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News Article | February 22, 2017
Site: www.nature.com

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
Site: www.businesswire.com

WALTHAM, Mass.--(BUSINESS WIRE)--Thermo Fisher Scientific Inc. (NYSE: TMO), the world leader in serving science, today announced it has elected Dion Weisler to its board of directors, effective today. Mr. Weisler’s appointment brings the total number of Thermo Fisher board members to 12. Mr. Weisler has been president and chief executive officer of HP Inc. since November 2015, following the separation of Hewlett-Packard into two independent companies. He also serves on the HP board of directors. Prior to that, Mr. Weisler was executive vice president of Hewlett-Packard’s Printing and Personal Systems Business for four years. During his more than 25 years of experience in the information technology industry, Mr. Weisler has led businesses in at least eight international markets. Before joining HP, he was with Lenovo as vice president and chief operating officer of its Product and Mobile Internet Digital Home groups, and led the company’s Global Transaction Model worldwide. Previously, Mr. Weisler served as general manager of Lenovo’s operations in Korea, Southeast Asia, Australia and New Zealand. Earlier in his career, he was general manager at Telstra Corporation, Australia’s leading telecommunications company, and also had an 11-year career at Acer Inc., where he became managing director of the company’s operations in the United Kingdom after establishing and leading the company’s businesses in Central and Eastern Europe. He holds a Bachelor’s Degree in Applied Science - Computing from Monash University in Australia. “We are delighted to welcome Dion to our board,” said Jim Manzi, chairman of the board of Thermo Fisher Scientific. “He brings many years of experience in international business and digital technology strategies. Given his background and global perspective, Dion will add valuable insights as we focus more and more on leveraging new digital capabilities to create a differentiated experience for our customers.” Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science, with revenues of $18 billion and more than 55,000 employees globally. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit www.thermofisher.com.


News Article | February 15, 2017
Site: www.nature.com

No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Cell lines used in this study were obtained from American Type Culture Collection (ATCC) and cultured under standard conditions. HCT116 cells were authenticated by karyotyping. All cell lines were confirmed to be free of mycoplasma contamination. EGFP was PCR amplified from EGFP-hAGO2 (Addgene catalogue number 21981) and cloned into pMSCV-Puro (Clontech) using the BglII and XhoI restriction sites. The puromycin resistance cassette was then removed by EcoRI and ClaI digestion and replaced with an insert containing eight imperfect miR-19 binding sites (modelled from ref. 39), synthesized as a gBlock (IDT) (sequence in Supplementary Table 8). For the EGFP-only reporter, the puromycin resistance cassette was removed by EcoRI and ClaI digestion followed by re-ligation after filling-in overhangs. Reporters for miR-16 and miR-200c were generated by replacing the puromycin cassette in the pMSCV-Puro vector containing EGFP by digesting with EcoRI and ClaI and ligating in oligonucleotides containing single miRNA binding sites (sequences in Supplementary Table 8). Multiple cloning cycles were performed using MfeI and ClaI to generate the final reporters containing eight total binding sites. MSCV-EGFP, MSCV-EGFP-miR-19, MSCV-EGFP-miR-16, and MSCV-EGFP-miR-200 retrovirus was generated by first seeding 6 × 105 cells per well in a six-well dish. The following day, cells were transfected using 1 μg of plasmid (MSCV-EGFP or MSCV-EGFP-miR-19), 3 μl of FuGENE HD (Promega), and 200 μl Opti-MEM (Thermo Fisher) per well according to the manufacturer’s instructions. Media were changed the next day. Two days after transfection, media were collected and passed through a 0.45 μm SFCA sterile filter. Recipient HCT116 cells were transduced overnight at a multiplicity of infection (MOI) of approximately 0.2 using media supplemented with 8 μg/ml polybrene (EMD Millipore). Cells expressing EGFP were enriched by FACS and single-cell clonal lines were derived. Heterogeneous knockout cell populations were generated using lentiCRISPR v2 (Addgene catalogue number 52961) or lentiCRISPR-hygro. lentiCRISPR-hygro was constructed by replacing the puromycin resistance open reading frame (ORF) in lentiCRISPR v2 with a hygromycin resistance ORF. A silent mutation was introduced into a BsmBI restriction site within the hygromycin resistance ORF to prevent fragmentation of the vector when cloning sgRNA oligonucleotides. sgRNA sequences (Supplementary Table 8) were cloned as described previously12. An sgRNA targeting an irrelevant gene (PPID) or a non-targeting guide were used as negative controls. To generate active lentivirus, 6 × 105 293T cells were first seeded in six-well dishes and transfected the following day using a 5:3:2 ratio of lentiCRISPR:psPAX2 (Addgene catalogue number 12260):pMD2.G (Addgene catalogue number 12259) using FuGENE HD and 1 μg of total plasmid per well. Media were changed the next day. Two days after transfection, media were collected and passed through a 0.45 μm SFCA sterile filter. Media containing the virus were diluted 1:1 with fresh media and used to transduce recipient cells overnight in a final polybrene concentration of 8 μg/ml. Media were changed 24 h later, and cells were split into fresh media containing 1 μg/ml puromycin 48 h after transduction. To generate clonal knockout lines, single-cell cloning was performed after infection with lentiCRISPR v2, lentiCRISPR-hygro, or after transient transfection of PX330 (Addgene catalogue number 42230) targeting the gene of interest. lentiCRISPR v2-derived clones were used in Figs 2d, 4d, e and 5a, e and Extended Data Figs 2, 4e, 5c, and 9a, b. A lentiCRISPR-hygro derived ANKRD52–/– clone was used in Fig. 2e. PX330-derived clones were used in Figs 2a, f and Extended Data Figs 3, 4c, 6, 7, 8c, d, 9a, b, d and 10. Three hundred thousand reporter cells were seeded per well in six-well dishes. Cells were transfected the following day with a mixture of inhibitors for miR-19a and miR-19b at 5 nM each (MiRIDIAN microRNA Hairpin Inhibitors, GE Dharmacon) using Lipofectamine RNAiMAX (Thermo Fisher). Fluorescence was assessed by flow cytometry 48 h after transfection. Lentiviral sgRNA library production. The human GeCKO v2 library was obtained from Addgene (catalogue number 1000000048) and amplified according to the provided instructions. Plasmid was purified from bacterial pellets using a Qiagen Plasmid Maxi Kit. Active lentivirus was prepared in 293T cells by first seeding 3.2 × 106 cells per 10-cm dish. GeCKO library A and library B were prepared independently using 15 dishes per library. The day after seeding, each dish was transfected using 10 μg of total plasmid (5:3:2 ratio of GeCKO library:psPAX2:pMD2.G), 30 μl of FuGENE HD, and 900 μl of Opti-MEM. Medium was exchanged the following day. Media collections at 48 and 72 h after transfection were pooled before filtering through a 0.45 μm SFCA sterile filter. Aliquots of the library were snap frozen on dry ice and ethanol before being stored at −80 °C. Library titre was determined as described12. Transduction of reporter cell lines with lentiCRISPR library. Genome-wide CRISPR–Cas9 screens using HCT116EGFP-miR-19, HCT116EGFP, or ANKRD52–/– HCT116EGFP-miR-19 cells were performed using both GeCKO v2 libraries A and B. Biological replicates were performed for all screens. For each transduction, five 12-well plates were seeded with 5 × 105 reporter cells per well. An overnight transduction was performed the following day by diluting virus to an MOI of 0.2–0.4 in 8 μg/ml polybrene. Cells were then trypsinized and pooled before being plated into fresh medium in six 15-cm dishes. Forty-eight hours later, cells were trypsinized, pooled, counted, and seeded into five 15-cm dishes with 1 μg/ml puromycin using 2.4 × 107 cells per dish. In parallel, a small aliquot of cells was used to confirm that an MOI of 0.2–0.4 was achieved. Cells were passaged for 12–14 days before sorting. At every passage, 1 × 107 cells were seeded per dish into four 15-cm dishes with medium containing puromycin. At least 2 × 107 cells were transduced with each library for each screen, corresponding to ~300× or greater coverage. Cell sorting. Two days before sorting, ten 15-cm dishes with 1.2 × 107 cells per dish were seeded for each library–reporter pair. Samples were prepared for FACS by trypsinization in 0.25% trypsin-EDTA (Thermo Fisher) for 7 min. Cells were dissociated by pipetting up and down approximately 20 times with a P1000 pipet to minimize doublets. Dissociated cells were pipetted directly into media, pelleted at 300g for 5 min, and washed once with PBS. Cells were resuspended at 1.4 × 107 cells per millilitre in PBS supplemented with 3% FBS. Cells were sorted at the University of Texas Southwestern Flow Cytometry Core Facility using a MoFlo cell sorter (Beckman Coulter). The brightest or dimmest 0.5% of cells were collected on the basis of EGFP fluorescence. Cell sorting was performed on approximately 9 × 107 cells, and typical yields ranged from 2 × 105 to 3 × 105 sorted bright/dim cells. Cells were pelleted at 300g and frozen at −80 °C for genomic DNA (gDNA) extraction. Unsorted cells were similarly collected. Genomic DNA extraction. gDNA was extracted from the unsorted cells using a Qiagen DNeasy Blood & Tissue Kit according to the manufacturer’s instructions. Extractions were performed on 4 × 107 cells using 5 × 106 cells per column to ensure enough gDNA for 300× coverage of the library. DNA was eluted by adding 125 μl of water to each column. The same eluate was added back to the column for a second elution. The DNA concentration in the final eluate was assessed using a Qubit dsDNA BR assay kit (Thermo Fisher). To facilitate maximum recovery of gDNA from the sorted cells, a previously described method40 was used with the following modifications: sorted cell pellets were resuspended in 500 μl of tissue lysis buffer, consisting of 460 μl of STE buffer (1 mM EDTA (pH 8.0), 10 mM Tris-HCl (pH 8.0), 100 mM NaCl) supplemented with 10 μl of 0.5 M EDTA, 10 μl of proteinase K (10 mg/ml in TE buffer containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA), and 20 μl of 10% SDS. Pellets were digested overnight at 55 °C while shaking at 1,000 r.p.m. on a Thermomixer (Eppendorf). The following day, 5 μl of 2 mg/ml RNase A was added to each tube and incubated at 37 °C for 1 h while shaking at 1,000 r.p.m. Extractions were performed with an equal volume of pH 7.9-buffer saturated phenol, followed by phenol:chloroform:isoamyl alcohol (25:24:1), followed by chloroform. Twenty micrograms of glycogen (Roche) and 1.5 ml of 100% ethanol were added to each tube and DNA was precipitated at −80 °C for 1 h followed by centrifugation at 18,000g for 10 min at 4 °C. Pellets were washed with 1 ml of 75% ethanol, dried, and resuspended in 21 μl of water by incubating at 37 °C for a minimum of 4 h. DNA concentration was determined with the Qubit dsDNA BR assay kit. Sequencing library preparation. Methods to prepare PCR amplicon libraries for deep sequencing were adapted from a previously published protocol12. All primer sequences are provided in Supplementary Table 8. For unsorted cells, an initial round of PCR (PCR I) was performed using 6.6 μg of gDNA per 100 μl PCR reaction. To maintain 300× coverage, 20 reactions were assembled for each sample. For sorted cells, all extracted gDNA for a given sample was distributed into two 100 μl reactions. In both cases, 18 cycles of amplification were performed using Herculase II Fusion polymerase (Agilent). All reactions for a given sample from PCR I were then pooled together and a second round of PCR (PCR II) was performed to add the necessary adapters for Illumina sequencing. Owing to variable PCR efficiency between samples, the cycle number for PCR II was