News Article | January 27, 2016
C57BL/6J mice (CD45.2) and B6.SJL-PtprcaPep3b/BoyJ (CD45.1) were purchased from The Jackson Laboratory (Bar Harbour, ME). Prdm16Gt(OST67423)Lex knockout mice31 were obtained from Lexicon Genetics. Conditional Mito-Dendra2 transgenic (Pham) mice32 (B6;129S-Gt(ROSA)26Sortm1(CAG-COX8A/Dendra2)Dcc/J) and E2A-Cre mice33 (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J) were purchased from Jackson Laboratory. Pham mice contain a mitochondrially targeted Dendra preceded by a stoplox sequence in the Rosa locus. These mice were crossed with E2A-Cre mice to effect ubiquitous induction of the MitoDendra2 reporter. Conditional Mfn2 knockout mice34 (B6/129SF1Mfn2tm3Dcc/Mmucd) were obtained from MMRRC and crossed to Vav-Cre transgenic mice35 (B6.Cg-Tg(Vav1-Cre)A2Kio/J) to obtain a homozygous floxed allele Mfn2 allele which generated a B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed mouse strain. All mouse strains were rederived by in vitro fertilization at the Jackson Laboratory. Animals were housed in a specific pathogen-free facility. Experiments and animal care were performed in accordance with the Columbia University Institutional Animal Care and Use Committee. All mice were used at age 8–12 weeks, except in experiments that involved fetal liver cells, when E14.4 embryos were used. Both sexes were used for experiments. Results were analysed in non-blinded fashion. In all experiments, randomly chosen wild type and littermates were used. MEFs were established from approximately 14.5 days post coitum embryos as previously described36 from Prdm16+/− breeder pairs. Briefly, dissected embryo trunks were minced into 1–2 mm fragments, resuspended in 3 ml 0.25% trypsin/EDTA (Gibco, Carlsbad, CA) and passed 20–30 times through a 16 gauge needle. Cell suspensions were incubated at 37 °C for 1 h with frequent agitation. Erythrocytes were lysed with ACK buffer, washed and cells were plated for 3 h in 10% FBS/DMEM. Cells remaining in suspension were aspirated and adherent cells were cultured with fresh media. MEFs were passaged 1:3 every 3 days and cells between passage 2 and 5 were used for all experiments. 293 cells and NIH-3T3 cells were purchased from ATCC (Manassas, VA) and sub-cultured in 10% FBS/DMEM or 10% calf serum/DMEM, respectively. WT and Mfn2−/− MEFs were a kind gift from E. Schon (Columbia University). All lines are tested yearly for mycoplasma contamination and found negative. Prdm16 constructs were generated by subcloning the murine full length (flPrdm16) or truncated (sPrdm16) cDNA into the XhoI/EcoRI sites of the pMSCV-IRES-GFP retroviral expression plasmid. The Mito-dsRed construct was purchased from Addgene (Cambridge, MA) (plasmid 11151). Mfn2 constructs were generated by subcloning the murine Mfn2 cDNA into the EcoRI/BamHI sites of the pLVX-EF1α-IRES-GFP or pLVX-EF1α-IRES-mCherry lentiviral expression plasmid (Clontech). The pGreenFire-Nfat and pGreenFire-CMV gene reporter constructs were purchased from System Biosciences (San Jose, CA) and contained three canonical Nfat response elements (5′- -3′) driving the expression of copGFP and luciferase reporters. The DNDrp1-pcDNA3.1 construct was purchased from Addgene (#45161) and subcloned using the BamHI/EcoRI restriction sites into the pLVX-IRES-GFP vector. Lentiviral 2nd generation packaging construct ΔR8.2 (8455) and pDM2.6 (12259) were purchased from Addgene. The −950/+22 murine MFN2 promoter was constructed by PCR amplification of the RP23-458J18 BAC clone (CHORI, Oakland, CA) and subcloned into the pGL4 luciferase reporter vector (Promega, Madison, WI). All cloning was carried out using KOD hot-start polymerase (Novagen, Billerica, MA) and subcloned for screening and sequencing into the pCR2.1 shuttle vector (Invitrogen, Carlsbad, CA). For peripheral blood analyses, erythrocytes were lysed twice with ACK lysis buffer and nucleated cells were stained with antibody cocktail (Supplementary Table 1) in FACS buffer for 15 min on ice, washed and analysed on a BD FACSCantoII flow cytometer (Becton Dickinson, Mountain View, CA). For bone marrow analyses, cells were isolated using the crushing method and erythrocytes were lysed with ACK lysis buffer followed by 40 μm filtration. bone marrow cells were stained with antibody cocktail in FACS buffer for 30 min on ice, washed and analysed on a BD LSRII flow cytometer (Becton Dickinson, Mountain View, CA). Dead cells were excluded from analyses by gating out 7AAD-positive cells. To isolate purified haematopoietic populations, bone marrow cells were isolated, stained and sorted using a BD Influx cell sorter (Becton Dickinson, Mountain View, CA) into complete media. Data were analysed using FlowJo9.6 (TreeStar Inc., Ashland, OR). Mfn2fl/fl-Vav-Cre fetal liver cells, bone marrow cells or purified LT-HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) were transplanted into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) recipients together with 2 × 105 competitor cells. As Mfn2fl/fl-Vav-Cre mice were not fully backcrossed onto the C57BL/6 background, recipient mice and competitor bone marrow cells were from the B6.Cg-Tg(Vav1-Cre)A2Kio/J;B6/126SF1 Mfn2tm3Dcc/Mmucd mixed background mouse strain crossed to B6.SJL-Ptprca Pep3b/BoyJ (CD45.1) to generate a CD45.1+CD45.2+ mixed background mouse. Competitor cells were T-cell depleted using MACS beads. For all competitive transplantation experiments, at least two independent transplants, each with at least 4 recipients per condition of genotype were performed, and result of all recipients pooled for statistical analysis. Power calculation was based on results of the first experiment. In limiting dilution assays, cohorts of recipients received 20 or 50 HSCs together with 2 × 105 competitor cells, allowing calculation of HSC frequency based on the number of non-repopulated mice (<1% donor contribution) using Poisson statistics 15 weeks after reconstitution. For Mfn2 KO single cell transplantation, LT-HSCs were sorted directly into complete media (StemPro34, 100 ng ml−1 SCF, 100 ng ml−1 TPO, 50 ng ml−1 IL-6) and single cells were visually confirmed. Positive single cell wells were combined with 2 × 105 CD45.1 competitor bone marrow cells and transplanted into lethally irradiated CD45.1 recipient mice. Recipients showing ≥ 0.1% CD45.2 donor contribution were considered positive and GM/(B+T) ratios were calculated as previously described for characterizing heterogeneous HSC phenotypes37. In transplantations using WT or Prdm16−/− HSCs (Lin−cKit+Sca1+CD48−Flt3−CD150+) B6.CD45.2 cells were mixed with 2 × 105 freshly isolated B6.CD45.1 bone marrow cells and injected via tail vein into lethally irradiated (two doses of 478 cGy over 3 h using a Rad Source RS-2000 X-ray irradiator (Brentwood, TN)) B6.CD45.1+CD45.2+ F1 hybrid recipients. After 8 to 15 weeks, peripheral blood (PB) and bone marrow were analysed. Lentiviral particles were produced by seeding 293 cells at 7 × 105 per cm2, or PlatE cells (Cell Biolabs, San Diego, CA), in Ultra Culture serum-free media (Lonza, Basel, Switzerland) overnight followed by transfection of each packaging and expression construct (1:1:1) using Trans-It 293 (Mirus, Madison, WI) for 2 h. Media were pooled after 36–48 h, clarified and concentrated by ultracentrifugation (100,000g), resuspended in StemPro-34 media and stored at −80 °C. Virus titre was calculated from transduction of NIH-3T3 fibroblasts serial dilutions of the viral preparation. Sorted LT-HSCs were transduced with ≥ 150 MOI lentivirus particles in the presence of 6 μg ml−1 polybrene (Sigma) and spun at 900g for 20 min at 20 °C. Supernatant was aspirated and replaced with complete media and cultured overnight. Transduction efficiency of cells was confirmed after 24 h. To assess proviral copy number 15 weeks post-transplantation in vivo, splenocytes were harvested and sorted into donor (CD45.2) or competitor (CD45.1) populations and gDNA was isolated as previously described38. Amplification of the proviral WPRE region was achieved using SYBR Green qPCR assay using the primer pair WPREFor: 5′- -3′ and WPRERev: 5′- -3′. Quantification of proviral copies was derived from the linear regression of serial dilutions of viral vector and normalized to input cell number. Sorted or cultured cell populations (2–5 × 103 cells) were lysed in TRIzol LS reagent (Invitrogen, Carlsbad, CA) and RNA was isolated according to manufacturer’s instructions. cDNA was synthesized using Superscript III Reverse Transcriptase (Invitrogen) and target CT values were determined using inventoried TaqMan probes (Applied Biosystems, Carlsbad, CA, see Supplementary Table 2) spanning exon/exon boundaries and detected using a Viia7 Real Time PCR System (Applied Biosystems). Relative quantification was calculated using the ΔΔC method. To estimate