News Article | August 31, 2016
All materials were molecular biology grade. Unless noted otherwise, all were from Sigma. MCF7, MCF10A, A549, H1299, SHSY5Y, Hep G2, Hep 3B2, HT-1080, NCI-H358, LLC, Neuro-2a, 4T1 and SK-N-Be2c cell lines were obtained from the American Type Culture Collection and their identity was not further authenticated. These cell lines are not listed in the database of commonly misidentified cell lines maintained by ICLAC. LLC, Neuro-2a, 4T1, Hep G2, HT-1080, Hep 3B2, MCF7 and A549 cells were cultured at 37 °C in DMEM with 10% fetal bovine serum (FBS), 5 ml of 100 U ml−1 penicillin–streptomycin (Life Technologies) and 5 ml of l-glutamine 200 mM. NCI-H358, H1299 and SK-N-Be2c cell lines were cultured at 37 °C in RPMI 1640 Medium with 10% FBS 1% penicillin–streptomycin and 1% L-glutamine. MCF10A cells were cultured at 37 °C in DMEM/F-12 supplemented with 5% horse serum (Life Technologies), 20 ng ml−1 human epidermal growth factor (Prepotec), 0.5 μg ml−1 hydrocortisone, 100 ng ml−1 cholera toxin, 10 μg ml−1 insulin, and 100 U ml−1 penicillin-streptomycin. The SHSY5Y cell line was cultured at 37 °C in DMEM/F-12 supplemented with 10% FBS, 2% penicillin–streptomycin and 1% non-essential amino acids (MEM). Mouse J1 ES cells were cultured feeder-free in fibroblast-conditioned medium. Cell cultures were confirmed to be mycoplasma-free every month. Control cell cultures were grown at atmospheric oxygen concentrations (21%) with 5% CO . To render cultures hypoxic, they were incubated in an atmosphere of 0.5% O , 5% CO and 94.5% N . Where indicated, IOX2 (50 μM), ascorbate (0.5 mM, a dose known to support TET activity19) or dimethyl-α-ketoglutarate (0.5 mM) was added to fresh culture medium, using an equal volume of the carrier (DMSO) as a control for IOX2. Cells were plated at a density tailored to reach 80–95% confluence at the end of the treatment. Fresh medium was added to the cells just before hypoxia exposure. For glutamine-free culture experiments, dialysed FBS was added to glutamine-free DMEM, and supplemented with glutamine (4 mM) for the control. Mouse J1 ES cells and Tet1-gene-trap ES cells were cultured feeder-free in fibroblast-conditioned medium. After exposure to the aforementioned stimuli, cultured cells were washed on ice with ice-cold PBS with deferoxamin (PBS-DFO, 200 μM), detached using cell scrapers and collected by centrifugation (400g, 4 °C). Nucleic acids were subsequently extracted using the Wizard Genomic DNA Purification kit (Promega) according to instructions. All buffers were supplemented with DFO (200 μM) and DNA was dissolved in 80 μl PBS-DFO with RNase A (200 U, NEB) and incubated for 10 min at 37 °C. After proteinase K addition (200 units) and incubation for 30 min at 56 °C, DNA was purified using the QIAQuick blood and tissue kit (all buffers supplemented with DFO). It was eluted in 100 μl of a 10 mM Tris, 1 mM EDTA solution (pH 8) and stored at −8 °C until further processing. To measure the cytosine, 5mC, 5hmC and 8-oxoG content of the DNA samples, three technical replicates were run for each sample. More specifically, 0.5–2 μg DNA in 25 μl H O were digested in an aqueous solution (7.5 μl) of 480 μM ZnSO , containing 42 U nuclease S1, 5 U Antarctic phosphatase, and specific amounts of labelled internal standards were added and the mixture was incubated at 37 °C for 3 h in a Thermomixer comfort (Eppendorf). After addition of 7.5 μl of 520 μM [Na] -EDTA solution containing 0.2 U snake venom phosphodiesterase I, the sample was incubated for another 3 h at 37 °C. The total volume was 40 μl. The sample was then kept at −20 °C until the day of analysis. Samples were then filtered by using an AcroPrep Advance 96-filter plate 0.2 μm Supor (Pall Life Sciences) and then analysed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), which are performed using an Agilent 1290 UHPLC system and an Agilent 6490 triple quadrupole mass spectrometer coupled with the stable isotope dilution technique. DNA samples were digested to give a nucleoside mixture and spiked with specific amounts of the corresponding isotopically labelled standards before LC–MS/MS analysis. The nucleosides were analysed in the positive ion selected reaction monitoring mode (SRM). In the positive ion mode, [M + H]+ species were measured. The resulting cytosine, 5mC, 5hmC and 8-oxoG peak areas were normalized using the isotopically labelled standards, and expressed relative to the total cytosine content (that is, C + 5mC + 5hmC). Concentrations were depicted as averages of independent replicates grown on different days, and compared between hypoxia and normoxia (21% O ), or between control and treated conditions, using a paired Student’s t-test. No statistical methods were used to predetermine sample size. For RNA extraction, cell culture medium was removed, TRIzol (Life Technologies) added and processed according to manufacturer’s guidelines. Reverse transcription and qPCR were performed using 2 × TaqMan Fast Universal PCR Master Mix (Life Technologies), TaqMan probes and primers (IDT, sequence in Supplementary Table 12). Thermal cycling and fluorescence detection were done using a LightCycler 480 Real-Time PCR System (Roche). Taqman assay amplification efficiencies were verified using serial cDNA dilutions, and estimated to be >95%. Cycle threshold (C ) values were determined for each sample and gene of interest in technical duplicates, and normalized according to the corresponding amplification efficiency. Per sample, TET expression was expressed relative to β-2-microglobulin (human) or hypoxanthine phosphoribosyltransferase 1 (Hprt mouse) levels by subtraction of their average C values. Concentrations were expressed as averages of at least 5 replicates extracted on different days. For Fig. 1a, copy number estimates for TET1, TET2 and TET3 were expressed for each cell line, relative to the summed copy number estimates of TET1, TET2 and TET3 under control conditions (21% O ). Concentrations were compared between hypoxia and normoxia, or between control and treatment conditions using a Student’s t-test. No statistical methods were used to predetermine sample size. To verify further induction of the hypoxia response program, hypoxia marker gene expression was verified. We analysed mRNA levels of genes encoding the E1B 19K/Bcl-2-binding protein Nip3 (BNIP3) and fructose-bisphosphate aldolase (ALDOA), 2 established hypoxia marker genes33. Reverse transcriptase–quantitative PCR (RT–qPCR) was performed as described for the TET mRNA concentration assays, and differential expression was calculated using the ΔΔ C method34. We ruled out transcriptional upregulation as the cause of the increase in HIF1α protein concentrations by assessing HIF1A mRNA expression in parallel. mRNA concentrations were expressed relative to normoxic controls (21% O ). Differences in mRNA concentration were assessed using a Student’s t-test on 5 or more independent replicates grown on different days. To assess Hif1α protein stabilization, proteins were extracted from cultured cells as follows: cells were placed on ice, and washed twice with ice-cold PBS. Proteins were extracted with extraction buffer (50 mM Tris HCl, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% SDS) with 1× protease inhibitor cocktail. Protein concentrations were determined using a bicinchoninic acid protein assay (BCA, Thermo Scientific) following the manufacture’s protocol. An estimated 60 μg protein was loaded per well on a NuPAGE Novex 3–8% Tris-Acetate Protein gel (Life Technologies), separated by electrophoresis and blotted on polyvinylidene fluoride membranes. Membranes were activated with methanol, washed and incubated with antibodies targeting β-actin (4967, Cell Signaling), Tet1 (09-872, Millipore) and Tet3 (61395, Active Motif), at 1:1,000 dilution, targeting Tet2 (124297, Abcam) at 1:250 dilution, and targeting Hif1α (C-Term) (Cayman Chemical Item 10006421) at 1:3,000 dilution. Secondary antibodies and detection were according to routine laboratory practices. Western blotting was performed on 6 independent replicates grown on different days. To confirm that hypoxia-associated differential expression of TET genes is induced by the HIF pathway, we performed HIF1β ChIP–seq. Because HIF1β is the obligate binding partner of all three HIFα proteins stabilized and activated upon hypoxia35, HIF1β ChIP–seq reveals all direct HIF-target genes. Approximately 25 × 106 –30 × 106 cells were incubated