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News Article | November 16, 2016

We analysed TCGA data for association between mRNA expression level of 16 candidate immune-related genes (ARG1, IL10, FOXP3, CD68, IL12A, IL12B, IFNG, CD8A, CD4, ITGAM (also known as CD11B), CD14, TNF, IL1A, IL1B, IL6 and CCL5) and 5 year overall survival. Illumina HiSeq RNaseqV2 mRNA expression and clinical data for 520 head and neck squamous cell carcinoma samples were downloaded from the TCGA data portal. Median follow-up from diagnosis was 1.8 years with a range of 0.01 years to 17.6 years. Follow-up time was truncated at 5-years for analysis and 200 deaths occurred in this period. For each of the 16 candidate immune response genes, we scored subjects as above (high) or below (low) the median expression and compared survival using a log-rank test at 5% significance. HPV+ patients were stratified into a favourable immune profile if they had expression above the median for the significant genes IL12A, IL12B, IFNG, CD8A and below the median for IL6. Kaplan–Meier curves were plotted for these two groups. Similar methods were used to examine association of these 16 genes with 720 lung adenocarcinoma and 876 gastric carcinoma samples using the publically available data from KM Plotter29. In lung adenocarcinomas, 12 genes were significantly associated with survival; patients were scored as having a favourable immune profile if 7 or more of the 12 significant genes had expression in the favourable direction. In 876 gastric cancer samples, 8 genes were significantly associated with survival. Patients were scored as having a favourable immune profile if 5 out of the 8 genes had expression in the favourable direction. We investigated 66 immune-related genes in four functional classes, 17 genes related to antigen presentation (HLA class I and II molecules), 24 genes surveying T cell activation, 20 innate immune response genes (IL6, CCL7 and others) and 5 genes related to cancer cell signalling. These genes changed expression in response to PI3Kγ inhibition for association with survival in HPV+ and HPV− TCGA HNSCC and lung adenocarcinoma cohorts. Within each cancer type, we scored subjects as above or below the median expression for each gene and compared survival using a log-rank test, using 10% false discovery rate (FDR) within each class as the significance threshold. HPV+ and HPV− HNSCC survival were investigated separately, as HPV− HNSCC generally has a worse prognosis. Within each cohort, patients were classified as having a favourable PI3Kγ immune response profile if they had expression levels above or below the median in the direction of low PI3Kγ activity for the genes identified as significant. We compared the survival experience of favourable versus less-favourable profiles of patients using Kaplan–Meier curves. Out of the 66 experimentally identified PI3Kγ-regulated genes, 43 showed significant association with overall survival in the HPV+ cohort (FDR < 10% within each functional class). Comparison of these genes between HPV+ and HPV− cohorts showed that HPV− samples generally had significantly (P < 0.05) lower expression of 42 genes in the antigen presentation and T cell activation classes, consistent with a pattern of adaptive immune suppression, and higher expression of genes in the innate immune response and cancer cell signalling classes, which were negatively associated with survival. Only MALT1 was not differentially expressed between the two groups (P = 0.7). Pik3cg−/− and Pik3cg−/−,PyMT mice were generated as previously described13. Cd8−/− and Cd4−/− mice with a C57Bl/6J background were purchased from the Jackson Laboratory and crossed with syngeneic Pik3cg−/− mice. All animal experiments were performed with approval from the Institutional Animal Care and Use Committee of the University of California. Animals were euthanized before the IACUC maximum allowable tumour burden of 2 cm3 per mouse was exceeded. Wild-type or Pik3cg−/− 6–8 week-old female or male syngeneic C57Bl/6J (LLC lung, PyMT breast and MEER HPV+ HNSCC) or C3He/J (SSCVII HPV− HNSCC) mice were implanted with 106 tumour cells by subcutaneous injection (LLC, MEER, SCCVII) or by orthotopic injection (PyMT) (n = 10–15) and tumour growth was monitored for up to 30 days. Tumour dimensions were measured once when tumours were palpable. Tumour volumes were calculated using the equation (l2 × w)/2. In some studies, wild-type and Pik3cg−/− mice with LLC tumours were treated with gemcitabine (150 mg kg−1) or saline by intraperitoneal (i.p.) injection on day 7 and day 14 (n = 10). LLC were acquired from ATCC, PyMT were from L. Ellies (University of California), HPV+ MEER were from J. Lee (Cancer Biology Research Center, Sanford Research/USD) and SCCVII squamous carcinoma cells were from S. Schoenberger (La Jolla Institute for Allergy and Immunology). All cell lines were tested for mycoplasma and mouse pathogens and checked for authenticity against the International Cell Line Authentication Committee (ICLAC; list. In some studies, mice bearing LLC, PyMT, HPV+ MEER or HPV− HNSCC tumour cells were treated once daily by oral gavage with vehicle (5% NMP and 95% PEG 400), 15 mg kg−1 per day of the PI3Kγ inhibitor IPI-549 or by i.p. injection with 2.5 mg kg−1 twice per day of TG100-115 (ref. 13) beginning on day 8 post-tumour injection and continuing daily until euthanasia. IPI-549 is an orally bioavailable PI3Kγ inhibitor with a long plasma half-life and a K value of 0.29 nM for PI3Kγ with >58-fold weaker binding affinity for the other class I PI3K isoforms17. Enzymatic and cellular assays confirmed the selectivity of IPI-549 for PI3Kγ (>200-fold in enzymatic assays and >140-fold in cellular assays over other class I PI3K isoforms17). To study the effect of IPI-549 on lung tumour growth, LLC tumour cells were passaged three times in C57BL/6 albino male mice. When tumour volume reached 1,500 mm3, tumours were collected and single-cell suspensions were prepared. This tumour cell suspension was implanted subcutaneously in the hind flank of C57BL/6 albino male mice at 106 cells per mouse. Prior to initiating treatment with once daily IPI-549 (15 mg kg−1 orally), groups were normalized on the basis of tumour volume. In some studies, wild-type- and Pik3cg−/−-tumour-bearing mice were treated with 100 μg of anti-CD8 (clone YTS 169.4) or an isotype-control clone (LTF-2) from Bio X Cell administered by i.p. injections on day 7, 10 and 13 of tumour growth. For all tumour experiments, tumour volumes and weights were recorded at death. C57Bl/6J (wild-type) or Pik3cg−/− 6–8 week-old male or female mice (MEER HPV+ HNSCC) or C3He/J (SCCVII HPV− HNSCC) were implanted with tumour cells by subcutaneous injection (106 MEER or 105 SCCVII). In HPV+ MEER studies, wild-type and Pik3cg−/− mice were treated with four doses of 250 μg of anti-PD-1 antibody (clone RMP-14, Bio X Cell) or rat IgG2a isotype control (clone 2A3, Bio X Cell) every 3 days, starting when tumours became palpable on day 11 (n = 12–14 mice per group). Wild-type mice bearing HPV+ tumours were also treated with the PI3Kγ inhibitor TG100-115 (ref. 13) twice per day