News Article | February 22, 2017
Pre-B acute lymphoblastic leukaemia (ALL) cells were obtained from patients who gave informed consent in compliance with the guidelines of the Internal Review Board of the University of California San Francisco (Supplementary Table 2). Leukaemia cells from bone marrow biopsy of patients with ALL were xenografted into sublethally irradiated NOD/SCID (non-obese diabetic/severe combined immunodeficient) mice via tail vein injection. After passaging, leukaemia cells were collected. Cells were cultured on OP9 stroma cells in minimum essential medium-α (MEMα; Invitrogen), supplemented with 20% fetal bovine serum (FBS), 2 mM l-glutamine, 1 mM sodium pyruvate, 100 IU/ml penicillin and 100 μg/ml streptomycin. Primary chronic myeloid leukaemia (CML) cases were obtained with informed consent from the University Hospital Jena in compliance with institutional internal review boards (including the IRB of the University of California San Francisco; Supplementary Table 3). Cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM; Invitrogen) supplemented with 20% BIT serum substitute (StemCell Technologies); 100 IU/ml penicillin and 100 μg/ml streptomycin; 25 μmol/l β-mercaptoethanol; 100 ng/ml SCF; 100 ng/ml G-CSF; 20 ng/ml FLT3; 20 ng/ml IL-3; and 20 ng/ml IL-6. Human cell lines (Supplementary Table 2) were obtained from DSMZ and were cultured in Roswell Park Memorial Institute medium (RPMI-1640; Invitrogen) supplemented with GlutaMAX containing 20% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cell cultures were kept at 37 °C in a humidified incubator in a 5% CO atmosphere. None of the cell lines used was found in the database of commonly misidentified cell lines maintained by ICLAC and NCBI Biosample. All cell lines were authenticated by STR profiles and tested negative for mycoplasma. BML275 (water-soluble) and imatinib were obtained from Santa Cruz Biotechnology and LC Laboratories, respectively. Stock solutions were prepared in DMSO or sterile water at 10 mmol/l and stored at −20 °C. Prednisolone and dexamethasone (water-soluble) were purchased from Sigma-Aldrich and were resuspended in ethanol or sterile water, respectively, at 10 mmol/l. Stock solutions were stored at −20 °C. Fresh solutions (pH-adjusted) of methyl pyruvate, OAA, 3-OMG (an agonist of TXNIP), d-allose (an agonist of TXNIP) and recombinant insulin (Sigma-Aldrich) were prepared for each experiment. DMS was obtained from Acros Organics, and fresh solutions (pH-adjusted) were prepared before each experiment. For competitive-growth assays, 5 mmol/l methyl pyruvate, 5 mmol/l dimethyl succinate (DMS) and 5 mmol/l OAA were used. The CNR2 agonist HU308 was obtained from Cayman Chemical. To avoid inflammation-related effects in mice, bone marrow cells were extracted from mice (Supplementary Table 4) younger than 6 weeks of age without signs of inflammation. All mouse experiments were conducted in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Bone marrow cells were obtained by flushing cavities of femur and tibia with PBS. After filtration through a 70-μm filter and depletion of erythrocytes using a lysis buffer (BD PharmLyse, BD Biosciences), washed cells were either frozen for storage or subjected to further experiments. Bone marrow cells were cultured in IMDM (Invitrogen) with GlutaMAX containing 20% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 50 μM β-mercaptoethanol. To generate pre-B ALL (Ph+ ALL-like) cells, bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-7 (PeproTech) and retrovirally transformed by BCR–ABL1. BCR–ABL1-transformed pre-B ALL cells were propagated only for short periods of time and usually not for longer than 2 months to avoid acquisition of additional genetic lesions during long-term cell culture. To generate myeloid leukaemia (CML-like) cells, the myeloid-restricted protocol described previously30 was used. Bone marrow cells were cultured in 10 ng/ml recombinant mouse IL-3, 25 ng/ml recombinant mouse IL-6, and 50 ng/ml recombinant mouse SCF (PeproTech) and retrovirally transformed by BCR–ABL1. Immunophenotypic characterization was performed by flow cytometry. For conditional deletion, a 4-OHT-inducible, Cre-mediated deletion system was used. For retroviral constructs used, see Supplementary Table 5. Transfection of retroviral constructs (Supplementary Table 5) was performed using Lipofectamine 2000 (Invitrogen) with Opti-MEM medium (Invitrogen). Retroviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pHIT60 (gag-pol) and pHIT123 (ecotropic env). Lentiviral supernatant was produced by co-transfecting HEK 293FT cells with the plasmids pCDNL-BH and VSV-G or EM140. 293FT cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen) with GlutaMAX containing 10% fetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin, 25 mmol/l HEPES, 1 mmol/l sodium pyruvate and 0.1 mmol/l non-essential amino acids. Regular medium was replaced after 16 h by growth medium containing 10 mmol /l sodium butyrate. After incubation for 8 h, the medium was changed back to regular growth medium. After 24 h, retroviral supernatant was collected, filtered through a 0.45-μm filter and loaded by centrifugation (2,000g, 90 min at 32 °C) onto 50 μg/ml RetroNectin- (Takara) coated non-tissue 6-well plates. Lentiviral supernatant produced with VSV-G was concentrated using Lenti-X Concentrator (Clontech), loaded onto RetroNectin-coated plates and incubated for 15 min at room temperature. Lentiviral supernatant produced with EM140 was collected, loaded onto RetroNectin-coated plates and incubated for 30 min at room temperature. Per well, 2–3 × 106 cells were transduced by centrifugation at 600g for 30 min and maintained for 48 h at 37 °C with 5% CO before transferring into culture flasks. For cells transduced with lentiviral supernatant produced with EM140, supernatant was removed the day after transduction and replaced with fresh culture medium. Cells transduced with oestrogen-receptor fusion proteins were induced with 4-OHT (1 μmol/l). Cells transduced with constructs carrying an antibiotic-resistance marker were selected with its respective antibiotic. For loss-of-function studies, dominant-negative variants of IKZF1 (DN-IKZF1, lacking the IKZF1 zinc fingers 1–4) and PAX5 (DN-PAX5; PAX5–ETV6 fusion) were cloned from patient samples. Expression of DN-IKZF1 was induced by doxycycline (1 μg/ml), while activation of DN-PAX5 was induced by 4-OHT (1 μg/ml) in patient-derived pre-B ALL cells carrying IKZF1 and PAX5 wild-type alleles, respectively. Inducible reconstitution of wild-type IKZF1 and PAX5 in haploinsufficient pre-B ALL cells carrying deletions of either IKZF1 (IKZF1∆) or PAX5 (PAX5∆) were also studied. Lentiviral constructs used are listed in Supplementary Table 5. A doxycycline-inducible TetOn vector system was used for inducible expression of PAX5 in mouse BCR–ABL1 pre-B ALL. The retroviral constructs used are listed in Supplementary Table 5. To study the effects of B-cell- versus myeloid-lineage identity in genetically identical mouse leukaemia cells, a doxycycline-inducible TetOn-CEBPα vector system31 was used to reprogram B cells. Mouse BCR–ABL1 pre-B ALL cells expressing doxycycline-inducible CEBPα or an empty vector were induced with doxycycline (1 μg/ml). Conversion from the B-cell lineage (CD19+Mac1−) to the myeloid lineage (CD19−Mac1+) was monitored by flow cytometry. For western blots, B-lineage cells (CD19+Mac1−) and CEBPα-reprogrammed cells (CD19−Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively, following doxycycline induction. For metabolic assays, sorted B-lineage cells and CEBPα-reprogrammed cells were cultured (with doxycycline) for 2 days following sorting, and were then seeded in fresh medium for measurement of glucose consumption (normalized to cell numbers) and total ATP levels (normalized to total protein). To study Lkb1 deletion in the context of CEBPα-mediated reprogramming, BCR–ABL1-transformed Lkb1fl/fl pre-B ALL cells expressing doxycycline-inducible CEBPα were transduced with 4-OHT(1 μg/ml) inducible Cre-GFP (Cre-ERT2-GFP). Without sorting for GFP+ cells, cells were induced with doxycycline and 4-OHT. Viability (expressed as relative change of GFP+ cells) was measured separately in B-lineage (gated on CD19+ Mac1−) and myeloid lineage (gated on CD19− Mac1+) populations. To study whether Lkb1 deletion causes CEBPα-dependent effects on metabolism and signalling, Lkb1fl/fl BCR–ABL1 B-lineage ALL cells expressing doxycycline-inducible CEBPα or an empty vector were transduced with 4-OHT-inducible Cre-GFP. After sorting for GFP+ populations, cells were induced with doxycycline. B-lineage cells (CD19+ Mac1−) and CEBPα-reprogrammed cells (CD19− Mac1+) were sorted from cells expressing an empty vector or CEBPα, respectively. Sorted cells were cultured with doxycycline and induced with 4-OHT. Protein lysates were collected on day 2 following 4-OHT induction. For metabolomics, sorted cells were re-seeded in fresh medium on day 2 following 4-OHT induction and collected for metabolite extraction. For CRISPR/Cas9-mediated deletion of target genes, all constructs including lentiviral vectors expressing gRNA and Cas9 nuclease were purchased from Transomic Technologies (Supplementary Table 5; see Supplementary Table 6 for gRNA sequences). In brief, patient-derived pre-B ALL cells transduced with GFP-tagged, 4-OHT-inducible PAX5 or an empty vector were transduced with pCLIP-hCMV-Cas9-Nuclease-Blast. Blasticidin-resistant cells were subsequently transduced with pCLIP-hCMV-gRNA-RFP. Non-targeting gRNA was used as control. Constructs including lentiviral vectors expressing gRNA and dCas9-VPR used for CRISPR/dCas9-mediated activation