relative copy number of Mfn1 and Mfn2 transcripts (Fig. 4a), copy numbers were derived from the linear regression of serial dilutions of respective cDNA plasmids and normalized to GAPDH-VIC values. To estimate relative copy number of flPrdm16 transcripts (Fig. 4d), a probe was designed to span the SET methyltransferase domain of Prdm16 (exon2/3 junction) and copy number was derived from the linear regression of serial dilutions of respective cDNA plasmids. Another probe (exon 14/15 junction) was used to quantify total Prdm16 copy numbers derived from the linear regression of serial dilutions of respective cDNA plasmids. The values derived from total Prdm16 probe was subtracted from flPrdm16-specific probe to determine sPrdm16 transcript quantity. All values were normalized to relative multiplexed GAPDH-VIC values. Culture of sorted LT-HSCs was carried out using StemPro34 media (Invitrogen) supplemented with 10 mM HEPES and 50 ng ml−1 of recombinant murine SCF, TPO, IL-6 (Peptrotech, Rocky Hill, NJ) and cultured in 5% O at 37 °C. In some experiments, LT-HSCs were cultured in the presence of 500 ng ml−1 VIVIT (Millipore, Billerica, MA) or 30 μM mDivi1 (MolPort, Riga, Latvia). To demonstrate a mitochondrial fusion activity, cell fusion experiments were performed using MEFs as previously described37. Briefly, BacMam baculovirus constructs (Invitrogen) expressing the signalling peptide from cytochrome c fused to either GFP or RFP were transduced separately into MEF cells. Sorted GFP+ and RFP+ MEFs were co-cultured for 24 h and plasma membranes were fused using PEG-1500 (Roche, Basel, Switzerland. Fused cells were cultured in DMEM containing cyclohexamide (Sigma, St. Louis, MO) for 4 h and analysed for colocalization of mitochondrial labels. Early passage Prdm16−/− MEFs were transduced with 10 MOI retrovirus for 72 h and fixed with 4% paraformaldehyde for 10 min. Protein lysates were isolated and chromatin immunoprecipitation was carried out using the ChIP-IT Express Enzyme kit (Active Motif, Carlsbad, CA). Antibodies used for ChIP include anti-Flag and anti-TF2D. Primer probes were designed to span regions of the Mfn2 promoter previously shown to regulate Mfn2 transcriptional activity (see Supplementary Table 3)39. Quantification of precipitated Mfn2 promoter regions were derived from the linear regression of serial dilutions of bone marrow genomic DNA, normalized to input DNA concentration and quantifiable IgG detection was subtracted from sample values. Bone marrow was freshly isolated and lineage depleted with the MACS Lineage Depletion Kit (Miltenyi Biotech, San Diego, CA). Cells were cultured for 30 min in complete medium supplemented with 1 μM Indo-1 prepared as stock supplemented with Pluronic-F127 and incubated at 37 °C for 30 min. Cells were washed and stained for surface markers for 15 min, washed and allowed to rest in for 15 min PBS in PBS with Ca2+. FACS tubes were run at 37 °C in the sample port of the LSRII flow cytometer equipped with a 355 nm excitation laser. Events were collected for 40 s before incubation with 25 μM ATP or 1 μM SDF1 to induce calcium transients. The average ratio, R, of bound/free Indo-1 (405 nm/485 nm emission) before simulation was used to determine baseline values. Identical samples were equilibrated in 10 mM EGTA PBS without Ca2+ to determine R or stimulated with 1 μM ionomycin to determine R . The Indo-1 dissociation constant (K ) was assumed to be 237 nM at 37 °C based on previous studies40. The following equation was then used to relate Indo-1 intensity ratios to [Ca2+] levels; Sorted or cultured haematopoietic populations (2–5 × 103 cells) were collected in complete media and plated on onto MicroWell 96-well glass-bottom plates (Thermo, Waltham, MA) coated with 1ug ml−1 poly-d-lysine. Cells were allowed to adhere for 10 min and fixed with 4% PFA for 15 min. Cells were then permeabilized with 0.1% TritonX-100/PBS for 5 min and blocked with 2% BSA/PBS for 1 h at 4 °C. Cells were incubated with anti-Nfat1 (1:100), anti-Mfn2 (1:200), anti-tubulin (1:200), anti CD150-APC (1:100) or anti-Flag (1:250) (see Supplementary Table 1) overnight, washed and incubated with AlexaFluor secondary antibodies (Invitrogen) for 1 h. Cell nuclei were counterstained with DAPI and mounted with fluorescent mounting media (Vector Labs, Burlingame, CA). Confocal images were acquired with a Zeiss LSM 700 confocal microscope or a Leica DMI 6000B and images were deconvoluted and processed with Leica AF6000 software package. NIH-3T3, WT or Mfn2−/− MEF cells were plated at 2 × 104 cells per cm2 in triplicate overnight and transfected with 500 ng of pGF-Nfat, pGF-CMV or −950/+22 Mfn2-pGL4 reporter construct, 500 ng of cDNA plasmids as indicated and 500 ng of either pSV-βGal or pLVX-IRES-mCherry plasmids with Lipofectamine 3000 according to manufacturer’s instructions for 24 or 48 h. Cells were lysed in reporter lysis buffer (Promega, Madison, WI) and analysed for luciferase activity using BrightGlo luciferase (Promega) and detected on a Synergy H2 plate reader (BioTek, Winooski, VT). To visualize βGal activity, cell lysate was incubated in Buffer Z (1mg ml−1 ONPG, 0.1 M phosphate, pH 7.5, 10 mM KCl, 1 mM βME, 1 mM MgSO ) at 37 °C for 1 h. Absorbance values were measured at 405 nm and used to normalize for transfection efficiency. In WT and Prdm16−/− MEFs, gene reporter luciferase values were normalized to mCherry excitation values. For total cell lysate experiments, MEF cultures were lysed in RIPA buffer, 50 mM Tris pH 7.5, 137 mM NaCl, 0.1% SDS, 0.5% deoxycholate and protease inhibitors (Roche). For subcellular fractionation studies, cells were scraped, washed in PBS. Cell pellets were lysed in 5× packed cell volume (pcv) Buffer A for 10 min on ice and vortexed for 15 s in the presence of 1/10 volume 3% NP-40. Plasma membrane lysis was verified by trypan blue staining. Lysate was spun at 15,000g for 10 min at 4 °C and the cytoplasmic fraction was saved. The remaining nuclear pellet was resuspended in 2.5× pc Buffer C and incubated at 4 °C for 1 h with rotation and spun at 15,000g for 10 min. The nuclear fraction was diluted with 2.5× volume of Nuclear Diluent Buffer and stored at −80 °C. To achieve even fractionation loading, equivalent percentages of nuclear and cytoplasmic fractions were loaded on each gel. All protein samples were denatured in 4× sample buffer at 95 °C and loaded onto 4–12% Bis-Tris SDS–PAGE gradient gels (Invitrogen). Gels were transferred onto 0.22 μm nitrocellulose membrane and stained with Ruby Red (Molecular Probes, Carlsbad, CA) to confirm transfer. Membranes were blocked with 3% non-fat milk or BSA in 0.1%Tween-20/TBS and incubated with anti-Mfn2 (1:200), anti-βGal (1:1,000), anti-Nfat1 (1:250), anti-tubulin (1:1,000), anti-lamin A/C (1:500) and anti-β-actin (1:5,000) overnight (see Supplementary Table 1). Membranes were washed, incubated with HRPO-conjugated secondary antibodies and exposed to X-ray film (Denville) after incubation with Super Signal West Fempto ECL reagent (Pierce). For mitochondrial length measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ (NIH, Bethesda, MD). Individual mitochondria were manually traced, binned into length categories and expressed as percent of cellular mitochondria. The mean ± s.e.m. number of mitochondria falling into each length category collected from ≥ 15 fields (30–50 cells) are expressed. For Nfat nuclear localization quantification, confocal or deconvoluted z-stacks were collected and a 1-μm section in the centre of the cell was projected as a z-project in ImageJ. Nuclear boundaries were constructed using DAPI staining. The ratio of staining within the nuclear boundary to total staining was expressed as percent of Nfat signal. The mean ± s.e.m. for ≥ 10 fields (20–40 cells) are expressed. For immunofluorescence intensity measurements, confocal or deconvoluted z-stacks were collected and projected as a z-project in ImageJ. Thresholds were set based on IgG-stained negative control cells and the integrated density value of each signal per cell was recorded. The mean ± s.e.m. for ≥ 15 fields (30–50 cells) are expressed. For statistical analysis between two groups, the unpaired Student’s t-test was used. When more than two groups were compared, one-way ANOVA was used. Results are expressed as mean ± s.e.m. The Bonferroni and Dunnett multiple comparison tests were used for post-hoc analysis to determine statistical significance between multiple groups. All statistics were calculated using Prism5 (GraphPad, La Jolla, CA) software. Differences among group means were considered significant when the probability value, P, was less than 0.05. Sample size (‘n’) always represents biological replicates. Cochran test was used for exclusion of outliers. 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.