by i.p. injection, beginning on day 11. Tumour regressions were calculated as a percentage of the difference in tumour volume between the date treatment was initiated and the first date of death of the control group. For HPV− SCCVII studies, C3He/J mice were treated with PI3Kγ inhibitor (2.5 mg kg−1 TG100-115 i.p.) beginning on day 6 post-tumour inoculation and with six doses of anti-PD-1 antibody (250 μg clone RMP-14, Bio X Cell) or rat IgG2a isotype control (clone 2A3, Bio X Cell) every 3 days beginning on day 3 (n = 12 mice per group) or with a combination of the two. Alternatively, mice were treated with 5 mg kg−1 TG100-115 twice per day ± anti-PD-1 (250 μg every 3 days) beginning on day 1 (Fig. 4). Mice that completely cleared HPV+ MEER tumours were re-injected with HPV+ tumour cells contralateral to the initial tumour injection and tumour growth was monitored. The growth and metastasis of spontaneous mammary tumours in female PyMT+ (n = 13) and Pik3cg−/−,PyMT+ (n = 8) mice was evaluated over the course of 0–15 weeks. Total tumour burden was determined by subtracting the total mammary gland mass in PyMT− mice from the total mammary gland mass in PyMT+ mice. Lung metastases were quantified macroscopically and microscopically in H&E tissue sections at week 15. Septic shock was induced in wild-type and Pik3cg−/− mice via i.p. injection of 25 mg kg−1 LPS (Sigma, B5:005). Survival was monitored every 12 h and liver, bone marrow and serum were collected 24 h after LPS injection. C57Bl/6J female mice were implanted with 106 LLC tumour cells by subcutaneous injection. When the average tumour size was 250 mm3, mice were treated by i.p. injection with 1 mg per mouse clodronate or control liposomes ( every 4 days for 2 weeks in combination with daily administration of vehicle or IPI-549 (15 mg kg−1 per day orally). In other studies, 6-week-old female BALB/c mice were injected subcutaneously with 2.5 × 105 CT26 mouse colon carcinoma cells in 100 μl phosphate buffered saline (PBS) in the right flank. Eight days later, tumour-bearing mice were arranged into four groups (n = 15) with an average tumour volume of 70 mm3. Oral administration of IPI-549 (15 mg kg−1) or vehicle (5% NMP and 95% PEG 400) and anti-CSF-1R antibody (50 mg kg−1 i.p. 3× per week, clone AFS98, Bio X Cell) began on day 8 after tumour injection via oral gavage at a 5 ml kg−1 dose volume and continued daily for a total of 18 doses. Six-week-old female BALC/c mice were injected subcutaneously with 2.5 × 105 CT26 mouse colon carcinoma cells in 100 μl PBS in the right flank. On day 8 after tumour injection, tumour-bearing mice were grouped and treated daily with IPI-549 (15 mg kg−1, orally) or vehicle (5%NMP and 95% PEG 400). In addition, mice were injected i.p. with 50 mg kg−1 anti-CD115 (Bio X Cell clone AFS98) or 50 mg kg−1 rat IgG2a isotype control (Bio X Cell clone 2A3) antibodies as described above for a total of three injections. Two days after the final injection mice were euthanized, tumours were digested in a mixture of 0.5 mg ml−1 collagenase IV and 150 U ml−1 DNase I in RPMI-1640 for 30 min at 37 °C and tumour-infiltrating myeloid cells were analysed by flow cytometry. CD11b+Gr1− cells were isolated from single-cell suspensions of LLC tumours from donor mice by fluorescence-activated cell sorting (FACS) or serial magnetic bead isolation. Additionally, for some experiments, primary bone-marrow-derived macrophages were polarized and collected into a single-cell suspension. Purified cells were admixed 1:1 with LLC tumour cells and 5 × 105 total cells were injected subcutaneously into new host mice. Tumour dimensions were measured three times per week beginning on day 7. In antibody blocking studies, CD11b+Gr1− cells were incubated with 5 μg anti-IL12 (clone RD1-5D9) or isotype (clone LTF-2, Bio X Cell) for 30 min before the addition of tumour cells. Mice were additionally treated intradermally with 5 μg of antibody 3 and 6 days after tumour cell inoculation. In some studies, CD11b+Gr1− cells were pre-incubated with inhibitors of arginase (nor-NOHA, 50 μM, Cayman Chemical), iNOS (1400W dihydrocholoride, 100 μM, Tocris), mTOR (rapamycin, 10 μM Calbiochem), or IκKβ (ML120B, 30 μM, Tocris) for 30 min before the addition of tumour cells. Inoculated mice were further treated by intradermal injection with inhibitors at 3 and 6 days after inoculation. Donor C57Bl/6J (WT) or Pik3cg−/− mice were implanted with 106 LLC tumour cells by subcutaneous injection. On day 14 after tumour implantation, CD90.2+, CD4+ or CD8+ cells were harvested by magnetic bead isolation (Miltenyi Biotec). T cells were mixed 1:1 with viable LLC tumour cells. Cell mixtures containing 5 × 105 total cells were injected into the flanks of naive wild-type or Pik3cg−/− mice (n = 8–10 per group). Tumour growth, intratumoral apoptosis and necrosis were investigated over 0–16 days. In other studies, wild-type T cells were incubated at 37 °C and 5% CO for 6 h with 10 or 100 nM IPI-549 (Infinity Pharmaceuticals) or Cal-101 (Selleck Chem). After 6 h, T cells were washed, admixed 1:1 with LLC tumour cells, and 106 total cells were injected subcutaneously into recipient mice. Tumour growth was monitored for 14 days. Tumours were isolated, minced in a Petri dish on ice and then enzymatically dissociated in Hanks balanced salt solution containing 0.5 mg ml−1 collagenase IV (Sigma), 0.1 mg ml−1 hyaluronidase V (Sigma), 0.6 U ml−1 dispase II (Roche) and 0.005 MU ml−1 DNase I (Sigma) at 37 °C for 5–30 min. The duration of enzymatic treatment was optimized for greatest yield of live CD11b+ cells per tumour type. Cell suspensions were filtered through a 70-μm cell strainer. Red blood cells were solubilized with red cell lysis buffer (Pharm Lyse, BD Biosciences) and the resulting suspension was filtered through a cell strainer to produce a single-cell suspension. Cells were washed once with PBS before use in flow cytometry analysis or magnetic bead purification. Thioglycollate-elicited peritoneal macrophages were collected 96 h after i.p. injection of a 3% thioglycollate solution. Cells were collected from the peritoneal cavity in 10 ml of PBS and macrophage enrichment was performed by plating cells in RPMI with 10% FBS and 1% penicillin/streptomycin for 2 h at 37 °C and 5% CO . After 2 h, non-adherent cells were removed with three PBS washes, and cells were analysed via flow cytometry and qPCR analysis. Single-cell suspensions (106 cells in 100 μl total volume) were incubated with aqua live dead fixable stain (Life Technologies), FcR-blocking reagent (BD Biosciences) and fluorescently labelled antibodies and incubated at 4 °C for 1 h. Primary antibodies to cell surface markers directed against F4/80 (BM8), CD45 (30-F11), CD11b (M1/70), Gr1 (RB6-8C5), CD3 (145-2C11), CD4 (GK1.5), CD8 (53-6.7), CD273 (B7-DC), CD274 (B7-H1) were from eBioscience; Ly6C (AL-21), Ly6G (1A8), CD11c (HL3), and MHC-II (AF6-120.1) from BD Pharmingen, CCR2 (475301) from R&D Systems and CD206 (MR5D3) from AbD Serotech. For intracellular staining, cells were fixed, permeabilized using transcription factor staining buffer set (eBioscience) and then incubated with