in hypoxic conditions for 16 h. Cultured cells were subsequently immediately fixed by adding 1% formaldehyde (16% formaldehyde (w/v), Methanol-free, Thermo Scientific) directly to the medium and incubating for 8 min. Fixed cells were incubated with 150 μM of glycine for 5 min to revert cross-links, washed twice with ice-cold PBS 0.5% Triton X-100, scraped and collected by centrifugation (1,000g for 5 min at 4°C). The pellet was re-suspended in 1,400 μl of RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS, 1% protease inhibitors) and transferred to a new Eppendorf tube. The lysate was homogenized by passing through an insulin syringe, and incubated on ice for 10 min. The chromatin was sonicated for 3 min by using a Branson 250 Digital Sonifier with 0.7 s ‘On’ and 1.3 s ‘Off’ pulses at 40% power amplitude, yielding a size of 100 to 500 bp. The sample was kept ice cold at all times during the sonication. The samples were centrifuged (10 min at 16,000g at 4 °C) and the supernatant were transferred in a new Eppendorf tube. The protein concentration was assessed using a BCA assay. Fifty microlitres of shared chromatin was used as ‘input’ and 1.4 μg of primary ARNT/HIF-1β monoclonal antibody (NB100-124, Novus) per 1 mg of protein was added to the remainder of the chromatin, and incubated overnight at 4 °C in a rotator. Pierce Protein A/G Magnetic Beads (Life Technologies) were added to the samples in a volume four times the volume of the primary antibody and incubated at 4 °C for at least 5 h. A/G Magnetic Beads were collected and the samples were washed five times with the washing buffer (50 mM Tris-HCl, 200 mM LiCl, 2 mM EDTA, pH 8, 1% Triton, 0.5% sodium deoxycholate, 0.1% SDS, 1% protease inhibitors), and twice with a 10 mM Tris, 1 mM EDTA (TE) buffer. The A/G magnetic beads were re-suspended in 50 μl of TE buffer, and 1.5 μl of RNase A (200 units, NEB) were added to the A/G beads samples and to the input, incubated for 10 min at 37 °C. After addition of 1.5 μl of proteinase K (200 U) and overnight incubation at 65 °C, the DNA was purified using 1.8× volume of Agencourt AMPure XP (Beckman Coulter) according to the manufactory instructions, and then eluted in 15 μl of TE buffer. The input DNA was quantified on NanoDrop. In total, 5 μg of input and all of the immunoprecipitated DNA was converted into sequencing libraries using the NEBNext DNA library prep master mix set. A single end of these libraries was sequenced for 50 bases on a HiSeq 2000, mapped using Bowtie and extended for the average insert size (250 bases). ChIP peaks were called by model-based analysis for ChIP–Seq36, with standard settings and using a sequenced input sample as baseline. To assess whether tumour-associated hypoxia reduces 5hmC levels in vivo, redundant material from two endometrial tumours and a breast tumour, removed during surgery, was grafted in the interscapular region of nude mice. Informed consent was obtained from the patient, following the ethical approval of the local ethical committee. All animal experiments were approved by the local ethical committee (P098/2014). Each tumour was allowed to grow to 1 cm3, after which it was collected. 10% of this tumour was re-implanted in a nude mouse, and the tumour was propagated for three generations until it was used for this experiment. To mark hypoxic areas, mice were injected with pimonidazole (60 mg kg−1, Hypoxyprobe) i.p. 1 h before killing. Tumours were collected, fixed in formaldehyde and embedded in paraffin using standard procedures. Paraffin was removed and slides were rehydrated in two xylene baths (5 min), followed by five 3-min ethanol baths at decreasing concentrations (100%, 96%, 70%, 50% and water) and a 3-min TBS (50 mM Tris, 150 mM NaCl, pH 7.6) bath. The following antibodies were used for immunofluorescence staining: primary antibodies were FITC-conjugated mouse anti-pimonidazole (HP2-100, Hydroxyprobe), rabbit anti-5hmC (39791, Active Motif), rat anti-polyoma middle T (AB15085, Abcam), rat anti-CD31 (557355, BD Biosciences), rat anti-CD45 (553076, BD Biosciences), rabbit anti-Ki67 (AB15580, Abcam) and mouse anti-pan cytokeratin (C2562, Sigma). Secondary antibodies were Alexa Fluor 405-conjugated goat anti-rabbit (A31556, Thermo Fisher), Alexa Fluor 647 conjugated goat anti-rat (A-21247, Life Technologies), peroxidase-conjugated goat anti-FITC (PA1-26804, Pierce), biotinylated goat anti-rat (A10517, Thermo Fisher) and biotinylated goat anti-rabbit (E043201, Dako). Signal amplification was performed using the TSA Fluorescein System (NEL701A001KT, Perkin Elmer) or the TSA Cyanine 5 System (NEL705A001KT, Perkin Elmer). Different protocols were implemented depending on the epitopes of interest. Staining for the following epitopes was combined: CD45, 5hmC, pimonidazole and DNA; PyMT, 5hmC, pimonidazole and DNA; Ki67, pimonidazole and DNA; CD31 and pimonidazole; and pan-cytokeratin, 5hmC, pimonidazole and DNA. Antigen retrieval for CD31, CD45 and pan-cytokeratin was done by a 7-min trypsin digestion, for pimonidazole and Ki67 using AgR at 100 °C for 20 min, followed by cooling for 20 min. Slides were washed in TBS for 5 min, permeabilized in 0.5% Triton X-100 in PBS for 20 min. For 5hmC antigen retrieval, slides were denatured in 2 M HCl for 10 min; HCl was neutralized for 2 min in borax, 1% in PBS pH 8.5, and washed twice for 5 min in PBS. For all slides, endogenous peroxidase activity was quenched using H O (0.3% in methanol), followed by three 5-min washes in TBS. Slides were blocked using pre-immune goat serum (X0907, Dako; 20% in TNB; TSA Biotin System kit, Perkin Elmer). Binding of primary antibodies (anti-5hmC, anti-CD45, anti-CD31 and anti-pan cytokeratin or FITC-conjugated anti-pimonidazole; all 1:100 in TNB) was allowed to proceed overnight. Slides were washed 3 times in TNT (0.5% Triton-X100 in TBS) for 5 min, after which the following secondary antibodies (all 1:100 in TNB with 10% pre-immune sheep serum) were allowed to bind for 45 min: sheep-anti-FITC-PO (for pimonidazole), goat anti-rabbit-Alexa Fluor 405 (for 5hmC), goat anti-rat-Alexa Fluor 647 (for CD45), and biotinylated goat anti-mouse (for pan-cytokeratin). Slides were washed three times for 5 min in TNT, after which signal amplification was performed for 8 min using Fluorescein Tyramide (1:50 in amplification diluent). Slides stained for pimonidazole that required co-staining for Ki67 or PyMT, or slides stained for pan-cytokeratin that required co-staining for pimonidazole were subjected to a second indirect staining for the latter epitopes. After 5 min of TNT and 5 min of TBS, slides were quenched again for peroxidase activity using H O and blocked using pre-immune goat serum, prior to a second overnight round of primary antibody binding (anti-Ki67, FITC-anti-pimonidazole or anti-PyMT, all 1/100). The next day, three 5-min washes with TNT were followed by a 1-h incubation with a biotinylated goat anti-rabbit antibody (for Ki67) or goat anti-rat (for PyMT), another three 5-min washes with TNT, a 30-min incubation with peroxidase conjugated to streptavidin (for Ki67 and PyMT) or to anti-FITC (for pimonidazole), another three 5-min washes with TNT and signal amplification for 8 min using, for pimonidazole, Fluorescein Tyramide and for others Cyanine 5 Tyramide (1:50 in amplification diluent). Slides were then stained with propidium iodide with RNase (550825; BD biosciences) for 15 min, washed for 5 min in PBS and mounted with Prolong Gold (Life Technologies). Slides were imaged on a Nikon A1R Eclipse Ti confocal microscope. Three to five sections per slide were imaged, and processed using Image J. Nuclei were identified using the propidium iodide signal and nuclear signal intensities for Fluorescein and Cy3 (pimonidazole and 5hmC) measured. Analyses were exclusively performed on slide regions showing a regular density and shape of nuclei, in order to avoid inclusion of acellular or necrotic areas. The pimonidazole signal will also not stain necrotic/acellular areas37, and was used to stratify viable cell nuclei into normoxic (pimonidazole negative) and hypoxic (pimonidazole positive) regions. The 5hmC signals in each population were compared using ANOVA. PyMT-negative and CD45-positive cells were counted directly. The fraction of pimonidazole and CD31-positive areas was directly quantified using ImageJ across ten images per slide. For metabolite extractions, 12-well cell culture dishes were placed on ice and washed twice