of gene expression are listed in Supplementary Table 5. Nuclease-null Cas9 (dCas9) fused with VP64-p65-Rta (VPR) was cloned from SP-dCas9-VPR (a gift from G. Church; Addgene plasmid #63798) and then subcloned into pCL6 vector with a blasticidin-resistant marker. gRNA sequences (Supplementary Table 6) targeting the transcriptional start site of each specific gene were obtained from public databases (http://sam.genome-engineering.org/ and http://www.genscript.com/gRNA-database.html)32. gBlocks Gene Fragments were used to generate single-guide RNAs (sgRNAs) and were purchased from Integrated DNA Technologies, Inc. Each gRNA was subcloned into pCL6 vector with a dsRed reporter. Patient-derived pre-B ALL cells transduced with either GFP-tagged inducible PAX5 or an empty vector were transduced with pCL6-hCMV-dCas9-VPR-Blast. Blasticidin-resistant cells were used for subsequent transduction with pCL6-hCMV-gRNA-dsRed, and dsRed+ cells were further analysed by flow cytometry. For each target gene, 2–3 sgRNA clones were pooled together to generate lentiviruses. Non-targeting gRNA was used as control. To elucidate the mechanistic contribution of PAX5 targets, the percentage of GFP+ cells carrying gRNA(s) for each target gene was monitored by flow cytometry upon inducible activation of GFP-tagged PAX5 or an empty vector in patient-derived pre-B ALL cells in competitive-growth assays. Cells were lysed in CelLytic buffer (Sigma-Aldrich) supplemented with a 1% protease inhibitor cocktail (Thermo Fisher Scientific). A total of 20 μg of protein mixture per sample was separated on NuPAGE (Invitrogen) 4–12% Bis-Tris gradient gels or 4–20% Mini-PROTEAN TGX precast gels, and transferred onto nitrocellulose membranes (Bio-Rad). The primary antibodies used are listed in Supplementary Table 7. For protein detection, the WesternBreeze Immunodetection System (Invitrogen) was used, and light emission was detected by either film exposure or the BioSpectrum Imaging system (UPV). Approximately 106 cells per sample were resuspended in PBS blocked using Fc blocker for 10 min on ice, followed by staining with the appropriate dilution of the antibodies or their respective isotype controls for 15 min on ice. Cells were washed and resuspended in PBS with propidium iodide (0.2 μg/ml) or DAPI (0.75 μg/ml) as a dead-cell marker. The antibodies used for flow cytometry are listed in Supplementary Table 7. For competitive-growth assays, the percentage of GFP+ cells was monitored by flow cytometry. For annexin V staining, annexin V binding buffer (BD Bioscience) was used instead of PBS and 7-aminoactinomycin D (7AAD; BD Bioscience) instead of propidium iodide. Phycoerythrin-labelled annexin V was purchased from BD Bioscience. For BrdU staining, the BrdU Flow Kit was purchased from BD Bioscience and used according to the manufacturer’s protocol. Methylcellulose colony-forming assays were performed with 10,000 BCR–ABL1 pre-B ALL cells. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers were counted after 14 days. Cell viability upon the genetic loss of function of target genes and/or inducible expression of PAX5 was monitored by flow cytometry using propidium iodide (0.2 μg/ml) as a dead-cell marker. To study the effects of an AMPK inhibitor (BML275), glucocorticoids (dexamethasone and prednisolone), CNR2 agonist (HU308), or TXNIP agonists (3-OMG and d-allose), 40,000 human or mouse leukaemia cells were seeded in a volume of 80 μl in complete growth medium on opaque-walled, white 96-well plates (BD Biosciences). Compounds were added at the indicated concentrations giving a total volume of 100 μl per well. After culturing for 3 days, cells were subjected to CellTiter-Glo Luminescent Cell Viability Assay (Promega). Relative viability was calculated using baseline values of cells treated with vehicle control as a reference. Combination index (CI) was calculated using the CalcuSyn software to determine interaction (synergistic, CI < 1; additive, CI = 1; or antagonistic, CI > 1) between the two agents. Constant ratio combination design was used. Concentrations of BML275, d-allose, 3-OMG and HU308 used are indicated in the figures. Concentrations of Dex used were tenfold lower than those of BML275. Concentrations of prednisolone used were twofold lower than those of BML275. To determine the number of viable cells, the trypan blue exclusion method was applied, using the Vi-CELL Cell Counter (Beckman Coulter). ChIP was performed as described previously33. Chromatin from fixed patient-derived Ph+ ALL cells (ICN1) was isolated and sonicated to 100–500-bp DNA fragments. Chromatin fragments were immunoprecipitated with either IgG (as a control) or anti-Pax5 antibody (see Supplementary Table 7). Following reversal of crosslinking by formaldehyde, specific DNA sequences were analysed by quantitative real-time PCR (see Supplementary Table 8 for primers). Primers were designed according to ChIP–seq tracks for PAX5 antibodies in B lymphocytes (ENCODE, Encyclopedia of DNA Elements, GM12878). ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown. CD19 and ACTA1 served as a positive and a negative control gene, respectively. The y axis represents the normalized number of reads per million reads for peak summit for each track. The ChIP–seq peaks were called by the MACS peak-caller by comparing read density in the ChIP experiment relative to the input chromatin control reads, and are shown as bars under each wiggle track. Gene models are shown in UCSC genome browser hg19. Extracellular glucose levels were measured using the Amplex Red Glucose/Glucose Oxidase Assay Kit (Invitrogen), according to the manufacturer’s protocol. Glucose concentrations were measured in fresh and spent medium. Total ATP levels were measured using the ATP Bioluminescence Assay Kit CLS II (Roche) according to the manufacturer’s protocol. In fresh medium, 1 × 106 cells per ml were seeded and treated as indicated in the figure legends. Relative levels of glucose consumed and total ATP are shown. All values were normalized to cell numbers (Figs 1b, c, 2c (glucose uptake), 3a and Extended Data Figs 2c, 4f, 6d) or total protein (Fig. 2c, ATP levels). Numbers of viable cells were determined by applying trypan blue dye exclusion, using the Vi-CELL Cell Counter (Beckman Coulter). Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using a Seahorse XFe24 Flux Analyzer with an XF Cell Mito Stress Test Kit and XF Glycolysis Stress Test Kit (Seahorse Bioscience) according to the manufacturer’s instructions. All compounds and materials were obtained from Seahorse Bioscience. In brief, 1.5 × 105 cells per well were plated using Cell-Tak (BD Biosciences). Following incubation in XF-Base medium supplemented with glucose and GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, OCR was measured at the resting stage (basal respiration in XF Base medium supplemented with GlutaMax and glucose) and in response to oligomycin (1 μmol/l; mitochondrial ATP production), mitochondrial uncoupler FCCP (5 μmol/l; maximal respiration), and respiratory chain inhibitor antimycin and rotenone (1 μmol/l). Spare respiratory capacity is the difference between maximal respiration and basal respiration. ECAR was measured under specific conditions to generate glycolytic profiles. Following incubation in glucose-free XF Base medium supplemented with GlutaMAX for 1 h at 37 °C (non-CO incubator) for pH stabilization, basal ECAR was measured. Following measurement of the glucose-deprived, basal ECAR, changes in ECAR upon the sequential addition of glucose (10 mmol/l; glycolysis), oligomycin (1 μmol/l; glycolytic capacity), and 2-deoxyglucose (0.1 mol/l) were measured. Glycolytic reserve was determined as the difference between oligomycin-stimulated glycolytic capacity and glucose-stimulated glycolysis. All values were normalized to cell numbers (Extended Data Fig. 2c) or total protein (Extended Data Figs 3a, 7a, b 8f) and are shown as the fold change relative to basal ECAR or OCR. Metabolite extraction and mass-spectrometry-based analysis were performed as described previously34. Metabolites were extracted from 2 × 105 cells per sample using the methanol/water/chloroform method. After incubation at 37 °C for the indicated time, cells were rinsed with 150 mM ammonium acetate (pH 7.3), and 400 μl cold 100% methanol (Optima* LC/MS, Fisher) and then 400 μl cold water (HPLC-Grade, Fisher) was added to cells. A total of 10 nmol norvaline (Sigma) was added as internal control, followed by 400 μl cold chloroform (HPLC-Grade, Fisher). Samples were vortexed three times over 15 min and spun down at top speed for 5 min at 4 °C. The top layer (aqueous phase) was transferred to a new Eppendorf tube, and samples were dried on Vacufuge Plus (Eppendorf) at 30 °C. Extracted metabolites were stored at −80 °C. For mass spectrometry-based analysis, the metabolites were resuspended in 70% acetonitrile and 5 μl used for analysis with a mass spectrometer. The mass spectrometer (Q Exactive, Thermo Scientific) was coupled to an UltiMate3000 RSLCnano HPLC. The chromatography was performed with 5 mM NH AcO (pH 9.9) and acetonitrile at a flow rate of 300 μl/min starting at 85% acetonitrile, going to 5% acetonitrile at 18 min, followed by an isocratic step to 27 min and re-equilibration to 34 min. The separation was achieved on a Luna 3u NH2 100A (150 × 2 mm) (Phenomenex). The Q Exactive was run in polarity switching mode (+3 kV/−2.25 kV). Metabolites were detected based on retention time (t ) and on accurate mass (± 3 p.p.m.). Metabolite quantification was performed as area-under-the-curve (AUC) with TraceFinder 3.1 (Thermo Scientific). Data analysis was performed in R (https://www.r-project.org/), and