Sant'Ambrogio di Torino, Italy
Sant'Ambrogio di Torino, Italy

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No statistical methods were used to predetermine sample size for biochemical or cell-based assays, or for pharmacokinetic studies. Investigators were not blinded to outcome assessment during these investigations. For GS-5734 efficacy assessments in nonhuman primates, statistical power analysis was used to predetermine sample size, and subjects were randomly assigned to experimental group, stratified by sex and balanced by body weight. Study personnel responsible for assessing animal health (including euthanasia assessment) and administering treatments were experimentally blinded to group assignment of animals and outcome. GS-5734, Nuc, and NTP were synthesized at Gilead Sciences, Inc., and chemical identity and sample purity were established using NMR, HRMS, and HPLC analysis (Supplementary Information). The radiolabelled analogue [14C]GS-5734 (specific activity, 58.0 mCi mmol−1) was obtained from Moravek Biochemicals (Brea, California) and was prepared in a similar manner described for GS-5734 using [14C]trimethylsilylcyanide (Supplementary Information). Small molecule X-ray crystallographic coordinates and structure factor files have been deposited in the Cambridge Structural Database ( and accession numbers are supplied in the Supplementary Information. RSV A2 was purchased from Advanced Biotechnologies, Inc. EBOV (Kikwit and Makona variants), Sudan virus (SUDV, Gulu), Marburg virus (MARV, Ci67), Junín virus (JUNV, Romero), Lassa virus (LASV, Josiah), Middle East respiratory syndrome virus (MERS, Jordan N3), Chikungunya virus (CHIV, AF 15561), and Venezuelan equine encephalitis virus (VEEV, SH3) were all prepared and characterized at the United States Army Medical Research Institute for infectious diseases (USAMRIID). EBOV containing a GFP reporter gene (EBOV–GFP), EBOV Makona (Liberia, 2014), and MARV containing a GFP reporter gene (MARV–GFP) were prepared and characterized at the Centers for Disease Control and Prevention26, 27. HEp-2 (CCL-23), PC-3 (CCL-1435), HeLa (CCL-2), U2OS (HTB-96), Vero (CCL-81), HFF-1 (SCRC-1041), and HepG2 (HB-8065) cell lines were purchased from the American Type Culture Collection. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in assays. HEp-2 cells were cultured in Eagle’s Minimum Essential Media (MEM) with GlutaMAX supplemented with 10% fetal bovine serum (FBS) and 100 U ml−1 penicillin and streptomycin. PC-3 cells were cultured in Kaighn’s F12 media supplemented with 10% FBS and 100 U ml−1 penicillin and streptomycin. HeLa, U2OS, and Vero cells were cultured in MEM supplemented with 10% FBS, 1% l-glutamine, 10 mM HEPES, 1% non-essential amino acids, and 1% penicillin/streptomycin. HFF-1 cells were cultured in MEM supplemented with 10% FBS and 0.5 mM sodium pyruvate. HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 0.1 mM non-essential amino acids. The MT-4 cell line was obtained from the NIH AIDS Research and Reference Reagent Program and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and 2 mM l-glutamine. The Huh-7 cell line was obtained from C. M. Rice (Rockefeller University) and cultured in DMEM supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin, and non-essential amino acids. Primary human hepatocytes were purchased from Invitrogen and cultured in William’s Medium E medium containing cell maintenance supplement. Donor profiles were limited to 18- to 65-year-old nonsmokers with limited alcohol consumption. Upon delivery, the cells were allowed to recover for 24 h in complete medium with supplement provided by the vendor at 37 °C. Human PBMCs were isolated from human buffy coats obtained from healthy volunteers (Stanford Medical School Blood Center, Palo Alto, California) and maintained in RPMI-1640 with GlutaMAX supplemented with 10% FBS, 100 U ml−1 penicillin and streptomycin. Rhesus fresh whole blood was obtained from Valley Biosystems. PBMCs were isolated from whole blood by Ficoll-Hypaque density gradient centrifugation. Briefly, blood was overlaid on 15 ml Ficoll-Paque (GE Healthcare Bio-Sciences AB), and centrifuged at 500g for 20 min. The top layer containing platelets and plasma was removed, and the middle layer containing PBMCs was transferred to a fresh tube, diluted with Tris buffered saline up to 50 ml, and centrifuged at 500g for 5 min. The supernatant was removed and the cell pellet was resuspended in 5 ml red blood cell lysis buffer (155 mM ammonium chloride, 10 mM