with ice-cold 0.9% NaCl, after which 500 μl of ice-cold 80% methanol was added to each well. Cells were scraped and 500 μl was transferred to a vial on ice. Wells were washed with 500 μl 80% methanol, which was combined with the initial cell extracts. The insoluble fraction was pelleted at 4 °C by a 10-min 21,000g centrifugation. The pellet (containing the proteins) was dried, dissolved in 0.2 N NaOH at 96 °C for 10 min and quantified using a bicinchoninic acid protein assay (BCA, Pierce), whereas the supernatant fraction was processed for metabolite profiling. The supernatant fraction containing the metabolites was transferred to a new vial and dried in a Speedvac. The dried supernatant fraction was dissolved in 45 μl of 2% methoxyamine hydrochloride in pyridine and held for 90 min at 37 °C in a horizontal shaker, followed by derivatization through the addition of 60 μl of N-(tert-butyldimethylsilyl)-n-methyl-trifluoroacetamide with 1% tert-butyldimethylchlorosilane and a 60-min incubation at 60 °C. Samples were subsequently centrifuged for 5 min at 21,000g and 85 μl was transferred to a new vial and analysed using a gas-chromatography based mass spectrometer (triple quadrupole, Agilent) operated in Multiple Reaction Monitoring (MRM) mode. For each sample, metabolite measurements were normalized per sample to the corresponding protein concentration estimates and expressed relative to control-treated samples. Four technical replicates were run for each sample, and the experiment was repeated 4 times using independent samples (n = 16). Differences in metabolite concentration were assessed using a two-tailed paired Student’s t-test or using analysis of variance with post-hoc Tukey HSD when repeated measures were compared. MCF7 cells were cultured in 24-well plates and exposed to 21% (control) or 0.5% O (hypoxia) for 24 h. DMEM used for staining was pre-equilibrated to the required O tension, and all steps performed at 21% (control) or 0.5% O (hypoxia) using a glove box. The cells were washed twice with 500 μl DMEM, and incubated for 30 min in 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA; 10 μM) in 500 μl DMEM, keeping 2 wells unstained by DMEM without DCF-DA. Cells were treated with the indicated concentrations of H O in DMEM for 30 min at 37 °C, and fixed by adding 33.3 μl of 16% methanol-free paraformaldehyde (Thermo Fisher) for 8 min at room temperature. The fixative was quenched using glycine (150 μM), cells were washed twice in ice-cold PBS, scraped to detach them and transfer them to pre-cooled FACS tubes over cell strainers. Cells were kept on ice until they were analysed by flow cytometry using a FACSVerse (BD Biosciences). MCF7 cells were seeded on 12-well glass-bottom plates and after 24 h exposed to 21% (control) or 0.5% O (hypoxia) for 24 h. PBS used for subsequent staining was pre-equilibrated to the required O tension, and all washing, treatment and staining steps were performed at the appropriate O tension (21% or 0.5%) using a glove box. Cells were loaded with nuclear peroxy emerald 1 (NucPE1; 5 μM)38, 39 and Hoechst 33342 (10 μg ml−1) in PBS for 15 min at 37 °C. After washing three times in PBS, control cells were incubated with H O (0.5 mM in PBS) as a positive control, or with water (control and hypoxia cells) in PBS at 37 °C for 20 min. Cells were washed three times in PBS, placed on ice and immediately imaged by confocal microscopy. The nuclear NucPE1 signal was measured, and averaged across >100 nuclei per replicate using ImageJ. This experiment was repeated 5 times on different days, and signals compared using a t-test. 5,000 cells/well were seeded in three 96-well plates. After 48 h, one plate was fixed using trichloroacetic acid (3.3% w/v) for 1 h at 4 °C, one plate incubated for 24 h at 37 °C under hypoxic and one under control conditions (0.5% and 21% O , respectively). The latter 2 plates were subsequently also fixed using trichloroacetic acid (3.3% wt/vol) for 1 h at 4 °C, and all 3 plates were next analysed using the In vitro Toxicology Assay Kit, Sulforhodamine B-based (Sigma) as per the manufacturer’s instructions. Growth inhibition was calculated as described40. siRNA ON-TARGETplus SMART pools (Thermo) were diluted in Optimem I reduced serum medium using Lipofectamine RNAiMAX (Life technologies) to reverse-transfect MCF7 cells in 10-cm dishes (for DNA) or 6-well plates (for RNA). Cells were transfected 72 h before RNA and DNA extraction as described. MCF7 cells were cultured for 24 h under control or hypoxic conditions (21% or 0.5% O , respectively), chilled on ice and processed for extraction of nuclear proteins using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific). The activity of control and hypoxic extracts was assessed in parallel using the Colorimetric Epigenase 5mC-Hydroxylase TET Activity/Inhibition Assay Kit (Epigentek) according to manufacturer’s instructions. Reactions were allowed to proceed for one hour, after which washing and detection of 5hmC were done according to manufacturer’s instructions. Differences between hypoxia and control were analysed using ANOVA, for 5 independent experiments. The genomic DNA used in this assay was extracted from Tet triple-knockout ES cells (G. -L. Xu), and it therefore was devoid of 5hmC41. To enable efficient denaturation, it was digested using MseI before the assay and purified using solid phase reversible immobilisation paramagnetic beads (Agencourt AMPure XP, Beckman Coulter). The assays were performed in Whitley H35 Hypoxystations (don Whitley Scientific) at 37 °C, 5% CO , N , with the following oxygen tensions: 0.1%, 0.3%, 0.5%, 1%, 2.5%, 5%, 10% and 21%. Hypoxystations were calibrated less than 1 month before all experiments. Optimized assay components were as follows: 1.0 μg μl−1 bovine serum albumin (New England Biolabs), 50 mM Tris (pH 7.8), 100 μM dithiothreitol (Life Technologies), 2 ng μl−1 digested gDNA, 250 μM α-ketoglutarate, 830 μM ascorbate, 200 μM FeSO and 45 ng μl−1 Tet1 enzyme (Wisegene). The major assay components (H O, BSA and Tris) used for all samples were allowed to pre-equilibrate at 0.1% O for 1 h. These and the remaining assay buffer components (<100 μl) were then pre-equilibrated at the desired oxygen tension for 15 min, and mixed before addition of Tet1 enzyme in a total reaction volume of 25 μl. Reactions were allowed to proceed for 3 min, longer incubations showed a decrease in activity. Reactions were stopped with 80 mM EDTA and stored at −80 °C. To measure the resulting 5hmC content of the DNA, reactions were diluted to 100 μl, denatured for 10 min at 98 °C and analysed in duplicate using the Global 5-hmC Quantification Kit (Active Motif) following manufacturer’s instructions. Michaelis–Menten and Lineweaver–Burk plots and the resulting K values were estimated using R. To assess where in the genome the levels of 5mC and 5hmC were altered, we performed DNA immunoprecipitations coupled to high-throughput sequencing (DIP-seq). MCF7 cells were selected for these experiments as they were a cancer cell line with high levels of 5hmC and expression of TET genes under control conditions, and a cell growth that is unaffected by hypoxia. This enabled us to study the effects of hypoxia on TET activity in a cell line that shows high endogenous activity, but that is isolated from hypoxia-induced changes in cell proliferation. MCF7 cell culture and DNA extractions were as described for LC–MS analyses. Library preparations and DNA immunoprecipitations were performed as described42, using established antibodies targeting 5mC (clone 33D3, Eurogentec,) and 5hmC (Active Motif catalogue number 39791). For 5hmC-DIP-seq, paired barcoded libraries prepared from DNA of hypoxic and control samples were mixed before capture, to enable a direct comparison of 5hmC-DIP-seq signal to the input. A single end of these libraries was sequenced for 50 bases on a HiSeq 2000, mapped using Bowtie and extended for the average insert size (150 bases). Mapping statistics are summarized in Supplementary Information Table 11. For analysis of sequencing data, MACS peak calling, read depth quantification and annotation with genomic features as annotated in EnsEMBL build 77 was performed using SeqMonk. Differential (hydroxy-)methylation was quantified by EdgeR43, using either 3 or 5 independent pairs of control and hypoxic samples (for 5hmC-DIP-seq and 5mC-DIP-seq, respectively). These cells were cultured and exposed to hypoxia (0.5% O ) or control