News Article | March 23, 2016
The data on TP53 mutations (including allele frequency) and CNVs in pan-tumours and AML are derived from The Cancer Genome Atlas (TCGA) data in the cBioPortal for Cancer Genomics (http://www.cbioportal.org/; accessed on 29 October 2014). Only sequenced samples with allele frequency information provided were included in our analysis. Considering potential normal tissue contamination, samples with TP53 mutation allele frequency above 0.6 were considered as a homozygous mutation. The SNP data were visualized in IGV and statistics for AML outcome were analysed in Prism 6. Since cBioPortal only has a few non-Hodgkin lymphoma cases available, we used published data to extract TP53 mutation and deletion information18, 30, 31, 32, 33, 34. Clinical outcomes were annotated from follow-up data available within the Gene Expression Omnibus GSE34171 series. CNV analysis was performed using published AML and DLBCL tumour copy number data in Affymetrix SNP Array 6.0 .cel format (http://cancergenome.nih.gov/)18, 35, 36, 37 according to GISTIC2.0 (ref. 14). Specifically, the following GISTIC parameters and values were used following the latest TCGA Copy Number Portal analysis version (3 November 2014 stddata__2014_10_17; http://www.broadinstitute.org/tcga/gistic/browseGisticByTissue): core GISTIC version 2.0.22; reference genome build hg19; amplification threshold 0.1; deletion threshold 0.1; high-level amplification threshold 1.0; high-level deletion threshold 1.0; broad length cut-off 0.50; peak confidence level 0.95; cap 1.5; gene-GISTIC, true; arm-level peel-off, true; significance threshold 0.25; join segment size 8; X chromosome removed, false; maximum segments per sample 2,000; minimum samples per disease 40. To create a conditional 11B3 chromosome deletion, the MICER strategy was used15. Briefly, MICER clones MHPN91j22 (centromeric to Sco1) and MHPP248j19 (telomeric to Alox12) (Sanger Institute) were introduced into AB2.2 ES cells (129S5 strain, Sanger Institute) by sequential electroporation, followed by G418 (neomycin; 180 μg ml−1) and puromycin (1 μg ml−1) selection, respectively. Successful recombination events were confirmed by Southern blotting using the hybridized probes designated in Supplementary Table 2 as described38. The cis- and trans-localizations of two loxP sites in doubly targeted ES cells were further distinguished by PCR with df-F and df-R, or dp-F and dp-R (Supplementary Table 2), respectively, after Adeno-cre infection and HAT (Gibco) selection. Correct cis-ES clones in which two loxP sites were integrated into the same allele were used to generate chimaera mice by blastocyst injection. The F1 pups were genotyped with 11B3-F and 11B3-R primers (Supplementary Table 2) and those positive backcrossed to C57BL/6 mouse strains for more than 10 generations. All of the mouse experiments were approved by the Institutional Animal Care and Use Committee at the Memorial Sloan Kettering Cancer Center. Eμ-Myc, Vav1-cre, Ella-cre, Trp53LSL-R270H/+, Trp53LSL-R72H/+, Trp53+/−, Trp53fl/+ and Rag1−/− mice were ordered from Jackson Laboratories21, 39, 40, 41, 42, 43, 44 and the Arf+/− mouse strain is a gift from C. Sherr45. Eμ-Myc mice with different Trp53 alterations were monitored weekly with disease state being defined by palpable enlarged solid lymph nodes and/or paralysis. Tumour monitoring was done as blinded experiments. For lymphoma generated by transplantation, 1 million Eμ-Myc HPSCs from embryonic day (E)13.5 fetal liver or autoMACS-purified B220+ B progenitor cells isolated from 6–8-week mouse bone marrow were transduced with retroviruses, followed by tail-vein injection into sublethally irradiated (6 Gy, Cs137) C57BL/6 mice (Taconic; 6–8-week old, female, 5–10 mice per cohort)11, 46. All recipient mice were randomly divided into subgroups before transplantation and monitored as described earlier. The generation of AML proceeded as previously reported29. Briefly, retrovirally infected c-Kit+ haematopoietic stem and progenitor cells were transplanted into sublethally irradiated (6 Gy, Cs137) C57BL/6 mice, followed by routine monitoring of peripheral blood cell counts and Giemsa–Wright blood smear staining. For secondary transplantation experiments, 1 million leukaemia cells were transplanted into sublethally irradiated (4.5 Gy) mice. The immunophenotypes of resulting lymphomas and leukaemias were determined by flow cytometry as previously reported using antibodies purchased from eBioscience11, 29. Statistical analysis of all survival data was carried out using the log-rank test from Prism 6. No statistical methods were used to predetermine sample size. MSCV-Myc-IRES-GFP and MLS-based retroviral constructs harbouring a GFP or mCherry fluorescent reporter and targeting Ren, Trp53, Eif5a, Nf1 or Mll3 have all been reported before11, 29, 47. For the tandem shRNA experiments performed in Fig. 3, mirE-based shRNAs targeting two different genes were cloned into an MLS-based vector in an analogous fashion to what has been previously described48, 49. Retrovirus packaging and infection of HSPCs was done as previously reported11, 29. B220+ cells were isolated from the bone marrow of 6-week-old Eμ-Myc mice by autoMACS positive selection with anti-B220 microbeads (Militeny Biotech). After overnight culture, cells were infected with retroviruses carrying the indicated shRNAs. Two days after infection, 0.5 × 106 cells were washed with PBS followed by annexin V buffer (10 mM HEPES, 140 mM NaCl, 25 mM CaCl , pH 7.4), and incubated at room temperature with Pacific Blue annexin V (BD Biosciences) and propridium iodide (PI; 1 μg ml−1; Sigma-Aldrich) for 15 min and analysed on a LSR II flow cytometer (BD Biosciences). For arachidonic acid treatment, pre-B cells were cultured out from bone marrow cells in pre-B cell medium (RPMI1640, 10% FBS, 1% penicillin/streptomycin, 50 μM β-mercaptoethanol, 3 ng ml−1 IL-7). After 3 days culture, pre-B cells were treated with a series concentration of arachidonic acid (Cayman Chemical) for 20 h, followed by annexin V staining as described earlier. Lymphoma cells isolated from lymph nodes of diseased animals were treated with vehicle (PBS) or 1 μg ml−1 adriamycin for 4 h. Whole cell lysates were extracted in cell lysis buffer (Cell Signaling Technology) supplemented with protease inhibitors (Roche), followed by SDS–PAGE gel electrophoresis and blotting onto PVDF membranes (Millipore). Eμ-Myc;Arf−/− lymphoma cell lines were used as a positive control for p53 induction. The p53 antibody used was obtained from Novocastra (NCL-p53-505) and horseradish peroxidase (HRP)-conjugated β-actin antibody from Sigma (AC-15). Alox15b expressions were examined in NIH3T3 cells, which were infected by shRNAs targeting Ren or Alox15b and then selected by G418. Anti-Alox15b antibody is from Sigma (SAB2100110), and HRP-conjugated GAPDH antibody is from ThermoFisher Scientific (MA5-15738-HRP). RNA-seq and data analysis were performed by the Integrated Genomic and