adjusted so that each library was amplified in a 50 μl reaction to a common endpoint with respect to DNA quantity (approximately 50 ng of DNA library in a 50 μl PCR sample). DNA was purified for sequencing using AMPure XP beads (Agencourt) according to the manufacturer’s instructions with the following modifications: each 50 μl PCR II reaction was mixed with 25 μl of beads and incubated for 5 min. Magnetic separation was used to collect the supernatant. The supernatant was mixed with 90 μl of beads and incubated for 5 min. The supernatant was collected and discarded. Beads were washed twice with 200 μl of 70% ethanol and then dried for approximately 12 min. Bound DNA was eluted from the beads using 40 μl of water. Next-generation sequencing. Before sequencing, all DNA libraries were analysed using a Bioanalyzer High Sensitivity DNA Analysis Kit (Agilent). Library concentration was then determined by qPCR using a KAPA Library Quantification Kit for Illumina platforms. All samples were sequenced on an Illumina HiSeq 2500 or a NextSeq 500 with 75 bp single reads. Approximately 15 million to 20 million reads were sequenced per library. Sequencing data analysis. A reference file for all sgRNAs in the library was acquired from Addgene, and identical sgRNAs targeting more than one protein-coding gene were removed. Demultiplexed FASTQ files were mapped to the reference file using Bowtie 2 requiring unique alignments with no mismatches. Normalized read counts were calculated as described previously12. Screen hits were identified using RIGER16 with the following parameters: log(fold-change ranking), 1 × 106 permutations, second-best rank (SBR) scoring algorithm. RNA was extracted from cells using a miRNeasy Mini Kit (Qiagen) with an on-column DNase digestion. cDNA was generated using either the SuperScript IV First-Strand Synthesis System (Thermo Fisher) or MultiScribe Reverse Transcriptase (Thermo Fisher). SYBR Green assays were performed using SYBR Green PCR Master Mix (Applied Biosystems) with custom primer pairs or qRT–PCR assays for mature miRNAs or mRNAs were performed using pre-designed assays and the TaqMan Universal Master Mix II (Applied Biosystems). Primer sequences and catalogue numbers provided in Supplementary Table 8. A custom Taqman assay was designed for pri-miR-17-92 (sequences provided in Supplementary Table 8). For all co-immunoprecipitation assays, 3.2 × 106 293T cells were seeded 1 day before transfection. Cells were transfected using FuGENE HD with 10 μg of total plasmid. Media were changed the following day. Cells were harvested 48 h after transfection. Cells were washed once, scraped in PBS, and lysed on ice for 10 min in 1 ml of lysis buffer composed of 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl , 0.5% NP-40, 1 mM DTT, and a protease inhibitor cocktail (cOmplete EDTA-free, Roche). Lysates were spun at 10,000g for 10 min. Supernatants were collected and diluted with 0.5 volumes of fresh lysis buffer. One and a half microlitres of immunoprecipitation antibody (anti-V5 (Invitrogen catalogue number 46-0705) or anti-HA (Cell Signaling catalogue number 2367S)) were added to each sample and rotated at 4 °C for 30 min. Thirty microlitres of washed Dynabeads Protein G (Thermo Fisher) were added to each sample and incubated for 6 h. RNase A (Thermo Fisher) was added to a final concentration of 20 μg/ml where indicated. Samples were washed four times in ice-cold lysis buffer. Fifty microlitres of 2× Laemmli sample buffer were added to each sample and aliquots were used for western blot analysis. Antibodies used for western blotting included anti-HA (2367S, Cell Signaling), anti-V5 (46-0705, Invitrogen), anti-AGO2 (SAB4200085, Sigma), anti-GAPDH (2118S, Cell Signaling), anti-α-tubulin (T6199-200UL, Sigma), anti-BRD4 (13440S, Cell Signaling), anti-CTNNB1 (9587S, Cell Signaling), anti-POU2F1 (8157S, Cell Signaling), anti-ANKRD52 (A302-372A, Bethyl), and anti-CSNK1A1 (sc-6477, Santa Cruz). SDS–PAGE gels (7%) were supplemented with Phos-tag AAL solution (Wako) according to the manufacturer’s recommendations. Gels were run at 100 V in an XCELL SureLOCK Mini-Cell (Invitrogen) until the dye front completely exited the gel. Gels were incubated in transfer buffer supplemented with 1 mM EDTA for 10 min. Gels were then soaked in normal transfer buffer for 10 min. Proteins were transferred to a nitrocellulose membrane and standard western blotting procedures were subsequently followed. For lambda phosphatase treatments, lysates were generated as described in the co-immunoprecipitation assays. Lysate (50 μl) was mixed with 10× MnCl buffer and 10× reaction buffer provided with the lambda protein phosphatase kit (NEB). Samples treated with enzyme received 1 μl of purified lambda protein phosphatase. Incubations were performed for 45 min at 30 °C, and samples were subjected to chloroform–methanol precipitation41 before Phos-tag electrophoresis. Endogenous AGO2 was purified from ANKRD52+/+ and ANKRD52–/– HCT116 cells. AGO2–/– cells were used as a control. Ten million cells were seeded per 15-cm dish, and eight dishes were used per cell line. AGO2 was immunoprecipitated using methods adapted from an established protocol42 with 100 μl of Dynabeads Protein G loaded with 18 μg of anti-AGO2 antibody (SAB4200085, Sigma) per purification. Immunoprecipitation eluates were resuspended in 5× Laemmli sample buffer. FH-AGO2 constructs (WT, T830A, S824A/T830A) were stably expressed using MSCV-puro in ANKRD52–/– cells. Ten million cells were seeded per 15-cm dish, and eight dishes were used per cell line. Media were changed 48 h later. Cells were scraped in PBS 72 h after plating. Lysates were generated using methods similar to the co-immunoprecipitation assays, with the exception that a phosphatase inhibitor cocktail (PhosStop, Roche) was included and lysate supernatants were diluted with one volume of lysis buffer. Proteins were immunoprecipitated using 100 μl of Dynabeads Protein G loaded with 20 μg of anti-Flag antibody (F1804, Sigma). Beads were rotated at 4 °C for 3 h. Beads were washed five times in lysis buffer. Proteins were eluted using 70 μl of 2× Laemmli sample buffer per 100 μl of beads. Purified AGO2 proteins were separated by SDS–PAGE and stained using InstantBlue (Expedeon). Gel slices containing AGO2 bands were reduced by DTT, alkylated by iodoacetic acid, and digested with trypsin (Trypsin Gold; Promega). The digestion was stopped by adding formic acid, followed by peptide extraction in acetonitrile. Extracted peptides were desalted by C18 ZipTip (Millipore). Peptide mixtures were separated by C-18 resin (100 Å, 3 μm, MICHROM Bioresources) in-house packed into a silica capillary emitter (100 μm ID, 100 mm resin length). LC gradient was generated by a Dionex Ultimate 3000 nanoLC system (Thermo Scientific), with mobile phase A: 0.1% formic acid and B: 0.1% formic acid in acetonitrile. Mobile phase gradient: 2% B at 0–15 min, 30% B at 81 min, 35% B at 85 min, 40% B at 87 min, 60% B at 95 min, 80% B at 96–107 min and 2% B at 108–120 min. Flow rate: 600 nl/min at 0–13.5 min, 250 nl/min at 13.5–120 min. Peptide eluents were sprayed online with a nano-electrospray ion source (Thermo Scientific) at spray voltage of 1.5 kV and capillary temperature of 250 °C. High-resolution MS analysis was performed on a QExactive Quadrupole-Orbitrap Hybrid mass spectrometer (Thermo Scientific), operating in data-dependent mode with dynamic exclusion of 30 s. Full-scan MS was acquired at an m/z range of 300–1650, resolution of 70,000, and automatic gain control target of 3 × 106 ions. The top 15 most intense ions were subsequently selected for higher-energy collisional dissociation fragmentation at resolution of 17,500, collision energy of 27 eV, and automatic gain control target of 1 × 105 ions. Proteome data analysis used Mascot (Matrix Science) and Proteome Discoverer (1.4, Thermo Scientific). The raw data were searched against the human proteome database (Uniprot, UP000005640) plus common contaminants. Static modification was cysteine carbamidomethylation; variable modifications were serine or threonine phosphorylation, methionine oxidation, and glutamine or asparagine deamination. Precursor mass tolerance was 20 p.p.m. and fragment mass tolerance, 0.05 Da. The maximum number of miscleavage sites allowed was 2. After peptide identification, precursor ion intensities were quantified manually in XCalibur using extracted ion chromatogram. Sequences of all primers used for cloning are provided in Supplementary Table 8. Flag–HA-AGO2 (FH-AGO2) was PCR amplified from pIRES-neo-Flag/HA AGO2 (Addgene catalogue number 10822) and subcloned into pcDNA3.1+. FH-AGO2 mutants were generated using a QuikChange II XL Site-Directed Mutagenesis Kit (Agilent) or by cloning customized gBlocks (IDT) into the parental pcDNA3.1+ vector containing FH-AGO2 (sequence of all mutants provided in Supplementary Table 8). Stable expression of wild-type or mutant FH-AGO2 was achieved in one of two ways. In one, constructs were subcloned into pMSCV-puro (Clontech). In another, stable expression of AGO2 for RNA-seq and eCLIP experiments was achieved by cloning individual mutants into a modified pLJM1-EGFP vector (Addgene catalogue number 19319) where EGFP was resected using AgeI and BsrGI before blunt-end ligation. AGO2 constructs were introduced at the EcoRI cloning site. Flag–HA-AGO1 was subcloned from pIRESneo-Flag/HA AGO1 (Addgene catalogue number 10820) into pMSCV-PIG (Addgene catalogue number 21654). V5-tagged ANKRD52 (corresponding to NP_775866.2) was constructed by PCR amplification from HCT116 cDNA followed by cloning into pcDNA3.1+. cDNA clones for human PPP6C and CSNK1A1 were obtained from the Invitrogen Ultimate ORF LITE Library (Clone ID IOH7224 and IOH59150, respectively) and subcloned into pCAGIG (Addgene catalogue number 11159) using Gateway LR Clonase (Thermo Fisher). For tethering assays, a 5× BoxB sequence adapted from a previous report32 was designed as a gBlock (IDT) and cloned in the XbaI site of pGL3-Control (Promega) (sequence in Supplementary Table 8). For the λN constructs, a gBlock containing the λN peptide sequence with an HA tag32 was subcloned into pcDNA3.1-FH-AGO2, replacing the Flag–HA tag. To generate control plasmid expressing λN-HA peptide alone, the λN-HA sequence was PCR amplified and cloned into pcDNA3.1+. Active lentivirus was generated using FH-AGO2 mutants (WT, 5XA, S828A, and empty vector) cloned into a modified pLJM1 vector with EGFP resected. A viral packaging protocol analogous to that used for the lentiCRISPR lentivirus preparations was used. Recipient ANKRD52–/– HCT116EGFP-miR-19 cells were transduced at an MOI of approximately 0.2. Transduced cells were selected in puromycin for at least 10 days, before use in flow cytometry experiments (Fig. 2f). For experiments involving endogenous AGO2, HCT116EGFP-miR-19 cells were used. For analysis of FH-AGO2 miRNA or mRNA binding, cells stably expressing the indicated wild-type or mutant FH-AGO2 protein were first generated by infecting AGO2–/– HCT116 cells with MSCV retroviruses. Then, for each immunoprecipitation sample, 6 × 106 cells were seeded per 10-cm dish. Cells were harvested 48 h later by scraping in PBS. Pelleted cells were resuspended in 1 ml of a lysis buffer consisting of 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl , 0.5% NP-40, 1 mM DTT, a protease inhibitor cocktail (cOmplete, EDTA-free, Roche), and 250 U/ml Recombinant RNasin Ribonuclease Inhibitor (Promega). Cells were lysed on ice for 10 min. Samples were spun at 10,000g for 10 min. Supernatant fractions were retained. Protein concentration was determined using a Bio-Rad DC Protein Assay Kit, and all samples were adjusted to the same concentration with lysis buffer. Dynabeads Protein G (Thermo Fisher) were prepared by pre-incubating with 1.5 μg of antibody (either anti-Flag (F1804, Sigma) or anti-AGO2 (SAB4200085, Sigma)) and pre-blocking with 0.5 mg/ml BSA, 0.5 mg/ml yeast tRNA, and 0.2 mg/ml heparin. Each sample was incubated with 25 μl of prepared Dynabeads Protein G for 3 h at 4 °C. Samples were washed three times in lysis buffer. Captured protein was eluted from the beads using either 2.5 mg/ml 3× Flag peptide (Sigma) or 3.5 mg/ml