conditions (21% O ) on different days. Results were reported for 5hmC peak areas that exhibited a change significant at a P < 0.05 and 5% FDR. To confirm enrichment of 5mC at gene promoters using an independent method, DNA libraries were prepared using methylated adapters and the NEBNext DNA library prep master mix set following manufacturer recommendations. Libraries were bisulfite-converted using the Imprint DNA modification kit (Sigma) as recommended, and PCR amplified for 12 cycles using barcoded primers (NEB) and the KAPA HiFi HS Uracil+ ready mix (Sopachem) according to manufacturer’s instructions. Fragments were selected from these libraries using the SeqCapEpi CpGiant Enrichment Kit (Roche) following the manufacturer’s instructions, sequenced from both ends for 100 bases on a HiSeq 2000. For analysing these sequences, sequencing reads were trimmed for adapters using TrimGalore and mapped on a bisulfite-converted human genome (GRCh37) using BisMark. The number of methylated and un-methylated cytosines in captured regions was quantified using Seqmonk for each experiment. Differential methylation of regions of interest was assessed by Fisher’s exact test and for 5 independent replicates grown on different days. t-scores were averaged following Fisher’s method. Mapping statistics are summarized in Supplementary Table 11. To assess the effect of the increased 5mC occupancy at gene promoters on their expression, RNA-seq was performed. Briefly, total RNA was extracted using TRIzol (Invitrogen), and remaining DNA contaminants in 17–20 μg of RNA was removed using Turbo DNase (Ambion) according to the manufacturer’s instruction. RNA was repurified using RNeasy Mini Kit (Qiagen). Ribosomal RNA present was depleted from 5 μg of total RNA using the RiboMinus Eukaryote System (Life technologies). cDNA synthesis was performed using SuperScript III Reverse Transcriptase kit (Invitrogen). 3 μg of Random Primers (Invitrogen), 8 μl of 5× First-Strand Buffer and 10 μl of RNA mix was incubated at 94 °C for 3 min and then at 4 °C for 1 min. 2 μl of 10 mM dNTP Mix (Invitrogen), 4 μl of 0.1 M DTT, 2 μl of SUPERase In RNase Inhibitor 20U μl−1 (Ambion), 2 μl of SuperScript III RT (200 U μl−1) and 8 μl of Actinomycin D (1 μg μl−1) were then added and the mix was incubated for 5 min at 25 °C, 60 min at 50 °C and 15 min at 70 °C to heat-inactivate the reaction. The cDNA was purified by using 80 μl (2× volume) of Agencourt AMPure XP and eluted in 50 μl of the following mix: 5 μl of 10× NEBuffer 2, 1.5 μl of 10 mM dNTP mix (10 mM dATP, dCTP, dGTP, dUTP, Sigma), 0.1 μl of RNaseH (10 Uμl−1, Ambion), 2.5 μl of DNA Polymerase I Klenov (10 U μl−1, NEB) and the remaining volume of water. The eluted cDNA was incubated for 30 min at 16 °C, purified by Agencourt AMPure XP and eluted in 30 μl of dA-Tailing mix (2 μl of Klenow Fragment, 3 μl of 10× NEBNext dA-Tailing Reaction Buffer and 25 μl of water). After 30 min incubation at 37 °C, the DNA was purified by Agencourt AMPure XP, eluted in TE buffer and quantified on NanoDrop. Subsequent library preparation was performed using the DNA library prep master mix set and sequencing was performed as described for ChIP-seq. Expression levels (reads per million) of genes displaying significant increases in methylation at their gene promoter, as determined using SeqCapEpi, was compared between control and hypoxic samples using a t-test. Mapping statistics are summarized in Supplementary Table 11. From the TCGA pan-cancer analysis, we selected all solid tumour types for which >100 tumours were available with both gene expression data (RNA-seq) and DNA methylation data (Illumina Infinium HumanMethylation450 BeadChip). These were 408 bladder carcinomas, 691 breast carcinomas, 243 colorectal adenocarcinomas, 520 head and neck squamous cell carcinomas, 290 kidney renal cell carcinomas, 430 lung adenocarcinomas, 371 lung squamous cell carcinomas, and 188 uterine carcinomas, representing in total 3,141 unique patients. Corresponding RNA-seq read counts as well as DNA methylation data from Infinium HumanMethylation450 BeadChip arrays were downloaded from the TCGA server. Breast tumour subtypes were annotated for 208 tumours and, for the remaining tumours, imputed by unsupervised hierarchical clustering of genes in the PAM50 gene expression signature44. Other clinical and histological variables were available for >95% of tumours, and missing values were encoded as not available. Gene mutation data was available for 129 bladder carcinomas, 646 breast carcinomas, 200 colorectal adenocarcinomas, 306 head and neck squamous cell carcinomas, 241 kidney renal cell carcinomas, 182 lung adenocarcinomas, 74 lung squamous cell carcinomas, and 3 uterine carcinomas. To identify which of these tumour samples were hypoxic or normoxic, we performed unsupervised hierarchical clustering based a modification (Ward.D of the clusth function in R’s stats package) of the Ward error sum of squares hierarchical clustering method45, on normalized log -transformed RNA-seq read counts for 14 genes that make up the hypoxia metagene signature (ALDOA, MIF, TUBB6, P4HA1, SLC2A1, PGAM1, ENO1, LDHA, CDKN3, TPI1, NDRG1, VEGFA, ACOT7 and ADM)25. In each case the top 3 sub-clusters identified were annotated as normoxic, intermediate and hypoxic. To identify which of these tumour samples were high- or low-proliferative, we performed unsupervised hierarchical clustering based a modification (Ward.D of the clusth function in R’s stats package) of the Ward error sum of squares hierarchical clustering method45, and this for all genes annotated to an established tumour proliferation signature (MKI67, NDC80, NUF2, PTTG1, RRM2, BIRC5, CCNB1, CEP55, UBE2C, CDC20 and TYMS)46. Tumours in the top 2 sub-clusters identified were labelled as high- or low-proliferative. To identify tumour-associated hypermethylation events, we compared 450k methylation data from tumours and normal tissues. All available DNA methylation data from normal tissue (matched or unmatched to tumour samples, on average 59 per tumour type, representing 472 in total, range = 21–160) were downloaded. For each of the 8 tumour types investigated, we selected the top 1,000 CpGs that showed the highest average tumour-associated increases in DNA methylation. Per tumour type, unsupervised hierarchical clustering based on a modification of the Ward error sum of squares hierarchical clustering method (Ward.D of the clusth function in R’s stats package)45 annotated the first 3 clusters identified as having low, intermediate and high hypermethylation. Cluster co-membership for methylation and hypoxia metagene expression were analysed using the Cochran–Armitage test for trend. Analyses using the top 100, 500, 5,000 or 10,000 CpGs yielded near identical results (not shown). We next applied a method to identify those CpGs that exhibit exceptional increases in hypermethylation but that are hypermethylated only in a subset of all tumours. Such rare events are typically found in cancer, where hypermethylation inactivates a gene in only a subset of tumours. Hypermethylation of individual CpGs at gene promoters (that is, on average 3.7 CpGs per promoter are represented on the 450K array) in individual tumours was assessed as follows: To achieve a normal distribution, all β-values were transformed to M-values47 using M = log (β/(1 − β)). For each tumour type, the mean μ and standard deviation σ of the M value across all control (normoxic) tumours was next calculated, irrespective of mutational status, for each CpG, and used to assign Z-values to each CpG in each tumour using Z = (M − μ)/σ. These Z-values describe the deviation in normal methylation variation for that probe. To identify CpGs that display an extreme deviation, we selected those for which the Z-value exceeded 5.6 (that is, μ + (5.6 × σ), corresponding to a Bonferroni-adjusted P value of 0.01); they were considered as hypermethylation events in that particular tumour. This analysis was preferred over Wilcoxon-based models that assess differences in the average methylation level between subgroups, as the latter do not enable the identification or quantification of the rarer hypermethylation events in individual promoters or CpGs. To identify genes with frequently hypermethylated CpGs in their promoter, the number of hypermethylation events in that promoter was counted in all tumours, and contrasted to the expected number of hypermethylation events in that promoter (that is, the