Bioinformatics core at the Memorial Sloan Kettering Cancer Center. Briefly, total RNA from 11B3fl/Trp53fl;shNf1;shMll3;Vav1-cre or Trp53fl/fl;shNf1;shMll3;Vav1-cre leukaemia cells (four lines per cohort), isolated from the bone marrow of moribund mice, was isolated by Trizol extraction (Life Technologies). After ribogreen quantification (Life Technologies) and quality control on an Agilent BioAnalyzer, 500 ng of total RNA (RNA integrity number > 8) underwent polyA selection and Truseq library preparation according to instructions provided by Illumina (TruSeq RNA Sample Prep Kit v.2) with 6 cycles of PCR. Samples were barcoded and run on a Hiseq 2500 in a 50 bp/50 bp paired-end run, using the TruSeq SBS Kit v.3 (Illumina). An average of 45 million paired reads were generated per sample. At the most the ribosomal reads represented 0.1% and the percentage of mRNA bases was close to 65% on average. The output from the sequencers (FASTQ files) was mapped to the mouse genome (mm9) using the rnaStar (https://code.google.com/p/rna-star/) aligner, with the two-pass mapping methods. After mapping, the expression counts of each individual gene were computed using HTSeq (http://www-huber.embl.de/users/anders/HTSeq), followed by normalization and differential expression analysis among samples using the R/Bioconductor package DESeq (http://www-huber.embl.de/users/anders/DESeq). Gene set enrichment analysis (GSEA) was performed with Broad’s GSEA algorithm. A list of all primers used for PCR analysis is given in Supplementary Table 2. For detection and quantification of 11B3 recombination/deletion two methods were employed. In both cases genomic DNA (gDNA) was extracted from lymphoma or leukaemia cells using Puregene DNA purification kit (Qiagen). Initially, semi-quantitative PCR was used to detect the recombined 11B3 allele using primers df-F and df-R, generating a 2.2 kb product (Fig. 2d). The estimated frequency of recombination was determined by dropping gDNA from 11B3+/− into 11B3fl/+ at various ratios. For qPCR of the 11B3 deletion (Fig. 2e), SYBR Green PCR Master Mix (Applied Biosystems) was used and cycling and analysis was carried out on a ViiA 7 (Applied Biosystems). Primers 11B3-Q-F and 11B3-Q-R were used to detect the floxed allele, and to estimate the frequency of 11B3 deletion. Allelic frequency in UPD analysis (Extended Data Fig. 5a) was determined similarly, in this case with serial dilution of wild-type gDNA into DNase-free water to construct a standard curve. Two-tailed t-test is used for statistics analysis by Prism 6. For p21 gene expression examination by RT–qPCR, RNA was isolated with Trizol, cDNA was synthesized with SuperScript III First-Strand Synthesis System (Life Technologies) and qPCR was performed as described earlier with primers p21-Q-F and p21-Q-R. Trp53 exons (2–10) were amplified from genomic DNAs of 11B3-deleted lymphomas by PCR (see Supplementary Table 2 for primer sequences) and subjected to Sanger sequencing. Mutations were called only if detected in sequencing reads carried out in the forward and reverse direction. SNP analysis of isolated lymphoma (tumour) or tail (normal) genomic DNAs from the same tumour-bearing mouse were carried out by Charles River laboratory. Briefly, a SNP Taqman assay with competing FAM- or VIC-labelled probes was used to detect the relevant C57BL/6 and 129S SNPs (D11Mit4 and D11NDS16) as described previously50. Genomic DNA was extracted from freshly isolated lymphoma cells from one Eμ-Myc;11B3fl/+;Vav-cre mice. One microgram of DNA was sonicated (17 W, 75 s) on an E220 sonicator (Covaris). Samples were subsequently prepared using standard Illumina library preparation (end repair, poly A addition, and adaptor ligation). Libraries were purified using AMPure XP magnetic beads (Beckman Coulter), PCR enriched, and sequenced on an Illumina HiSeq instrument in a multiplexed format. Sequencing reads per sample were mapped using Bowtie with PCR duplicates removed. Approximately 2.5 million uniquely mappable reads were further processed for copy number determination using the ‘varbin’ algorithm51, 52 with 5,000 bins, allowing for a median resolution of ~600 kb. GC content normalization, segmentation and copy number estimation was calculated as described53. A custom shRNA library was designed to target mouse homologues (six shRNAs for one gene) to all human protein-coding genes on chromosome 17p13.1 from ALOX12 to SCO1, except TP53 and EIF5A. shRNAs were cloned into a retrovirus-based vector MLS by pool-specific PCR as previously described11. Eμ-Myc HSPCs infected with pooled shRNAs were transplanted into sublethally irradiated recipient mice. Resulting tumours were harvested, and used to extract contained shRNAs, followed by HiSeq in HiSeq 2500 (Illumina). Twenty-two oligonucleotides of shRNAs used in this study are listed in Supplementary Table 3. Total lipids were extracted using Folch’s method54 and analysed by LC-MS as previously described55. Briefly, freshly harvested cells were homogenized by chloroform/methanol (2:1, v-v). After being washed by water, the lipid-containing chloroform phase is evaporated. Dried lipids were dissolved in 100 μl 95% acetonitrile (in H O), sonicated for 3–5 min, and spiked with 10 μl of 500 ng ml−1 deuterated internal standard solution (IS; arachidonic acid-d8; Cayman Chemical, 390010). Then, 5 μl samples were injected into Acquity ultra performance liquid chromatography (UPLC) system (Waters), equipped with Acquity UPLC BEH C18 column (100 mm × 2.1 mm I.D., 1.7 μm; Waters). Samples were washed through the column with a gradient 0.1% formic acid: acetonitrile mobile elution from 35:65 (v:v) to 5:95 for 10 min. Flow rate was 0.25 ml min−1. Right after HPLC, samples were analysed in a Quattro Premier EX triple quadrupole mass spectrometer (Waters), which has electrospray negative mode and MasslynxV4.1 software. For each run, a standard curve was generated with different concentration of arachidonic acid lipid maps MS standard (Cayman Chemical, 10007268) mixed with IS (50 ng ml−1 final concentration). Arachidonic acid standard m/z is 303.2, and IS is 311.3. Three Eμ-Myc lymphoma cell lines generated from Trp53fl/+;Vav1-cre or 11B3fl/+;Vav1-cre tumour-bearing mice were cultured in BCM medium (45% DMEM, 45% IMDM, 10% FBS, 2 mM glutamine, 50 μM β- mercaptoethanol, 1× penicillin/streptomycin) in 96-well plates. Cells were treated with the indicated concentrations of 4-hydroxycyclophosphamide (Toronto Research Chemicals) or vincristine (Bedford Laboratories) for 3 days. The number of living cells was determined by PI staining and cell counting on a Guava EasyCyte (EMD Millipore). Leukaemia cell lines from Trp53∆/∆ or 11B3∆/Trp53∆;shNf1;shMll3 mice were treated with cytarabine (araC; Bedford Laboratories) or JQ1 (a gift from J. Bradner) in stem cell medium (BCM medium supplemented with 1 ng ml−1 IL-3, 4 ng ml−1 IL-6 and 10 ng ml−1 SCF) and cell viability after 3 days was determined similarly. All cytokines are from Invitrogen.