AGO2 peptide (sequence derived from ref. 42, synthesized at the University of Texas Southwestern Protein Chemistry Technology Core) dissolved in lysis buffer. Eighty per cent of the eluate was harvested for RNA extraction and 20% was diluted with 2× Laemmli sample buffer for western blot analysis. For each immunoprecipitation, qRT–PCR assays were performed to determine input and immunoprecipitation levels for mature miRNAs and mRNA targets of interest. Western blot analysis determined the relative amount of AGO2 in the immunoprecipitation eluate. RNA quantity as a percentage of input was determined for all immunoprecipitation eluates and then normalized to the relative amount of protein captured in each eluate. Experiments to capture AGO2 loaded with miRNA were adapted from a previously published method30. ANKRD52+/+ and ANKRD52–/– HCT116EGFP-miR-19 cells were seeded at 1.35 × 107 cells per dish in six 15-cm dishes per cell line. Forty-eight hours later, cells from each dish were scraped in PBS, pelleted, and lysed on ice for 10 min in 1 ml of a buffer containing 25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl , 0.5% NP-40, 1 mM DTT, a protease inhibitor cocktail (cOmplete, EDTA-free, Roche), a phosphatase inhibitor cocktail (PhosStop, Roche), and 250 U/ml Recombinant RNasin Ribonuclease Inhibitor (Promega). Lysates were spun at 10,000g for 10 min and supernatants were further diluted with one volume of lysis buffer. To assess binding of AGO2 to the target mimic, 1.8 ml of each lysate was incubated with 50 μl of washed Dynabeads MyOne Streptavidin C1 (Thermo Fisher) pre-loaded with 300 pmol of wild-type or mutant RNA oligonucleotide (Supplementary Table 8) and pre-blocked with 1 mg/ml BSA, 0.5 mg/ml yeast tRNA, and 0.2 mg/ml heparin. To assess AGO2 phosphorylation after immunoprecipitation, 1.8 ml of each lysate was incubated with 50 μl of washed Dynabeads Protein G (Thermo Fisher) pre-incubated with 5 μl of anti-AGO2 antibody (SAB4200085, Sigma42) and pre-blocked as noted previously. Lysates were incubated with beads for 3 h at room temperature. Beads were washed four times in lysis buffer before 50 μl of 2× Laemmli sample buffer was added. Phos-tag electrophoresis was performed on captured protein complexes and on input protein samples subjected to chloroform–methanol precipitation41. The 293T cells were seeded in 24-well plates using 7.5 × 104 cells per well. Cells were transfected the following day using FuGENE HD and 301 ng of total plasmid. Each transfection consisted of 1 ng of phRL-SV40 (Promega), 20 ng of pGL3-Control or pGL3-BoxB, 150 ng of pcDNA3.1+ (expressing tethered or untethered proteins), and 130 ng of empty pcDNA3.1+. Cells were harvested 24 h later for luciferase activity assays using a Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized to Renilla luciferase activity in each well to control for variation in transfection efficiency. Biological triplicates were performed for each transfection. ANKRD52–/– HCT116EGFP-miR-19 cells were seeded in six-well dishes at 6 × 105 cells per well. The following day, cells were treated with 10, 50, or 200 nM rapamycin for 72 h (fresh medium with rapamycin was exchanged at 48 h). Cells were harvested in 2× Laemmli sample buffer at the experimental endpoint. AGO2–/– cells were infected with MSCV retroviral constructs to stably express FH-AGO2WT or FH-AGO25XA. FH-AGO2-expressing cells were seeded using 1.5 × 107 cells per dish in 15-cm dishes with three dishes per cell line. Lysates were generated using methods similar to the co-immunoprecipitation assays, with the exception that 2 ml of lysis buffer was used per dish. Lysates were diluted with one volume of lysis buffer. FH-AGO2 was immunoprecipitated using 9 μg of anti-Flag antibody (F1804, Sigma) and 150 μl of washed Dynabeads. Samples were rotated at 4 °C overnight. Beads were washed three times with lysis buffer and then treated with lambda protein phosphatase (NEB) for 45 min. Beads were washed three times with lysis buffer and then resuspended in 100 μl reaction buffer composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl , 2.5 mM DTT, 0.01% Triton X-100, 0.5 mg/ml BSA, 0.5 mM EGTA, 0.5 mM Na VO , 5 mM β-glycerophosphate, 170 ng of recombinant CSNK1A1 (PV3850, Thermo Fisher), and 200 μM [γ-32P]ATP (SA = 100–500 c.p.m./pmol). Reactions were incubated at 37 °C for 2 h. Beads were separated and mixed with 50 μl of 2× Laemmli sample buffer. SDS–PAGE was performed, and gels were stained using SimplyBlue SafeStain (Invitrogen). 32P signal was detected using a phosphor screen (GE Healthcare) and Typhoon FLA 7000 (GE Healthcare). In vitro CSNK1A1 kinase assays were performed using assay conditions adapted from the manufacturer’s recommendations (Recombinant CSNK1A1, PV3850, Thermo Fisher). All reactions were performed in a 50 μl volume for 90 min at 30 °C. Assay buffer was composed of 25 mM Tris-HCl (pH 7.5), 10 mM MgCl , 2.5 mM DTT, 0.01% Triton X-100, 0.5 mg/ml BSA, 0.5 mM EGTA, 0.5 mM Na VO , 5 mM β-glycerophosphate, 1 mM peptide (Supplementary Table 8), 170 ng of recombinant CSNK1A1, and 200 μM [γ-32P]ATP (SA = 100–500 c.p.m./pmol). Reactions were terminated using 75 mM H PO and spotted onto P81 phosphocellulose squares. Samples were washed four times in 75 mM H PO for 5 min per wash and immersed in acetone for 5 min before drying. 32P incorporation was assessed by Cerenkov counting. The linear form of ciRS-7 was constructed by amplifying the endogenous ciRS-7 locus from human genomic DNA (Roche) by PCR (Phusion Polymerase, Thermo Scientific) using primer sequences described previously36 (Supplementary Table 8). The PCR fragment was then cloned into the HindIII and NotI cloning sites of pcDNA3.1+ (Invitrogen). To generate the ciRS-7 construct capable of circularization, an ~800-bp region upstream of the splice acceptor was amplified using previously described primers36 (Supplementary Table 8) and inserted in the inverse orientation downstream of the linear ciRS-7 sequence at the XhoI cloning site of pcDNA3.1+. The effect of ciRS-7 expression on AGO2 phosphorylation was assessed through co-transfection experiments. Cells were seeded at a density of 9 × 105 cells per well in six-well dishes. Cells were transfected according to the manufacturer’s recommendations using Lipofectamine 2000 (Thermo Fisher). Where indicated, each well received 2 μg of plasmid and 10 nM miRNA mimics (miRIDIAN miRNA mimics, GE Dharmacon). Cells were harvested 28 h later for western blot analysis. Parental HCT116EGFP-miR-19, AGO2–/– HCT116EGFP-miR-19, ANKRD52–/– HCT116EGFP-miR-19, and ANKRD52–/–;CSNK1A1–/– HCT116EGFP-miR-19 cells were used for RNA-seq. Three independent clonal AGO2–/–, ANKRD52–/–, and ANKRD52–/–;CSNK1A1–/– knockout cell lines and three biological triplicates of parental cells were sequenced. Five hundred thousand cells were seeded per well in six-well dishes. Cells were harvested 48 h later, and RNA was extracted using a RNeasy Mini Kit (Qiagen) with an on-column DNase digestion. Sequencing libraries were generated using a TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) and run on a NextSeq 500 using a NextSeq 500/550 High Output v2 Kit, 75 cycle (Illumina). AGO2–/– HCT116EGFP-miR-19 cells generated using PX330 were reconstituted with either empty pLJM1 vector (with EGFP previously resected), FH-AGO2-WT (AGO2WT), or FH-AGO2-5XA (AGO25XA). Biological triplicates for each cell line were seeded with 5.0 × 105 cells per well in six-well dishes. Cells were collected 48 h later, and RNA was extracted using a miRNeasy Mini Kit (Qiagen) with an on-column DNase digestion. Sequencing libraries were generated using a TruSeq Stranded Total RNA with Ribo-Zero Human/Mouse/Rat Low-throughput (LT) kit (Illumina) and run as performed in the previous RNA-seq experiment. Quality assessment of the RNA-seq data was done using the NGS-QC-Toolkit43 with default settings. Quality-filtered reads generated by the tool were then aligned to the human reference genome hg19 (for AGO2–/–, ANKRD52–/–, and ANKRD52–/–;CSNK1A1–/– RNA-seq experiments) or hg38 (for FH-AGO2 reconstitution experiments) using the TopHat2 (version 2.0.12) aligner44 using default settings. Read counts obtained from featureCounts45 were used as input for edgeR (version 3.8.6)46 for differential expression analysis. Genes with FDR ≤ 0.05 were regarded as differentially expressed for comparisons of each sample group. Cell culture, library preparation, and deep sequencing. AGO2–/– cells or AGO2–/– cells reconstituted with FH-AGO2WT or FH-AGO25XA via lentiviral expression (described above) were seeded in 15-cm dishes with five dishes per cell line at 1.0 × 107 cells per dish. Cells were cultured for 48 h and subsequently ultraviolet crosslinked at 400 mJ/cm2. Aliquots of 2.0 × 107 cells were then frozen at −80 °C. eCLIP was performed using the frozen samples as previously described37, using anti-Flag antibody for immunoprecipitations (F1804, Sigma). For each cell line, duplicate input and immunoprecipitation samples were prepared and sequenced. The RiL19 RNA adaptor (Supplementary Table 8) was used as the 3′ RNA linker for all samples. PAGE-purified DNA oligonucleotides were obtained from Sigma for the PCR library amplification step (Supplementary Table 8). PCR amplification was performed using between 11 and 15 cycles for all samples. Paired-end sequencing was performed on a NextSeq 500 using a NextSeq 500/550 High Output v2 Kit, 75 cycle (Illumina). Mapping deep sequencing reads. Adapters were trimmed from original reads using Cutadapt (version 1.9.1)47 with default settings. Next, the randomer sequence from the rand103Tr3 linker (Supplementary Table 8) was trimmed and recorded. TopHat2 (version 2.0.12)44 was used to align mate 2 to hg38. Only the uniquely mapped reads were retained. PCR duplicates were then removed using the randomer information with an in-house script. All reads remaining after PCR duplicate removal were regarded as usable reads and used for cluster calling. eCLIP cluster calling and annotation. eCLIP clusters were identified using a previously described method6 with the following modifications. Genome coverage by usable reads was determined at nucleotide resolution for each data set, and regions of continuous coverage greater than expected from a Poisson noise distribution were identified (P ≤ 0.001). For each region, read counts were obtained using Bedtools (version 2.17)48. If 50% of a read overlapped a region on the same strand, it was counted as a read covering that region. For each region, normalization to total usable reads was performed and a fold change between immunoprecipitation and input samples was calculated. Significant CLIP clusters in each data set were defined by (1) the presence of significantly greater coverage in the region than expected by chance on the basis of the Poisson distribution, and (2) log (fold change) of normalized reads in the cluster was ≥2 comparing immunoprecipitation to input. The final CLIP clusters for FH-AGO2WT and FH-AGO25XA were identified by first identifying significant clusters present in both experimental replicates. A region was considered to be present in both replicates if it occurred on the same strand and the replicate clusters overlapped by at least one-third of their total length. Significant clusters from both replicates were then merged to define the final cluster length. Lastly, all clusters identified in the AGO2–/– samples were subtracted to generate the final CLIP cluster calls (Supplementary Table 7). Clusters were annotated on the basis of their genomic locations (Ensembl GRCh38.85) if 55% of the cluster overlapped with a given genomic region. If a cluster was assigned to multiple annotations, the annotation was selected using the following priority: CDS exon > 3′ UTR > 5′ UTR > protein-coding gene intron > noncoding RNA exon > noncoding RNA intron > intergenic. Identification of active miRNA seed families and calculation of CLIP coverage at miRNA binding sites. Active miRNAs in HCT116 were identified using an approach similar to that described previously6 with the following modifications. The top 100 most highly expressed miRNAs in HCT116 cells were identified on the basis of a previously published small RNA