general hypermethylation frequency multiplied by the number of CpGs assessed in that promoter multiplied by the number of tumours) using Fisher’s exact test. Genes with an associated Bonferroni-adjusted P value below 0.01 were retained and considered as frequently hypermethylated in that tumour type. To assess what fraction of these hypermethylation events are hypoxia-related, we assumed that the fraction of events detected under normoxia was hypoxia-unrelated, and that all excess events detected in intermediate and hypoxic tumours were hypoxia-related. For example, in 691 breast carcinomas, 0.25% of CpGs were hypermethylated in 251 normoxic tumours, 0.81% in 350 intermediate and 1.40% in 90 hypoxic tumours. So, 0.56% and 1.15% of hypermethylation events in respectively intermediate and hypoxic tumours were hypoxia-related. Taking into account the number of tumours, 0.25% of hypermethylation events (that is, (0.25% × 251 + 0.25% × 350 + 0.25% × 90)/691) are not hypoxia-related, and 0.43% are hypoxia related (that is, (0% × 251 + 0.56% × 350 + 1.15% × 90)/691). So, 63% of all hypermethylation events combined (that is, 0.43/(0.43 + 0.25)) are hypoxia related. To assess the contribution of hypoxia to hypermethylation relative to other covariates, partial R2 values were calculated for the contribution of each covariate in a linear model, and compared to the total R2 achieved by the model. To identify genes with CpGs in their promoter that are more frequently hypermethylated in hypoxic than normoxic tumours, the number of hypermethylation events in that promoter was counted in all hypoxic tumours, and contrasted to the number found in normoxic tumours. Differences in frequencies were assessed using Fisher’s exact test, and genes with a Bonferroni-adjusted P < 0.01 were retained and considered hypermethylated upon hypoxia. Enrichment of ontologies associated with these genes was assessed using Fisher’s exact test as implemented in R’s topGO package. To enable a direct comparison between the expression of different genes, we transformed gene expression values (reads per million) to their respective z-scores. To assess the impact of hypermethylation events on the expression of associated genes, we compared the expression z-scores of all frequently hypermethylation genes that contain one or more hypermethylation events in their promoter (on average each promoter contains 3.7 CpGs; if one of these is hypermethylated the associated gene is considered hypermethylated in that particular tumour), to the expression of all frequently hypermethylated genes that do not contain a hypermethylation event. The effect of hypermethylation on gene expression was plotted for the 8 main tumour types stratified into normoxic, intermediately hypoxic and hypoxic tumours, and for glioblastomas was stratified into normoxic, intermediately hypoxic, hypoxic and IDH-mutant tumours (n = 4). The difference in expression z-scores between genes not carrying and carrying a hypermethylation event in their promoter was assessed using a t-test. To assess the impact of somatic mutations on hypoxia-associated hypermethylation frequencies, we analysed the top 20 genes described to be most frequently mutated in the pan-cancer analysis24, and supplemented this list with genes known to cause hypermethylation upon mutation (that is, IDH1, IDH2, SDHA, FH, TET1, TET2 and TET3). Mutations in IDH1 and IDH2 were retained if they respectively affected amino acid R132, and amino acids R140 or R172. Mutations in other genes were scored using Polyphen, and only mutations classified as probably damaging were retained. 7 mutations were found in lung tumours, 3 mutations in colorectal tumours, 8 mutations in breast tumours and 6 mutations (all IDH1R132) in glioblastomas. None of these mutations were enriched in hypoxic subsets. In multivariate analyses of variance, in each of the tumour types analysed, mutations in these genes were significantly associated with increased hypermethylation frequencies. Hypoxia was independently and significantly associated with the hypermethylation frequency. Gene expression values (reads per million) of DNMT and TET enzymes were determined for each tumour using available RNA-seq data. The number of hypermethylation events at significantly hypermethylated genes in each tumour was determined as described above. Hypermethylation in each tumour was subsequently correlated to TET or DNMT gene expression in that tumour, correcting for hypoxia and proliferation status using ANOVA. Newly diagnosed and untreated non-small-cell lung cancer patients scheduled for curative-intent surgery were prospectively recruited. Included subjects had a smoking history of at least 15 pack-years. The study protocol was approved by the Ethics Committee of the University Hospital Gasthuisberg (Leuven, Belgium). All participants provided written informed consent. In the framework of a different project48, RNA-seq was performed on 39 tumours from these patients. Gene expression for these samples was clustered for their hypoxia metagene signature25. This yielded 2 clear clusters, containing 24 and 15 normoxic and hypoxic tumours, respectively. Twelve samples were randomly selected from each cluster, in order to perform 5hmC and 5mC profiling. For TAB–ChIP, DNA was glycosylated and oxidized as described49, using the 5hmC TAB-Seq Kit (WiseGene). Subsequently, bisulfite conversion, DNA amplification and array hybridization were done following manufacturer’s instructions. Data processing was largely as described50. In brief, intensity data files were read directly into R. Each sample was normalized using Subset-quantile within array normalization (SWAN) for Illumina Infinium HumanMethylation450 BeadChips49. Batch effects between chips and experiments were corrected using the runComBat function from the ChAMP bioconductor package51. For obtaining 5mC-specific beta values, TAB-ChIP generated normalized beta values were substracted from the standard 450K generated normalized beta values, exactly as described50. All the experimental procedures were approved by the Institutional Animal Care and Research Advisory Committee of the KU Leuven. For sFlk1-overexpression studies, male Tg(MMTV-PyMT) FVB mice were intercrossed with wild-type FVB female mice. Female pups of the Tg(MMTV-PyMT) genotype were retained, and tumours allowed to develop for 9 weeks. Subsequently, 2.5 μg of plasmid (sFlk1-overexpressing or empty vector; randomly assigned within litter mates) per gram of mouse body weight was introduced in the bloodstream using hydrodynamic tail vein injections52. sFlk1 overexpression was monitored at 4 days after injection and at the day of killing (18 days after the injection), by eye bleeds followed by an ELISA assay for sFlk1 (R&D Systems) in blood plasma. At 12 weeks of age, mice were killed and mammary tumours collected were blinded for treatment. For the Phd2+/− experiments, male Tg(MMTV-PyMT) FVB mice were intercrossed with female Phd2−/+ mice, yielding litters of which half have either a Tg(MMTV-PyMT) genotype or a Tg(MMTV-PyMT);Phd2−/+ genotype. For the Phd2WT/fl experiments, male Tg(MMTV-PyMT) FVB mice were intercrossed with female Tie2-Cre;Phd2WT/fl mice as described27, yielding litters of which half have either a Tie2-cre;Tg(MMTV-PyMT);Phd2WT/WT genotype or a Tie2-cre;Tg(MMTV-PyMT);Phd2−/+ genotype. At 16 weeks of age, female mice were killed and mammary tumours collected. RNA was extracted from fresh-frozen tumours (stored at −8 °C) using TRIzol (Life Technologies), and converted to cDNA and quantified as described for the cell lines. TaqMan probes and primers (IDT or Life Technologies) are described under Supplementary Table 12. TAB-seq libraries were prepared as described, using the 5hmC TAB-Seq Kit (WiseGene). DNA was bisulfite-converted using the EZ DNA Methylation-Lightning Kit (Zymo Research) and sequenced as described for SeqCapEpi experiments. Reads were mapped to the mouse genome (build Mm9) and further data processing was as for SeqCapEpi experiments. DNA from 3 independent tumours was selected per condition. TET oxidation efficiency was required to exceed 99.5% as estimated using a fully CG-methylated plasmid spike-in, 5hmC protection by glycosylation was 65% as estimated using a fully hydroxymethylated plasmid spike-in, bisulfite conversion efficiencies were estimated to exceed 99.8% based on nonCG methylation (equal to percentage hypermethylated CpG). Mapping statistics are