data were normalized to the number of cells. Relative amounts were log -transformed, median-centred and are shown as a heat map. To generate a model for pre-leukaemic B cell precursors expressing BCR–ABL1, BCR–ABL1 knock-in mice were crossed with Mb1-Cre deleter strain (Mb1-Cre; Bcr+/LSL-BCR/ABL) for excision of a stop-cassette in early pre-B cells. Bone marrow cells collected from Mb1-Cre; Bcr+/LSL-BCR/ABL mice cultured in the presence of IL-7 were primed with vehicle control or a combination of OAA (8 mmol/l), DMS (8 mmol/l) and insulin (210 pmol/l). Following a week of priming, cells were maintained and expanded in the presence of IL-7, supplemented with vehicle control or a combination of OAA (0.8 mmol/l) and DMS (0.8 mmol/l) for 4 weeks. Pre-B cells from Mb1-Cre; Bcr+/LSL-BCR/ABL mice expressed low levels of BCR–ABL1 tagged to GFP, and were analysed by flow cytometry for surface expression of GFP and CD19. The methylcellulose colony-forming assays were performed with 10,000 cells treated with vehicle control or metabolites. Cells were resuspended in mouse MethoCult medium (StemCell Technologies) and cultured on 3-cm diameter dishes, with an extra water supply dish to prevent evaporation. Images were taken and colony numbers counted after 14 days. For in vivo transplantation experiments, cells were treated with vehicle control or metabolites (OAA/DMS) for 6 weeks. One million cells were intravenously injected into sublethally irradiated (250 cGy) 6–8-week-old female NSG mice (n = 7 per group). Mice were randomly allocated into each group, and the minimal number of mice in each group was calculated by using the ‘cpower’ function in R/Hmisc package. No blinding was used. Each mouse was killed when it became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move). The bone marrow and spleen were collected for flow cytometry analyses for leukaemia infiltration (CD19, B220). After 63 days, all remaining mice were killed and bone marrow and spleens from all mice were analysed by flow cytometry. Statistical analysis was performed using the Mantel–Cox log-rank test. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Following cytokine-independent proliferation, BCR–ABL1-transformed Lkb1fl/fl or AMPKa2fl/fl pre-B ALL cells were transduced with 4-OHT-inducible Cre or an empty vector control. For ex vivo deletion, deletion was induced 24 h before injection. For in vivo deletion, deletion was induced by 4-OHT (0.4 mg per mouse; intraperitoneal injection). Approximately 106 cells were injected into each sublethally irradiated (250 cGy) NOD/SCID mouse. Seven mice per group were injected via the tail vein. We randomly allocated 6–8-week-old female NOD/SCID or NSG mice into each group. The minimal number of mice in each group was calculated using the ‘cpower’ function in R/Hmisc package. No blinding was used. When a mouse became terminally sick and showed signs of leukaemia burden (hunched back, weight loss and inability to move), it was killed and the bone marrow and/or spleen were collected for flow cytometry analyses for leukaemia infiltration. Statistical analysis was performed by Mantel–Cox log-rank test. In vivo expansion and leukaemia burden were monitored by luciferase bioimaging. Bioimaging of leukaemia progression in mice was performed at the indicated time points using an in vivo IVIS 100 bioluminescence/optical imaging system (Xenogen). d-luciferin (Promega) dissolved in PBS was injected intraperitoneally at a dose of 2.5 mg per mouse 15 min before measuring the luminescence signal. General anaesthesia was induced with 5% isoflurane and continued during the procedure with 2% isoflurane introduced through a nose cone. All mouse experiments were in compliance with institutional approval by the University of California, San Francisco Institutional Animal Care and Use Committee. Data are shown as mean ± s.d. unless stated. Statistical significance was analysed by using Grahpad Prism software or R software (https://www.r-project.org/) by using two-tailed t-test, two-way ANOVA, or log-rank test as indicated in figure legends. Significance was considered at P < 0.05. For in vitro experiments, no statistical methods were used to predetermine the sample size. For in vivo transplantation experiments, the minimal number of mice in each group was calculated through use of the ‘cpower’ function in the R/Hmisc package. No animals were excluded. Overall survival and relapse-free survival data were obtained from GEO accession number GSE11877 (refs 35, 36) and TCGA. Kaplan–Meier survival analysis was used to estimate overall survival and relapse-free survival. Patients with high risk pre-B ALL (COG clinical trial, P9906, n = 207; Supplementary Table 10) were segregated into two groups on the basis of high or low mRNA levels with respect to the median mRNA values of the probe sets for the gene of interest. A log-rank test was used to compare survival differences between patient groups. R package ‘survival’ Version 2.35-8 was used for the survival analysis and Cox proportional hazards regression model in R package for the multivariate analysis (https://www.r-project.org/). The investigators were not blinded to allocation during experiments and outcome assessment. Experiments were repeated to ensure reproducibility of the observations. Gel scans are provided in Supplementary Fig. 1. Gene expression data were obtained from the GEO database accession numbers GSE32330 (ref. 12), GSE52870 (ref. 37), and GSE38463 (ref. 38). Patient-outcome data were derived from the National Cancer Institute TARGET Data Matrix of the Children’s Oncology Group (COG) Clinical Trial P9906 (GSE11877)35, 36 and from TCGA (the Cancer Genome Atlas). GEO accession details are provided in Supplementary Tables 9 and 10. ChIP–seq tracks for PAX5, IKZF1, EBF1 and TCF3 antibodies in a normal B-cell sample (ENCODE GM12878, UCSC genome browser) on INSR, GLUT1, GLUT3, GLUT6, HK2, G6PD, NR3C1, TXNIP, CNR2 and LKB1 gene promoter regions are shown in UCSC genome browser hg19. All other data are available from the corresponding author upon reasonable request.
News Article | March 1, 2017
The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. ARC-Net, University of Verona: approval number 1885 from the Integrated University Hospital Trust (AOUI) Ethics Committee (Comitato Etico Azienda Ospedaliera Universitaria Integrata) approved in their meeting of 17 November 2010, documented by the ethics committee 52070/CE on 22 November 2010 and formalized by the Health Director of the AOUI on the order of the General Manager with protocol 52438 on 23 November 2010. APGI: Sydney South West Area Health Service Human Research Ethics Committee, western zone (protocol number 2006/54); Sydney Local Health District Human Research Ethics Committee (X11-0220); Northern Sydney Central Coast Health Harbour Human Research Ethics Committee (0612-251M); Royal Adelaide Hospital Human Research Ethics Committee (091107a); Metro South Human Research Ethics Committee (09/QPAH/220); South Metropolitan Area Health Service Human Research Ethics Committee (09/324); Southern Adelaide Health Service/Flinders University Human Research Ethics Committee (167/10); Sydney West Area Health Service Human Research Ethics Committee (Westmead campus) (HREC2002/3/4.19); The University of Queensland Medical Research Ethics Committee (2009000745); Greenslopes Private Hospital Ethics Committee (09/34); North Shore Private Hospital Ethics Committee. Baylor College of Medicine: Institutional Review Board protocol numbers H-29198 (Baylor College of Medicine tissue resource), H-21332 (Genomes and Genetics at the BCM-HGSC), and H-32711(Cancer Specimen Biobanking and Genomics). Patients were recruited and consent obtained for genomic sequencing through the ARC-Net Research Centre at Verona University, Australian Pancreatic Cancer Genome Initiative (APGI), and Baylor College of Medicine as part of the ICGC (www.icgc.org). A patient criterion for admission to the study was that they were clinically sporadic. This information was acquired through direct interviews with participants and a questionnaire regarding their personal history and that of relatives with regard to pancreas cancers and any other cancers during anamnesis. Clinical records were also used to clarify familial history based on patient indications. Samples were prospectively and consecutively acquired through institutions affiliated with the Australian Pancreatic Cancer Genome Initiative. Samples from the ARC-Net biobank are the result of consecutive collections from a single centre. All tissue samples were processed as previously described5151. Representative sections were reviewed independently by at least one additional pathologist with specific expertise in pancreatic diseases. Samples either had full face frozen sectioning performed in optimal cutting temperature (OCT) medium, or the ends excised and processed in formalin to verify the presence of tumour in the sample to be sequenced and to estimate the percentage of neoplastic cells in the sample relative to stromal cells. Macrodissection was performed if required to excise areas that did not contain neoplastic epithelium. Tumour cellularity was determined using SNP arrays (Illumina) and the qpure tool9. PanNET is a rare tumour type and the samples were collected via an international network. We estimate that with 98 unique patients in the discovery cohort, we will achieve 90% power for 90% of genes to detect mutations that occur at a frequency of ~10% above the background rate for PanNET (assuming a somatic mutation frequency of more than 2 per Mb)52. Cancer and matched normal colonic mucosa were collected at the time of surgical