News Article | February 22, 2017
The study was approved by the ethics committee of the medical faculty and the university clinics of the University of Tübingen and strictly adhered to Good Clinical Practice and the principles of the Declaration of Helsinki. The study is registered at ClinicalTrials.gov (https://clinicaltrials.gov/ct2/show/NCT02115516) and in the EudraCT database, number 2013-003900-38. The study was carried out under FDA IND 15862 and with approval of the Paul-Ehrlich-Institute. Volunteers were healthy, 18–45 years old, malaria-naive adults. The full list of inclusion and exclusion criteria is given in the clinical trial protocol (Supplementary Information). All volunteers, except those in the second part who received the every 5-day regimen, received 10 mg kg−1 or 620 mg chloroquine base (Resochin, Bayer) loading dose 2 days before the first dose of PfSPZ Challenge, whichever dose was less, followed by weekly doses of 5 mg kg−1 or 310 mg through 5 days after the last dose of PfSPZ Challenge. Volunteers who were immunized on days 0, 5, and 10 received chloroquine on days 0 (loading dose), 5, 10, and 15. Randomization was performed on the day of first immunization by an independent party through provision of sealed envelopes to the syringe preparation team members, who diluted PfSPZ Challenge12, 13, 34 or loaded placebo (normal saline) into syringes at an allocation ratio of 9:5, PfSPZ:placebo. Only the syringe preparation team was aware of allocation to the intervention and had no other role in the trial. The rest of the team remained blinded until completion of CHMI of group III. PfSPZ Challenge dose escalation for groups I, II, and III was staggered by at least four weeks and in each group three sentinel volunteers (PfSPZ:placebo, 2:1) received injections one to seven days before the main group. For group I, II, and III the three immunizations were given at 28-day intervals and CHMI by DVI of 3.2 × 103 PfSPZ was done 8–10 weeks following the last immunization. In the second part the three immunizations were administered at 14-day and 5-day intervals and CHMI was done at 10 weeks post immunization. Chloroquine concentrations were measured by mass spectrometry in the blood of selected volunteers on the day before CHMI to exclude carry-over from the immunization period. Following CHMI volunteers were regularly visited for 20 weeks. The primary efficacy endpoint was the proportion of volunteers with thick blood smears positive for Pf within 21 days of CHMI, primary safety endpoint was occurrence of related grade 3 or serious adverse events from first chloroquine dose until the end of the follow-up. We performed a randomized, placebo-controlled, double-blind trial in healthy, malaria-naive, 18–45 year olds (TÜCHMI-002; ClinicalTrials.gov ID: NCT02115516). Between 1 May and 4 July 2014, 42 volunteers were randomized to receive either three doses of normal saline (placebo) or 3.2 × 103 PfSPZ (PfSPZ Challenge12, 13, 34) (group I), 1.28 × 104 PfSPZ (group II), or 5.12 × 104 PfSPZ (group III) by DVI12, 13 at four-week intervals (Extended Data Fig. 2). All volunteers received oral chemoprophylaxis with chloroquine starting with a loading dose (10 mg kg−1 chloroquine base or 620 mg, whichever dose was less) two days before first PfSPZ inoculation followed by weekly maintenance doses (5 mg kg−1) through five days after last PfSPZ inoculation; total of 10 doses. Chloroquine is not active against SPZ or liver-stage parasites35 and Pf asexual blood-stage parasites leave the liver between days 5 and 6 following inoculation36; hence dosing five days following inoculation ensured high drug levels upon liver egress. Dose-escalation was staggered in four-week intervals and at each dose escalation the ratio of placebo-immunized to PfSPZ-CVac-immunized subjects was 5:9. Following PfSPZ dose escalation, accelerated regimens (14- and 5-day intervals) were assessed using essentially the same procedures. A full report will be published separately. Eight to ten weeks after last vaccine or placebo dose (51–67 days after last chloroquine dose), protective efficacy was assessed by CHMI using DVI with PfSPZ Challenge12, 13. Daily thick blood smears were performed as previously described37 from day 6 to 21, following each DVI for immunization and CHMI, during antimalarial treatment and at each late follow-up visit. Slides were considered positive when at least two readers detected two unambiguous parasites, each. A negative slide was defined as no observed parasites in the volume of blood required to detect with 95% probability less than 10 parasites per μl (~0.5 μl). In case of discordance, a third reading was performed. In addition, 1.2 ml blood was sampled in EDTA tubes (Sarstedt) for nucleic acid extraction at the same time-points. DNA extraction control 610 (Bioline) was added and total nucleic acid (DNA and RNA) was isolated from 0.5 ml blood using the QIAamp DNA blood mini kit (Qiagen) according to the manufacturer’s specifications but without addition of RNase. Parasitaemia was calculated from a standard curve generated from serially diluted (2–20,000 parasites per ml) Pf 3D7 ring-stage synchronized cultured parasites, counted by microscopy and cytometry. All purified nucleic acid samples were stored at –20 °C until time of use. Reverse transcription quantitative polymerase chain reaction (RT–qPCR) was performed using TaqMan RNA-to-CT 1-Step Kit using published primers and probes38, with a different fluorophore and addition of a minor groove binder (probe: VIC-ATGGCCGTTTTTAGTTCGTG-NFQMGB; primers: 5′-GCTCTTTCTTGATTTCTTGGATG-3′ and 5′-AGCAGGTTAAGATCTCGTTCG-3′). Reactions were done in 384 wells at 48 °C for 20 min (reverse transcription), 96 °C for 10 min (enzyme activation), and 45 cycles of 95 °C for 15 s, 62 °C 1 min. Samples were run as triplicates with no-template control, no-RT control and positive controls in the same plate. Amplification controls were assessed manually and cycle values (C ) were calculated with the second derivative maximum method (LightCycler 480 software; version 184.108.40.206). The assay was validated in accordance to MIQE guidelines38, 39 and had a lower limit of quantification of 3 parasites per ml. qPCR results were not reported to the clinical and microscopy teams during the study period to maintain blinding of the study. Sample size was calculated with the intention to show, with a power of 80% and a two-tailed alpha of 5%, a difference in proportion of infected volunteers of 25% or less of immunized volunteers and 99% in controls, allocated in a 2:1 ratio (8 PfSPZ:4 controls), allowing for one dropout in each group (9:5). Clinical data were captured on paper case report forms and transferred to an electronic database (OpenClinica; version 3.2) by double data entry. Efficacy data were reported as proportions (primary) and time to parasitaemia. Safety and tolerability data were listed and reported as summary statistics. Results of immunological assays were explored by post hoc analyses and used to generate hypotheses about correlates and immunological mechanisms of protection. Analyses were coded in R (version 3.2.3)40 when not otherwise stated. Where possible, estimate and 95% confidence interval are given. Box plots display median (middle line), 25th (lower hinge) and 75th (upper hinge) quartile. Whiskers extend to values within 1.5× the inter-quartile ranges of the lower and upper hinges, respectively. A two-sided P value less than 5% was considered statistically significant. Flow cytometry data were analysed with Pestle v1.7, SPICE v5.3 (ref. 41) and Prism 6 (GraphPad). Graphs were rendered in FlowJo, SPICE, and Prism. For vaccine immunogenicity, comparisons to pre-vaccine were performed using Wilcoxon signed rank test with Bonferroni correction for multiple comparisons or two-way ANOVA with Bonferroni correction, as specified in the text. Immune responses assessed at baseline, two weeks after final immunization, and at the time of CHMI were compared to outcome (parasitaemia or no parasitaemia) after CHMI. Assessment of immune responses that correlated with sterile protection was made using a stratified Wilcoxon test controlling for vaccine dose group as a covariate. Sera were assessed for vaccine-induced antibodies by ELISA (enzyme-linked immunosorbent assay), immunofluorescence assay, inhibition of sporozoite invasion assay and protein arrays representing 91% of the Pf proteome. ELISAs were performed for antigens first expressed in PfSPZ (PfCSP, PfSSP2/TRAP, PfCelTOS, PfMSP5, PfAMA1), early liver stages (PfEXP1 and PfLSA1) and late liver stages (PfMSP1 and PfEBA175). The ELISA methods for each antigen are described below. Recombinant (r) proteins used in ELISA assays are listed in Supplementary Table 7. 