fluorescently labelled antibodies to FoxP3 (FJK-16 s) from eBioscience. Multicolour FACS analysis was performed on a BD Canto RUO 11 colour analyser. All data analysis was performed using the flow cytometry analysis program FloJo (Treestar). Single-cell preparations from bone marrow or tumours were incubated with FcR-blocking reagent (BD Biosciences) and then with 20 μl magnetic microbeads conjugated to antibodies against CD11b, Gr1, CD90.2, CD4 and CD8 (Miltenyi Biotech MACS Microbeads) per 107 cells for 20 min at 4 °C. Cells bound to magnetic beads were then removed from the cell suspension according to the manufacturer’s instructions. For cell sorting, single-cell suspensions were stained with aqua live dead fixable stain (Life Technologies) to exclude dead cells and anti-CD11b-APC (M1/70, eBioscience) and anti-Gr1-FITC (RB6-8C5, eBioscience) antibodies. FACS sorting was performed on a FACS Aria 11 colour high speed sorter at the Flow Cytometry Core at the UC San Diego Center for AIDS Research. Live cells were sorted into the following populations: CD11b+Gr1−, CD11b+Gr1lo, CD11b+Gr1hi and CD11b−Gr1− cells. CD11b-positive cells were defined by increased staining over the isotype control, and Gr1 levels were defined both by comparison to the isotype control and relative staining to other populations. Bone-marrow-derived cells were aseptically collected from 6–8 week-old female mice by flushing leg bones of euthanized mice with PBS, 0.5% BSA, 2 mM EDTA, incubating in red cell lysis buffer (155 mM NH Cl, 10 mM NaHCO and 0.1 mM EDTA) and centrifuging over Histopaque 1083 to purify the mononuclear cells. Approximately 5 × 107 bone-marrow-derived cells were purified by gradient centrifugation from the femurs and tibias of a single mouse. Purified mononuclear cells were cultured in RPMI + 20% serum + 50 ng ml−1 mCSF (PeproTech). Human leukocytes from apheresis blood products were obtained from the San Diego Blood Bank. Cells were diluted in PBS, 0.5% BSA, 2 mM EDTA, incubated in red cell lysis buffer (155 mM NH Cl, 10 mM NaHCO and 0.1 mM EDTA) and centrifuged over Histopaque 1077 to purify mononuclear cells. Approximately 109 bone-marrow-derived cells were purified by gradient centrifugation from one apheresis sample. Purified mononuclear cells were cultured in RPMI + 20% serum + 50 ng ml−1 Human mCSF (PeproTech). Non-adherent cells were removed after 2 h by washing and adherent cells were cultured for 6 days to differentiate macrophages fully. Bone-marrow-derived macrophages were polarized with IFNγ (20 ng ml−1, Peprotech) + LPS (100 ng ml−1, Sigma) or LPS alone for 24 h, or IL4 (20 ng ml−1, Peprotech) for 24–48 h. For inhibitor studies, PI3Kγ inhibitors (1 μM) (IPI-549, Infinity Pharmaceuticals and TG100-115, Targegen/Sanofi-Aventis), rapamycin (10 μM, Selleck), or ML120B (30 μM) were incubated with macrophages 1 h before the addition of polarizing stimuli. Total RNA was harvested from macrophages using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Freshly isolated mouse bone marrow cells from nine wild-type and nine Pik3cg−/− mice were pooled into three replicates sets of wild-type or Pik3cg−/− cells and differentiated into macrophages for 6 days in RPMI + 20% FBS+ 1% penicillin/streptomycin + 50 ng ml−1 mCSF. Each replicate set of macrophages was then treated with mCSF, IL4 or IFNγ/LPS. Macrophages were removed from dishes, and RNA was collected using Qiagen Allprep kit. In addition, RNA was harvested from day 14 (500 mm3) LLC tumours or purified CD11b+Gr1-F480+ TAMs from wild-type (C57BL/6) and Pik3cg−/− mice. RNA was collected using the Qiagen Allprep kit. RNA libraries were prepared from 1 μg RNA per sample for sequencing using standard Illumina protocols. RNA sequencing was performed by the University of California, San Diego Institute for Genomic Medicine. mRNA profiles were generated by single read deep sequencing, in triplicate, using Illumina HiSeq2000. Sequence analysis was performed as previously described16. Sequence files from Illumina HiSeq that passed quality filters were aligned to the mouse transcriptome (mm9 genome build) using the Bowtie2 aligner4. Gene-level count summaries were analysed for statistically significant changes using DESeq. Individual P values were adjusted for multiple testing by calculating Storey’s q values using fdrtooltrimmer. For each gene, the q value is the smallest false discovery rate at which the gene is found significant. We analysed biological processes as defined by the Gene Ontology Consortium. Each gene ontology term defines a set of genes. The entire list of genes, sorted by the q value in ascending order, was subjected to a non-parametric variant of the gene set enrichment analysis (GSEA), in which the parametric Kolmogorov–Smirnov P value was replaced with the exact rank-order P value. We perform a Bonferroni adjustment of gene set P values for the number of gene sets tested. Heat maps of expression levels were created using in-house hierarchical clustering software that implements Ward clustering. The colours qualitatively correspond to fold changes. cDNA was prepared using 1 μg RNA with the qScript cDNA Synthesis Kit (Quanta Biosciences). Sybr green-based qPCR was performed using human and mouse primers to Arg1, Ifng, Il10, Il12p40, Il1b, Il6, Ccl2, Vegfa, Gapdh, Nos2, Tgfb1, Tnfa and mouse H2-Aa, H2-Ab1, H2-Eb1, and H60a (Qiagen QuantiTect Primer Assay). mRNA levels were normalized to Gapdh and reported as relative mRNA expression or fold change. Freshly isolated bone-marrow-derived CD11b+ myeloid cells or differentiated macrophages were transfected by electroporation using an AMAXA mouse macrophage nucleofection kit with 100 nM of siRNA or 2 μg Pik3cgCAAX or pcDNA control plasmid. Non-silencing (Ctrl_AllStars_1) siRNA and Cebpb (MmCebpb_4 and MmCebpb_6), and Mtor (Mm_Frap1_1 and Mm_Frap1_2) siRNAs were purchased from Qiagen. After transfection, cells were cultured for 36–48 h in RPMI containing 10% serum and 10 ng ml−1 mCSF (PeproTech) or polarized as described above. Whole tumours, CD11b+Gr1− cells, CD90.2+ cells, CD4+ cells and CD8+ cells isolated from LLC tumours were lysed in RIPA buffer and total protein concentrations were determined using a BCA protein assay (Pierce). Macrophage supernatants (100 μl) or 500 μg of total protein lysate from tumours were used in ELISAs to detect CCL2, TGFβ, IL1β, TNFα, IL6, IFNγ, IL10, IL12 and granzyme B (ready set go ELISA, eBioscience). Protein expression was normalized to total volume (supernatants) or mg total protein (tumour lysates). The QuantiChrom arginase assay kit (DARG-200, BioAssay Systems) was used to measure arginase activity in primary mouse bone-marrow-derived macrophages from wild-type and Pik3cg−/− mice according to the manufacturer’s instructions. For all conditions, cells were harvested and lysed in 10 mM Tris (pH 7.4) containing 1 μM pepstatin A, 1 μM leupeptin, and 0.4% (w/v) Triton X-100. Samples were centrifuged at 20,000g at 4 °C for 10 min. To measure NFκB and C/EBPβ activation, TransAM NFκB family and C/EBP transcription factor assay kits (43296 and 44196, Active Motif) were used according to the manufacturer’s protocol. Briefly, wild-type