resection from the Royal Brisbane and Women’s Hospital and snap frozen in liquid nitrogen. A biallelic germline mutation in the MUTYH gene was detected by restriction fragment length polymorphism analysis and confirmed by automated sequencing to be the G382D mutation (or ENST00000450313.5 G396D, ClinVar#5294) in both alleles53. The primary antibodies used for immunohistochemical staining were: cytokeratin 8/18 (5D3, Novocastra), chromogranin A (DAK-A3, Dako), and CD99 (O13, Biolegend). Antibodies and staining conditions have been described elsewhere39. Whole-genome sequencing with 100-bp paired reads was performed with a HiSEQ2000 (Illumina). Sequence data were mapped to a GRCh37 using BWA and BAM files are available in the EGA (accession number: EGAS00001001732). Somatic mutations and germline variants were detected using a previously described consensus mutation calling strategy11. Mutations were annotated with gene consequence using SNPeff. The pathogenicity of germline variants was predicted using cancer-specific and locus-specific genetic databases, medical literature, computational predictions with ENSEMBL Variant Effect Predictor (VEP) annotation, and second hits identified in the tumour genome. Intogen27 was used to find somatic genes that were significantly mutated. Somatic structural variants were identified using the qSV tool as previously described10, 11, 17. Coding mutations are included in supplementary tables and all mutations have been uploaded to the International Cancer Genome Consortium Data Coordination Center. Mutational signatures were predicted using a published framework14. Essentially, the 96-substitution classification was determined for each sample. The signatures were compared to other validated signatures and the prevalence of each signature per megabase was determined. Somatic copy number was estimated using high density SNP arrays and the GAP tool12. Arm level copy number data were clustered using Ward’s method, Euclidian distance. GISTIC13 was used to identify recurrent regions of copy number change. The whole genome sequence data was used to determine the length of the telomeres in each sample using the qMotif tool. Essentially, qMotif determines telomeric DNA content by calculating the number of reads that harbour the telomere motif (TTAGG), and then estimates the relative length of telomeres in the tumour compared to the normal. qMotif is available online (http://sourceforge.net/projects/adamajava). Telomere length was validated by qPCR as previously described54. RNASeq library preparation and sequencing were performed as previously described55. Essentially, sequencing reads were mapped to transcripts corresponding to ensemble 70 annotations using RSEM. RSEM data were normalized using TMM (weighted trimmed mean of M-values) as implemented in the R package ‘edgeR’. For downstream analyses, normalized RSEM data were converted to counts per million (c.p.m.) and log transformed. Genes without at least 1 c.p.m. in 20% of the sample were excluded from further analysis55. Unsupervised class discovery was performed using consensus clustering as implemented in the ConsensusClusterPlus R package56. The top 2,000 most variable genes were used as input. Differential gene expression analysis between representative samples was performed using the R package ‘edgeR’57. Ontology and pathway enrichment analysis was performed using the R package ‘dnet’58. PanNET class enrichment using published gene signatures44 was performed using Gene Set Variation Analysis (GSVA) as described previously55. Two strategies were used to verify fusion transcripts. For verification of EWSR1–BEND2 fusions, cDNAs were synthesized using the SuperScript VILO cDNA synthesis kit (Thermofisher) with 1 μg purified total RNA. For each fusion sequence, three samples were used: the PanNET sample containing the fusion, the PanNET sample without that fusion, and a non-neoplastic pancreatic sample. The RT–PCR product were evaluated on the Agilent 2100 Bioanalyzer (Agilent Technologies) and verified by sequencing using the 3130XL Genetic Analyzer (Life Technologies). Primers specific for EWSR1–BEND2 fusion genes are available upon request. To identify the EWSR1 fusion partner in the case ITNET_2045, a real-time RT–PCR translocation panel for detecting specific Ewing sarcoma fusion transcripts was applied as described59. Following identification of the fusion partner, PCR amplicons were subjected to sequencing using the 3130XL Genetic Analyzer. EWSR1 rearrangements were assayed on paraffin-embedded tissue sections using a commercial split-signal probe (Vysis LSI EWSR1 (22q12) Dual Colour, Break Apart Rearrangement FISH Probe Kit) that consists of a mixture of two FISH DNA probes. One probe (~500 kb) is labelled in SpectrumOrange and flanks the 5′ side of the EWSR1 gene, extending through intron 4, and the second probe (~1,100 kb) is labelled in SpectrumGreen and flanks the 3′ side of the EWSR1 gene, with a 7-kb gap between the two probes. With this setting, the assay enables the detection of rearrangements with breakpoints spanning introns 7–10 of the EWSR1 gene. Hybridization was performed according to the manufacturer’s instructions and scoring of tissue sections was assessed as described elsewhere60, counting at least 100 nuclei per slide. Recurrently mutated genes identified by whole-genome sequencing were independently evaluated in a series of 62 PaNETs from the ARC-Net Research Centre, University of Verona. Four Ion Ampliseq Custom panels (Thermofisher) were designed to target the entire coding regions and flanking intron–exon junctions of the following genes: MEN1, DAXX, ATRX, PTEN and TSC2 (panel 1); DEPDC5, TSC1 and SETD2 (panel 2); ARID1A and MTOR (panel 3); CHEK2 and MUTYH (panel 4). Twenty nanograms of DNA were used per multiplex PCR amplification. The quality of the obtained libraries was evaluated by the Agilent 2100 Bioanalyzer on chip electrophoresis. Emulsion PCR was performed with the OneTouch system (Thermofisher). Sequencing was run on the Ion Torrent Personal Genome Machine (PGM, Thermofisher) loaded with 316 or 318 chips. Data analysis, including alignment to the hg19 human reference genome and variant calling, was done using Torrent Suite Software v4.0 (Thermofisher). Filtered variants were annotated using a custom pipeline based on the Variant Effector Predictor (VEP) software. Alignments were visually verified with the Integrative Genomics Viewer: IGV v2.3 (Broad Institute). There is no contiguous structure available for CHEK2, so we produced a model of isoform C using PDBid 3i6w61 as a template for predicting the structure of sequence O96017. Modelling was carried out within the YASARA suite of programs62 and consisted of an initial BLAST search for suitable templates followed by alignment, building of loops not present in selected template structure and energy minimization in explicit solvent. Modelling was carried out in the absence of a phosphopeptide ligand, which was added on completion by aligning the model with structure 1GXC and merging the ligand contained therein with the model structure. Similarly, MUTYH is represented by discontinuous structures and so this too was modelled using PDBids 3N5N and 4YPR as templates together with sequence NP_036354.1. Having constructed both models, amino acid substitutions were carried out to make the wild-type sequences conform to the variants described above. Each substitution was carried out independently and the resulting variant structures were subject to simulated annealing energy minimization using the AMBER force field. The resulting energy-minimized structures formed the basis of the predictions. CHEK2 site mutants were generated by site-directed mutagenesis of wild-type pCMV–FLAG CHEK2 (primer sequences in Supplementary Table 16). Proteins were expressed in HEK293T, a highly transfectable derivative of HEK293 cells that were retrieved from the cell culture bank at the QIMR Berghofer medical research institute. Cells were authenticated by STR profiling and were negative for mycoplasma. Transfected cells were lysed in NP-40 modified RIPA with protease and phosphatase inhibitors. Protein expression levels were analysed by western blotting with anti-FLAG antibodies and imaging HRP luminescent signal on a CCD camera (Fuji) and quantifying in MultiGauge software (Fuji). Kinase assays were performed using recombinant GST–CDC25C (amino acids 200–256) as substrate, essentially as described63. Kinase assay quantification was performed by scintillation counting of excised gel bands in OptiPhase scintillant (Perkin Elmer) using a Tri-Carb 2100TR beta counter (Packard). Counts for each reaction set were expressed as a fraction of the wild type. All experiments were performed at least three times. The date of diagnosis and the date and cause of death for each patient were obtained from the Central Cancer Registry and treating clinicians. Median survival was estimated using the Kaplan–Meier method and the difference was tested using the log-rank test. P values of less than 0.05 were considered statistically significant. The hazard ratio and its 95% confidence interval were estimated using Cox proportional hazard regression modelling. The correlation between DAXX or ATRX mutational status and other clinico-pathological variables was calculated using the χ2 test. Statistical analysis was performed using StatView 5.0 Software (Abacus Systems). Disease-specific survival was used as the primary endpoint. Genome sequencing data presented in this study have been submitted to the European Genome-Phenome Archive under accession number EGAS00001001732 (https://www.ebi.ac.uk/ega/search/site/EGAS00001001732).