summarized in Supplementary Table 11. As no mouse capture kit was available for targeted BS-seq, a specific ampliconBS was developed for a set of 15 tumour suppressor gene promoters and 5 oncogene promoters. More specifically, DNA was bisulfite-converted using the Imprint DNA modification kit and amplified using the MegaMix Gold 2× mastermix and validated primer pairs. Per sample, PCR products were mixed to equimolar concentrations, converted into sequencing libraries using the NEBNext DNA library prep master mix set and sequenced to a depth of ~500×. Mapping and quantification were done as described for SeqCapEpi. The average and variance of methylation level M values in normal mammary glands were used as baseline, and amplicons displaying over 3 standard deviations more methylation (FDR-adjusted P < 0.05) than this baseline were called as hypermethylated. At least 9 different tumours, each from different animals, were profiled per genotype or treatment, and differences in hypermethylation frequencies between sets of tumours were assessed using Mann–Whitney’s U-test. Data entry and analysis were performed in a blinded fashion. Statistical significance was calculated by two-tailed unpaired t-test (Excel) or analysis of variance (R) when repeated measures were compared. Data were tested for normality using the D’Agostino–Pearson omnibus test (for n > 8) or the Kolmogorov–Smirnov test (for n ≤ 8) and variation within each experimental group was assessed. Data are presented as mean ± s.e.m. DNA methylation and RNA-seq gene expression data distributions were transformed to a normal distribution by conversion to M values and log transformation respectively. Sample sizes were chosen based on prior experience for in vitro and mouse experiments, or on sample and data availability for human tumour analyses. Other statistical methods (mostly related to specific sequencing experiments) are described together with the experimental details in other sections of the methods.
News Article | December 30, 2016
As many as 20,000 dead fish and other sea animals have washed on the western shores of Nova Scotia, leaving Canadian authorities worried about the cause of such a massive fish kill. Officials from the Department of Fisheries and Oceans (DFO) investigated reports on Wednesday, Dec. 28, of thousands of dead sea creatures ending up on the beaches of Annapolis and Digby counties. Among those that washed up along the coast were carcasses of crabs, lobsters, scallops, starfish, and various species of fish. Authorities have begun testing for low oxygen levels or potential pesticide contamination in the water. They've also issued a warning to the public to buy their seafood only from authorized vendors as a precaution. In a post on Twitter, the DFO advised residents of affected areas not to collect any of the dead fish and sea creatures that wash up on the shore. While authorities have yet to determine the exact cause of the fish kill, the event appears to be connected to a recent spate of marine animal die-offs along the Bay of Fundy in Nova Scotia over the past few weeks. Earlier this month, thousands of herring were found dead on shores along the Annapolis Basin. This came just a few weeks after masses of the same fish species ended up on St. Marys Bay. The latest die-offs were discovered on the beach near Savary Provincial Park in Plympton on Boxing Day, Dec. 26. Residents found thousands of crabs, lobster, bar clams, scallops, starfish, herring carcasses lining up the coast. DFO regional director general Doug Wentzell said they have asked the help of the Canadian Food Inspection Agency and Environment as well as the Climate Change Canada to get to the bottom of the mysterious fish kills. The agencies, however, haven't determined the cause of the mass die-offs as of yet. Wentzell said they weren't able to detect any evidence of infections or deadly agents in the water. The DFO are now planning to test some of the other species that have turned up dead on the shores in recent weeks. Tests on possible viral outbreaks in the water are still ongoing. These could take a longer time to finish since it involves the cultivation of specimens in the laboratory. The U.S. Geological Survey (USGS) said a number of factors could trigger a mass fish kill. One of the most common causes is low levels of dissolved oxygen in the water. This is believed to be the cause of similar mass die-offs of fish in the Indian River Lagoon in Florida and the Hongcheng Lake in China. © 2017 Tech Times, All rights reserved. Do not reproduce without permission.
News Article | February 16, 2017
Note to editors: There is a photo associated with this press release. The Honourable Dominic LeBlanc, P.C., Q.C., M.P., Minister of Fisheries, Oceans and the Canadian Coast Guard, toured Seaspan's Vancouver Shipyards (VSY) for a first-hand review of the progress on the Coast Guard's three new Offshore Fisheries Science Vessels (OFSVs). The three new OFSVs are the first ships to be built at VSY under the National Shipbuilding Strategy (NSS). Ships as large as these are built in blocks which are then fit together to form the whole vessel. The main blocks for the first OFSV are almost completely joined - the basic structure of the ship is in place and the vessel resembles its final form. The blocks forming the hull of the second OFSV are now being assembled, allowing the Minister to participate in the ship's the keel-laying while he was onsite. Keel-laying is a significant milestone. For the first time, steel sections, modules, and blocks are joined into a recognizable part of the ship. VSY has recently commenced production on the third OFSV. The new OFSVs will enable Fisheries and Oceans and the Canadian Coast Guard to continue conducting important science and research work such as collecting information about the distribution, abundance and biology of our species on the Atlantic and Pacific coasts. "I am pleased that progress is being made on construction of the Offshore Fisheries Science Vessels at Seaspan's Vancouver Shipyards. The keel laying of the second OFSV was a highlight as the pieces are starting to resemble the actual ship. This and other projects under the National Shipbuilding Strategy and the Coast Guard Fleet Renewal Plan will provide the men and women of the Coast Guard and our scientists with the equipment they need to conduct their important work for Canadians." The Honourable Dominic LeBlanc, P.C., Q.C., M.P., Minister of Fisheries, Oceans and the Canadian Coast Guard "Seaspan's Vancouver Shipyards is honoured to celebrate the laying of the keel for the second Offshore Fisheries Science Vessel (OFSV) with Minister LeBlanc. Today's ceremony signifies an important milestone in the National Shipbuilding Strategy and serves as a testament to the continuing momentum of the OFSV program for our Coast Guard customers and the many thousands of Canadians involved across the country." For more information about the Canadian Coast Guard, visit www.ccg-gcc.gc.ca. To view the photo associated with this press release, please visit the following link: http://media3.marketwire.com/docs/DFO.jpg
News Article | November 10, 2016
OTTAWA, ONTARIO--(Marketwired - Nov. 9, 2016) - The successful implementation of the government's National Oceans Protection Plan will require the reversal of previous government's cuts to scientists. "We welcome the government's announcement of the National Oceans Protection Plan," said PIPSC President, Debi Daviau. "We strongly believe that hiring at least 150 scientists should be included as a priority to make up for the professional positions that were eliminated at the Canadian Coast Guard and the Department of Fisheries and Oceans in recent years." The government's initiative will provide $1.5 billion in funding over five years to the Canadian Coast Guard (CCG) and the Department of Fisheries and Oceans (DFO). This announcement is long overdue and absolutely necessary if there is going to be more shipping traffic in the coming years. The new spending is especially positive given the legacy of cuts inherited from the last government. PIPSC has been vocal in our opposition to the closing of Coast Guard facilities, diminishing research capacity at science based departments like DFO, and the weakening of emergency preparedness units. Protecting our coast lines and waterways is of vital importance today and for future generations. In doing so it is essential that the Coast Guard's invaluable front-line response and enforcement function work in a coordinated manner with researchers and scientific regulatory experts at DFO to ensure evidence is driving the decision making process. "The lifework of these public service professionals is to ensure our coastal communities are safe and our aquatic ecosystems are protected," said Daviau. "If the cuts to the professional positions at DFO and CCG are reversed, this investment will increase capacity in critical areas and assist these professionals in carrying out the organization's mandate." Follow us on Facebook and on Twitter (@pipsc_ipfpc)