and Pik3cg−/− bone-marrow-derived macrophages were stimulated with LPS (100 ng ml−1) or IL4 (20 ng ml−1) and nuclear extracts were prepared in lysis buffer AM2 (Active Motif). Nuclear extracts were incubated with the immobilized consensus sequences and RelA, cRel or C/EBPβ were detected using specific primary antibodies. Quantification was performed via colourimetric readout of absorbance at 450 nm. IL4 and LPS macrophage cultures were solubilized in RIPA buffer containing protease and phosphatase inhibitors. Thirty micrograms of protein was electrophorezed on Biorad precast gradient gels and electroblotted onto PVDF membranes. Proteins were detected by incubation with 1:1,000 dilutions of primary antibodies, washed and incubated with goat anti-rabbit-HRP antibodies and detected after incubation with a chemiluminescent substrate. Primary antibodies directed against Akt (11E7), p-Akt (244F9), IκBα (L35A5), IκKβ (D30C6), p-IκKα/β (16A6), RelA (D14E12), pRelA (93H1), C/EBPβ (#3087), p-CEBPβ (#3082), IRAK1 (D51G7), TBK1 (D1B4) and PI3Kγ (#4252) were from Cell Signaling Technology and pTBK1 (EPR2867(2)) was from Abcam. CD90.2+ tumour-derived T cells were purified from LLC tumour-bearing wild-type and Pik3cg−/− or TG100-115 and control treated mice and then co-incubated with LLC tumour cells (target cells) at 2.5:1, 5:1 and 10:1 ratios of T cells to tumour cells (2 × 103 LLC tumour cells per well) for 6 h. Target cell killing was assayed by collecting the supernatants from each well for measurement of the lactate dehydrogenase release (Cytotox96 non-radioactive cytotoxicity assay kit, Promega). Tumour samples were collected and cryopreserved in OCT, sections (5 μm) were fixed in 100% cold acetone, blocked with 8% normal goat serum for 2 h, and incubated anti-CD8 (53-6.7, 1:50 BD Biosciences) for 2 h at room temperature. Sections were washed three times with PBS and incubated with Alexa594-conjugated secondary antibodies. Slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to identify nuclei. Immunofluorescence images were collected on a Nikon microscope (Eclipse TE2000-U) and analysed using Metamorph image capture and analysis software (Version 6.3r5, Molecular Devices). The detection of apoptotic cells was performed using a TUNEL-assay (ApopTag fluorescein in situ apoptosis detection kit, Promega) according to the manufacturer’s instructions. Slides were washed and mounted in DAKO fluorescent mounting medium. Immunofluorescence images were collected on a Nikon microscope (Eclipse TE2000-U) and analysed with MetaMorph software (version 6.3r5) or SPOT software (version 4.6). Pixels per field or cell number per field were quantified in five 100× fields from ten biological replicates. Primary tumour samples with mRNA expression data were scored as above or below the median expression level, and tested for association with patient survival using a log-rank test at 5% significance. For studies evaluating the effect of drugs on tumour size, tumour dimensions were measured directly before the start of treatment, tumour volumes were computed and mice were randomly assigned to groups so that the mean volume ± s.e.m. of each group was identical. A sample size of ten mice per group provided 80% power to detect a mean difference of 2.25 standard deviation (s.d.) between two groups (based on a two-sample t-test with two-sided 5% significance level). Sample sizes of 15 mice per group provided 80% power to detect one s.d. difference between two groups. Data were normalized to the standard (control). Analysis for significance was performed by one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise testing with more than two groups and by parametric or nonparametric Student’s t-test when only two groups were compared. We used a two-sample t-test (two groups) and ANOVA (multiple groups) when data were normally distributed and a Wilcoxon rank sum test (two groups) when data were not normally distributed. All mouse studies were randomized and blinded; assignment of mice to treatment groups, tumour measurement and tumour analysis was performed by coding mice with randomly assigned mouse number, with the key unknown to operators until experiments were completed. In tumour studies for which tumour size was the outcome, mice removed from the study owing to health concerns were not included in endpoint analyses. All experiments were performed at least twice; n refers to biological replicates. RNA sequencing data can be accessed using numbers GSE58318 (in vitro macrophage samples) and GSE84535 (in vivo tumour and tumour-associated macrophages samples) at

Over the last 15 years, several dozen deep boreholes (depths in excess of 500 m) have been drilled in the Maurienne Valley as part of the survey work for the new Lyons-Turin rail link. A number of parameters have been logged in these boreholes as a matter of routine, including data on fluids, such as temperature, electrical conductivity and vertical flow velocity. Because several logging campaigns have been carried out in each borehole, it has generally been possible to separate disturbances caused by the drilling (creation of links between water-bearing fractures, establishment of transitory thermal regimes, drilling-related heating, etc.) from the natural initial thermal state. Consequently, most natural thermal state descriptions are based on reconstructions, rather than on raw data. In many cases, in particular in the Houillère Briançonnaise Zone, this natural thermal state is governed by the circulation of hot and cold fluids. We suggest that these two types of circulation actually form a single circuit, in which cold waters infiltrating in the north-south trending Evaporite Zone and in the carbonates of the Sub-Briançonnais Zone flush deep waters so that they rise preferentially along the axis of the valley in the east-west trending Houillère Zone. Focal mechanism data and leveling comparisons show that the front of the Houillère Zone (including the evaporites and the Sub-Briançonnais carbonates), which controls infiltration into the system, is currently subject to WNW-ESE extension. The rising hot waters are controlled by the valley, which is an extensional structure perpendicular to the Houillère Zone. This interpretation is in line with the increased heat flow towards the axis of the valley, and with the occurrence of rising thermal waters further down the valley, for example at Echaillon and Les Chavannes. The interaction between these two extensional systems, which has been observed at least on a regional scale, gives a highly dynamic character to the current deformation, as is shown by the new a-seismic tectonic regime that has affected the region since 1995. Given this dynamic character, it is unlikely that a hydraulic-thermal regime has existed undisturbed for more than 15,000 years. The example described in the present article shows how the repeated logging of thermal data in boreholes over a period of time can help delineate deep circulations. Combined with detailed information about the current deformation regime, this logging data allows a detailed picture of circulation patterns to be drawn.