News Article | February 15, 2017
No statistical methods were used to predetermine sample size. Affinity-purified antibodies against total and Ser886 and Ser999 phospho-sites of EPRS were generated as described7, 31. Antibody against phospho-Ser was from Meridian Life Science. Antibodies specific for the C terminus of S6K1 and N terminus of S6K2 were purchased from Abcam and LifeSpan, respectively. Antibodies against PKA, DMPK, PKN, GAPDH, caveolin1, CD36/FAT, GLUT4, His-tag, β-actin and FATP1, FATP3, and FATP4 were from Santa Cruz. Antibody specific for FABP4 and FABP5 were from R&D and for FABPpm/GOT2 was from GeneTex. All other antibodies and rapamycin were from Cell Signaling. SignalSilence siRNAs targeting RSK1, AKT and S6K1 were from Cell Signaling, and those targeting raptor and rictor were from Santa Cruz. The 3′-UTR-specific duplex siRNAs, 5′-UGAUACGAAGAUCUUCUCAG-3′ and 5′-GCCUAAAUUAACAGUGGAA-3′, targeting mouse EPRS were from Origene. Smart pool siRNA targeting the coding sequence of mouse FATP1 (SLC27a1) was from Dharmacon and 3′-UTR-specific trilencer siRNA targeting human S6K1 was from Origene. Recombinant wild-type and Ser-to-Ala (S886A and S999A) mutant His-tagged linker proteins spanning Pro683 to Asn1023 of human EPRS were expressed and purified as described7, 8. Recombinant active S6K1 (ref. 32) and RSK1–3 were from Cell Signaling; Akt1 and Akt2 were from EMD Millipore. Mouse EPRS domains ERS (Met1 to Gln682), linker (Pro683 to Asn1023), and PRS (Leu1024 to Tyr1512) were cloned into pcDNA3 vector with an N terminus Flag tag using full-length mouse EPRS cDNA (Origene) as template. Flag-tagged mouse wild-type linker and linker with Ser999-to-Ala (S999A) and Ser999-to-Asp (S999D) mutations were generated as described33. Full-length human S6K1 cDNA in pCMV6-entry vector was purchased from Origene and recloned, deleting the 23-amino acid N terminus nuclear localization signal, and adding an in-frame upstream 6-His tag and a downstream Myc tag in pcDNA3. Specific Thr389-to-Ala (T389A) and Thr389-to-Glu (T389E) mutations were introduced using primers with the desired mutation and GENEART Site-Directed Mutagenesis System (Invitrogen). Human U937 monocytic cells (CRL 1593.2; ATCC authenticated by STR DNA profiling) were cultured in RPMI 1640 medium and 10% fetal bovine serum (FBS) with penicillin and streptomycin at 37 °C in 5% CO . Bone-marrow-derived macrophages (BMDM) were flushed from femur and tibia marrows of S6K1−/−S6K2−/−, and double-knockout S6K1−/−S6K2−/− mice (from G. Thomas and S. Kozma), and then cultured for one week in RPMI 1640 medium containing 10% FBS and 20% L929 cell-conditioned medium at 37 °C in 5% CO . 1 × 107 cells were treated with 500 U ml−1 IFNγ (R&D) for up to 24 h, as described previously34, 35. 3T3-L1 fibroblasts (CL-173; ATCC-certified) were cultured in high glucose containing Dulbecco’s modified Eagle’s medium (DMEM), 10% FBS and antibiotics/antimycotic at 37 °C in 10% CO to near 75% confluence. Confluent fibroblasts were induced to differentiate in medium containing DMEM and 10% FBS supplemented with 1× solutions of insulin:dexamethasone:3-isobutyl-1-methylxanthine (Cayman). After 72 h, the medium was replaced with 10% FBS and DMEM containing only insulin and maintained for a week with 3 changes in the same medium. Adipocytes were maintained in DMEM medium with 10% calf serum and antibiotics/antimycotic for at least 3 d before utilization. Differentiated adipocytes were serum-deprived for 4 h followed by treatment with 100 nM insulin (Sigma-Aldrich) for 4 h, or as indicated. Cell lysates were prepared using Phosphosafe Extraction buffer (Novagen) supplemented with protease inhibitors. As certified U937 monocytes and 3T3-L1 fibroblasts were directly procured from ATCC, they were not subjected to any further testing for contamination. Primary adipocytes from white adipose tissue (WAT) were prepared as described14, 36. Briefly, after mouse sacrifice, fat pads were removed and minced in Krebs-Ringer-bicarbonate-HEPES (KRBH) buffer (pH 7.4) containing 10 mM sodium bicarbonate, 30 mM HEPES, 200 nM adenosine, and 1% fatty acid-free bovine serum albumin (BSA, Sigma). WATs were digested with collagenase (2 mg g−1) in KRBH buffer at 37 °C for 1 h. Digested WATs were suspended in DMEM supplemented with 10% FBS, and filtered through 100-μm mesh cell strainer (BD Falcon) to remove undigested material. The cell suspension was incubated for 10 min at room temperature, and adipocytes collected from the floating layer after centrifugation. Adipocytes were incubated for 1 h at room temperature with gentle shaking and washed three times with DMEM. Differentiated human adipocytes in adipocyte maintenance medium were obtained from Cell Applications. Adipocytes were maintained in DMEM medium with 10% calf serum and antibiotics/antimycotic for 2 d before utilization, and 5 × 106 cells were serum-deprived for 4 h followed by treatment with 100 nM insulin for 4 h. Hepatocytes were isolated by collagenase perfusion of mouse livers and cells seeded for 4 h on collagen-coated 6-well plates (1 × 106 cells per well) in Williams’ medium E with 10% FBS, 25 mM HEPES, 100 nM insulin, and 100 nM triiodothyronine37, 38, 39. Cells were cultured for 48 h in serum-free Williams medium E with two medium changes. Before experiments, hepatocytes were pre-incubated overnight in serum-, insulin-, and triiodothyronine-free DMEM, and then with 100 nM insulin. Adult mouse cardiac cells were isolated by sequential plating using non-perfusion adult cardiomyocyte isolation kit (Cellutron)40. After isolation, 1 × 106 cardiac cells were incubated for 24 h in serum containing AS medium, and then with serum-free AW medium for another 24 h. Before experiments, cells were incubated for 4 h in serum-free DMEM, and then with 100 nM insulin. All studies using cultured cells were repeated at least three times. The number of replicates was estimated from comparable published studies that gave statistically significant results. U937 cells (1 × 107), PBMs and differentiated 3T3-L1 adipocytes (5 × 106 cells for both) were transfected with endotoxin-free plasmid DNAs or siRNAs (target-specific and scrambled control) using nucleofector (100 μl solution V for U937 cells and PBMs and 100 μl solution L for 3T3-L1 adipocytes) from Amaxa nucleofection kit (Lonza) following the manufacturer’s protocol. Transfected cells were immediately transferred to pre-warmed Opti-MEM media for 6 h and then to RPMI 1640 (for U937 cells and PBMs) and DMEM (for 3T3-L1 adipocytes) containing 10% FBS supplemented with penicillin, streptomycin, and geneticin (G418; 20 μg ml−1) for 18 to 24 h before treatment with insulin and inhibitors. Cell lysates or purified active kinases were pre-incubated with recombinant EPRS linker (wild-type and mutant) for 5 min in kinase assay buffer (50 mM Tris-HCl (pH 7.6), 1 mM dithiotheitol, 10 mM MgCl , 1 mM CaCl , and phosphatase inhibitor cocktail)7, 8, 33. Phosphorylation was initiated by addition of 5 μCi [γ-32P]ATP (Perkin-Elmer) for 15 min, and terminated using SDS gel-loading buffer and heat denaturation. Phosphorylated proteins were detected after resolution on Tris-glycine SDS–PAGE, fixation in 40% methanol and 10% acetic acid, and autoradiography. Immunoblot with anti-His tag antibody to detect EPRS linker served as control. To assay kinase activity using peptide substrates, 50 μM of synthetic peptides were phosphorylated with 1 μCi [γ-32P]ATP in kinase assay buffer. Equal volumes were spotted onto P81-phosphocellulose squares, washed in 0.5% H PO , and 32P incorporation determined by scintillation counting. U937 cell lysates were pre-cleared using protein A-sepharose, and target AGC kinase members and a non-member, MK2 were immunoprecipitated by incubation with specific antibodies for 4 h. The immunocomplex was captured by incubating with protein A-sepharose beads for 4 h, and washed three times with kinase assay buffer supplemented with 0.1% Triton X-100. The immunocomplex was resuspended in kinase assay buffer and used to phosphorylate EPRS linker as above, and 32P incorporation into peptide substrates was determined by scintillation counting41. Target peptides for S6K1, RSK1, MSK1, SGK494, NDR1, MRCKα, CRIK, RSKL1, ROCK1 and 2 (RRRLSSLRA), GRK2 (CKKLGEDQAEEISDDLLEDSLSDEDE), LATS1 (CKKRNRRLSVA), MAST1 (KKSRGDYMTMQIG), PRKX (RRRLSFAEPG), DMPK (KKSRGDYMTMQIG), and PDK1 (KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC) were from SignalChem; for MK2 (KKLNRTLSVA) from Enzo Life Sciences; for PKA (RRKASGP), SGK1/AKT (RPRAATF), PKC/PKN (HPLSRTLSVAAKK), PKG, (RKISASEFDRPLR), and Cdk5 (PKTPKKAKKL) were from Santa Cruz. All mice were housed in microisolator cages (maximum 5 per cage of same-sex littermates) and maintained in climate/temperature- and photoperiod-controlled barrier rooms (22 ± 0.5 °C, 12–12 h dark–light cycle) with unrestricted access to water and standard rodent diet (Harlan Teklad 2918) deriving 24, 18 and 58 kcal% from protein, fat and carbohydrate, respectively. Mice were fed standard rodent diet unless otherwise indicated. The number of animals used in each experiment was estimated from examination of comparable published studies that gave statistically significant results. All mouse studies were performed in compliance with procedures approved by the Cleveland Clinic Lerner Research Institute Institutional Animal Care and Use Committee. Genetically-modified EPRS phospho-deficient S999A and phospho-mimetic S999D knock-in mice were generated (Xenogen Biosciences, Taconic). The RP23-86H18 BAC clone from mouse chromosome 1 containing full-length mouse Eprs gene was used to generate 5′ and 3′ homology arms, the knock-in region for the gene targeting vector, and Southern blot probes for screening targeted events. The homology arms and the knock-in region were generated by high-fidelity PCR, and cloned into the pCR4.0 vector. The S999A and S999D mutations (TCA to GCA or GAT, respectively) in exon 20 were introduced by PCR-based site-directed mutagenesis. The final vector also contained Frt sequences flanking the Neo expression cassette for positive embryonic stem cell selection, and a DTA expression cassette for negative selection. The targeting vector was electroporated into C57BL/6 embryonic stem cells and screened with G418. Positive expanded clones with confirmed mutation were selected. Neo was deleted by Flp electroporation, and blastocysts injected. Male chimaeras were bred with C57BL/6 wild-type females, and resulting F1 heterozygotes interbred to