sequencing experiment in this cell line49 and collapsed to 66 7-nucleotide seed families with identical sequence from nucleotides 2–8. Eight-nucleotide binding sites for these seeds, defined as in ref. 3, were identified in the 3′ UTRs of all expressed genes (FPKM > 0) using seqMap (version 1.0.12)50. The locations were then transformed to genomic coordinates and extended 10 nucleotides upstream and downstream to obtain a seed match region (excluding sites on exon–exon junctions). The numbers of crosslinking sites in these seed match regions for each miRNA seed family in FH-AGO2WT CLIP data were counted, normalized to the total usable reads in each replicate library, and averaged across replicates. To determine the significance cut-off, all possible 8-nucleotide sequences except for known miRNA seeds and those with four consecutive A, C, G, or T nucleotides were used to generate a null distribution. These background 8-nucleotide sequences were divided into 13 groups with 1,000 8-nucleotide sequences in the first 12 groups and 678 8-nucleotide sequences in the final group. CLIP crosslinking to each 8-nucleotide sequence in expressed 3′ UTRs was quantified as described above for actual miRNA seeds. An mRNA seed family was considered to be active in HCT116 cells if it obtained more crosslinking events than expected by chance, defined by the average number of crosslinking events from each of the 13 background 8-nucleotide groups above which P < 0.01. On the basis of this analysis, 15 active miRNA seed families were identified (representative miRNA: miR-423-5p, miR-17-5p, miR-200a-3p, miR-19a-3p, miR-23a-3p, miR-148a-3p, miR-221-3p, miR-125-5p, miR-182-5p, miR-21-5p, miR-30a-5p, miR-25-3p, let-7a-5p, miR-27a-3p, miR-24-3p). To quantify CLIP coverage of miRNA binding sites in FH-AGO2WT and FH-AGO25XA CLIP data (Fig. 6c), 8-, 7-, and 6-nucleotide binding sites, defined as in ref. 3, for all active miRNAs were identified within FH-AGO2WT CLIP clusters in 3′ UTRs using seqMap. Clusters with only a single type of binding site (8, 7, and 6 nucleotides) were identified. If an 8-nucleotide binding site was identified, this site was excluded from 7- or 6-nucleotide categories. Likewise, 7-nucleotide sites were excluded from the 6-nucleotide sites. Clusters were further filtered for those that were present in transcripts with FPKM > 0 in both FH-AGO2WT and FH-AGO25XA cell lines, yielding 228, 89, and 80 clusters containing 6, 7, or 8 nucleotides, respectively. For each cluster with a given type of binding site, CLIP coverage was calculated by determining the average number of CLIP reads in the cluster in each replicate normalized to the total number of reads in all clusters in each replicate, divided by FPKM of the transcript. The final reported CLIP coverage is the average of both replicates. To quantify CLIP coverage of miRNA binding sites in FH-AGO25XA-unique clusters versus FH-AGO2WT/FH-AGO25XA-common clusters (Extended Data Fig. 10e), 8-, 7-, and 6-nucleotide binding sites for all active miRNAs were identified within each class of CLIP cluster. Windows around each site were then extended 10 nucleotides upstream and downstream to obtain a seed match region. The numbers of crosslinking sites within these regions were counted and normalized to the total number of reads in clusters of each class (FH-AGO25XA-unique or FH-AGO2WT/FH-AGO25XA-common) to derive the CLIP coverage used to draw the CDF plots. CLIP coverage of FH-AGO25XA rescued versus non-rescued transcripts. Genes whose repression in AGO2–/– cells was rescued by FH-AGO25XA were defined by first identifying the genes that were significantly upregulated in AGO2–/– cells compared with parental HCT116 (FDR ≤ 0.05), then, among these genes, those that were significantly downregulated in FH-AGO25XA versus AGO2–/– (FDR ≤ 0.05). All other genes upregulated in AGO2–/– cells were considered not-rescued. The FH-AGO2WT CLIP coverage for each gene in these classes was calculated as the sum of all reads in CLIP clusters in a given 3′ UTR, normalized to total reads in all clusters, divided by the FPKM of the transcript. The final reported CLIP coverage (Extended Data Fig. 10d) is the average of both FH-AGO2WT replicates. mRNA half-life analysis. Half-lives of transcripts with FH-AGO25XA CLIP clusters in their 3′ UTRs were obtained from a previously published study38. Genes that had half-lives assigned to more than one RefSeq mRNA isoform were removed to avoid ambiguity. Genes in the top quartile of half-lives were defined as having a long half-life (n = 273) and genes in the bottom quartile of half-lives were defined as having a short half-life (n = 274). The total numbers of CLIP reads in clusters in a given 3′ UTR were obtained for each replicate, and edgeR (version 3.8.6)46 was used to calculate the normalized fold change of CLIP coverage comparing FH-AGO25XA with FH-AGO2WT (Extended Data Fig. 10f). All high-throughput sequencing data generated in the course of this study (CRISPR–Cas9 screens, RNA-seq, eCLIP) have been deposited in Gene Expression Omnibus under accession number GSE89946. All other data are available from the corresponding author upon reasonable request.


News Article | February 21, 2017
Site: globenewswire.com

Dublin, Feb. 21, 2017 (GLOBE NEWSWIRE) -- Research and Markets has announced the addition of Jain PharmaBiotech's new report "Therapeutic Drug Monitoring - Technologies, Markets, and Companies" to their offering. This report deals with therapeutic drug monitoring, a multi-disciplinary clinical specialty, aimed at improving patient care by monitoring drug levels in the blood to individually adjust the dose of drugs for improving outcome. TDM is viewed as a component of personalized medicine that interacts with several other disciplines including pharmacokinetics and pharmacogenetics. One chapter is devoted to monitoring of drugs of abuse (DoA). Various technologies used for well-known DoA are described. A section on drug abuse describes methods of detection of performance-enhancing drugs. TDM market is analyzed from 2015 to 2025 according to technologies as well as geographical distribution. Global market for DoA testing was also analyzed from 2016 to 2026 and divided according to the area of application. Unmet needs and strategies for development of markets for TDM are discussed. The report contains profiles of 27 companies involved in developing tests and equipment for drug monitoring along with their collaborations. The text is supplemented with 18 tables, 6 figures and 190 selected references from literature. Benefits of this report: - Up-to-date one-stop information on therapeutic drug monitoring - Description of 27 companies involved with their collaborations in this area - Market analysis 2016-2026/ - Market values in major regions - Strategies for developing markets for therapeutic drug monitoring - A selected bibliography of 190 publications - Text is supplemented by 18 tables and 6 figures Who should read this report? - Biotechnology companies developing assays and equipment for drug monitoring - Reference laboratories providing drug monitoring services - Pharmaceutical companies interested in companion tests for monitoring their drugs - Clinical pharmacologists interested in integrating therapeutic drug monitoring with pharmacogenetics for development of personalized medicine Key Topics Covered: Executive Summary 1. Introduction Definitions Historical Landmarks in the development of TDM Pharmacology relevant to TDM Pharmacokinetics Pharmacodynamics Pharmacogenetics Pharmacogenomics Pharmacoproteomics Drug receptors Protein binding Therapeutic range of a drug Variables that affect TDM Indications for TDM Multidisciplinary nature of TDM 2. Technologies for TDM Introduction Sample preparation Proteomic technologies Mass spectrometry Liquid chromatography MS Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Combining capillary electrophoresis with MS Gas-liquid chromatography Tissue imaging mass spectrometry New trends in sample preparation Pressure Cycling Technology Desorption electrospray ionization imaging High Performance Liquid Chromatography (HPLC) Ultra performance LC TDM using dry blood spots Analysis of dried blood spots for drugs using DESI Quantitative analysis of drugs in dried blood spot by paper spray MS Immunoassays Enzyme-linked immunosorbent assay Cloned Enzyme Donor Immunoassay Enzyme Multiplied Immunoassay Technique Fluorescence Polarization Immunoassay Particle Enhanced Turbidimetric Inhibition Immunoassay Radioimmunometric assays Biosensors Nanosensors Biochips & Microarrays Introduction Microchip capillary electrophoresis Phototransistor biochip biosensor Microchip-based fluorescence polarization immunoassay for TDM Cellular microarrays Microfluidics for TDM Lab-on-a-chip Micronics' microfluidic technology Rheonix CARD technology Nano-interface in a microfluidic chip Levitation of nanofluidic drops with physical forces Nanoarrays Nanobiotechology NanoDx Biomarkers Applications of biomarkers in drug safety studies Genomic technologies for toxicology biomarkers Proteomic technologies for toxicology biomarkers Metabonomic technologies for toxicology biomarkers Integration of genomic and metabonomic data to develop toxicity biomarkers Toxicology studies based on biomarkers Biomarkers of hepatotoxicity Biomarkers of nephrotoxicity Cardiotoxicity Neurotoxicity Biomarkers in clinical trials Molecular diagnostics 3. Drug Monitoring Instruments Introduction Description of important instruments AB SCIEX instruments AB SCIEX LC/MS/MS Abbott instruments ARCHITECT c16000 ARCHITECT c4000 ARCHITECT c8000 ARCHITECT ci16200 Integrated System ARCHITECT ci4100 Integrated System ARCHITECT ci8200 integrated with the ARCHITECT i2000SR ARCHITECT i1000SR ARCHITECT i4000SR AxSYM Agilent's 6400 Series Triple Quadrupole LC/MS Alfa Wassermann's ACE Alera AMS Diagnostics' LIASYS Awareness Technology's STAT FAX 4500 Beckman Coulter instruments Beckman Coulter Unicel Series AU5800 automated chemistry systems AU480 Binding Site ESP600 bioMerieux Mini Vidas Carolina BioLis 24i Chromsystems' HPLC instruments Grifols Triturus ABX Pentra 400 Medica EasyRA Nova Biomedical Critical Care Xpress Ortho Clinical Diagnostics' VITROS® family of systems Immunodiagnostic systems Randox intruments Randox RX Imola Roche instruments Cobas® 8000 COBAS INTEGRA® Systems Siemens instruments ADVIA 1200 ADVIA Centaur XP immunoassay system EMIT® II Plus Syva® Viva® Drug Testing Systems Dimension® Xpand® Plus Integrated Chemistry System Thermo Scientific instruments Indiko Tosoh AIA-Series 4. Applications of TDM Introduction Pharmaceutical research and drug development Clinical trials Computerized clinical decision support systems for TDM and dosing Medication-related interferences with measurements of catecholamines Polymorphisms of genes affecting drug metabolism TDM for drug safety TDM in special groups The aged Children Pregnancy TDM of prophylactic therapy Monitoring of vitamin D levels Monitoring of RBC folic acid levels during pregancy Personalized medicine Role of TDM in personalized medicine Applications according to various conditions Anesthesia and critical care Optimizing antimicrobial dosing for critically ill patients TDM monitoring of thiopental continuous infusion in critical care Role of TDM in critical care cardiac patients. Cancer Epilepsy Personalized approach to use of AEDs Infections Virus infections Fungal infections Pain management Role of TDM in pain management Monitoring of analgesic drugs in urine samples AEDs as analgesics Triptans for migraine Psychiatric disorders Guidelines for use of TDM in psychiatric patients TDM of psychotropic drugs Transplantation TDM of Tacrolismus in transplantation TDM of cyclosporine A in transplantation Monitoring of immunosuppression with mycophenolate mofetil Emergency toxicology Future prospects of TDM 5. Drugs Requiring Monitoring Introduction Antiepileptics Carbamazepine TDM of carbamazepine Gabapentin Lacosamide Lamotrigine TDM of lamotrigine Levetiracetam TDM of levetiracetam Phenobarbital TDM of phenobarbital Phenytoin TDM of phenytoin Primidone TDM of primidone Topiramate TDM of topiramate Valproic acid TDM of valproic acid TDM of multiple antiepileptic