The murine cancer cell lines for melanoma (B16F10, referred to as B16), breast cancer (4T1) and colon cancer (CT26) were obtained from ATCC. The colon cancer cell line MC38 was obtained from the NCI and the lung cancer (LLC-Brei) from Caliper Life Sciences. Cells were maintained in RPMI medium supplemented with 10% fetal calf serum (FCS) and penicillin with streptomycin (complete RPMI media). The GMCSF-secreting B16 cell line (referred as to B16-GMCSF) has been reported and was used to increase the number of myeloid cells recruited to the tumour14. The cell lines have been mycoplasma tested. cDNA was prepared using 1 μg RNA with the qScript cDNA Synthesis Kit (Quanta Biosciences). Sybr green-based qPCR was performed using murine primers to Arg1, Ifng, Il10, Il12p40, Il1b, Il6, Ccl2, Gapdh, Nos2, Tgfb1, Tnfa, IL4ra, Indo, Ctla4, Pd-l1, CD86, CxCr2, Fizz1, Ymd1 (Qiagen QuantiTect Primer Assay). mRNA levels were normalized to Gapdh (ΔC  = C gene of interest – C Gapdh) and reported as relative mRNA expression or fold change. Tumours were excised, snap-frozen in liquid nitrogen, and pulverized using a tissue grinder. Tumour protein lysates were prepared in MSD Tris Lysis Buffer (Meso Scale Discovery, Rockville, Maryland) containing 2× Halt Protease and Phosphatase Inhibitor Cocktail (Fisher Scientific). Total protein concentration was normalized to 4 mg ml−1 and cytokines were quantified using the MILLIPLEX map Mouse Cytokine/Chemokine Magnetic Bead 32 Plex Panel and MILLIPLEX map Mouse CD8+ T Cell Magnetic Bead Panel kits according to the manufacturer’s instructions (Millipore, Billerica, Massachusetts). Mice bearing CT26 tumours were treated with vehicle or IPI-549 (15 mg kg−1 day−1, PO) for 6 or 9 days. Tumours were isolated, and frozen until needed; tumours then thawed and RNA was extracted from all cells. RNA-seq was done at Expression Analysis (Q2 Solutions). Sequence reads were aligned to the mouse B38 reference genome using OmicSoft ArrayStudio and the UCSC gene model. log [FPKM] was calculated for each gene, and data were mean centred for display in heat maps. The analysis focused on a compilation of about 4,200 mouse genes related to cancer immunology and PI3K pathway signalling compiled from numerous sources including BioCarta pathways, GO gene ontologies, KEGG pathways, WikiPathways, and literature23. Female C57BL/6J and Balb/c mice (6–8 weeks old) were purchased from Jackson Laboratory. Pmel-1 TCR transgenic mice have been previously reported24and were provided by N. Restifo (National Cancer Institute, Bethesda, Maryland). All mice were maintained in micro-isolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the Memorial Sloan Kettering Cancer Center Institutional Animal Care and Use Committee. No statistical methods were used to predetermine sample size. Ten to fifteen mice per treatment strategy were used to allow 90% power, and a 5% significance level, and detect differences in tumour-free survival from 10% to 80%. Typically, tumours grew in 100% of control animals. An additional 5 mice per group were used for tissue harvest at day 7 and day 14 on treatment. Mice cage and treatment allowance were randomized at day 7 after tumour implants. On day 0 of the experiments, tumour cells were injected intradermally (i.d.) in the right flank. For the B16 model, 2.5 × 105 B16-WT or B16-GMCSF cells were injected into C57BL/6J mice. For 4T1 model and for the CT26 model, 5 × 105 cells were used subcutaneously in Balb/c mice. For studies in immune compromised mice, the CT26 study was done in the Balb/c NU/NU strain and the B16-GMCSF in C57Bl.6 Rag1−/− mice. Mice were obtained from Jackson Labs and Charles River Labs. Treatments were given as single agents or in combinations with the following regimen for each drug. The PI3Kγ inhibitor drug IPI-549 was dissolved at 5% 1-methyl-2-pyrrolidinone in polyethylene glycol 400 and administered by oral gavage once a day at 15 mg kg−1. Treatment was initiated on day 7 ending on day 21 post tumour implant. Control groups received vehicle (5% NMP, 95% PEG) without the active product. Anti-CTLA4 antibody (100 μg per mouse, clone 9H10, Bio X cell) and anti-PD-1 antibody (250 μg per mouse, clone RPM1-14, Bio X cell) were injected intraperitoneally (i.p.) on days 7, 10, 13 and 16 for the B16, B16-GMCSF and 4T1 models and on days 10, 13 and 16 for the CT26 model. Tumours were measured every second or third day with a calliper, and the volume (length × width × height) was calculated. Mice that had no visible and palpable tumours that could be measured on consecutive measurement days were considered complete regressions. Animals were euthanized for signs of distress or when the total tumour volume reached 2,500 mm3. For the re-challenge study: mice with complete responses in the anti-PD-1 treatment group and the anti-PD-1 and IPI-549 combination group were re-challenged with 2.5 × 105 CT26 WT tumour cells (on day 106 of the study since original tumour implant). Additional mice with complete responses from an additional IPI-549 and anti-PD-1 group were implanted with 1 × 105 4T1 tumour cells. Female BALB/c mice were depleted of CD8+ T cells by intraperitoneal (IP) injection of a CD8-depleting antibody beginning 3 days before tumour implantation and continuing every 3 days (Q3D) for the duration of the study. Control animals were injected IP with an isotype control antibody according to the same dosing schedule. CD8+ T cell depletion was verified by flow cytometry of splenic cells from a separate cohort of mice at the time of tumour implant. Anti-CD8 antibody (BioXCell, in vivo MAb Rat IgG2b anti-mouse CD8, clone 169.4) and isotype control antibody (BioXCell, in vivo MAb Rat IgG2b, κ isotype control, clone LTF-2) were used. CD8+ T-cell-depleted and isotype contro-treated mice were implanted with 2.5 × 105 CT26 WT cells subcutaneously to the dorsal right flank. Eight days after implant, tumour-bearing animals with average tumour volumes of 50 to 60 mm3 began treatment. Animals were dosed daily with vehicle or IPI-549 (15 mg kg−1, PO) and dosed every three days with 100 μg antibody, either isotope control or anti-CD8. Tumour measurements were taken every second or third day during the 13 days of dose administration. For CD11b+ cell depletion, murine Lewis Lung Carcinoma tumour brei (here referred to as LLC) was propagated into ten C57BL/6 Albino male mice. When tumours reached an average of 1,500 mm3, tissue was collected and made into a single-cell suspension. Tissue was dissociated in a glass dunce, filtered through a 100 μm filter and washed with cold buffer of PBS pH 7.2, 0.5% BSA and 2 mM EDTA. An aliquot of cells was separated, and placed on ice until the time of re-transplant. Cells were counted and adjusted to 2 × 107 total cells. To deplete the CD11b cells from the tumour homogenate, CD11b micro beads (MACS #130-049-601) were added to the sample and incubated on ice for 15 min. Cells were washed and re-suspended with buffer. A negative depletion was preformed following the Miltenyi autoMACS protocol. The positive selected cells and starting tumour inoculum were counted and cell numbers adjusted to the tumour inoculum used for historical studies. For each cell condition 30 C57BL/Albino male mice were inoculated. Treatment of IPI-549 or vehicle started when tumours for each cell condition reached an average of 200 mm3 regardless of what day post implant this occurred. Mouse tumour samples were minced with scissors before incubation with 1.67 U ml−1 Liberase (Roche) and 0.2 mg ml−1 DNase (Roche) in RPMI for 30 min at 37 °C. Tumour samples were homogenized by repeated pipetting and filtered through a 100-μm nylon filter (BD Biosciences) in RPMI supplemented with 7.5% FCS to generate single-cell suspensions. Cell suspensions were washed once with complete RPMI and purified on a Ficoll gradient to eliminate dead cells. Cells from mouse spleens were isolated by grinding spleens through 40-μm