potassium bicarbonate, 0.1 mM EDTA, pH 7.5). To generate stimulated PBMCs, freshly isolated quiescent PBMCs were seeded into a T-150 (150 cm2) tissue culture flask containing fresh medium supplemented with 10 U ml−1 of recombinant human interleukin-2 (IL-2) and 1 μg ml−1 phytohaemagglutinin-P at a density of 2 × 106 cells ml−1 and incubated for 72 h at 37 °C. Human macrophage cultures were isolated from PBMCs that were purified by Ficoll gradient centrifugation from 50 ml of blood from healthy human volunteers. PBMCs were cultured for 7 to 8 days in in RPMI cell culture media supplemented with 10% FBS, 5 to 50 ng ml−1 granulocyte-macrophage colony-stimulating factor and 50 μM β-mercaptoethanol to induce macrophage differentiation. The cryopreserved human primary renal proximal tubule epithelial cells were obtained from LifeLine Cell Technology and isolated from the tissue of human kidney. The cells were cultured at 90% confluency with RenaLife complete medium in a T-75 flask for 3 to 4 days before seeding into 96-well assay plates. Immortalized human microvascular endothelial cells (HMVEC-TERT) were obtained from R. Shao at the Pioneer Valley Life Sciences Institute28. HMVEC-TERT cells were cultured in endothelial basal media supplemented with 10% FBS, 5 μg of epithelial growth factor, 0.5 mg hydrocortisone, and gentamycin/amphotericin-B. RNA POLII was purchased as part of the HeLaScribe Nuclear Extract in vitro Transcription System kit from Promega. The recombinant human POLRMT and transcription factors mitochondrial transcription factors A (mtTFA or TFAM) and B2 (mtTFB2 or TFB2M) were purchased from Enzymax. RSV ribonucleoprotein (RNP) complexes were prepared according to a method modified from ref. 29. The intracellular metabolism of GS-5734 was assessed in different cell types (HMVEC and HeLa cell lines, and primary human and rhesus PBMCs, monocytes and monocyte-derived macrophages) following 2-h pulse or 72-h continuous incubations with 10 μM GS-5734. For comparison, intracellular metabolism during a 72-h incubation with 10 μM of Nuc was completed in human monocyte-derived macrophages. For pulse incubations, monocyte-derived macrophages isolated from rhesus monkeys or humans were incubated for 2 h in compound-containing media followed by removal, washing with 37 °C drug-free media, and incubated for an additional 22 h in media which did not contain GS-5734. Human monocyte-derived macrophages, HeLa and HMVEC were grown to confluence (approximately 0.5, 0.2, and 1.2 × 106 cells per well, respectively) in 500 μl of media in 12-well tissue culture plates. Monocyte and PBMCs were incubated in suspension (approximately 1 × 106 cells ml−1) in 1 ml of media in micro centrifuge tubes. For adherent cells (HMVEC, HeLa, and monocyte-derived macrophages), media was removed at select time points from duplicate wells, cells washed twice with 2 ml of ice-cold 0.9% normal saline. For non-adherent cells (monocytes and PBMCs), duplicate incubations were centrifuged at 2,500g for 30 s to remove media. The cell pellets were re-suspended with 500 μl cell culture media (RPMI with 10% FBS) and layered on top of a 500 μl oil layer (Nyosil M25; Nye Lubricants) in a microcentrifuge tube. Samples were then centrifuged at room temperature at 13,000 r.p.m. for 45 s. The media layer was removed and the oil layer was washed twice with 500 μl water. The oil layer was then carefully removed using a Pasteur pipet attached to vacuum. A volume of 0.5 ml of 70% methanol containing 100 nM of the analytical internal standard 2-chloro-adenosine-5′-triphosphate (Sigma-Aldrich) was added to isolated cells. Samples were stored overnight at −20 °C to facilitate extraction, centrifuged at 15,000g for 15 min and then supernatant was transferred to clean tubes for drying in a MiVac Duo concentrator (Genevac). Dried samples were then reconstituted in mobile phase A containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine (DMH) in water for analysis by liquid chromatography coupled to triple quadrupole mass spectrometry (LC-MS/MS). LC-MS/MS was performed using low-flow ion-pairing chromatography, similar to methods described previously30. Briefly, analytes were separated using a 50 × 2 mm × 2.5 μm Luna C18(2) HST column (Phenomenex) connected to a LC-20ADXR (Shimadzu) ternary pump system and HTS PAL autosampler (LEAP Technologies). A multi-stage linear gradient from 10% to 50% acetonitrile in a mobile phase containing 3 mM ammonium formate (pH 5.0) with 10 mM dimethylhexylamine over 8 min at a