News Article | February 20, 2017
Editors Note: There is a photo associated with this press release. The Canadian Coast Guard is informing residents of Repentigny and those living along the shores of the L'Assomption, Des-Prairies, Mille-Îles and Châteauguay Rivers that spring icebreaking operations will begin around February 21, 2017. The date is subject to change with no notice, as activities could begin before or after that period, according to operational requirements or prevailing weather conditions. The purpose of this annual operation is to break up ice at the entrance of the tributaries in order to prevent ice jams and flooding that may result from the spring thaw. Owners of facilities or equipment on the ice should move them safely ashore before operations begin. The Canadian Coast Guard strongly recommends that pedestrians, fishers and snowmobilers leave the ice when they see the Canadian Coast Guard hovercraft in the vicinity. The ice may move, creating a real danger for anyone in the area of Canadian Coast Guard operations. The operation will be carried out by a Canadian Coast Guard hovercraft, either the CCGS Mamilossa or the CCGS Sipu Muin, an air-cushioned vehicle with engines that produce a noise similar to those of an aircraft. For more information about the Canadian Coast Guard, visit www.marinfo.gc.ca or www.ccg-gcc.gc.ca. To view the photo associated with this press release, please visit the following link: http://media3.marketwire.com/docs/DFO_22017.jpg
News Article | February 16, 2017
Note aux rédacteurs : Une photo est associée à ce communiqué de presse. L'honorable Dominic LeBlanc, c.p., c.r., député et ministre des Pêches, des Océans et de la Garde côtière canadienne, a visité le chantier naval Vancouver Shipyards (VSY) de Seaspan pour y effectuer directement un examen sur place des progrès réalisés à l'égard de la construction des trois nouveaux navires hauturiers de sciences halieutiques (NHSH) de la Garde côtière. Ces trois nouveaux NHSH sont les premiers bâtiments à être construits dans le chantier naval VSY dans le cadre de la Stratégie nationale de construction navale (SNCN). Les navires de cette envergure sont construits par sections qui sont par la suite assemblées pour former le navire au complet. Les sections principales du premier NHSH sont presque complètement assemblées; la structure centrale du bâtiment est en place et celui-ci revêt son apparence finale. Les sections formant la coque du deuxième NHSH sont en voie d'être assemblées; le Ministre a ainsi pu observer la pose de la quille alors qu'il était sur place. La construction du troisième NHSH a récemment été entamée au chantier naval VSY. Les nouveaux NHSH permettront à Pêches et Océans Canada et à la Garde côtière canadienne de poursuivre d'importants travaux scientifiques et de recherche comme la collecte de renseignements concernant la répartition, l'abondance et la biologie de nos espèces sur les côtes de l'Atlantique et du Pacifique. « Je suis heureux des progrès réalisés concernant la construction des navires hauturiers de sciences halieutiques au chantier naval VSY de Seaspan. La pose de la quille du second NHSH a été un moment phare puisque les parties assemblées nous montrent déjà à quoi va ressembler le navire. Ce projet, parmi d'autres projets ayant trait à la Stratégie nationale de construction navale et au Plan de renouvellement de la flotte de la Garde côtière, fournira aux hommes et aux femmes de la Garde côtière et à nos scientifiques, l'équipement nécessaire qui leur permettra de mener leurs travaux de grande importance pour les Canadiens. » L'honorable Dominic LeBlanc, c.p., c.r., député et ministre des Pêches, des Océans et de la Garde côtière canadienne « C'est un honneur pour le chantier naval Vancouver Shipyards de Seaspan de célébrer la pose de la quille pour le second navire hauturier de sciences halieutiques (NHSH) en présence du ministre LeBlanc. La cérémonie d'aujourd'hui marque un important jalon pour la Stratégie nationale de construction navale et sert de testament à la dynamique continue du programme des NHSH pour nos clients de la Garde côtière et les milliers de Canadiens partout au pays qui font partie intégrante de cette impulsion. » Pour de plus amples renseignements au sujet de la Garde côtière canadienne, visitez le www.ccg-gcc.gc.ca. Pour voir la photo associée à ce communiqué, veuillez visiter le lien suivant : http://media3.marketwire.com/docs/DFO.jpg
News Article | November 16, 2016
MISSISSAUGA, ON--(Marketwired - November 16, 2016) - Gay Lea Foods Co-operative Limited ("Gay Lea Foods") today announced a significant investment of $140 million over four years to establish an innovative, nutrition and nutraceutical-grade dairy ingredients hub in Canada. This unprecedented investment in dairy processing in Canada delivers on the Co-operative's mission to transform more Canadian milk by building an innovative and market-driven ingredients business that caters to our customers' needs. As a 100% Canadian owned and operated co-operative, Gay Lea Foods is committed to strong, sustainable rural communities where our members and employees live and prosper with their families. Phase one will commence in early 2017, with a $60 million expansion plan in the village of Teeswater in Bruce County, Ontario. Phase one also includes a $3 million investment to build a Research & Development Centre of Excellence in Hamilton, Ontario. This working laboratory and innovation incubator will be the nexus between R&D and commercialization throughout Gay Lea Foods' operations, and also service our partners in the dairy, food and health sectors. The first phase also includes upgrades and expansion at our Toronto area food manufacturing facilities to increase our capabilities and competitiveness, improving cost efficiencies, while working to reduce our environmental footprint. "As a dairy farmer and co-operative member owner, I am excited that Gay Lea Foods is driving growth through innovation and the development of new markets that will increase demand for milk from Canadian dairy farms. I am also proud that Gay Lea Foods is once again leading the way by demonstrating that rural Ontario is capable of world class innovation and food manufacturing." - Steve Dolson, Chair, Gay Lea Foods "Dairy Farmers of Ontario (DFO) congratulates and supports Gay Lea Foods in their continued commitment to growing Ontario dairy, through the construction of new, state-of-the-art processing capacity and R&D Centre, to expand markets for Canadian dairy products and ingredients." - Ralph Dietrich, Chair, Dairy Farmers of Ontario "Gay Lea Foods is motivated to shape the Canadian dairy industry of tomorrow while nourishing our farmer-owned co-operative today. Our co-operative is proving that successful and continuous growth as a wholly owned and operating Canadian dairy and food processor is possible. We are pleased to contribute to a growing Canadian economy, creating middle class jobs while sustaining our local communities." - Michael Barrett, President and CEO, Gay Lea Foods ABOUT GAY LEA FOODS In 1958 a group of farmers came together with a common vision -- to better the lives of Ontario farming families and co-operatives. Today, Gay Lea Foods the largest dairy co-operative in Ontario, with members on more than 1,300 dairy farms producing 35% of Ontario's cow milk, and more than 4,000 members in total. 100% Canadian owned and operated, Gay Lea Foods has both licensed dairy cow and goat producer members in Ontario, and processes both types of milk into a range of dairy products. The co-operative is driven by innovation and growing the market for Canadian cow and goat milk, with products ranging from the consumer favourite Spreadables; North America's first Smooth™ Cottage Cheese; and an innovative snack made with 100% cheese, Nothing But Cheese™.