drugs in plasma/serum Antimicrobials Antibiotics Amikacin Anti-tuberculosis drugs Chloramphenicol Gentamicin Tobramycin Vancomycin Norvancomycin Antiviral agents Anti-HIV drugs Antifungal agents Voriconazole Antidepressants TDM of selective serotonin reuptake inhibitors Antipsychotics Aripiprazole Quetiapine TDM of risperidone TDM of AEDs in psychiatric disorders TDM of multiple drugs in psychiatry Bronchodilators Theophylline Cardiovascular drugs Antiarrhythmic drugs Anticoagulants Dabigatran Antihypertensive drugs ß-blockers Cardiotonic drugs Digoxin TDM of statins for hypercholesterolemia Chemotherapy for cancer TDM of 5-FU TDM of Methotrexate TDM of imitanib Drugs used for treatment of Alzheimer disease Donepezil Galantamine Memantine Drugs used for treatment of Parkinson disease Monitoring of levodopa and carbidopa therapy Catechol-O-methyltransferase inhibitors Drugs for treatment of attention-deficit hyperactivity disorder Atomoxetine Methylphenidate Hypnotic-sedative drugs Benzodiazepines Propofol Immunosuppressive drugs TDM of mycophenolic acid for the treatment of lupus nephritis Steroids Prednisone Miscellaneous drugs Azathioprine Sildenafil 6. Monitoring of Biological Therapies Introduction Cell therapy In vivo tracking of cells Molecular imaging for tracking cells MRI technologies for tracking cells Superparamagnetic iron oxide nanoparticles as MRI contrast agents Visualization of gene expression in vivo by MRI Gene therapy Application of molecular diagnostic methods in gene therapy Use of PCR to study biodistribution of gene therapy vector PCR for verification of the transcription of DNA In situ PCR for direct quantification of gene transfer into cells Detection of retroviruses by reverse transcriptase (RT)-PCR Confirmation of viral vector integration Monitoring of gene expression Monitoring of gene expression by green fluorescent protein Monitoring in vivo gene expression by molecular imaging Monoclonal antibodies Natalizumab 7. Monitoring of Drug Abuse Introduction Tests used for detection of drug abuse Forensic applications of detection of illicit drugs in fingerprints by MALDI MS MS for doping control Randox assays for DoA Drugs of Abuse Array V Urine drug testing TDM of drugs for treatment of substance abuse-related disorders Drug testing to monitor treatment of drug abuse Minimum requirement for drug testing in patients Analgesic abuse ?-blockers as doping agents Detection of ß-blockers in urine Chronic alcohol abuse Cocaine CEDIA for cocaine in human serum Detection of cocaine molecules by nanoparticle-labeled aptasensors Infrared spectroscopy for detection of cocaine in saliva Marijuana Use of marijuana and synthetic cannabinoids Detection of cannabinoids ELISA for detection of synthetic cannabinoids Drug abuse for performance enhancement in sports Historical aspects of drug abuse in sports Drugs used by athletes for performance enhancement Techniques used for detection of drug abuse by athletes Mass spectrometry for detection of peptide hormones miRNAs for the detection of erythropoiesis-stimulating agents Detection of anabolic steroids Body fluids and tissues used for detection of drug abuse in sports Urine drug testing Spray (sweat) drug test kits Hair drug testing Gene doping in sports Gene transfer methods used for enhancing physical performance Misuse of cell therapy in sport Challenges of detecting genetic manipulations in athletes Drug abuse testing in race horses Limitations and future prospects Role of pharmaceutical industry in anti-doping testing 8. Markets for TDM Introduction Methods for market estimation and future forecasts Markets for TDM tests Markets for TDM and DoA testing equipment Geographical distribution of markets for TDM tests Drivers for growth of TDM markets Markets for DoA testing Unmet needs in TDM Cost-benefit studies Simplifying assays and reducing time and cost Strategies for developing markets Physician education Supporting research on TDM Biomarker patents for drug monitoring 9. Companies Profiles of companies Collaborations 10. References For more information about this report visit http://www.researchandmarkets.com/research/g4tq2x/therapeutic_drug


Research and Markets has announced the addition of the "Spectroscopy Equipment and Accessories - Global Strategic Business Report" report to their offering. The report provides separate comprehensive analytics for the US, Canada, Japan, Europe, Asia-Pacific, Latin America, and Rest of World. Annual estimates and forecasts are provided for the period 2015 through 2022. Also, a six-year historic analysis is provided for these markets. Market data and analytics are derived from primary and secondary research. This report analyzes the worldwide markets for Spectroscopy Equipment and Accessories in US$ Thousand by the following Product Segments: The report profiles 117 companies including many key and niche players such as: 1. OUTLOOK A Prelude Overview Current and Future Analysis Sustained Growth for Aftermarket Products and Services 2. MARKET OVERVIEW Government Funding and Research Activities Propel Molecular Spectroscopy Market Growing Preference for Handheld Instruments Infrared Spectroscopy - A Peek into Technology and Application Trends Raman Spectroscopy - A Review of Advanced Technologies and Applications Tip-enhanced Raman Spectroscopy (TERS) Surface-enhanced Raman Spectroscopy (SERS) Spatially Offset Raman Spectroscopy (SORS) Magnetic Photoacoustoic Raman (MPR) Raman Spectroscopy Emerges as Attractive Analytical Tool for Pharmaceutical Industry Fluorescence Spectroscopy - A Gold Standard Technology Atomic Spectroscopy - An Overview Mass Spectrometry: Technological Developments and Expanding End-Use Applications to Bolster Growth Review of Select MS Technologies The Way Ahead for FT-MS and Magnetic Sector MS Technologies Portability: A Major Driving Force for MS Systems Market Nanotube Coating to Enable Miniaturization in Mass Spectrometers High Prices of MS Systems Hold Down Sales Growth Purpose-Built Mass Spectrometers to Transform Personalized Medicine Leading End Users of Mass Spectrometry Devices A Peek into Regulatory and Competitive Landscape in MS Ion-Trap Mass Spectrometry Technology Losses Sheen Reduced Government Spending on Laboratory Testing Lack of Suitable Software and Diversity of MS Systems - A Major Challenge Smaller Clinical Laboratories Continue to Shy Away from Mass Spectrometers 3. MARKET TRENDS & ISSUES Rise in Drug Development Outsourcing to Fuel Global Spectroscopy Market Growing Significance of Miniaturization in Spectroscopy Shift of Analytical Instruments Industry to Mass Customization Improved Mobility Broadens the Role of Spectroscopy Improved Analyzer Reliability and Performance Hyphenated Technologies Exhibit Growth Spectroscopy Makes Inroads into Novel Applications Applications Extend to Defense and Civilian Areas Atomic and Molecular Spectrometers Benefit from Technological Improvements Demand for Used Spectroscopy Instruments to Grow Robustly in Future 4. COMPETITIVE LANDSCAPE Competition in the Spectrometry Market: An Insight Molecular Spectroscopy: A Highly Fragmented Market List of Leading Players in the Global Molecular Spectroscopy Market by Product Type Atomic Spectroscopy Market List of Leading Players in the Global Atomic Spectroscopy Market Mass Spectrometry Market List of Leading Players in the Global Mass Spectrometry Market 5. PRODUCT OVERVIEW Spectroscopy Spectrophotometer Fluorometer Different Types of Spectrophotometry Analysis Infrared Spectrophotometry Near-Infrared Spectrophotometry Visible Spectrophotometry Ion Mobility Spectrometry Color Spectrophotometry Near-Ultraviolet Spectrophotometry Spectrometry Spectrometers: A Broad Spectrum of Devices A. Molecular Spectroscopy - The Study of Absorption of Light by Molecules Ultraviolet/Visible (UV/VIS) Spectroscopy Raman Spectroscopy Nuclear Magnetic Resonance (NMR) Spectrometers Near Infrared (NIR) Spectroscopy Applications of Near-IR Techniques Process Analysis Applications of the NIR Spectroscopy Infrared Spectroscopy Fourier-Transform Infrared (FTIR) Spectroscopy B. Atomic Spectroscopy Arc/Spark Spectrometry ICP and ICP-MS Atomic Absorption Spectroscopy Atomic Absorption Analysis Instrumentation Atomizers Double Beam Systems Applications Trends and Future Developments X-Ray Fluorescence Spectrometers (XRF) C. Mass Spectrometry (MS) Mass Spectrometer Types of Mass Spectrometers and their Applications Liquid Chromatography-Mass Spectrometry (LC-MS) Gas Chromatography-Mass Spectrometry (GC-MS) MALDI-TOF Fluorescence Spectroscopy - An Overview Biomedical Applications of Fluorescence Spectroscopy Fluorescence Quenching Fluorescence Polarization Resonance Energy Transfer Filter Fluorimeters Spectrofluorimeters Monochromators Fluorescence-Lifetime Measurements 6. PRODUCT INNOVATIONS/INTRODUCTIONS Shimadzu Launches LCMS-8045 Mass Spectrometer in Europe JUKI Group Commercializes AY555 Spectrophotometer Avantes Launches AvaSpec-Hero Sensline Spectrometer Agilent Launches New 4210 Microwave Plasma-Atomic Emission Spectrometer Princeton Launches FERGIE Spectroscopy System Konica Minolta Introduces Automobile Specific Spectrophotometers, CM-25cG and CM-M6 Shimadzu Launches New GC-MS/MS System, GCMS-TQ8050 Datacolor Introduces Datacolor 20D for Paint Retailers StellarNet Launches Portable Research Grade Raman Spectroscopy System Jenway® Launches New Visible Scanning Spectrophotometer, 7200 IRsweep to Launch IRspectrometer Shimadzu Introduces Atomic Absorption Spectrophotometer, AA-6880F Bruker Launches timsTOF System Thermo Fisher Launches New Spectrometry Devices Techkon Launches Continuous Scanning Spectrophotometer, SpectroEdge ES7500 Sciex Introduces SCIEX QTRAP® 6500+ LC-MS/MS System StellarNet Launches RED-Wave-NIRX-SR Spectrometer Thermo Scientific Launches NanoDrop One Spectrophotometers Thermo Scientific Launches GENESYS 30 Visible-Range Spectrophotometer Thermo Scientific Launches New DXR2 Line of Raman Microscopes Bruker Launches Total Reflection X-Ray Fluorescence Spectrometer, S4 TStar Agilent Launches 5110 ICP-OES Magritek Introduces 60 MHz Benchtop NMR Spectrometers, Spinsolve 60 Thermo Scientific Launches New FT-NIR Spectrometer, Nicolet iS5N Rigaku Launches New Benchtop Variable Spot EDXRF Spectrometer, Rigaku NEX DE Advion Introduces TIDES EXPRESS Merck Introduces Spectroquant® Prove Spectrophotometers Shimadzu Introduces Inductively Coupled Plasma Mass Spectrometer, ICPMS-2030 AQULABO Group Introduces Uviline 9300 and Uviline 9600 Rigaku Launches Rigaku ZSX Primus IV StellarNet BLUE-Wave Compact Spectrometers Line SPECTRO Introduces New Line of SPECTRO XEPOS Spectrometers Thermo Fisher Introduces Thermo Scientific 253 Ultra HR-IRMS StellarNet Launches BLACK-Comet-HR Specac Introduces Pearl Liquid Analysis Accessory Agilent Introduces Agilent 5977B HES GC/MSD System in China Datacolor Introduces New Line of Color Measurement Spectrophotometers Bruker Introduces HH-LIBS Device, EOS 500 Bruker Launches 10 kHz rapifleX MALDI-TOF/TOF Mass Spectrometer Konica Minolta Introduces Auto-Scanning Spectrophotometer, FD-9 Rigaku Introduces Rigaku NANOHUNTER II TXRF Spectrometer SPECTRO Introduces New SPECTROLAB Arc/Spark Optical Emission Spectrometers SPECTRO Introduces SPECTROSCOUT X-ray Fluorescence Spectrometer Bruker Introduces S2 PUMA Shimadzu Introduces Shimadzu LCMS-8060 Triple Quadrupole Mass Spectrometer Thermo Scientific Launches Orbitrap Fusion Lumos Tribrid Mass Spectrometer Agilent Introduces Agilent 6470 Triple Quadrupole LC/MS Waters Introduces Vion IMS QTof Mass Spectrometer DeNovix Introduces DS-11 FX+ Spectrophotometer / Fluorometer Agilent Launches Agilent 6545 Q-TOF Mass Spectrometry System Implen Introduces Fourth Generation NanoPhotometer® Spectrophotometers Shimadzu Corporation Compact Monochromator System Package, SPG-120-REV JEOL Develops JPS-9030, X-ray Photoelectron Spectrometer Datacolor Introduces CHECK 3 Portable Spectrophotometer SPECTRO Launches SPECTRO xSort Handheld EDXRF Spectrometers Bruker Handheld Raman Spectrometer, BRAVO Shimadzu Launches RF-6000 Spectrofluorophotometer X-Rite Introduces X-Rite Ci7800 and X-Rite Ci7600 SPECTRO Introduces SPECTRO xSORT Line of Handheld EDXRF Spectrometers Analytik Jena Introduces New ICP-MS Products, PlasmaQuant® MS and PlasmaQuant® MS Elite Ocean Optics Introduces New Miniature Spectrometer, Flame Shimadzu Launches UV1280 UVVIS Spectrophotometer in the US SPECTRO Introduces SPECTRO ARCOS High-Resolution Spectrometer JEOL Launches JMS-T200GC High-end GC-TOFMS Thermo Scientific Launches Orbitrap Based Q Exactive Focus LC MS/MS Mass Spectrometer 7. RECENT INDUSTRY ACTIVITY Bruker Acquires Active Spectrum Inc. Wasatch Photonics Acquires Process Raman Spectroscopy Technology from Mustard Tree Instruments Shimadzu Institutes Shimadzu China Mass Spectrometry Center INSION Enters into Distribution Agreement with Digilab - The United States (75) - Canada (2) - Japan (11) - Europe (40) - France (3) - Germany (11) - The United Kingdom (18) - Italy (1) - Spain (1) - Rest of Europe (6) - Asia-Pacific (Excluding Japan) (9) - Middle East (1) For more information about this report visit http://www.researchandmarkets.com/research/gxbsmg/spectroscopy