flow rate of 150 μl min−1 was used to separate analytes. Detection was performed on an API 4000 (Applied Biosystems) MS/MS operating in positive ion and multiple reaction monitoring modes. Intracellular metabolites alanine metabolite, Nuc, nucleoside monophosphate, nucleoside diphosphate, and nucleoside triphosphate were quantified using 7-point standard curves ranging from 0.274 to 200 pmol (approximately 0.5 to 400 μM) prepared in cell extract from untreated cells. Levels of adenosine nucleotides were also quantified to assure dephosphorylation had not taken place during sample collection and preparation. In order to calculate intracellular concentration of metabolites, the total number of cells per sample were counted using a Countess automated cell counter (Invitrogen). Antiviral assays were conducted in biosafety level 4 containment (BSL-4) at the Centers for Disease Control and Prevention. EBOV antiviral assays were conducted in primary HMVEC-TERT and in Huh-7 cells. Huh-7 cells were not authenticated and were not tested for mycoplasma. Ten concentrations of compound were diluted in fourfold serial dilution increments in media, and 100 μl per well of each dilution was transferred in duplicate (Huh-7) or quadruplicate (HMVEC-TERT) onto 96-well assay plates containing cell monolayers. The plates were transferred to BSL-4 containment, and the appropriate dilution of virus stock was added to test plates containing cells and serially diluted compounds. Each plate included four wells of infected untreated cells and four wells of uninfected cells that served as 0% and 100% virus inhibition controls, respectively. After the infection, assay plates were incubated for 3 days (Huh-7) or 5 days (HMVEC-TERT) in a tissue culture incubator. Virus replication was measured by direct fluorescence using a Biotek HTSynergy plate reader. For virus yield assays, Huh-7 cells were infected with wild-type EBOV for 1 h at 0.1 plaque-forming units (PFU) per cell. The virus inoculum was removed and replaced with 100 μl per well of media containing the appropriate dilution of compound. At 3 days post-infection, supernatants were collected, and the amount of virus was quantified by endpoint dilution assay. The endpoint dilution assay was conducted by preparing serial dilutions of the assay media and adding these dilutions to fresh Vero cell monolayers in 96-well plates to determine the tissue culture infectious dose that caused 50% cytopathic effects (TCID ). To measure levels of viral RNA from infected cells, total RNA was extracted using the MagMAX-96 Total RNA Isolation Kit and quantified using a quantitative reverse transcription polymerase chain reaction (qRT–PCR) assay with primers and probes specific for the EBOV nucleoprotein gene. Antiviral assays were conducted in BSL-4 at USAMRIID. HeLa or HFF-1 cells were seeded at 2,000 cells per well in 384-well plates. Ten serial dilutions of compound in triplicate were added directly to the cell cultures using the HP D300 digital dispenser (Hewlett Packard) in twofold dilution increments starting at 10 μM at 2 h before infection. The DMSO concentration in each well was normalized to 1% using an HP D300 digital dispenser. The assay plates were transferred to the BSL-4 suite and infected with EBOV Kikwit at a multiplicity of infection of 0.5 PFU per cell for HeLa cells and with EBOV Makona at a multiplicity of infection of 5 PFU per cell for HFF-1 cells. The assay plates were incubated in a tissue culture incubator for 48 h. Infection was terminated by fixing the samples in 10% formalin solution for an additional 48 h before immune-staining, as described in Supplementary Table 1. Antiviral assays were conducted in BSL-4 at USAMRIID. Primary human macrophage cells were seeded in a 96-well plate at 40,000 cells per well. Eight to ten serial dilutions of compound in triplicate were added directly to the cell cultures using an HP D300 digital dispenser in threefold dilution increments 2 h before infection. The concentration of DMSO was normalized to 1% in all wells. The plates were transferred into the BSL-4 suite, and the cells were infected with 1 PFU per cell of EBOV in 100 μl of media and incubated for 1 h. The inoculum was removed, and the media was replaced with fresh media containing diluted compounds. At 48 h post-infection, virus replication was quantified by immuno-staining as described in Supplementary Table 1. For antiviral tests, compounds were threefold serially diluted in source plates from which 100 nl of diluted compound