96-well plates (Nunc Maxisorp Immuno Plate) were coated overnight at 4 °C with 0.5 μg to 2.0 μg recombinant proteins (Supplementary Table 7) in 50 μl per well in coating buffer (KPL). Plates were washed three times with 2 mM imidazole, 160 mM NaCl, 0.02% Tween 20, 0.5 mM EDTA and blocked with 1% Bovine Serum Albumin (BSA) blocking buffer (KPL) containing 1% or 5% non-fat dry milk (Supplementary Table 7) for 1 h at 37 °C. Plates were washed three times and serially diluted serum samples (in triplicates) were added and incubated at 37 °C for 1 h. After three washes, peroxidase labelled goat anti-human IgG (KPL) was added at a dilution of 0.05 μg ml−1 to 0.2 μg ml−1 (Supplementary Table 7) and incubated at 37 °C for 1 h. Plates were washed three times, ABTS peroxidase substrate was added for plate development, and the plates were incubated for defined periods at 22 °C room temperature (Supplementary Table 7). The plates were read with a Spectramax Plus384 microplate reader (Molecular Devices) at 405 nm. The data were collected using Softmax Pro GXP v5 and fit to a 4-parameter logistic curve, to calculate the serum dilution at OD 1.0. A negative control (pooled serum from non-immune individuals from malaria free area) was included in all assays. The following positive control sera were used for the different antigens: serum from an individual with anti-PfCSP antibodies for PfCSP; pooled sera from individuals immunized with PfLSA-1 and PfEBA-175 subunit vaccines respectively for PfLSA1 and PfEBA175; pooled sera from volunteers from a malaria-endemic area (acquired from a blood bank in Kenya) for PfAMA1, PfEXP1, and PfMSP1. No positive control sera were available for PfMSP5, PfSSP2/TRAP or PfCelTOS. Samples were considered positive if the difference between the post-immunization OD 1.0 and the pre-immunization OD 1.0 (net OD 1.0) was ≥50 and the ratio of post-immunization OD 1.0 to pre-immunization OD 1.0 (ratio) was ≥3. Purified PfSPZ (NF54 strain) from aseptic Anopheles stephensi mosquitoes produced by Sanaria were resuspended in phosphate buffered saline (PBS (pH 7.4)) to obtain 5 × 103 PfSPZ per 40 μl. 40 μl (5 × 103 PfSPZ) were added to each well of Greiner cellstar clear-bottom black 96-well plates (Sigma-Aldrich). After addition of the suspension, plates were left at room temperature for 12–18 h for air-drying. 50 μl of sera diluted in PBS with 2% BSA were added to each well of the 96-well plate containing air-dried PfSPZ. Sera samples were added at twofold dilutions starting at 1:50. After adding samples, plates were incubated at 37 °C for 1 h. Plates were washed in PBS three times on an Aquamax Microplate washer. Alexa Fluor 488 conjugated goat anti-human IgG (Molecular Probes) was diluted to 1:250 in PBS with 2% BSA and 50 μl was added to each well. The plates were then incubated for 1 h at 37 °C. Plates were washed three times with PBS on an Aquamax Microplate washer. 100 μl PBS was added to each well. The plates were sealed using a plate sealer and stored in the dark at 4 °C until data acquisition. Samples were assessed by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The positive control was pooled human serum taken two weeks after the last immunization from 12 uninfected volunteers immunized 4 or 5 times with 1.35 × 105 PfSPZ Vaccine5. The Acumen image cytometer scans the entire surface area of each well in a 96-well plate and the fluorescence intensity values (arbitrary units) therefore represent the signal from all 5 × 103 PfSPZ that were seeded in each well. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units (AFU) on the y axis. Over many iterations during development of this assay we have determined that sera from naive volunteers in the USA and Europe, including pre-immune sera, always register an arbitrary fluorescence value less than 2 × 105 even at the highest concentration (1:50 dilution, see above) used in this assay. Moreover, for sera that react to PfSPZ, 2 × 105 AFU falls in the exponential portion of their sigmoidal curves. Therefore, 2 × 105 was chosen as a threshold in the automated immunofluorescence assay and the results for each volunteer for antibodies to PfSPZ are reported as the reciprocal serum dilution at which fluorescence intensity was equal to 2 × 105 AFU. HC-04 (1F9) (ref. 42) cells (hepatocytes) were obtained from the Naval Medical Research Center. Master and working cell banks were produced, and after establishing they were free of mycoplasma, were quality control released. For the assay they were cultured in complete medium (10% FBS in DMEM/F12 with 100 units per ml penicillin and 100 μg per ml streptomycin; Gibco by Life Technologies) in Entactic-Collagen IV-Laminin (ECL)-coated 96-well clear bottom black well plates (Greiner Bio-One North America) at a density of 2.5 × 104 cells per well, and incubated for 24 h at 37 °C, 5% CO with 85% relative humidity. Twenty-four hours later cells were infected with 104 aseptic, purified, cryopreserved PfSPZ per well, without or with sera diluted in a 12-point series from subjects immunized with PfSPZ Vaccine. The assay control included PfSPZ added with media alone. All subjects were assessed at pre-immunization (baseline), post-immunization and pre-CHMI time points. Three hours later, PfSPZ that had not invaded the HC-04 cells were removed by washing three times with Dulbecco’s phosphate-buffered saline (DPBS), and the cultures were fixed using 4% paraformaldehyde for 15 min at room temperature. Differential immunostaining was performed to differentiate between PfSPZ inside the hepatocytes versus PfSPZ outside the hepatocytes. PfSPZ outside the hepatocytes were stained with an anti-PfCSP mAb (2A10, 6.86 μg ml−1) (Protein Potential LLC, with permission from New York University School of Medicine) conjugated with Alexa Fluor 633 (far-red) (1 μg ml−1; custom-conjugated at GenScript). The hepatocytes were then permeabilized with 0.1% Triton X-100 for 10 min at room temperature, and the PfSPZ inside the hepatocytes were stained with the anti-PfCSP mAb (2A10, 6.86 μg ml−1) conjugated with Alexa Fluor 488 (green; 1 μg ml−1, conjugated from Genscript). The numbers and intensity of infected hepatocytes (green only) were counted by scanning the plates using an Acumen eX3 laser scanning imaging cytometer. The data obtained from the Acumen image cytometer were plotted to fit a 4-parameter sigmoidal curve in GraphPad Prism software using serum dilution (log transformed) as the x axis variable and arbitrary fluorescence units on the y axis. 75% inhibition was interpolated from the sigmoidal curves as the reciprocal serum dilution at which the fluorescent intensity of infected wells with serum was 25% of the negative control without serum. The number of invaded PfSPZ scored in this assay in the absence of sera ranged from 400–600 (intensity of 1–3 × 106 fluorescence units) (4% to 6% of those added to the wells). A whole-proteome microarray with 91% coverage of the Pf proteome (PfWPM) was produced by Antigen Discovery, Inc. (ADI). Proteins were expressed as previously described43 from a library of Pf partial or complete open reading frames (ORFs) cloned into a T7 expression vector pXI using an in vitro transcription and translation (IVTT) system, the Escherichia coli cell-free Rapid Translation System (RTS) kit (5 Prime). The library was created through an in vivo recombination cloning process with PCR-amplified Pf ORFs, and a complementary linearized expressed vector transformed into chemically competent E. coli was amplified by PCR and cloned into pXI vector using a high-throughput PCR recombination cloning method described elsewhere44. Each expressed protein includes a 5′ polyhistidine (HIS) epitope and 3′ haemagglutinin (HA) epitope. After expressing the proteins according to manufacturer’s instructions, translated proteins were printed onto nitrocellulose-coated glass AVID slides (Grace Bio-Labs) using an Omni Grid Accent robotic microarray printer (Digilabs, Inc.). Microarray chip printing and protein expression were quality checked by probing random slides with anti-HIS and anti-HA monoclonal antibodies with fluorescent labelling. PfWPM chips contained 7,455 Pf peptide fragments, representing proteins from 4,805 unique genes, 302 IgG positive control spots and 192 spotted IVTT reactions without Pf ORFs (IVTT controls). For each PfWPM chip, 3 replicates were printed per microarray slide on 3 nitrocellulose pads. IgG-positive control spots were included as an assay control, whereas IVTT control spots were included as a sample-level normalization factor. Serum samples were diluted 1:100 in a 3 mg ml−1 E. coli lysate solution in protein arraying buffer (Maine Manufacturing) and incubated at room temperature for 30 min. Chips were rehydrated in blocking buffer for 30 min. Blocking buffer was removed, and chips were probed with pre-incubated serum samples using sealed, fitted slide chambers to ensure no cross-contamination of sample between pads. Chips were incubated overnight at 4 °C with agitation. Chips were washed five times with