The following antibodies and reagents were from BD Biosciences or eBiosciences: monoclonal antibodies (mAb) to CD11b-pacific-blue (M1/70), CD11c-APC, F4/80-FITC, CD3-pacific-blue, CD4-FITC, CD40-PE, CD80-PE, CD86-PE, CD40L-PE, CD69-PE, C5aR1-PE, and their corresponding isotypes antibodies (rat IgG2b pacific blue, Armenian hamster IgG-APC, rat IgG2a-PE, rat IgG2b PE), Fc blocking antibodies, and Cytofix/Cytopermkit. Anti-phospho-LAT (Tyr191), and anti-LAT clone 11B.12 were from Upstate cell signaling solutions. Rabbit GCS-specific antibody was from Abbiotec LLC. Rabbit affinity-purified GC-specific antibody was from Glycobiotech GmbH. The C5aR antagonist A8(Δ71−73) (C5aRA) was generated as described14. ELISA kits for the detection of human and mouse C5a and cytokines (IFNγ, TNF, IL-1β, IL-6, IL-12p40, IL-12p70, IL-17A/F, IL-23 and CCL18) were from R&D System or eBiosciences. Proteome Profiler A was from R&D System, anti-Profiler A, Bio-Rad Molecular Imager Gel Doc. Liberase Cl was from Roche. DNase (DNase), Diethanolamine (DEA), p-Nitrophenylphosphate (PNPP), MgCl , goat anti-mouse IgG2a, DNase-I kit, and anti-β actin antibody were from Sigma. Alkaline phosphatase-conjugated antibodies to mouse (IgG1, IgG2a/c, IgG2b, and IgG3), human IgG isotypes (IgG1, IgG2, IgG3, and IgG4), and rabbit IgG were from Southern Biotech. Tween 20, Nunc plates, Aminolink Plus Coupling Resin, and BCA protein assay reagents were from Thermo Scientific, RIPA buffer containing sodium orthovanadate and protease inhibitors were from Roche Diagnostics. GM-CSF and M-CSF were from Peprotech. Conduritol B epoxide (CBE) was from Calbiochem. Anti-CD11c, anti-CD11b and anti-CD4 microbeads were from Miltenyi Biotec. Horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-mouse IgG and biotinylated protein ladder detection pack were from Cell Signaling Technology Inc. GC and C12-GC standards were from Matreya, LLC and Avanti Polar lipids, Inc. The 4–12% BisTris gel, sample loading, reducing, running buffer, standard protein molecular weight marker, iBlot 2 dry blotting system, iBind western system, and enzyme-linked chemiluminescence (ECL) chemiluminescent substrate reagent kit, RPMI, DMEM, BSA, FBS, penicillin, streptomycin, HEPES, sodium pyruvate, Trizol, Gel apparatus, Xcell SureLock, and TRIzol reagent were from Invitrogen, Life Technology. RNeasy plus mini kit was from Qiagen. The U937 (ATCC CRL-1593.2TM) cell line, dimethylsulfoxide, and growth medium were from American Type Culture Collection. The U937 cell line has been thoroughly tested and authenticated by the supplier through DNA profiling. It has not been tested for mycoplasma contamination. High capacity RNA-cDNA kit, Taqman universal mastermixII, human and mouse pre-developed primer/probe sets for UGCG/Ugcg and Hypoxanthin phosphoribosyltransferase 1 (HPRT1/hprt) and the real-time PCR system (7500 fast) were from Applied Biosystem, Life Technology and Thermo Fisher Scientific, Inc. (NYSE: TMO). OCT freezing medium was from Sakura Finetek and Vectashield was from Vector Laboratories. The Fortessa-I, -II, and LSRII flow cytometers were from BD Biosciences. FCS Express software version 4 was from DeNovo Software. The plate reader was from Molecular Devices. The D409V/null mice (Gba19V/−) and wild-type controls were both on the mixed FVB/C57BL 6J/129SvEvBrd (50:25:25) backgrounds. Male and female mice were used at 20–24 weeks of age7. To directly assess the role of C5aR1-mediated effects, Gba19V/− mice were backcrossed to C5aR1-deficient mice for at least 10 generations. Out of these backcrosses, we generated double mutant mice (Gba19V/−C5ar1−/−) and Gba19V/−, wild-type and C5ar1−/− background-matched littermates. To assess the role of C5aR1, C5aR2 and FcγRs in pharmacologically induced Gaucher disease, wild-type mice and those lacking C5aR1, C5aR2, and activating FcγRs (Fcer1g−/−) or the inhibitory FcγRIIB (Fcgr2b−/−) of both sexes were used at ~12 weeks of age. Mice were bred and maintained in the specific-pathogen free facility at the Cincinnati Children’s Research foundation. Mice of the appropriate genotype were randomly assigned to groups. No specific randomization was performed. The investigators were not blinded to allocation during experiments and outcome assessment. Animal care was provided in accordance with National Institute of Health guidelines and was approved by Cincinnati Children’s Hospital Medical Center IACUC. Frozen sera from human patients with untreated Gaucher disease (n = 10) and healthy volunteers (n = 15) were de-identified. Patients with Gaucher disease were diagnosed at Cincinnati Children’s Hospital Medical Center. They did not receive any specific-enzyme therapy or substrate reduction therapy for Gaucher disease and are designated as untreated. The study was approved by the ethics committee at Cincinnati Children’s Hospital Medical Center. Protocols for human studies were approved by the Institutional Review Board, and patients with Gaucher disease and controls gave written, informed consent for the use of their serum for the studies described here. To assess the effect of genetic or pharmacological targeting of C5aR1 on the inflammatory response in Gaucher disease, wild-type (n = 10) and C5ar1−/− mice (n = 10) were treated with CBE, which is an irreversible inhibitor of acid β-glucosidase21. More specifically, both mouse strains were injected i.p. with 100 mg CBE per kg body weight or vehicle (PBS) per day for up to 60 days, which was the termination point of these experiments. After 60 days of the indicated treatment with CBE, immune cells (macrophages, DCs, and T cells) were purified from lung of these mouse strains and used for measurement of GC, costimulatory molecules, and several of the proinflammatory cytokines. In additional experiments, wild-type (n = 15) or Gba19V/− mice (n = 15) were injected with 100 μl of the C5aRA A8(Δ71−73) (i.p. 0.5 mg per kg) or vehicle (100 μl, PBS) on five consecutive days. Five days after the final C5aRA treatment, liver, spleen and lung were separated and measured for GC accumulation. In addition, DCs and CD4+ T cells were purified from the lung of the indicated mouse strains, co-cultured and measured for costimulatory molecule expression and the production of proinflammatory cytokines. Liver, spleen and lung of vehicle- or CBE-treated wild-type or C5ar1−/− mice were homogenized in 1% sodium taurocholate/1% Triton X-100. The protein concentrations of cells from such tissue lysates were determined by BCA assay using BSA as standard. GCase activities were determined fluorometrically with 4MU-Glc in 0.25% Na taurocholate and 0.25% Triton X-100 as described7. Liver, spleen, lung and bone marrow were collected aseptically. Single-cell suspensions from liver and lung were obtained from minced pieces that were treated with Liberase Cl (0.5 mg ml−1) and DNase (0.5 mg ml−1) in RPMI (45 min, 37 °C). Single-cell suspensions from spleen were obtained by grinding and then filtration through a 70-μm cell strainer. Similar suspensions of liver and lung were obtained from minced pieces that were treated with Liberase Cl (0.5 mg ml−1) and DNase (0.5 mg ml−1) in RPMI (45 min, 37 °C). For bone marrow cells, femurs, tibias and humeri were flushed with sterile PBS, followed by red blood cell lysis (155 mM NH Cl, 10 mM NaHCO , 0.1 mM EDTA), passage through a strainer. Cells were then pelleted by centrifugation at 350g. Viable cells were counted using a Neubauer chamber and trypan blue exclusion. DCs, macrophages and CD4+ T lymphocytes were purified from single-cell suspensions of liver, spleen and lung using CD11c, CD11b and CD4 (L3T4) microbeads according to the manufacturer’s protocol. The purity of the cells was ~90–95%. Bone marrow cells were used to differentiate macrophage as described22. Briefly, fresh bone marrow cells were stimulated with M-CSF (10 ng ml−1) in complete DMEM (FBS 10% + 100 U ml−1 penicillin, 100 μg ml−1 streptomycin, 10 mM HEPES and 1 mM sodium pyruvate). Cells were seeded in six-well tissue culture plates and incubated at 37 °C in a 5% CO atmosphere. Five days after cell seeding, supernatants were discarded and the attached cells were washed with 10 ml of sterile PBS. 10 ml of ice-cold PBS were added to each plate and incubated at 4 °C for 10 min. The macrophages were detached by gently pipetting the PBS across the dish. The cells were centrifuged at 200g for 5 min and resuspended in 10 ml of complete DMEM. The cells were counted, seeded and cultured for 12 h before they were used for further experiments. DCs were differentiated from bone marrow cells as described22. Briefly, bone marrow was flushed from the long bones of the limbs and depleted of red cells with ammonium chloride. Such bone marrow cells were plated in six-well plates (106 cells per ml, 3 ml per well) in RPMI 1640 medium supplemented with FBS (10%) and 100 U ml–1 penicillin, 100 μg ml−1 streptomycin, 10 mM HEPES and 1 mM sodium pyruvate and 10 ng ml−1 recombinant murine GM-CSF at days 0, 2, 4 and 6. Floating cells were gently removed and fresh medium was added. At day 7, nonadherent cells and loosely adherent proliferating DC aggregates were collected, counted, seeded and cultured for 12 h before they were used. Tissue cells were identified by flow cytometry. First, they were suspended in PBS containing 1% BSA. After incubation (15 min, 4 °C) with FcγR-blocking antibody 2.4G2, cells were stained (45 min, 4 °C) with the following antibodies to identify antigen-presenting cells and T cells: CD4 for T cells; CD11b and F4/80 for macrophages; and CD11b and CD11c for DCs. Cells were also stained with the respective isotype antibodies as controls. Macrophages were first identified by their typical FSC/SSC pattern, and F4/80 and CD11b expression. DCs were identified as CD11c+CD11b+ cells. Further, CD40, CD80 CD86 and C5aR1 expression was determined in tissue DCs. T cells were first characterized by their FSC/SSC pattern and CD3 staining. CD3+ T cells were further stained for CD4, CD40L and CD69 expression. A total of 106 events were acquired for each cell type isolated from the different organs. Specific surface expression was assessed relative to the expression of the corresponding isotype control antibody. Lipids were extracted from tissues (5 mg; liver, spleen, and lung), purified macrophages, DCs, CD4+ T lymphocytes, U937 cells and GC-specific IgG2a by chloroform and methanol1, 22. GC and GS species in IgG2a isolates were quantified by ESI-LC–MS/MS using a Waters Quattro Micro API triple quadrupole mass spectrometer interfaced with Acquity UPLC system7. Calibration curves were built for the GC species (C16:0, C18:0, C24:1) using C12-GC as standard. Quantification of GCs with various fatty acid chain lengths were realized by using the curve of each GC species with closest number of chain length. The total GCs in the tissues and purified IgG2a were normalized to 1 mg of tissue and protein, and immune cells to 1 × 106 cells. C5a concentrations were determined in sera or culture supernatants from bone-marrow-derived macrophages and DCs (each of 106 cells per 200 μl of complete RPMI media) of wild-type and Gba19V/− (n = 15 per group) mice, CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per of group), as well as in sera obtained