F. candida, and (2) this variation affects life-history and molecular endpoints relative to cadmium toxicity. This sheds new light on the sources of biological variability in test results, even when the test organisms are thought to be genetically homogeneous because of their parthenogenetic reproduction. Gene expression was measured in two different clones (TO1 and TO2) of the springtail Folsomia candida, after exposure of 2 days to soil containing cadmium (Cd+) and non-spiked (Cd-) soil. A 2 x 2 factorial analysis was performed, to examine the effect of clone (TO1, TO2), of treatment (Cd+, Cd-), and the clone x treatment interaction.