was transferred to a 384-well cell culture plate using an Echo acoustic transfer apparatus. HEp-2 cells were added at a density of 5 × 105 cells per ml, then infected by adding RSV A2 at a titer of 1 × 104.5 tissue culture infectious doses (TCID ) per ml. Immediately following virus addition, 20 μl of the virus and cells mixture was added to the 384-well cell culture plates using a μFlow liquid dispenser and cultured for 4 days at 37 °C. After incubation, the cells were allowed to equilibrate to 25 °C for 30 min. The RSV-induced cytopathic effect was determined by adding 20 μl of CellTiter-Glo Viability Reagent. After a 10-min incubation at 25 °C, cell viability was determined by measuring luminescence using an Envision plate reader. Antiviral assays were conducted in 384-or 96-well plates in BSL-4 at USAMRIID using a high-content imaging system to quantify virus antigen production as a measure of virus infection. A ‘no virus’ control and a ‘1% DMSO’ control were included to determine the 0% and 100% virus infection, respectively. The primary and secondary antibodies and dyes used for nuclear and cytoplasmic staining are listed in Supplementary Table 1. The primary antibody specific for a particular viral protein was diluted 1,000-fold in blocking buffer (1 × PBS with 3% BSA) and added to each well of the assay plate. The assay plates were incubated for 60 min at room temperature. The primary antibody was removed, and the cells were washed three times with 1 × PBS. The secondary detection antibody was an anti-mouse (or rabbit) IgG conjugated with Dylight488 (Thermo Fisher Scientific, catalogue number 405310). The secondary antibody was diluted 1,000-fold in blocking buffer and was added to each well in the assay plate. Assay plates were incubated for 60 min at room temperature. Nuclei were stained using Draq5 (Biostatus) or 33342 Hoechst (ThermoFisher Scientific) for Vero and HFF-1 cell lines. Both dyes were diluted in 1× PBS. The cytoplasm of HFF-1 (EBOV assay) and Vero E6 (MERS assay) cells were counter-stained with CellMask Deep Red (Thermo Fisher Scientific). Cell images were acquired using a Perkin Elmer Opera confocal plate reader (Perkin Elmer) using a ×10 air objective to collect five images per well. Virus-specific antigen was quantified by measuring fluorescence emission at a 488 nm wavelength and the stained nuclei were quantified by measuring fluorescence emission at a 640 nm wavelength. Acquired images were analysed using Harmony and Acapella PE software. The Draq5 signal was used to generate a nuclei mask to define each nuclei in the image for quantification of cell number. The CellMask Deep Red dye was used to demarcate the Vero and HFF-1 cell borders for cell-number quantitation. The viral-antigen signal was compartmentalized within the cell mask. Cells that exhibited antigen signal higher than the selected threshold were counted as positive for viral infection. The ratio of virus-positive cells to total number of analysed cells was used to determine the percentage of infection for each well on the assay plates. The effect of compounds on the viral infection was assessed as percentage of inhibition of infection in comparison to control wells. The resultant cell number and percentage of infection were normalized for each assay plate. Analysis of dose–response curve was performed using GeneData Screener software applying Levenberg–Marquardt algorithm for curve-fitting strategy. The curve-fitting process, including individual data point exclusion, was pre-specified by default software settings. R2 value quantified goodness of fit and fitting strategy was considered acceptable at R2 > 0.8. All virus infections were quantified by immuno-staining using antibodies that recognized the relevant viral glycoproteins, as described in Supplementary Table 1. HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 1 PFU per cell MARV, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.08 PFU SUDV per cell, which resulted in 50% to 70% of the cells expressing virus antigen in a 48-h period. HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.3 PFU per cell JUNV, which resulted in ~50% of the cells expressing virus antigen in a 48-h period. HeLa cells were seeded at 2,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell LASV, which resulted in >60% of the cells expressing virus antigen in a 48-h period. African green monkey (Chlorocebus sp.) kidney epithelial cells (Vero E6) were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of MERS virus, which