generate homozygotes in C57BL/6 background. Genotyping was done using forward primer 5′-CAGCATAAGAACAGTTGCCAAATAAAGG-3′ and reverse primer 5′-TTCTTGAACACACACATGCACAGACTC-3′. For all experiments the wild-type (EprsS/S), EprsA/A and EprsD/D were generated exclusively by breeding heterozygotes (EprsS/A and EprsS/D), and most experiments shown use male mice unless otherwise indicated. Mice were not randomized and studies were performed unblinded with respect to mouse genotype. S6K1−/− mice in C57BL/6 background were generated at the National Jewish Medical and Research Centre (Denver, Colorado) by blastocyst injection of embryonic stem cells with targeted disruption of the S6K1 gene as described previously19, 42. Briefly, neomycin (Neo) selection cassette was inserted to disrupt the exon corresponding to amino acids 207–237 in the catalytic domain of S6K1, thereby frame-shifting the downstream coding region. S6K1−/− mice exhibited phenotypes consistent with the previously reported mice that were generated by similar approach that is, replacing the catalytic domains of S6K1 with a Neo selection cassette9, 20. EprsD/DS6K1−/− and EprsS/SS6K1−/− were generated by EprsS/DS6K1−/− × EprsS/DS6K1−/− crosses. Mice wild-type for both Eprs and S6K1 genes (EprsS/SS6K1+/+) were generated from crosses of S6K1+/− heterozygotes. Male and female mice of EprsS/S and EprsA/A genotypes were recruited (n = 212 total mice) exclusively from crosses of heterozygotes (EprsS/A). All mice were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance as above. Weaned mice (>21 days), born between June 2010 and December 2012 from 40 heterozygous parents, were monitored daily and weighed biweekly for the entire duration of their life. Mice that spontaneously developed conditions common in the C57BL/6 strain, such as malocclusion and hydrocephalus, were sacrificed and excluded from the study43. Assessments of deterioration in general health and quality of individual life were made in consultation with veterinary services of the Biological Resources Unit (BRU) of the Cleveland Clinic Lerner Research Institute. Severely sick and moribund mice that were judged to not survive another 48 h were euthanized with this date considered date of death, and included in the longevity analysis. Mice euthanized owing to imminent death include 11.5% (6 out of 52) male and 11.1% (6 out of 54) female of EprsS/S genotype, and 7.7% (4 out of 52) male and 9.3% (5 out of 54) female of EprsA/A genotype. Longevity was analysed by Kaplan–Meier survival curves from 212 mice (52 male and 54 female of each genotype, EprsS/S and EprsA/A) using known birth and death dates. Statistical differences were evaluated by log-rank Mantel–Cox and Gehan–Breslow–Wilcoxon tests using GraphPad Prism 5. Male and female mice of EprsS/S and EprsD/D genotypes were recruited (n = 89 total mice) exclusively from crosses of heterozygotes (EprsS/D). All weaned mice (>21 days born between February, 2011 and September, 2014 from 23 EprsS/D parents) were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance and health monitoring as above. Mice killed owing to imminent death (as described above) include 8.7% (2 out of 23) male and 9.5% (2 out of 21) female of EprsS/S genotype, and 8.3% (2 out of 24) male and 4.8% (1 out of 21) female of EprsA/A genotype. Longevity was analysed by Kaplan–Meier survival curves from 89 mice (23, 21 male and 24, 21 male of genotype, EprsS/S and EprsA/A, respectively) using known birth and death dates and statistical analysis, as above. Male and female mice of S6K1+/+ and S6K1−/− genotypes were recruited (n = 112 total mice) exclusively from crosses of heterozygotes (S6K1+/−). All weaned mice (>21 days born between February 2011 and December 2013 from 23 S6K1+/− parents) were housed in microisolator cages (maximum 5 per cage of same-sex littermates) with routine cage maintenance and health monitoring as above. Mice killed owing to imminent death (as described above) include 13.8% (4 out of 29) male and 10.3% (3 out of 29) female of S6K1+/+ genotype, and 14.3% (4 out of 28) male and 14.3% (3 out of 21) female of S6K1−/− genotype. Longevity estimation was analysed by Kaplan–Meier survival curves from 112 mice (29, 29 male and 28, 26 female of genotype, S6K1+/+ and S6K1−/−, respectively) using known birth and death dates and statistical analysis as above. Univariate and multivariate CPH regression models were performed to analyse the effects of 4 variables; genotype, date of birth (DOB), gender, and parental identity (PID), on longevity of mice recruited for the study. The independent variables were fitted as categorical variables in the model. Genotype and gender were coded as binary variables. DOB and PID were coded as multiple categories. For CPH regression analysis of EprsS/S and EprsA/A mice (n = 212), the data were coded as follows: genotype, EprsS/S (1) and EprsA/A (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 79 (0–78, 0 being the DOB for oldest mice in the study) and 40 (1–40) categories, respectively. Oldest DOB category represents the reference for DOB. PID-1 was considered reference for PID variable. Models were fit using Cox proportional hazards regression in R package ‘survival’ using coxph function. Univariate model was built fitting each of the four variables individually and multivariate model was built fitting all four variables simultaneously. For CPH regression analysis of EprsS/S and EprsD/D mice (n = 89), the data were coded as follows: genotype, EprsS/S (1) and EprsD/D (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 38 (0–37, 0 being the DOB for oldest mice in the study) and 23 (1–23) categories, respectively. For CPH regression analysis of S6K1+/+ and S6K1−/− mice (n = 112), the data were coded as follows: genotype, S6K1+/+ (1) and S6K1−/− (0); gender, male (0) and female (1). On the basis of unique occurrences, DOB and PID were categorized into 36 (0–35, 0 being the DOB for oldest mice in the study) and 23 (1–23) categories, respectively. Scanning electron microscopy was performed by the Cleveland Clinic Imaging Core. WAT from 20-week-old male mice was fixed using 2.5% glutaraldehyde and 4% paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C. Tissues were washed three times in PBS followed by post-fixation with 1% osmium tetroxide in PBS for 1 h at 4 °C. Finally, the tissues were dehydrated through graded alcohol (50, 70, 90, and 100%), twice in ethanol:hexamethyldisilizane (HMDS; 1:1), and three times in 100% HMDS for 10 min each, and dried at room temperature. Samples were mounted on aluminium stubs and coated with palladium-gold using a sputter-coater, and viewed at X500 magnification with a Jeol JSM 5310 Electron Microscope (EOL). Adipose tissues from 20-week-old male mice were fixed in formalin, dehydrated in ethanol, embedded in paraffin, and cut at 5-μm thickness. Sections were deparaffinized, rehydrated, and stained with haematoxylin and eosin by the Cleveland Clinic Histology Core. Stained tissues were visualized with Leica DM2500 microscope, captured with Micropublisher 5.0 RTV digital camera (QImaging) using a 5X objective lens for magnification, and QCapture Pro 6.0 (QImaging) software for image acquisition. Adipocytes from 100 mg EWAT of 20-week male mice were isolated as described above and suspended in DMEM. Cells were counted in a haemocytometer. Basal lipolysis in primary adipocytes from EprsS/S, EprsA/A, and EprsD/D EWAT was measured by glycerol release using adipolysis assay kit (Cayman). Fatty acid oxidation in EWAT of 20-week-old male mice was performed as described13, 44. Explants were placed in an Erlenmeyer flask (Kimble-chase Kontes) containing the reaction mixture (DMEM with 0.1 μCi of [14C]oleic acid, 100 mM l-carnitine, and 0.2% fat-free BSA), and conditioned for 5 min in a 37 °C CO incubator. The flask was sealed with a rubber stopper containing a centre-well (Kimble-chase Kontes) fitted with a loosely folded filter paper moistened with 0.2 ml of 1 N NaOH, and incubated for 5 h at 37 °C. 14CO in the filter paper was trapped by addition of 200 μl of perchloric acid to the reaction mixture followed by incubation at 55 °C for 1 h. Radioactivity in the filter paper was determined by scintillation counting. At 16 weeks, mice were individually housed and given standard rodent diet and water ad libitum. Cumulative food intake was measured by weighing the mouse and food every second day for 30 consecutive days. Intraperitoneal glucose tolerance test (GTT) and insulin tolerance test (ITT) in EprsS/S, EprsA/A, and EprsD/D mice were determined as described22, 39. Briefly, GTT was done after an overnight (12 h) fast followed by peritoneal injection of glucose (2 mg g−1 body weight, Sigma). ITT was performed in 6-h fasted mice by injection of 0.75 U kg−1 body weight of insulin (Sigma). Blood glucose was determined using a commercial glucometer (Contour, Bayer). Serum triglycerides, free fatty acids, glucose, and insulin in 12-h fasted and in 1-h post-prandial (fed) mice were determined using commercially available kits. Serum triglycerides, free fatty acids, and glucose kits were from Wako. Insulin was determined using enzyme-linked immunoassay-based, ultra-sensitive mouse insulin kit (Crystal). Determination of serum β-hydroxybutyrate (for ketone body analysis) from 6-h fasted mice was done using colorimetric assay kit from Cayman. White blood cell counts in blood freshly collected by cardiac puncture in the presence of 10 mM EDTA were determined using Advia hematology system. Lipid content in mouse faeces was determined after extraction with chloroform:methanol (2:1)45, 46. GAIT system activity in insulin-treated adipocytes was determined by in vitro translation of capped poly(A)-tailed Luc-Cp GAIT and T7 gene 10 reporter RNAs as described35, 47. Gel-purified RNAs were incubated with lysates from U937 monocytes and differentiated 3T3-L1 adipocytes in the presence of rabbit reticulocyte lysate and [35S]methionine. Translation of the two transcripts was determined following resolution on 10% SDS–PAGE and autoradiography. Cytokine levels in mouse serum (100 μg protein) were determined using mouse cytokine antibody array C3 kit (RayBiotech). Mouse liver triglyceride content was determined by measurement of glycerol following saponification in ethanolic KOH (2:1, ethanol: 30% KOH)48. For assessment of total neutral lipid, freshly isolated liver slices were frozen in OCT, 5-μm sections stained with Oil Red O, and analysed by densitometry using NIH image J as