filters. After red blood cell (RBC) lysis (ACK Lysing Buffer, Lonza) when required, all samples were washed and re-suspended in FACS buffer (PBS/0.5% albumin) or RPMI depending on further use. Cells isolated from mouse tumours and spleens were pre-incubated (15 min, 4 °C) with anti-CD16/32 monoclonal antibody (Fc block, clone 2.4G, BD Biosciences) to block nonspecific binding and then stained (30 min, 4 °C) with appropriate dilutions of various combinations of the following fluorochrome-conjugated antibodies: anti-CD45-AF 700 (clone 30-F11), anti-CD11b-APC-Cy7 (clone M1/70), anti-CD11b-PE-TR(M1/70.15), anti-Ly6G-APC (clone 1A8), anti-F4/80-PercP-Cy5.5 (clone BM8), anti-Ly6C-PE-Cy7 (clone AL-21), anti-MHC Class II-eFluor 450 (clone M5/114.15.2), anti-CD206-PE (clone 19.2), anti-CD8-Percp-Cy5.5 (clone 53-6.7), anti-CD8-PE Texas Red (clone 5H10), anti-CD4-PE-Cy7 (clone RM4-5), anti-CD4-Pacific Blue (clone RM4-5), anti-Foxp3-APC (clone FJK-16 s), anti-Foxp3-PE-Cy7 (clone FJK-16 s), anti-CD25-APC-Cy7 (clone PC61), anti-CD44-PE-Cy7 (clone IM7), anti-CD62L-PE (clone MEL-14), anti-Ki67 (clone B56), anti-CTLA4-APC (clone), anti-Granzyme B-PE-TR (clone GB11), antibodies, all purchased from BD Biosciences, eBioscience or invitrogen. For intracellular stain, cells were further permeabilized using a FoxP3 Fixation and Permeabilization Kit (eBioscience) and stained for Foxp3 (clone FJK-16 s, Alexa-Fluor-700-conjugated, eBioscience), Ki67 (clone SolA15, eFluor-450-conjugated, eBioscience) or CTLA4. The stained cells were acquired on a LSRII Flow Cytometer using BD FACSDiva software (BD Biosciences) and the data were processed using FlowJo software (Treestar). Dead cells and doublets were excluded on the basis of forward and side scatter and Fixable Viability Dye eFluor 506. Mouse tumour and spleen single-cell suspensions were generated as described in the previous section. Tumour cells were subsequently separated from debris over a Ficoll gradient (Sigma-Aldrich). B cells were depleted from splenocytes using CD19 microbeads and LD columns according to the manufacturer’s instructions (Miltenyi Biotec) to enrich the myeloid fractions. Cells were stained with anti-CD45.2-Alexa- Fluor-700, anti-CD11b-APC-Cy7, anti CD8-PE antibodies for flow sorting on a FACSAria II Cell Sorter (BD Biosciences). Dead cells were excluded using DAPI (Invitrogen). Purity of flow-sorted populations was above 90%. Spleens and lymph nodes from pmel-1 TCR transgenic mice were isolated and ground through 100-μm filters. After RBC lysis, CD8+ T cells were purified by positive selection using Miltenyi magnetic beads. The isolated cells were loaded with CellTrace Far Red DDAO-s.e. (Thermo Fisher Scientific) and injected into recipient animals via tail vein at indicated numbers. Activated CD8+ T cells were generated by culturing splenocytes with soluble anti-CD3 (1 μg ml−1, clone 145-2C11, eBioscience) and anti-CD28 (2 μg ml−1, clone 37.51, eBioscience) for 72 h. Recombinant mouse IL-2 (30 U ml−1, Chiron) was added for the final 24 h of culture. CD8+ T cells were subsequently positively selected with Miltenyi magnetic beads before injection via tail vein, as described above. The frequency and proliferation of Pmel cells were measured 2 weeks after tumour challenge and 7 days after adoptive transfer of 1 × 106 in vitro activated CD8+ Pmel T cells using Thy1.1 antibody and by assessing CellTrace Far Red DDAO-s.e. dilution by flow cytometry, respectively. Spleens from naive mice were isolated and ground through 40-μm filters to generate a single-cell suspension. After RBC lysis, CD8+ cells were purified using anti-CD8 (Ly-2) microbeads (Miltenyi Biotech) according to the manufacturer’s protocol and labelled with 1 mM CFSE (Invitrogen) in pre-warmed PBS for 10 min at 37 °C. The CFSE-labelled CD8+ T cells were then plated in complete RPMI media supplemented with 0.05 M β-mercaptoethanol onto round bottom 96-well plates (25 × 103 cells per well) coated with 1 μg ml−1 anti- CD3 (clone 1454-2C11) and 5 μg ml−1 anti-CD28 (clone 37N) antibodies. Purified myeloid cells were added in indicated ratios and plates were incubated at 37 °C. After 48 h, cells were harvested and CFSE signal in the gated CD8+ T cells was measured by flow cytometry (LSRII Flow Cytometer, BD Biosciences). For the human MDSC suppression assay PBMCs were isolated using Lymphoprep from donor blood. T cells were isolated by CD3+ selection (Easysep, Stem Cell Technologies) and frozen in Sigma freezing media for later use. The remaining PBMCs without T cells were incubated with 20 ng ml−1 GMCSF and 20 ng ml−1 IL-6 for 6 days to differentiate the myeloid cells and were incubated with or without added IPI-549. After 6 days, the MDSC cells were isolated by CD33+ selection (Easysep, Stem Cell Technologies). These MDSC cells were then mixed with the autologous T cells (at a 1:4 ratio) that had been pre-stained with cell trace violet and activated with anti-CD3 and anti-CD28 beads (Dynal). The T cell proliferation is determined by Cell Trace Violet dye dilution measured by flow cytometry after 72 h. Blood was collected from CT26 tumour-bearing mice after 10 days of treatment with either vehicle or IPI-549 (15 mg kg−1). PBMCs were isolated using Lymphoprep density gradient media (Stem Cell Technologies, Vancouver, BC). IFNγ producing cells were quantified using the CTL Mouse Immunospot IFNγ Single Colour ELISPOT kit (CTL, Shaker Heights, Ohio) according to the manufacturer’s instructions. For in vitro re-stimulation, 1 × 105 PBMC were co-cultured with 1 × 105 irradiated (2,000 rad) CT26 colon carcinoma target cells in CTL test media (CTL) for 16 h. Irradiated (2,000 rad) 4T1 mammary carcinoma target cells were used as negative control targets to assess specificity. IFNγ spots were quantified using a CTL Immunospot S6 Micro Analyzer and Immunospot Professional Software (CTL). Bone marrow derived macrophages were prepared from C57Bl/6 mice femur and tibias. Red blood cells were lysed and then the remaining cells were plated in bone macrophage media (BMM) consisting of DMEM, 20% FBS plus penicillin/streptomycin and 50 ng ml−1 M-CSF and incubated for 6 days. Cells were polarized towards an M2 phenotype with the addition of 20 ng ml−1 IL4 and 50 ng ml−1 M-MCSF (both from R&D Systems) with or without added IPI-549. Cells were incubated for 48 h and then RNA was harvested from the cells (Qiagen RNeasy). qRT–PCR was performed using primers for mouse ARG1 (Mm00475988_m1, Life Technologies NY) and mouse β-actin (Mm00607939_s1 Life Technologies, New York). Biochemical and cell-based assays for the Class I and Class II PI3K isoforms were run as previously described19, 25. Whole blood from six healthy donors was pre-treated with a dose titration of IPI-549 and then stimulated for 2 min with 2.3 μg ml−1 CXCL12. Cells were lysed, fixed and stained. Human blood samples were collected after ICF approval. Response to stimulation was determined by measuring phosphorylation of AKT S473 in monocytes (CD14+) by flow cytometry and comparing the value to that of untreated controls. IC values for IPI-549 were calculated for each donor, measuring compound potency against PI3Kγ and in whole blood. Where indicated, data were analysed for statistical significance and reported as P values. Data were analysed with a Mann–Whitney test when comparing means of two independent groups and two-way ANOVA when comparing more than two groups. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001). Evaluation of survival patterns in tumour-bearing mice was performed using the Kaplan–Meier method, and results were ranked according to the Mantel–Cox log-rank test. P < 0.05was considered statistically significant. Survival was defined as mice with tumours <2,500 cm3. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Experiments have been repeated to ensure reproducibility of the observations. Source Data for the main figures are provided in the online version of this paper.