from patients with untreated Gaucher disease (n = 10) and healthy control humans (n = 15) by commercial ELISA kits according to the manufacturer’s instructions. C5aR1 expression in macrophages and DCs purified from liver, spleen and lung of wild-type or Gba19V/− mice was evaluated by flow cytometry using a C5aR1-specific antibody. For detection of cytokines and chemokines, blood from CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per group) was obtained by cardiac puncture. Sera were isolated after one-hour incubation at room temperature. Sera were diluted 1:10 with sterile PBS and used for detection of cytokines and chemokines with Proteome Profiler A Densitometry, which was performed with a Bio-Rad Molecular Imager Gel Doc system. To assess the effect of C5a on GC-induced costimulatory molecule expression, DCs and CD4+ T cells purified from lungs of Gba19V/− mice and background-matched wild-type mice (n = 15 per group) were stimulated ex vivo in the presence or absence of different C5a concentrations (0, 8, 16 and 32 nM) for 24 h at 37 °C. DCs and CD4+ T cells were purified from liver, spleen and lung of CBE-treated C5ar1−/− and background-matched wild-type mice (n = 10 per group) and stained with CD40-, CD80- and CD86- (DCs) or CD40L- and CD69-specific antibodies (CD4+ T cells). To assess the effect of C5a on GC-induced cytokine and chemokine production, DCs and CD4+ T cells (1:2.5 ratio), purified from lungs of Gba19V/− mice and background-matched wild-type mice (n = 15 per group), were cocultured in the presence and absence of C5a (32 nM) for 48 h in complete medium. In additional experiments, indicated ratios (1:25) of DCs and CD4+ T cells, purified from lungs of CBE-treated and untreated wild-type and C5ar1−/− mice (n = 10 per each group), were cocultured for 48 h in complete medium. Supernatant of these experiments were used to determine IFNγ, TNF, IL-1β, IL-6, IL-12p40, IL-12p70, IL-17A/F and IL-23 by ELISA. To determine the levels of GC-specific IgG antibodies in mice and patients with Gaucher disease, 10 μg of GC were dissolved in 1 ml of methanol and water to a final concentration of 10 μg ml−1. 100 ml of this GC solution (1 μg per well) were used to coat a 96-well ELISA plate. GC-coated plates were kept overnight at room temperature followed by three washings with PBS containing 1% Tween-20 (PBST). Test sera (100 μl; 1:100) isolated from wild-type and Gba19V/− mice (n = 15 per group), CBE-treated and untreated wild-type mice (n = 10 per group), as well as healthy humans (n = 15) and untreated patients with Gaucher (n = 10), and GC-specific IgG control antibody were loaded into the lipid-coated wells, followed by incubation for 1.5 h at room temperature. These plates were then washed three times with PBST and subsequently incubated with alkaline phosphatase-conjugated rat anti-mouse IgG1 (1:500 in PBS), IgG2a/c (1:1,000 in PBS), IgG2b (1:1,000 in PBS) or IgG3 (1:1,000 in PBS) or alkaline phosphatase-conjugated mouse anti-human IgG1, IgG2 (each 1:1,000 in PBS), IgG3 and IgG4 (each 1:500 in PBS) in triplicates. Then, the plates were incubated for 1.5 h at room temperature followed by two washing steps with PBST and one with 10 mM DEA. 100 ml of 1 mg ml−1 PNPP in 10 mM DEA containing 5 mM MgCl was added to each well and incubated for 30 min at room temperature in the dark. Finally, plates were read at 405 nm to detect the GC-specific IgG antibodies. To determine GS- and GC-specific IgG IC formation, IgG2a was purified from pooled sera that were prepared from wild-type and Gba19V/− mice (n = 15 per group). Briefly, pooled mouse sera (5–10 ml) were incubated with goat anti-mouse IgG2a (25–50 μg) that had been immobilized on 2 ml of Aminolink Plus coupling resin overnight at 4 °C according to the manufacturer’s instructions. After several washing steps with working buffer (20 mM PBS, pH 7.4), bound IgG2a antibody fractions were finally eluted using 3 ml of elution buffer (50 mM Gly–HCl, pH 2.8). The eluted fractions were then used to determine GS and GC species bound to IgG2a and to quantify them with an ESI-LC–MS/MS system as above. Protein separation of purified IgG2a was performed using a 12% NuPAGE Bis-Tris Mini gel and reducing SDS–PAGE system according to the manufacturer’s instruction. Briefly, 4 μl of IgG2a (2.5 mg ml−1) was mixed with 16 μl of reducing buffer, (for example, 5 μl of NuPAGE LDS Sample Buffer 4×, 2 μl of NuPAGE Reducing Agent 10×, and 13 μl of deionized water) and then boiled for 5 min in a water bath. 10 μg of protein were applied to each lane and PAGE (130–180 mA) was run for 1 h at room temperature. The gel was then stained with Coomassie blue R250 using standard techniques. A minimum of two sections from CBE-treated and CBE-untreated wild-type and C5ar1−/− mice (n = 10 per group), as well as C5aRA-treated and vehicle-treated wild-type and Gba19V/− mouse strains (n = 15 per group) were examined from each tissue. Liver, spleen and bone were collected after the mice had been perfused with PBS and the tissues fixed in 10% formalin or 4% paraformaldehyde, and processed for paraffin and frozen blocks, respectively. Paraffin sections of indicated tissues were stained with haematoxylin and eosin (H&E), whereas frozen sections were stained with rat anti-mouse CD68 (1:100) followed by biotinylated goat anti-rat and streptavidin-conjugated antibodies as described previously7, 23. To determine whether GC induces complement activation in Gaucher disease, we used freshly isolated liver, spleen and lung from CBE-treated and untreated wild-type and C5ar1−/− mice (n = 10 per group). These tissues were embedded in OCT freezing medium and snap-frozen in liquid nitrogen and eventually stored at −80 °C until use. Tissues were then sectioned at 5–7 μm and fixed with cold acetone and permeablized with 0.2% Triton X-100 in PBS. Tissue sections were blocked with 2% BSA and counter-stained with FITC-conjugated antibody to mouse C3/C3b (2 μg ml−1) and its isotype control overnight at 4 °C. Tissues were washed and coverslipped with Vectashield. Immunofluorescence images were captured with a Zeiss Apotome microscope (AxioV200). To investigate the direct effect of GC immune complexes on C5a release in Gaucher disease, macrophages (106 cells per 200 μl of complete RPMI media) purified from lung tissues of Gba19V/− mice (n = 15) were ex vivo stimulated in the presence or absence of GC (0.25, 0.5 and 1.0 μg) and anti-GC IgG (25 μg) for 2 h. Supernatants were used to determine C5a concentrations by ELISA. To evaluate the effect of GC immune complexes on C5a secretion in vivo, wild-type and Gba19V/− mice were injected i.p. with vehicle (ethanol), GC, anti-GC IgG or GC immune complexes (n = 15 per group). After 2 h, serum and peritoneal lavage fluid were collected and C5a was measured by ELISA according to the manufacturer’s instructions. After incubation of lung-derived F4/80+CD11b+ macrophages (5 × 106) from wild-type and Gba19V/− mice (n = 15 per group) with GC (1.0 μg), anti-GC IgG (25 μg of anti-GC IgG), GC immune complex or vehicle (1 μl methanol) per ml of media for 5 min at 37 °C, cells were collected and pellets were lysed with 1× RIPA buffer containing sodium orthovanadate and protease inhibitors. Protein concentrations were determined in cell lysates using BCA protein assay. Each 10 μg of cell lysates were loaded on an 10% SDS–PAGE and transferred onto a PVDF membrane and probed with antibodies to phosphorylated LAT (pLAT; 1:200) and non-phosphorylated LAT (linker of T cell activation; 1:1,000) using the iBlot 2 Gel transfer device and iBind western system according to the manufacturer’s instruction. pLAT and LAT (both ~36/38 kDa) proteins were visualized using anti-rabbit and anti-mouse secondary antibodies conjugated to HRP (1:1,000) and the Novex ECL chemiluminescent substrate reagent kit. Total RNA was extracted from mouse lung, liver, spleen and U937 macrophage-like cells using TRIzol reagent according to the manufacturer’s instructions. Reverse transcription was performed using the High capacity RNA to cDNAKit and qPCR was performed using Taqman assay reagents, primer/probes sets for both human and mouse UGCG/Ugcg and HPRT1/hprt. Amplifications were done using the ABI 7500 Real-Time PCR System and the calculations and analysis were based on the comparative C method24. For protein expression of GCS, mouse lung homogenates were lysed using mammalian protein extraction reagent. Protein concentration was determined by BCA according to the manufacturer’s instructions. Equal amounts of proteins (20 μg per lane) were loaded onto NuPAGE 4–12% Bis-Tris gradient SDS–PAGE. The mouse proteins were transferred to Hybond-ECL PVDF membranes and immunoblotted using iBind western blotting system with antibodies to mouse GCS diluted 1:100 with iBind solution and incubated over night at 4 °C. The GCS signals were detected using a HRP-conjugated anti-rabbit IgG (1:1,000) and ECL detection reagent as described25 with β-actin as a loading control. The intensities of protein bands were quantified using an NIH Image J. The GCS expression in the different treatment groups is depicted as the GCS/β-actin ratio normalized against the 100% value assigned to the wild-type group. To assess whether GC immune complexes causes C5a generation in patients with Gaucher disease, sera prepared from healthy humans (n = 15) and untreated patients with Gaucher disease (n = 10) were diluted 1:10,000 with saline and used to identify C5a by ELISA according to the manufacturer’s instructions. To determine the direct effect of GC immune complexes on C5a production and proinflammatory cytokine release in human Gaucher disease, the human macrophage-like cell line U937 (106 cells per 200 μl of complete RPMI media) was treated with CBE at 37 °C and 5% CO for 72 h. These cells were then stimulated in the presence or absence of GC (1 μg), anti-GC IgG (25 μg) or GC immune complex. Supernatants were used to determine C5a, CCL18, TNF, IL-1β, IL-6 and IL-23 concentrations by ELISA. All quantitative experiments were repeated at least three times. The sample sizes in all animal studies were estimated on the basis of effect sizes present in pilot studies to ensure we had sufficient power. The number of animals used in each experiment is outlined in the relevant sections in the Methods. An unpaired Student’s t-test (for two groups) or one-way analysis of variance (ANOVA) (for more than two groups) were used to determine significant differences between groups (Graph Pad Prism). To ensure that the statistical inference was appropriate, we evaluated the normality of the data distribution. Between group differences in many of the variables made, the overall distributions of many of the measures were non-normal. However, evaluation with non-parametric tests supported the inference of the parametric tests suggesting that the parametric tests were robust to these deviations from normality. For t- tests of the 40 cytokines, a simple Bonferroni correction is not appropriate as there is a high degree of correlation between the cytokines (average pairwise rho = 0.76). Thus, we employed a correlation corrected Bonferroni adjustment in SISA (http://www.quantitativeskills.com/sisa/calculations/bonfer.htm) resulting in a significance threshold of 0.021. As two conditions were considered for these 40 cytokines, the final correction was 0.021 / 2 = 0.0105. For the ANOVA, rather than considering all possible pairs of comparisons, we focused on a restricted set of a priori comparisons. Specifically, we performed analysis to determine the effect of (1) genotype, (2) C5aRA treatment, and (3) GCase targeting. Within each of these specific tests, we applied Bonferroni correction on the basis of the number of a priori comparisons made. For analyses, which were not pre-specified, the Bonferroni comparison was made on the number of possible comparisons. All data in the bar graphs are reported as mean ± s.d. *P < 0.05, **P < 0.01, ***P < 0.001 denote the uncorrected P values, with the significance thresholds denoted in the figure legend if multiple testing corrections were applied. The data generated during and/or analysed during the current study are available from the corresponding author on reasonable request.