described49. Mouse energy metabolism was determined by indirect calorimetry using the Oxymax CLAMS system (Columbus Instruments) in the Rodent Behavioural Core of the Cleveland Clinic Lerner Research Institute. Mice were housed individually in CLAMS cages and allowed to acclimate for 48 h with unrestricted excess to food and water. Thereafter, O consumption (VO ), CO release, RER and heat generation were recorded for 24 h spanning a single light–dark cycle. Adipocytes from 500 mg WAT from wild-type and EprsA/A mice were labelled with 150 μCi of 32P-orthophosphate (MP Biomedicals) in phosphate-free DMEM medium in absence or presence of insulin (100 nM) for 4 h. EPRS was immunoprecipitated with antibodies cross-linked to protein A-sepharose beads (Sigma) in 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Triton X-100, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease/phosphatase inhibitor cocktail. Immunoprecipitated beads were washed with 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and 0.1% Triton X-100, and then in 50 mM Tris-HCl (pH 7.6) and 150 mM NaCl. 32P incorporation in immunoprecipitated proteins was determined by Tris-glycine SDS–PAGE, fixation and autoradiography. Adipocytes (0.25 × 106 cells) were pre-incubated in serum-free DMEM for 4 h. Subsequently, the medium was supplemented with 2.5 μCi [14C]Glu or [14C]Pro (Perkin-Elmer), and cells incubated for additional 6 h. Adipocytes were lysed and 14C incorporation determined by trichloroacetic acid-precipitation and scintillation counting. Mouse adipocytes (0.25 × 106 cells) were pre-incubated in methionine-free RPMI medium (Invitrogen) with 10% FBS for 30 min. [35S]Met/Cys (250 μCi, Perkin-Elmer) was added and incubated at 37 °C with 5% CO for 15 min. Labelled cells were lysed in RIPA buffer (Thermo Fisher) and analysed by Tris-glycine SDS–PAGE, fixation and autoradiography. Cell lysates or immunoprecipitates were denatured in Laemmli sample buffer (Bio-Rad) and resolved on Tris-glycine SDS–PAGE (10, 12, or 15% polyacrylamide) prepared using 37.5:1 acrylamide:bis-acrylamide stock solution (National Diagnostics). After transfer to polyvinyl difluoride membrane, the membranes were probed with target-specific antibody, followed by incubation with horseradish peroxidase conjugated secondary antibody and detection with Amersham ECL prime western blotting detection reagent (GE Healthcare). Immunoblots shown are typical of experiments independently done at least three times. Pre-cleared cell lysates (1 mg) were incubated with antibody cross-linked to protein A-sepharose beads in detergent-free buffer containing 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, and EDTA-free protease/phosphatase inhibitor cocktail. Immunoprecipitates were analysed by Tris-glycine SDS–PAGE and immunoblotting either after washing the beads three times in the same buffer or after elution, followed by neutralization with 0.2 M glycine-HCl (pH 2.6) or 50 mM Tris-HCl (pH 8.5), respectively. Fatty acid uptake assay kit (QBT, Molecular Devices) that utilizes fluorescent bodipy-C , a LCFA analogue, was used to determine fatty acid uptake50. Differentiated 3T3-L1 adipocytes were plated at 5 × 104 cells per well in a 96-well plate. Adipocytes were first incubated in serum-free Hank’s balanced salt (HBS) solution for 4 h, and then with 100 nM insulin and bodipy-C for an additional 4 h. After 30 min, relative fluorescence was read at 485 nm excitation and 515 nm emission wavelength in bottom-read mode (SpectraMax GeminiEM, Molecular Devices). LCFA uptake was also determined in differentiated 3T3-L1 adipocytes as cellular accumulation of [14C]oleate (Perkin-Elmer). Adipocytes (10,000 cells) were seeded in a 24-well plate in DMEM with 10% calf serum overnight. Cells were serum-deprived for 4 h, treated with 100 nM insulin for 3.5 h, and then with 50 μM of [14C]oleate in HBS containing 0.1% fatty acid-free BSA for 30 min51, 52. Cells were washed extensively in cold HBS with 0.1% fatty acid-free BSA to remove unincorporated [14C]oleate, lysed in RIPA buffer (Thermo Fisher), and centrifuged at 2000 rpm for 5 min. Supernatant radioactivity was determined by scintillation counting and normalized to protein. LCFA uptake by mouse WAT, hepatocytes, cardiac cells, BMDM, and soleus muscle strips were measured using essentially the same method13. Adipocytes from wild-type and mutant mice were pre-incubated for 4 h in serum- and glucose-free DMEM and then rinsed with Krebs-Ringer buffer containing 20 mM HEPES (pH 7.4), 5 mM sodium phosphate, 1 mM MgSO , 1 mM CaCl , 136 mM NaCl, and 4.7 mM KCl53, 54. Adipocytes were incubated for 4 h in the presence of 1 μCi of [14C]2-deoxy-d-glucose (DG; Perkin-Elmer) and 100 nM insulin in the same buffer supplemented with 100 mM unlabelled 2-DG (Sigma). Uptake was stopped using ice-cold PBS containing 50 μM cytochalasin, followed by four washes with PBS. Lysate radioactivity was determined by scintillation counting. Membrane fraction from differentiated 3T3-L1 adipocytes was isolated by phase partitioning using Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo-Scientific). Plasma membrane fractions from 3T3-L1 adipocytes were prepared as described14. Differentiated 3T3-L1 adipocytes were washed in buffer containing 250 mM sucrose, 10 mM Tris (pH 7.4), and 0.5 mM EDTA. Lysates were prepared by homogenization in the same buffer supplemented with protease and phosphatase inhibitor cocktail, and centrifuged at 16,000g for 20 min at 4 °C. The re-suspended pellet was layered onto a solution containing 1.12 M sucrose, 10 mM Tris (pH 7.4), and 0.5 mM EDTA, and centrifuged at 150,000g for 20 min at 4 °C. The resulting pellet was suspended in RIPA buffer (Sigma) and plasma membrane was obtained by centrifugation at 74,000g for 20 min at 4 °C. All data generated are included in the published article and in the supplementary information files. Additional statistical data sets generated are available from the corresponding author upon request.
News Article | February 24, 2017
“A case of mistaken identity” may drive the plot of the latest spy film or crime novel, but it’s only a tale of trouble for geneticists, oncologists, drug manufacturers and others working with mouse cell lines, one of the most commonly used laboratory model systems for genetic research. Cell lines that have been contaminated or misidentified due to poor laboratory technique and human error lead to inaccurate research studies, retracted publications and wasted resources. In fact, many scientific funding organizations, such as the National Institutes of Health, now require scientists to verify their cell lines for identity and quality before research grants are awarded. To help address this challenge, the National Institute of Standards and Technology (NIST) is working with partners to design tools, establish datasets, and further develop and standardize NIST’s system to authenticate mouse cell lines. One of the first milestones in this effort is the recently granted U.S. patent (No. 9,556,482(link is external)) for an authentication method using NIST-identified short tandem repeat (STR) markers─tiny repeating segments of DNA found between genes─for mouse cell lines. The method can be used to verify that a cell line is derived from a particular mouse in the same way forensic experts can confirm the identify of a person using DNA evidence. Once upcoming interlaboratory tests of the STR markers are completed, this will become the world’s first validated method for the authentication of mouse cell lines. The new NIST authentication method uses STR markers that are non-coding (that is, do not provide instructions for protein production the way genes do) DNA segments with a specific sequence of nucleotide bases─the four key components of DNA known as adenine, cytosine, guanine and thymine. Each STR is considered a separate marker for genetic matching because the number of times it is repeated is unique to an individual within a species. For example, a cell line may have one STR sequence─such as G-A-T-A─that repeats five times, another─say G-T-A-T─six times, a third seven times and so on. If another cell line has a high percentage of the same STR sequences in the same numbers, it is considered likely that they share a common ancestry. Misidentified human cell lines and the disruption they cause have been documented for decades. For example, two tainted cell lines were responsible for invalidating approximately $700 million of research studies and some 7,000 publications between the late-1950s and mid-1960s(link is external). The International Cell Line Authentication Committee(link is external), a volunteer group that monitors and raises awareness of authentication issues, currently lists nearly 500 misidentified human cell lines in its database. Fortunately, there are now standards and public databases that labs can use to confirm the identity of their human cell lines. In contrast, the extent of misidentification in mouse cell lines is unknown and there are currently no guidelines for authenticating them, said NIST microbiologist Jamie Almeida. “That is why NIST has been working on STR markers that are unique, easy to interpret and capable of distinguishing between different mouse cell lines,” Almeida said. “Additionally, we have partnered with the ATCC(link is external) [formerly the American Type Culture Collection], a global leader in biological materials management and standards, to further develop the STR technology for authentication and establish the Mouse Cell Line Authentication Consortium. The consortium is made up of organizations that have agreed to work with NIST and ATCC to test and validate the patented authentication method using the NIST-identified STR markers.” The consortium also will create a consensus standard to unify how authentication is performed across laboratories and will establish a public database that defines which STR profiles identify which mouse cell lines. “In the future, we hope to see the NIST-identified mouse STR markers and authentication method incorporated into a commercially available assay kit,” Almeida said. “The proposed kit, combined with a consensus standard and a cell line database, would provide researchers worldwide with the tools needed to ensure the identity of their mouse cell lines.” The new mouse cell line authentication method using the NIST-identified STR markers is available for licensing for research and non-exclusive commercial purposes through the agency’s Technology Partnerships Office.