resulted in >70% of the cells expressing virus antigen in a 48-h period. U2OS cells were seeded at 3,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.5 PFU per cell of CHIK, which resulted in >80% of the cells expressing virus antigen in a 48-h period. HeLa cells were seeded at 4,000 cells per well in a 384-well plate, and compounds were added to the assay plates. Assay plates were transferred to the BSL-4 suite and infected with 0.1 PFU per cell VEEV, which resulted in >60% of the cells expressing virus antigen in a 20-h period. HEp-2 (1.5 × 103 cells per well) and MT-4 (2 × 103 cells per well) cells were plated in 384-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. PC-3 cells (2.5 × 103 cells per well), HepG2 cells (4 × 103 cells per well), hepatocytes (1 × 106 cells per well), quiescent PBMCs (1 × 106 cells per well), stimulated PBMCs (2 × 105 cells per well), and RPTEC cells (1 × 103 cells per well) were plated in 96-well plates and incubated with the appropriate medium containing threefold serially diluted compound ranging from 15 nM to 100,000 nM. Cells were cultured for 4–5 days at 37 °C. Following the incubation, the cells were allowed to equilibrate to 25 °C, and cell viability was determined by adding Cell-Titer Glo viability reagent. The mixture was incubated for 10 min, and the luminescence signal was quantified using an Envision plate reader. Cell lines were not authenticated and were not tested for mycoplasma as part of routine use in cytotoxicity assays. RNA synthesis by the RSV polymerase was reconstituted in vitro using purified RSV L/P complexes and an RNA oligonucleotide template (Dharmacon), representing nucleotides 1–14 of the RSV leader promoter31, 32, 33 (3′-UGCGCUUUUUUACG-5′). RNA synthesis reactions were performed as described previously, except that the reaction mixture contained 250 μM guanosine triphosphate (GTP), 10 μM uridine triphosphate (UTP), 10 μM cytidine triphosphate (CTP), supplemented with 10 μCi [α-32P]CTP, and either included 10 μM adenosine triphosphate (ATP) or no ATP. Under these conditions, the polymerase is able to initiate synthesis from the position 3 site of the promoter, but not the position 1 site. The NTP metabolite of GS-5734 was serially diluted in DMSO and included in each reaction mixture at concentrations of 10, 30, or 100 μM as specified in Fig. 1f. RNA products were analysed by electrophoresis on a 25% polyacrylamide gel, containing 7 M urea, in Tris–taurine–EDTA buffer, and radiolabelled RNA products were detected by autoradiography. Transcription reactions contained 25 μg of crude RSV RNP complexes in 30 μL of reaction buffer (50 mM Tris-acetate (pH 8.0), 120 mM potassium acetate, 5% glycerol, 4.5 mM MgCl , 3 mM DTT, 2 mM EGTA, 50 μg ml−1 BSA, 2.5 U RNasin, 20 μM ATP, 100 μM GTP, 100 μM UTP, 100 μM CTP, and 1.5 μCi [α-32P]ATP (3,000 Ci mmol−1)). The radiolabelled nucleotide used in the transcription assay was selected to match the nucleotide analogue being evaluated for inhibition of RSV RNP transcription. To determine whether nucleotide analogues inhibited RSV RNP transcription, compounds were added using a six-step serial dilution in fivefold increments. After a 90-min incubation at 30 °C, the RNP reactions were stopped with 350 μl of Qiagen RLT lysis buffer, and the RNA was purified using a Qiagen RNeasy 96 kit. Purified RNA was denatured in RNA sample loading buffer at 65 °C for 10 min and run on a 1.2% agarose/MOPS gel containing 2 M formaldehyde. The agarose gel was dried, exposed to a Storm phosphorimaging screen, and developed using a Storm phosphorimager. For a 25 μl reaction mixture, 7.5 μl 1 × transcription buffer (20 mM HEPES (pH 7.2–7.5), 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 20% glycerol), 3 mM MgCl , 100 ng CMV positive or negative control DNA, and a mixture of ATP, GTP, CTP and UTP was pre-incubated with various concentrations (0–500 μM) of the inhibitor at 30 °C for 5 min. The mixture contained 5–25 μM (equal to K ) of the competing 33P-labelled ATP and 400 μM of GTP, UTP, and CTP. The reaction was started by addition of 3.5 μl of HeLa and extract. After 1 h of incubation at 30 °C, the polymerase reaction was stopped by addition of 10.6 μl proteinase K mixture that contained final concentrations of 2.5 μg μl−1 proteinase K, 5% SDS, and 25 mM EDTA. After incubation at 37 °C for 3–12 h, 10 μl of the reaction mixture was mixed with 10 μl of the loading dye (98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue), heated at 75 °C for 5 min, and loaded