News Article | December 9, 2016

JACKSONVILLE, Fla.--(BUSINESS WIRE)--Borregaard ASA (BRG) and Rayonier Advanced Materials Inc. (NYSE:RYAM) announced today the companies have secured the necessary approvals from their Boards of Directors and the appropriate permits to proceed with the investment for construction of a new lignin facility at RYAM’s Fernandina Beach site in Florida. The venture, named LignoTech Florida (LTF), will serve the growing global demand for natural lignin-based products. Lignin, a natural component of wood, is a co-product of RYAM’s sulphite cellulose manufacturing process. The new operation will process the lignin into value-added products that provide environmentally-friendly alternatives to fossil fuel-based products used globally in construction, agriculture and other industrial applications. The plant is expected to be constructed at a cost of $135 million over two phases of the project. Phase one, which will deliver a lignin capacity of 100,000 metric tons, is estimated to cost $110 million. An estimated incremental $25 million will be required in phase two to increase the total capacity to 150,000 metric tons. Construction for the project is expected to begin shortly with operations beginning approximately 18 months after the commencement of construction. LTF received unwavering support from local city, county and state officials during the evaluation phase of the process. The economic and logistical support provided by local and state governments helped to bring this investment into the community and is expected to create more than 50 new high-paying jobs. A study commissioned by Nassau County determined the facility will produce an annual economic impact on the region of more than $28 million. LTF will be owned 55 percent by BRG and 45 percent by RYAM. BRG will provide its market leading technical knowledge and global sales distribution network, while RYAM will supply the raw material, site services and other support. The parties expect to finance about half of the $110 million investment for phase one. Financing will reduce the capital required by BRG and RYAM pro rata based on their ownership levels. Investment returns for the project are expected to exceed a mid-teens return hurdle for the investors. Rayonier Advanced Materials is the leading global supplier of high-purity, cellulose specialties natural polymers for the chemical industry. Working closely with its customers, the Company engineers natural polymeric chemical chains to create dozens of customized high-purity performance fibers at its plants in Florida and Georgia. Rayonier Advanced Materials’ intellectual property and manufacturing processes have been developed over 85 years, resulting in unique properties and very high quality and consistency. The Company’s facilities have the capacity to produce approximately 485,000 tons of cellulose specialties for use in a wide range of industrial and consumer products such as filters, cosmetics and pharmaceuticals and approximately 245,000 tons of commodity products. Rayonier Advanced Materials is consistently ranked among the nation’s top 50 exporters and delivers products to 79 ports around the world, serving customers in 20 countries across five continents. More information is available at Borregaard operates the world’s most advanced biorefinery. By using natural, sustainable raw materials, Borregaard produces advanced and environmentally friendly biochemicals, biomaterials and bioethanol that replace oil-based products. The Borregaard Group has 1050 employees in 16 countries and had revenues in 2015 of approximately $500 million. The lignin business constitutes close to 50% of the Group’s turnover with plants in 7 countries. Certain statements in this document regarding anticipated financial, business, legal or other outcomes including business and market conditions, outlook and other similar statements relating to Rayonier Advanced Materials’ or LignoTech Florida’s future or expected events, developments or financial or operational performance or results, are "forward-looking statements" made pursuant to the safe harbor provisions of the Private Securities Litigation Reform Act of 1995 and other federal securities laws. These forward-looking statements are identified by the use of words such as "may," "will," "should," "expect," "estimate," "believe," "intend," "anticipate" and other similar language. However, the absence of these or similar words or expressions does not mean that a statement is not forward-looking. While we believe that these forward-looking statements are reasonable when made, forward-looking statements are not guarantees of future performance or events and undue reliance should not be placed on these statements. Although we believe that the expectations reflected in any forward-looking statements are based on reasonable assumptions, we can give no assurance that these expectations will be attained and it is possible that actual results may differ materially from those indicated by these forward-looking statements due to a variety of risks and uncertainties. Other important factors that could cause actual results or events to differ materially from those expressed in forward-looking statements that may have been made in this document are described or will be described in our filings with the U.S. Securities and Exchange Commission, including our Annual Report on Form 10-K and Quarterly Reports on Form 10-Q. Rayonier Advanced Materials assumes no obligation to update these statements except as is required by law.

News Article | November 15, 2016
Site: adds new analysis report on Global Drilling Machines Market 2016-2020. Drilling is cutting method where a drill bit is used to make a circular cross-section in any solid material. A drill bit is a multi-point rotary cutting tool. It is attached to the machine that makes it rotate at speeds ranging from hundreds to thousands of revolutions per minute. This, when pressed against the workpiece, creates the desired hole by cutting off chips from the workpiece. This analyst forecast the global drilling machines market to grow at a CAGR of 5.7% during the period 2016-2020. This report covers the present scenario and the growth prospects of the global drilling machines market for 2016-2020. To calculate the market size, the report presents the vendor landscape and a corresponding detailed analysis of the top four vendors in the market. The market is divided into the following segments based on geography: Key players in the global drilling machines market: DATRON, DMTG, DMG MORI, and SMTCL. Other prominent vendors in the market are: Cameron Micro Drill Presses, Ernst Lenz Maschinenbau, Fehlmann, Fives Landis, Forma, Gate Machinery International, Hsin Geeli Hardware Enterprise, Kaufman, LTF, Microlution, Minitool, MTI, Roku, Scantool, Taiwan Winnerstech Machinery, Tongtai Machine & Tool, and Yamazaki Mazak. Commenting on the drilling machines market report, an analyst said: "One of latest trends in the market is high-speed steel versus carbide tools. The cutting tool plays a vital role in metal cutting technology. The major materials used for cutting tools are high-speed steel (HSS) and carbide. HSS is a tungsten-containing high-carbon alloys. Although over the last few years carbide tools have steadily taken over HSS tools, the latter remain popular in major segments of tools, including reamers and gear shaping cutters that are not limited to drills. This is mainly due to the advantages of HSS, like its cost-effectiveness and ability to withstand higher cutting forces." Inquire for Discount on the Report at According to the drilling machines market report, one of the primary drivers in the market is integration of CAD/CAM with machine fabrication. The metal fabrication industry is increasingly making use of advances in technology in the development of machine tools. By using new technologies, manufacturers design new machine tools that are easy to operate, consume less time, and are more efficient. Electrical discharge machines, ultrasonic, and electronic beam technologies have revolutionized manufacturing technology.