TBS-0.05% Tween 20, followed by incubation with biotin-conjugated goat anti-human IgG (Jackson ImmunoResearch) diluted 1:200 in blocking buffer at room temperature. Chips were washed three times with TBS-0.05% Tween 20, followed by incubation with streptavidin-conjugated SureLight P-3 (Columbia Biosciences) at room temperature protected from light. Chips were washed three times with TBS-0.05% Tween 20, three times with TBS, and once with water. Chips were air dried by centrifugation at 1,000g for 4 min and scanned on a ScanArray Express HT spectral scanner (Perkin-Elmer), and spot and background intensities were measured using an annotated grid file (.GAL). Data were exported and normalized using the IVTT control spots for statistical analysis in R40. Raw spot and local background fluorescence intensities, spot annotations and sample phenotypes were imported and merged in R, where all subsequent procedures were performed40. Foreground spot intensities were adjusted by local background by subtraction, and negative values were converted to 1. Next, all foreground values were transformed using the base 2 logarithm (log ). The dataset was normalized to remove systematic effects by subtracting the median signal intensity of the IVTT controls for each sample. As the IVTT control spots carry the chip, sample and batch-level systematic effects, but also antibody background activity to the IVTT system, this procedure normalizes the data and provides a relative measure of the specific antibody binding to the non-specific antibody binding to the IVTT controls (a.k.a. background). With the normalized data, a value of 0.0 means that the intensity is no different than the background and a value of 1.0 indicates a doubling with respect to background. A seropositivity threshold was established for each protein on the chip using the top 2.5th percentile of the pre-immunization samples for each protein. Reactive antigens were defined as those that had seropositive responses after immunization and before CHMI, but which did not show seropositive responses in the mock-immunization group. PBMCs were isolated by density-gradient centrifugation from heparinized whole blood. Assessment of cellular immune responses using multi-parameter flow cytometry was performed on PBMCs from cryopreserved samples at the completion of the study, as described6. In brief, PBMCs were thawed and rested in complete RPMI for 8 h followed by stimulation for 17 h with: (1) 1.5 × 105 viable, irradiated, aseptic, purified, cryopreserved PfSPZ from a single production lot; (2) PfSPZ Vaccine diluent (1% human serum albumin, HSA, CSL Behring); (3) 2 × 105 lysed RBC, >90% infected with late-stage schizonts (PfRBC) from a single production lot; or (4) 2 × 105 donor-matched uninfected erythrocytes from a single production lot. For the last 5 h of the stimulation, 10 μg ml−1 Brefeldin A (BD) was added to the culture. After stimulation, cells were stained as previously described45. The staining panels are shown in Supplementary Table 8 and the antibody clones and manufacturers are shown in Supplementary Table 9. Briefly, cells were surface stained with CCR7 at 37 °C for 20 min. Dead cells were identified by Aqua Live-Dead dye (Invitrogen), as per manufacturer’s instructions. This was followed by 15 min surface staining at room temperature for CD4, CD8, CD14, CD38, CD45RA, CD56, CD57, CD127, CD161, TCR-γδ, TCR-Vδ1, TCR-Vδ2, TCR-Vγ9, TCR-Vα7.2, CXCR6, or PD-1. Cells were washed, fixed, and permeabilized using Cytofix/Cytoperm kit (BD) and stained intracellularly for CD3, IFN-γ, IL-2, TNF-α, IL-4, IL-10, perforin, or Ki-67. Cells were washed, fixed in 0.5% paraformaldehyde, and measured on a modified LSR II (BD). Flow cytometry data were analysed using FlowJo v9.9 (Tree Star) blinded to vaccination group and CHMI outcome. All antigen-specific cytokine frequencies are reported after background subtraction of identical gates from the same sample incubated with the control antigen stimulation (HSA or uninfected erythrocytes). The data that support the findings of these studies are available in part on request from the corresponding author (S.L.H.) subject to restrictions. Some data are not publicly available, as they contain information that could compromise research participant privacy/consent.
News Article | November 28, 2016
Harris Teeter is proud to welcome shoppers to its Berewick Town Center location on Wednesday, December 7, 2016 at 8 a.m. as the company celebrates its grand opening with a ribbon cutting ceremony. In each of its stores, Harris Teeter strives to offer customers an excellent shopping experience, which begins with customer service and features high-quality products, variety and selection. Harris Teeter also works to be a true community partner by supporting local schools and youth sports organizations, among other non-profit organizations. Berewick Town Center will have a Harris Teeter Fuel Center, which will open at a later date. Once open, Harris Teeter customers can expect $0.03 off per gallon, every day with the use of a VIC card. Fast Facts Store Address: Berewick Town Center, 4821 Berewick Town Center Drive, Charlotte, NC 28278 Square Footage: 53,000 Grand Opening Date: Wednesday, December 7, 2016 Grand Opening Time: 8 a.m., ribbon cutting, store open immediately following Store Hours: 24 hours a day, 7 days a week Pharmacy Hours: Mon.-Fri. 9 a.m.-9 p.m.; Sat. 9 a.m.-7 p.m.; Sun. 10 a.m.-6 p.m. Check-Out Lanes: 9 checkouts and 4 express checkouts Store Features and Departments Full-service Butchers Market with Rancher Beef, HT Reserve Angus Beef and HT Naturals Beef • Expanded Service meat • Fresh store-made sausage and burgers •Full-service Fishermans Market • Farmers Market Produce • Full-Service Floral and Custom Floral Arrangements • Fresh Trail Mix Bar • Salad Bar • Expanded Organic and Specialty Produce • Full-service Fresh Foods Market Deli/Bakery • Sushi • Self-Serve Olives • Asian Hot Bar • International Cheeses • Custom Cakes • Sub Shop • Made to Order Sandwich Program • Artisan Breads • Boar’s Head Meats and Cheeses • Fresh Made Salads • Fresh Made Pizza • Organic and Natural Foods • International Foods • Beer and Wine • Local and Craft Beer • Build your own 6-pack • Drive Thru Pharmacy • Free Blood Pressure Testing • Private, Professional Consultations • Drug Interaction/ Allergy Screening • Double Coupons • Club 60 Discount • Carryout Service • Parcel Pick-up • USCAN • Western Union • Coinstar • Rug Doctor • Express Lane Online Shopping • Red Box DVD Rental Kiosk • ATM • Starbucks • Sit-down eating area
News Article | December 7, 2016
Beer drinkers in Vietnam may have a little more to celebrate as the local brew – Saigon Beer Alcohol Beverage JSC (Sabeco) has officially posted its 641,281,186 shares at the HCMC Stock Exchange (HOSE) under the code SAB. The shares are traded at the starting price of VND 110,000 per share (about US$ 4.80) and by the end of the first trading day, Sabeco stocks have jumped to its maximum of 20 percent up to VND 132,000 per share. This news has excited local and foreign investors, many of whom have been waiting for further shares to become available since Sabeco initial public offering in 2008.Both events are considered separate in Vietnam. Sabeco’s market value has also increased to 83.4 trillion dong (about US$3.6 million). This market capitalization ranked the company as the fifth largest listed firm on HOSE behind dairy firm Vinamilk (VNM), Vietcombank (VCB), Vietnam Petrol Gas Corp (GAS) and the Vingroup (VIC). Sabeco is the state-owned maker of Saigon Beer and 333 Beer and with this event, the second largest consumer company after Vietnam Dairy Products JSC on the exchange. It’s also the largest brewer in Vietnam, where brewing Vietnam’s biggest labels “Saigon”, has reported a sales revenue of VND 21.8 trillion for the first 9 months of 2016, an increase of 9 percent year on year. With this listing in HOSE, it will expand Vietnam’s US$77 billion stock market as it increases the size and liquidity of the benchmark VN-index, which is poised for a fifth straight year of gains. “Sabeco’s listing is a positive and important step for a country that is embracing and liberalizing its own corporation,” said Federico Parenti, a Milan-based fund manager at Sempione Sim Spa, “The listing will be successful because the market is hungry for these type of businesses.” In fact, seven companies including Heineken NV, Anheuser-Busch InBev NV and Asashi Group Holdings Ltd. have already registered to bid for Sabeco. The outlook on Vietnam beer market is an enthusiastic one as demand for beer is expected to continue growing in Vietnam in light of its population increases and the expansion of Vietnam middle class. VN Express even estimates the thirst for Saigon beer to increase by 30 percent during the upcoming Lunar New Year holiday, to a projection of 40 million liters of beer.