News Article | February 23, 2017
A fluorescent microscope image of NIH 3T3 fibroblast cells, a commonly used mouse cell line. Microtubules within the cell appear green while the nuclei show as red. NIST and partners are developing tools, datasets and a standardized authentication method to ensure the identity of mouse cell lines used in research. Credit: Jan Schmoranzer, Leibniz-Institut für Molekulare Pharmakologie "A case of mistaken identity" may drive the plot of the latest spy film or crime novel, but it's only a tale of trouble for geneticists, oncologists, drug manufacturers and others working with mouse cell lines, one of the most commonly used laboratory model systems for genetic research. Cell lines that have been contaminated or misidentified due to poor laboratory technique and human error lead to inaccurate research studies, retracted publications and wasted resources. In fact, many scientific funding organizations, such as the National Institutes of Health, now require scientists to verify their cell lines for identity and quality before research grants are awarded. To help address this challenge, the National Institute of Standards and Technology (NIST) is working with partners to design tools, establish datasets, and further develop and standardize NIST's system to authenticate mouse cell lines. One of the first milestones in this effort is the recently granted U.S. patent (No. 9,556,482) for an authentication method using NIST-identified short tandem repeat (STR) markers—tiny repeating segments of DNA found between genes—for mouse cell lines. The method can be used to verify that a cell line is derived from a particular mouse in the same way forensic experts can confirm the identify of a person using DNA evidence. Once upcoming interlaboratory tests of the STR markers are completed, this will become the world's first validated method for the authentication of mouse cell lines. The new NIST authentication method uses STR markers that are non-coding (that is, do not provide instructions for protein production the way genes do) DNA segments with a specific sequence of nucleotide bases—the four key components of DNA known as adenine, cytosine, guanine and thymine. Each STR is considered a separate marker for genetic matching because the number of times it is repeated is unique to an individual within a species. For example, a cell line may have one STR sequence—such as G-A-T-A—that repeats five times, another—say G-T-A-T—six times, a third seven times and so on. If another cell line has a high percentage of the same STR sequences in the same numbers, it is considered likely that they share a common ancestry. Misidentified human cell lines and the disruption they cause have been documented for decades. For example, two tainted cell lines were responsible for invalidating approximately $700 million of research studies and some 7,000 publications between the late-1950s and mid-1960s . The International Cell Line Authentication Committee, a volunteer group that monitors and raises awareness of authentication issues, currently lists nearly 500 misidentified human cell lines in its database. Fortunately, there are now standards and public databases that labs can use to confirm the identity of their human cell lines. In contrast, the extent of misidentification in mouse cell lines is unknown and there are currently no guidelines for authenticating them, said NIST microbiologist Jamie Almeida. "That is why NIST has been working on STR markers that are unique, easy to interpret and capable of distinguishing between different mouse cell lines," Almeida said. "Additionally, we have partnered with the ATCC [formerly the American Type Culture Collection], a global leader in biological materials management and standards, to further develop the STR technology for authentication and establish the Mouse Cell Line Authentication Consortium. The consortium is made up of organizations that have agreed to work with NIST and ATCC to test and validate the patented authentication method using the NIST-identified STR markers." The consortium also will create a consensus standard to unify how authentication is performed across laboratories and will establish a public database that defines which STR profiles identify which mouse cell lines. "In the future, we hope to see the NIST-identified mouse STR markers and authentication method incorporated into a commercially available assay kit," Almeida said. "The proposed kit, combined with a consensus standard and a cell line database, would provide researchers worldwide with the tools needed to ensure the identity of their mouse cell lines." Explore further: A call for consensus standards to ensure the quality of cell lines
News Article | February 15, 2017
MADISON, Wis.--(BUSINESS WIRE)--Soon scientists will be able to perform efficient DNA analysis right at their bench. Promega Corporation has announced the development of a benchtop capillary electrophoresis (CE) instrument in collaboration with Hitachi High-Technologies Corporation. The Spectrum Compact CE System meets small batch and single sample needs in DNA analysis and performs both sequencing and fragment analysis at a moment’s notice. The Spectrum Compact CE System allows laboratories of all sizes the freedom to carry out single nucleotide polymorphism, PCR sizing and microsatellite analysis, de novo sequencing, NGS validation, and mutation detection. This benchtop CE instrument runs up to 32 samples at once and features 4-capillary, 6-dye detection along with an integrated touch-screen for instrument operation. Plug-and-play prefilled reagent cartridges with a guided software user interface brings capillary electrophoresis capabilities to the hands of any scientist in the laboratory regardless of skill level or expertise. “This opens new possibilities to scientists seeking high quality DNA detection on a smaller scale,” says Doug Storts, Head of Research - Nucleic Acid Technologies at Promega. “This system supports the unique workflows of the lab versus the lab needing to batch samples as a work around and cost savings measure when using a high throughput instrument or service. When we ask for early input from labs worldwide, the consistent response we get from scientists is ‘thank you’.” The first prototype will be on display at the Advances in Genome Biology and Technology Meeting February 13-16, 2017. It will also be featured at the American Academy of Forensic Sciences Annual Meeting February 13-18, 2017. It is anticipated to be commercially available in the second half of 2017. The collaboration between Promega and Hitachi High-Technologies leverages the two companies’ respective strengths as the industry’s leading manufacturer of reagents and STR kits and the world’s chief developer of capillary electrophoresis, allowing them to offer a high-performing, high-value, compact capillary electrophoresis instrument. Promega will also release a new Sanger sequencing kit designed for the Spectrum Compact CE System that features increased base-calling accuracy and applies Promega expertise in enzymology, dye manufacturing and master mixes. The Spectrum Compact CE System joins the Promega Spectrum CE System to support laboratories worldwide and is for research use only. To learn more about the Spectrum Compact CE System, visit www.promega.com/NewSpectrumCompact. Hitachi High-Technologies Corporation, headquartered in Tokyo, Japan, is engaged in activities in a broad range of fields, including Science & Medical Systems, Electronic Device Systems, Industrial Systems, and Advanced Industrial Products. The company's consolidated sales for FY2015 were approx. JPY629 billion [USD6 billion]. For further information, visit http://www.hitachi-hitec.com/global/. Promega Corporation has provided products for DNA-based human identification for more than 25 years. Promega is a leader in providing innovative solutions and technical support to the life sciences industry. The company’s 3,500 products enable scientists worldwide to advance their knowledge in genomics, proteomics, cellular analysis, molecular diagnostics and human identification. Founded in 1978, the company is headquartered in Madison, WI, USA with branches in 16 countries and over 50 global distributors. For more information about Promega, visit www.promega.com.
News Article | February 24, 2017
According to images published by the Asia Maritime Transparency Initiative, structures to house surface-to-air missiles are being installed on Fiery Cross Reef, Mischief Reef and Subi Reef in the Spratly Islands (AFP Photo/STR) Recent satellite imagery appears to show China is completing structures intended to house surface-to-air missiles (SAMs) on a series of artificial islands in the South China Sea, a Washington think-tank said Thursday. According to images published by the Asia Maritime Transparency Initiative, the structures are being installed on Fiery Cross Reef, Mischief Reef and Subi Reef in the Spratly Islands. The AMTI, which is part of the Center for Strategic and International Studies, said China appears to have begun construction on the buildings between late September and early November 2016. "This indicates they are not reactions to the political cycle in Washington, but rather part of a steady pattern of Chinese militarization," the group wrote. China has already installed HQ-9 SAMs on Woody Island, but these are only covered by camouflage netting, AMTI said. The new structures would provide the SAMs with better protection from seawater and the elements. Beijing has created seven islets in the Spratly Islands in recent years, built up from smaller land protuberances and reefs. Although Beijing insists it does not wish to militarize the contested waters of the South China Sea, ongoing satellite imagery has shown the installation of military equipment and longer runways. In December, AMTI released images showing a series of hexagonal structures in place on each of the seven islets -- apparently housing large anti-aircraft guns and close-in weapons systems.