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No statistical methods were used to predetermine sample size. The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Uniformly [32P]-labelled, m7G(5′)ppp(5′)G-capped MINX pre-mRNA was synthesized in vitro by T7 runoff transcription. HeLa S3 cells were obtained from GBF, Braunschweig (currently Helmholtz Zentrum für Infektionsforschung, Braunschweig) and tested negative for mycoplasma. HeLa nuclear extract was prepared essentially as previously described48, but without the final dialysis step. To isolate C* complexes, splicing was performed with 5 nM of 32P-labelled pre-mRNA and 20% (v/v) HeLa nuclear extract, in buffer containing 3 mM MgCl , 50 mM NaCl, 4 mM HEPES-KOH pH 7.9, 12 mM MES-NaOH pH 6.4, 2 mM ATP and 20 mM creatine phosphate, and was incubated at 30 °C for different time periods. Samples were analysed on a denaturing 4–12% NuPAGE gel (Life Technologies) and pre-mRNA and splicing intermediates and products were visualized with a Typhoon phosphorimager (GE Healthcare). Spliceosomal complexes were isolated by MS2 affinity selection. In previous studies, affinity purified human C complexes were formed in vitro on mutated pre-mRNA substrates that are unable to undergo the second step of splicing49, 50, 51. Here, we used the MINX pre-mRNA substrate52 that contains an intron flanked by a 5′ and 3′ exon, allowing a functional analysis of complexes that assemble on it. In brief, MINX pre-mRNA containing three MS2 aptamers at its 3′ end RNA was incubated with a tenfold molar excess of MS2–MBP fusion protein and then added to a splicing reaction. After incubating at 30 °C for 5 h and centrifuging to remove aggregates, the reaction was loaded onto a MBP Trap HP column (GE Healthcare) after addition of 5 mM HEPES-KOH pH 7.9. The column was washed with G-150 buffer (20 mM HEPES-KOH pH 7.9, 1.5 mM MgCl , 150 mM NaCl) and complexes were eluted with G-150 buffer containing 1 mM maltose. Eluted complexes were loaded onto a 36 ml linear 10–30% (v/v) glycerol gradient containing G-150 buffer (20 mM HEPES-KOH pH 7.9, 1.5 mM MgCl , 150 mM NaCl), centrifuged at 23,000 r.p.m. for 15 h at 4 °C in a Surespin 630 (Thermo Scientific) rotor and fractions were harvested from the bottom. The distribution of 32P-labelled MINX RNA across the gradient was determined by Cherenkov counting. Fractions were analysed by denaturing 4–12% NuPAGE (Life Technologies) followed by autoradiography. Peak fractions containing the first step splicing intermediates were pooled, concentrated by centrifugation with an Amicon 50 kD cut-off unit, diluted to decrease the glycerol concentration and reloaded on the same gradients with glutaraldehyde as fixative53. For biochemical sample validation, the same procedure was performed but without fixation in the second gradient. The RNA and protein compositions of purified complexes were determined by denaturing 1D PAGE and 2D gel electrophoresis. Two-dimensional gel-electrophoresis of affinity-purified spliceosomal complexes was performed as described in ref. 54, using a 7% acrylamide mono gel in the second dimension for analysis of proteins larger than 25 kDa, or 15% acrylamide for proteins smaller than 25 kDa. For mass spectrometry, coomassie-stained protein-spots were cut out of the 1D or 2D gels, and proteins were digested in-gel with trypsin and extracted. The extracted peptides were analysed in a liquid-chromatography coupled electrospray ionization mass spectrometer (LTQ Orbitrap XL) under standard conditions. Proteins were identified by searching fragment spectra against the NCBI non-redundant (nr) database using Mascot as a search engine. Total IgGs against human PRP16 were purified as described previously55. Affinity-purified C* complexes formed on 32P-labelled MINX-MS2 pre-mRNA were incubated with splicing buffer alone (20 mM HEPES-KOH pH 7.9, 50 mM NaCl, 3 mM MgCl , 2 mM ATP, 20 mM creatine phosphate) or additionally in the presence of 20% HeLa nuclear extract prepared according to ref. 48. For antibody inhibition experiments, the splicing mixture was pre-incubated with 3 μg μl−1 of anti-PRP16 antibody at 30 °C for 15 min as described55. The splicing reaction was initiated by addition of 32P-labelled MINX-MS2 pre-mRNA or C* complex assembled on 32P-labelled MINX-MS2 pre-mRNA. The reaction was incubated at 30 °C for 0–60 min. RNA was recovered, separated by SDS–PAGE, and visualized with a Typhoon phosphorimager (GE Healthcare). Following MS2 affinity selection and the first density gradient centrifugation step, purified spliceosomal complexes were crosslinked with 150 μM BS3 for 40 min at 20 °C and purified further by a second density gradient centrifugation step. Approximately 25 pmol of C* complexes were pelleted by ultracentrifugation and analysed essentially as described before56 with the following modifications: precipitated material was dissolved in a solution containing 4 M urea and 50 mM ammonium bicarbonate, reduced with DTT, alkylated with iodoacetamide, diluted to 1 M urea and digested with trypsin (1:20 w/w). Peptides were reverse-phase extracted and fractionated by gel filtration on a Superdex Peptide PC3.2/30 column (GE Healthcare). 50-μl fractions corresponding to an elution volume of 1.2–1.8 ml were analysed in quadruplicate on a Thermo Scientific Q Exactive HF mass spectrometer. Protein–protein crosslinks were identified by pLink 1.23 search engine (http://pfind.ict.ac.cn/software/pLink) and filtered at FDR 1% according to the recommendations of the developer57. For simplicity, the crosslink score is represented as a negative value of the common logarithm of the original pLink score, that is Score = –log (pLink Score). For model building, a maximum distance of 30 Å between the Cα atoms of the crosslinked lysines was allowed. Approximately 97% of all crosslink-assigned spectra correspond to crosslink distances of 30 Å or less. Purified spliceosomes, stabilized by GraFix53, were allowed to adsorb on a thin carbon film before negative staining or rapid plunge freezing into liquid ethane at 100% humidity and 4 °C. Micrographs of negatively stained particles were recorded in a CM200 electron microscope (FEI/Phillips, the Netherlands) at room temperature and approximately 50,000 particles were picked by hand. The latter were then used to de novo build a negative stain starting structure that was refined to around 25 Å by using 3D maximum-likelihood alignment and 3D classification58. The resulting model was subsequently used as an initial reference in cryo-particle image processing and classification. Cryo-images were recorded at −193 °C in a Titan Krios electron microscope (FEI Company, The Netherlands) on a Falcon II direct electron detector at a nominal 88,000× magnification resulting in a calibrated pixel size of 1.59 Å on the specimen level. Seventeen frames were recorded for each micrograph with an average dose of 2.1 e− per frame per Å2. Motion correction and spatial frequency weighed frame summation was achieved using the unblur software suite59 (http://grigoriefflab.janelia.org/unblur). Summed micrograph images were then evaluated based on CTF parameters and only those revealing isotropic Thon rings were used for particle picking and extraction. Using the particle picking software Gautomatch (http://www.mrc-lmb.cam.ac.uk/kzhang/) and 40° projections of the negative stain model filtered to 40 Å as a reference, we extracted approximately 2.5 million particle images from the pre-sorted cryo-micrographs and applied several particle sorting steps at the 2D and 3D level. 2D multivariate statistics and classification was first applied to the non-aligned particle images and subsequently to the aligned particles. In each round, particles contributing to bad classes were excluded from further processing. The remaining ~1,708,000 particles were split into seven equally sized groups and subsequently applied to seven separate rounds of 3D classification in RELION featuring six classes each. The ~393,000 particles pooled from all satisfactory classes were then subjected to further rounds of 3D classification in RELION71. For the high resolution structure determination, the 136,534 particles finally contributing to the best 3D class were used for refinement revealing an 8.4 Å resolution structure (which we refer to as the unmasked EM density map). Roughly 20% of the spliceosome density was not clearly defined at this level of resolution. As these densities largely disappear during the higher-resolution structure calculations, we excluded them with a mask in the final rounds of the refinement. A soft mask with a cut-off of 7 voxels was used for the refinement and for the determination of resolution. We obtained the final map with a resolution of 5.9 Å as determined by Fourier shell correlation calculated from two independent data sets with a threshold of 0.143. A local resolution plot revealed that there are areas of higher resolution in the catalytic RNP core of the C* complex that approach 4.5 Å. Some peripheral regions have somewhat lower resolution (Fig. 1 and Extended Data Fig. 2c). More dynamic areas or those with components with non-stoichiometric occupancies are not visible in the 5.9 Å structure. Available X-ray or homology models of proteins were fit into the EM density by Chimera60. Individual models of substructures (for example, domains or structural motifs) were further fitted as rigid bodies by Coot61. After visual inspection, the models were adjusted manually in the density, the disordered regions were removed and regions that were reorganized or were not present in the initial models (for example, loops and various elements of secondary structure) were built in Coot. Homology models of proteins were either obtained by using the respective functions of the SWISS-MODEL suite62 or directly adapted from the SpliProt3D database63. Initial human snRNA and intron models were obtained by using the S. cerevisae C complex spliceosomal RNA14, 21 as reference, and homology modelled according to the human sequence using the ModeRNA package64 (http://genesilico.pl/moderna/). The snRNA fit was improved at rigid body level in Chimera and subsequently adjusted in Coot. The 5′ stem loop of U6 and the U2/U6 helix II were generated by rigid-body fitting of idealized double-stranded RNA helices. Exon 1 was modelled ab initio into available density in Coot and the resulting model later verified by comparison to the S. cerevisiae C spliceosome model22. Once the entire coordinate model was built up, all proteins were truncated to poly-alanine level and a global minimization real space refinement was conducted against the 5.9 Å cryo-EM density using the real space refine program from the PHENIX suite65. The RNA model was subsequently validated by the MolProbity server66 and had an all atom clash score of less than 12 and no bad bond lengths or angles. Final visualization was carried out with Chimera and PyMOL (http://www.pymol.org). See also Supplementary Table 1 for protein and model building information for all modelled human C* proteins. All data generated or analysed during this study are included in this published article and its Supplementary Information files. The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-3547 (8.4 Å map) and EMD-3545 (5.9 Å map). The atomic model has been deposited in the Protein Data Bank under accession code 5MQF.


News Article | February 28, 2017
Site: www.businesswire.com

WALTHAM, Mass.--(BUSINESS WIRE)--Thermo Fisher Scientific Inc. (NYSE: TMO), the world leader in serving science, today announced that its board of directors declared a quarterly cash dividend of $0.15 per share. The dividend will be paid on April 17, 2017, to shareholders of record as of March 15, 2017. Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science, with revenues of $18 billion and more than 55,000 employees globally. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit www.thermofisher.com.


News Article | February 15, 2017
Site: www.businesswire.com

WALTHAM, Mass.--(BUSINESS WIRE)--Thermo Fisher Scientific Inc. (NYSE: TMO), the world leader in serving science, today completed its previously announced acquisition of Finesse Solutions, Inc., a leader in the development of scalable control automation systems and software for bioproduction. The business will be integrated into Thermo Fisher’s Life Sciences Solutions Segment. Terms of the transaction were not disclosed. Based in Santa Clara, California, Finesse Solutions is a leader in bioprocess management technology, generating approximately $50 million in revenue in 2016. Its proprietary Smart™ technology, which consists of sensors, controllers and software, is designed to optimize the bioproduction workflow. The company has been a technology partner of Thermo Fisher Scientific since 2013. About Thermo Fisher Scientific Thermo Fisher Scientific Inc. (NYSE: TMO) is the world leader in serving science, with revenues of $18 billion and more than 55,000 employees globally. Our mission is to enable our customers to make the world healthier, cleaner and safer. We help our customers accelerate life sciences research, solve complex analytical challenges, improve patient diagnostics and increase laboratory productivity. Through our premier brands – Thermo Scientific, Applied Biosystems, Invitrogen, Fisher Scientific and Unity Lab Services – we offer an unmatched combination of innovative technologies, purchasing convenience and comprehensive support. For more information, please visit www.thermofisher.com.

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