News Article | December 4, 2016
LOS ANGELES and MELBOURNE, Australia, Dec. 4, 2016 /PRNewswire/ -- Live Nation Entertainment, Inc. (NYSE: LYV) today announced that it has acquired 51% in Secret Sounds Group, the Australian company led by Jessica Ducrou and Paul Piticco, which owns and produces two of Australia's leading festivals: Splendour in the Grass (located in Byron Bay New South Wales) and Falls Music & Arts Festival (located in Byron Bay, Lorne Victoria, Marion Bay Tasmania and Fremantle Western Australia). Splendour in the Grass and Falls are the first Australian festivals to join Live Nation, which has the largest and most diverse festival portfolio in the world, encompassing more than 80 global festivals, including the landmark Governors Ball, Lollapalooza, Bonnaroo, Austin City Limits, Electric Daisy Carnival, Reading, Leeds, Rock Werchter and many more. Live Nation continues to grow its unparalleled festival platform and currently operates over half of the top 25 festivals around the world. "Splendour in the Grass and Falls are the two most iconic festivals in Australia," said Michael Rapino, President and CEO, Live Nation Entertainment. "Jess and Paul have created events that attract the biggest artists in the world but still feel uniquely Australian. We look forward to partnering with them to find new ways to grow our live event footprint across Australia." Michael Coppel, President and CEO of Live Nation Australia and New Zealand added, "We wholeheartedly welcome Jess and Paul and their teams into the rapidly growing Live Nation Australia family. Our new relationship with the Secret Sounds group confirms Live Nation's position at the forefront of the Australian live performance industry." "We started Secret Sounds with the sole mission of introducing Australians to the best artists, events and music possible, and Live Nation is the perfect partner to help us take this goal to the next level. With their support we can continue to curate the lineups that fans love, while also experimenting with new exciting ways for fans to experience live music," said Ducrou and Piticco, Co-CEO's of Secret Sounds. After 16 successful years, Splendour in the Grass attracts a national audience to its home base at North Byron Parklands, Byron Bay. It is currently the largest contemporary music festival in Australia, and has had the pleasure of featuring artists such as Coldplay, Florence & The Machine, Blur, The Strokes, The Cure, Kanye West, Flume, Vance Joy, Mumford & Sons, Jack White, The Avalanches and many more. After 24 years in the making, The Falls Music & Arts Festival is one of Australia's longest running coastal New Year's Eve festivals and has now expanded to four destinations around the country – Lorne (VIC), Marion Bay (TAS), Byron Bay (NSW) and Fremantle (WA) hosting over 60,000 people per day nationally. Recent headliners Disclosure, Arctic Monkeys, Kings of Leon, Childish Gambino & London Grammar have made it a rite of passage for many young Australians. The acquisition also includes Secret Sounds' touring, sponsorship, PR, artist management and domestic agency businesses. The partnership excludes the Dew Process & Create Control Record Labels, Dew Process Publishing and its interests in the North Byron Parklands festival site and The Triffid venue in Brisbane. About Live Nation Entertainment Live Nation Entertainment (NYSE: LYV) is the world's leading live entertainment company comprised of global market leaders: Ticketmaster, Live Nation Concerts, Live Nation Media & Sponsorship and Artist Nation Management. For additional information, visit www.livenationentertainment.com. About Secret Sounds Secret Sounds is a multi-faceted music platform comprising of Splendour In The Grass & Falls Festivals, Dew Process/Create Control Record Labels and Publishing, Secret Sounds Touring, Village Sounds Agency, Secret Sounds Connect, Secret Service Artist Management & Secret Service Public Relations, Whole Lot of Love Creative Agency & interests in venues North Byron Parklands & Brisbane's The Triffid. www.secretsounds.com
News Article | February 27, 2017
Harris Teeter is proud to welcome customers to its Quarterpath Crossing Fuel Center on Friday, March 3, 2017 as the company celebrates its grand opening with a $0.20 off per gallon fuel promotion. The Fuel Center, which is located in close proximity to the Quarterpath Crossing Harris Teeter, will offer customers $0.03 off per gallon every day with the use of a VIC card, but shoppers are encouraged to fill up during the grand opening when the Center will feature a special $0.20 off per gallon discount March 3-5, 2017. This location is Harris Teeter’s first fuel center in Virginia. The company operates 15 other fuel centers throughout North and South Carolina. At each of its Fuel Centers, Harris Teeter strives to provide customers an excellent experience through high-quality products and great customer service. Fast Facts Store Address: Quarterpath Crossing Fuel Center, 1530 Quarterpath Rd., Williamsburg, VA 23185 Grand Opening Date: Friday, March 3, 2017 Store Hours: Staffed daily from 6 a.m. – 10 p.m.; fuel available for purchase by debit/credit card 24 hours Square Footage: 240 Fuel Dispensers: Four
News Article | November 24, 2016
The renowned High Dental practice has announced its new tooth whitening service using the unique and highly effective photo dynamic Smartbleach 3LT System to quickly, safely, gently and painlessly provide patients in Melbourne, Australia, with a brighter smile. More information is available at http://highdental.com.au. High Dental is a fully qualified and accredited dental clinic headed by the renowned Melbourne dentist Dr. Nicholas Hii, committed to providing the highest quality of dental care supported by a premier patient-centric approach for families and patients of all ages with its wide and popular range of preventive, pediatric, restorative and cosmetic dentistry treatments. The popular dental practice has now announced its new tooth whitening service using the unique and state-of-the-art Smartbleach 3LT System which combines a photo activated whitening gel and a patented green light to deliver a comfortable, safe, painless and highly effective procedure ensuring superior and long lasting teeth whitening results in less than hour with minimal or no sensitivity. The premier High Dental photo dynamic tooth whitening services are delivered by a team of experience and caring professionals with an established reputation for helping patients improve their smiles and confidence with the latest in cosmetic dentistry out of its comfortable and conveniently located offices close to the CBD and with on-site parking in 1187 High Street, Armadale VIC, Melbourne, Australia. Appointments and consultations with High Dental along with more information on its full suite of dentist treatments ranging from leading general dentistry, pediatric care or oral hygiene to dental implants and a wide variety of renowned cosmetic treatments including its state of the art teeth whitening or full smile makeovers can be requested at 03 9077 6363 or through the website link provided above along with multiple patient testimonials. High Dental states “we are a gentle and caring Melbourne dental practice providing a wide range of dental treatments. Dr. Nicholas Hii’s ethos is to put patients first and hence provide excellent care and attention to all. This patient centered approach and our track record helping our patients get that amazing and healthy looking smile that makes them feel confident in their personal and professional life differentiate us from other dentists in Melbourne.” For more information, please visit http://www.highdental.com.au/