WiseGuyReports.Com Publish a New Market Research Report On – “Drilling Machines Market by Manufacturers,Types,Regions and Applications Research Report Forecast to 2021”. This report studies Drilling Machines in Global market, especially in North America, Europe, China, Japan, Southeast Asia and India, focuses on top manufacturers in global market, with production, price, revenue and market share for each manufacturer, covering  DATRON  DMTG  DMG MORI  SMTCL  Cameron Micro Drill Presses  Ernst Lenz Maschinenbau  Fehlmann  Fives Landis  Forma  Gate Machinery International  Hsin Geeli Hardware Enterprise  Kaufman  LTF  Microlution  Minitool  MTI  Roku  Scantool  Taiwan Winnerstech Machinery  Tongtai Machine & Tool  Yamazaki Mazak For more information or any query mail at [email protected] Market Segment by Regions, this report splits Global into several key Regions, with production, consumption, revenue, market share and growth rate of Drilling Machines in these regions, from 2011 to 2021 (forecast), like  North America  Europe  China  Japan  Southeast Asia  India Split by product type, with production, revenue, price, market share and growth rate of each type, can be divided into  Type I  Type II  Type III Split by application, this report focuses on consumption, market share and growth rate of Drilling Machines in each application, can be divided into  Application 1  Application 2  Application 3 Global Drilling Machines Market Research Report 2016  1 Drilling Machines Market Overview  1.1 Product Overview and Scope of Drilling Machines  1.2 Drilling Machines Segment by Type  1.2.1 Global Production Market Share of Drilling Machines by Type in 2015  1.2.2 Type I  1.2.3 Type II  1.2.4 Type III  1.3 Drilling Machines Segment by Application  1.3.1 Drilling Machines Consumption Market Share by Application in 2015  1.3.2 Application 1  1.3.3 Application 2  1.3.4 Application 3  1.4 Drilling Machines Market by Region  1.4.1 North America Status and Prospect (2011-2021)  1.4.2 Europe Status and Prospect (2011-2021)  1.4.3 China Status and Prospect (2011-2021)  1.4.4 Japan Status and Prospect (2011-2021)  1.4.5 Southeast Asia Status and Prospect (2011-2021)  1.4.6 India Status and Prospect (2011-2021)  1.5 Global Market Size (Value) of Drilling Machines (2011-2021) 2 Global Drilling Machines Market Competition by Manufacturers  2.1 Global Drilling Machines Production and Share by Manufacturers (2015 and 2016)  2.2 Global Drilling Machines Revenue and Share by Manufacturers (2015 and 2016)  2.3 Global Drilling Machines Average Price by Manufacturers (2015 and 2016)  2.4 Manufacturers Drilling Machines Manufacturing Base Distribution, Sales Area and Product Type  2.5 Drilling Machines Market Competitive Situation and Trends  2.5.1 Drilling Machines Market Concentration Rate  2.5.2 Drilling Machines Market Share of Top 3 and Top 5 Manufacturers  2.5.3 Mergers & Acquisitions, Expansion 7 Global Drilling Machines Manufacturers Profiles/Analysis  7.1 DATRON  7.1.1 Company Basic Information, Manufacturing Base and Its Competitors  7.1.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.1.3 DATRON Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.1.4 Main Business/Business Overview  7.2 DMTG  7.2.1 Company Basic Information, Manufacturing Base and Its Competitors  7.2.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.2.3 DMTG Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.2.4 Main Business/Business Overview  7.3 DMG MORI  7.3.1 Company Basic Information, Manufacturing Base and Its Competitors  7.3.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.3.3 DMG MORI Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.3.4 Main Business/Business Overview  7.4 SMTCL  7.4.1 Company Basic Information, Manufacturing Base and Its Competitors  7.4.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.4.3 SMTCL Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.4.4 Main Business/Business Overview  7.5 Cameron Micro Drill Presses  7.5.1 Company Basic Information, Manufacturing Base and Its Competitors  7.5.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.5.3 Cameron Micro Drill Presses Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.5.4 Main Business/Business Overview  7.6 Ernst Lenz Maschinenbau  7.6.1 Company Basic Information, Manufacturing Base and Its Competitors  7.6.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.6.3 Ernst Lenz Maschinenbau Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.6.4 Main Business/Business Overview  7.7 Fehlmann  7.7.1 Company Basic Information, Manufacturing Base and Its Competitors  7.7.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.7.3 Fehlmann Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.7.4 Main Business/Business Overview  7.8 Fives Landis  7.8.1 Company Basic Information, Manufacturing Base and Its Competitors  7.8.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.8.3 Fives Landis Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.8.4 Main Business/Business Overview  7.9 Forma  7.9.1 Company Basic Information, Manufacturing Base and Its Competitors  7.9.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.9.3 Forma Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.9.4 Main Business/Business Overview  7.10 Gate Machinery International  7.10.1 Company Basic Information, Manufacturing Base and Its Competitors  7.10.2 Drilling Machines Product Type, Application and Specification Type I Type II  7.10.3 Gate Machinery International Drilling Machines Production, Revenue, Price and Gross Margin (2015 and 2016)  7.10.4 Main Business/Business Overview  7.11 Hsin Geeli Hardware Enterprise  7.12 Kaufman  7.13 LTF  7.14 Microlution  7.15 Minitool  7.16 MTI  7.17 Roku  7.18 Scantool  7.19 Taiwan Winnerstech Machinery  7.20 Tongtai Machine & Tool  7.21 Yamazaki Mazak For more information or any query mail at [email protected] Wise Guy Reports is part of the Wise Guy Consultants Pvt. Ltd. and offers premium progressive statistical surveying, market research reports, analysis & forecast data for industries and governments around the globe. Wise Guy Reports features an exhaustive list of market research reports from hundreds of publishers worldwide. We boast a database spanning virtually every market category and an even more comprehensive collection of market research reports under these categories and sub-categories.

Morabito D.,Branch of the Engineering Group Systra | Bufalini M.,LTF | Farinetti A.,LTF | Elia P.,LTF
Tunnels and Tunnelling International | Year: 2014

The article discusses plans for La Maddalena exploratory tunnel, the organizational structure of the stretches of line, and problems that have dogged the project since before site handover. The future high-speed rail 'Mediterranean Corridor' will be a line that will run for a total length of 3,000 km across several EU nations. The line is listed among the 10 top priority projects of the great Trans-European Network (TEN) for the period 2014-2020. The corridor starts from Algeciras in Spain and runs to Budapest in Hungary. This line includes the Spanish Algeciras-Madrid-Tarragona and Seville-Valencia-Tarragona stretches, before crossing Barcelona, where there is also a rail-port-interconnection. After this it runs through France from Perpignan, and through the Lyon-Turin route into Italy, where it should then join up with the Milan-Venice route and Trieste before connecting with Ljubljana in Slovenia and then Budapest.

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