News Article | February 20, 2017
Note aux rédacteurs : Une photo est associée à ce communiqué de presse. La Garde côtière canadienne avise les personnes habitant à Repentigny et le long des berges des rivières L'Assomption, des Prairies, des Mille Îles et Châteauguay, qu'elle entreprendra ses opérations de déglaçage printanier vers le 21 février 2017. La date peut changer sans préavis car les opérations pourraient avoir lieu avant ou après cette période, selon les besoins opérationnels ou les conditions météorologiques du moment. Ces opérations annuelles ont pour but de dégager de leurs glaces l'entrée des affluents et ainsi prévenir les embâcles et les inondations susceptibles de se produire lors du dégel printanier. Les personnes qui ont laissé des installations ou du matériel sur la glace doivent les rapporter sur la rive avant le début des opérations. La Garde côtière canadienne recommande aux promeneurs, pêcheurs et motoneigistes de ne pas demeurer sur la glace lorsqu'ils aperçoivent l'aéroglisseur dans les environs. Des mouvements de glace peuvent en effet se produire et présenter un danger pour toute personne se trouvant dans la zone opérationnelle de la Garde côtière canadienne. Le déglaçage sera effectué par un aéroglisseur de la Garde côtière canadienne, soit le NGCC Mamilossa ou le NGCC Sipu Muin, un véhicule à coussin d'air dont les moteurs font un bruit qui s'apparente à celui d'un avion. Pour de plus amples renseignements au sujet de la Garde côtière canadienne, visitez le www.marinfo.gc.ca ou www.ccg-gcc.gc.ca. Pour voir la photo associée à ce communiqué, veuillez visiter le lien suivant : http://media3.marketwire.com/docs/DFO_22017.jpg
News Article | February 21, 2017
Firemen put out a blaze from a light aircraft which exploded as it smashed into a shopping centre near Melbourne on February 21, 2017 killing five people aboard (AFP Photo/) A light aircraft smashed into shops and exploded into a "massive fireball" killing all five on board, including four American passengers reportedly golfers on the trip of a lifetime, officials in Australia said Tuesday. The twin-engined Beechcraft plane veered just after take-off into a shopping centre, that was still closed, next to Essendon Fields airport near Melbourne. "Five on the aircraft and looks like no one has survived the crash," said Victoria Police Assistant Commissioner Stephen Leane. Premier Daniel Andrews described it as "the worst civil aviation accident that our state has seen for 30 years". The private charter from Essendon, north of Melbourne, to King Island, 55 minutes to the south, came down just short of a major motorway packed with the heavy traffic of early morning commuters. Live television footage showed burned out wreckage, flames and major damage at the shopping centre and adjacent buildings. A column of thick black smoke rose into the air as witnesses spoke of an explosion. “The pilot unfortunately attempted to return to Essendon but has crashed into the DFO (Direct Factory Outlet) at Essendon Fields,” Leane told reporters. The centre was not due to open for another hour and the authorities confirmed no one inside was hurt. A taxi driver called ABC radio and told of the "massive fireball" and a landing wheel bouncing onto the motorway. "I saw this plane... when it hit the building there was a massive fireball," said the man called Jason. "I could feel the heat through the window of the taxi, and then a wheel -- it looked like a plane wheel -- bounced on the road and hit the front of the taxi as we were driving along." A shopworker called Ash told Sky News he saw "the fireball go up into the air", adding it "felt like a bomb had gone off". "The fire was just so hot we could not get anywhere near it," he said. "We could see the wreckage, or what was left of it." The US embassy in Canberra said the four passengers were American citizens. "We extend our deepest condolences to the families and loved ones of those who died in today's tragic crash," a spokeswoman said. Melbourne's Herald Sun identified two of the dead as Greg De Haven, 70, a retired FBI agent and lawyer Russell Munsch, both from Texas, who were travelling with two unnamed friends, the daily said. Plumber Michael Howard, 29, told the Australian Broadcasting Corporation he saw a "blue flash". "I was... just looking out the window... and then all of a sudden I just saw a blue flash come down and then all of a sudden there was a massive fireball." "It was like something from a movie," Howard said. Melbourne fire brigade chief Paul Stacchino tweeted that "more than 60 firefighters have worked hard to bring the fire... under control. Crews to remain on scene for some time". Essendon Fields was closed and all traffic diverted to Melbourne's two larger airports Tullamarine and Avalon.
News Article | February 17, 2017
VANCOUVER, BRITISH COLUMBIA--(Marketwired - Feb. 17, 2017) - The Government of Canada is committed to protecting our oceans and marine life for future generations. During his visit to Vancouver this week, the Honourable Dominic LeBlanc, Minister of Fisheries, Oceans and the Canadian Coast Guard, announced a suite of initiatives to ensure that our Pacific Coast remains healthy, prosperous and safe for generations to come. The Minister announced the establishment of the new Hecate Strait and Queen Charlotte Sound Glass Sponge Reefs Marine Protected Area, which will protect large colonies of unique glass sponges estimated to be 9000 years old. The reefs provide refuge, habitat and nursing grounds for many aquatic species such as rockfish, finfish and shellfish. The designation of this Marine Protected Area is a step forward in Canada's plan to protecting 5% of its marine and coastal areas by 2017 and 10% by 2020. In addition, the Minister signed, along with provinces, territories, Indigenous peoples and stakeholders, the Pacific North Coast Integrated Management Area (PNCIMA) Plan. This Plan will help protect the health of the North Pacific Coast by setting out a framework to manage the marine activities and resources in that area. Under the Oceans Protection Plan, the government is taking action to better understand and address the cumulative effects of shipping on marine mammals. While speaking to stakeholders at the Vancouver Aquarium, the Minister announced that Fisheries and Oceans Canada (DFO) will work with a coalition of partners to integrate underwater acoustic data to enhance our knowledge on the impacts of noise on marine mammals and make better decisions on how to mitigate these impacts. The Department is currently concluding an agreement with the Vancouver Fraser Port Authority to further support this project through the acquisition of hydrophones and other acoustic monitoring technology and systems. The Minister also announced that Fisheries and Oceans Canada has launched a science-based review of the effectiveness of current management and recovery actions under way for the southern resident killer whale, the North Atlantic right whale and the St. Lawrence estuary beluga. The science-based review will be completed this summer and will identify key additional measures and priorities for new or enhanced actions. At the Vancouver Aquarium, the Minister also announced over $1 million in support for two new research projects to monitor contaminants and investigate their impacts in the Pacific and Arctic Oceans. Fisheries and Oceans Canada is providing $399,000 to the Vancouver Aquarium to help implement Pollution Tracker, a new science program that will help identify the sources of contaminants in British Columbia and inform policies and management decisions. The Vancouver Aquarium is receiving a further $215,000 to study, for the first time, microplastics in the Arctic Ocean and their biological effects on marine life. An additional $520,000 in in-kind support, such as vessel use, will be provided to assist in the collection of samples. The Minister also highlighted the Government of Canada's commitment to enhancing the prevention and response capacity of the Canadian Coast Guard. New lifeboat stations, modern equipment, and emergency tow packages are among the measures that will be put in place under the Oceans Protection Plan. The Government of Canada will also be establishing a dedicated Primary Environmental Response Team (PERT) near Port Hardy, B.C. "Our Government is acting on its commitment to protect our coasts. While visiting beautiful British Columbia, I met with many partners and stakeholders and saw first-hand the accomplishments we can achieve by working together. With these initiatives, as well as other initiatives under the $1.5 billion Oceans Protection Plan, I know that we will make great strides in safeguarding our coasts for future generations." The Honourable Dominic LeBlanc, P.C., Q.C., M.P., Minister of Fisheries, Oceans and the Canadian Coast Guard "Pollution is a major issue for sea life and human health around the world. The Government's announcement addresses the urgent need for data on a wide range of pollutants in coastal British Columbia - including hydrocarbons, flame retardants, and heavy metals - as well the emerging issue of plastics in our oceans. This partnership will help us all understand what needs to be done to protect ocean life and human health for future generations." "This acoustic data project will complement and help expand the work already underway as part of the Vancouver Fraser Port Authority led ECHO program." Pacific North Coast Integrated Management Area (PNCIMA) Plan officially endorsed by all planning partners For more information about the Canadian Coast Guard, visit www.ccg-gcc.gc.ca.