onto a 6% polyacrylamide gel (8 M urea). The gel was dried for 45 min at 70 °C and exposed to a phosphorimager screen. The full length product, 363 nucleotide runoff RNA, was quantified using a Typhoon Trio Imager and Image Quant TL Software. Twenty nanomolar POLRMT was incubated with 20 nM template plasmid (pUC18-LSP) containing POLRMT light-strand promoter region and mitochondrial (mt) transcription factors TFA (100 nM) and mtTFB2 (20 nM) in buffer containing 10 mM HEPES (pH 7.5), 20 mM NaCl, 10 mM DTT, 0.1 mg ml−1 BSA, and 10 mM MgCl 34. The reaction mixture was pre-incubated to 32 °C, and the reactions were initiated by addition of 2.5 μM of each of the natural NTPs and 1.5 μCi of [32P]GTP. After incubation for 30 min at 32 °C, reactions were spotted on DE81 paper and quantified. A homology model of RSV A2 and EBOV polymerases were built using the HIV reverse transcriptase X-ray crystal structure (PDB:1RTD). Schrödinger Release 2015-1: Prime, version 3.9 (Schrödinger, LLC), default settings with subsequent rigid body minimization and side-chain optimization. Loop insertions not in 1RTD of greater than 10 amino acids were not built. For quantitative assessment of viral RNA nonhuman primate plasma samples, whole blood was collected using a K3 EDTA Greiner Vacuette tube (or equivalent) and sample centrifuged at 2500 (± 200) relative centrifugal force for 10 ± 2 min. To inactivate virus, plasma was treated with 3 parts (300 μl) TriReagent LS and samples were transferred to frozen storage (−60 °C to −90 °C), until removal for RNA extraction. Carrier RNA and QuantiFast High Concentration Internal Control (Qiagen) were spiked into the sample before extraction, conducted according to manufacturer’s instructions. The viral RNA was eluted in AVE buffer. Each extracted RNA sample was tested with the QuantiFast Internal Control RT–PCR RNA Assay (Qiagen) to evaluate the yield of the spiked-in QuantiFast High Concentration Internal Control. If the internal control amplified within manufacturer-designated ranges, further quantitative analysis of the viral target was performed. RT–PCR was conducted using an ABI 7500 Fast Dx using primers specific to EBOV glycoprotein. Samples were run in triplicate using a 5 μl template volume. For quantitative assessments, the average of the triplicate genomic equivalents (GE) per reaction were determined and multiplied by 800 to obtain GE ml−1 plasma. Standard curves were generated using synthetic RNA. The limits of quantification for this assay are 8.0 × 104 − 8.0 × 1010 GE ml−1 of plasma. Acceptance criteria for positive template control (PTC), negative template control (NTC), negative extraction control (NEC), and positive extraction control (PEC) are specified by standard operating procedure. For qualitative assessments, the limit of detection (LOD) was defined as C 38.07, based on method validation testing. An animal was considered to have tested positive for detection of EBOV RNA when a minimum of 2 of 3 replicates were designated as ‘positive’ and PTC, NTC, and NEC controls met specified method-acceptance criteria. A sample was designated as ‘positive’ when the C value was

News Article | February 15, 2017

Oklahoma City based Sigma Blood Systems has established a relationship with Central Jersey Blood Center as the newest client for the firm’s PERFEQTA software and legacy product QC Manager 2.0. Sigma Blood Systems CEO, Max Doleh, stated “We are pleased to work with the dynamic team at CJBC and thrilled that they have decided to implement PERFEQTA and QC Manager 2.0, with PERFEQTA launching across multiple departments within their organization. Having the ability to link together different areas of the business is key to their ongoing mission of saving lives.” PERFEQTA is the world’s first fully flexible software for managing processes, procedures, and forms in any size business. The web-based platform gives users the ability to automate, track, and audit multiple processes throughout their business, all in a collaborative setting, complete with compliance standards that meet FDA guidance such as 21 CFR Part 11 for electronic records and eSignatures. With a fully integrated reporting dashboard that allows the user insight into data from any area of their operation, PERFEQTA provides vision into the business never before possible. Central Jersey Blood Center has been in operation for more than 50 years and services 16 hospitals in the state. CJBC will look to compliment other software systems as well as unify data from their day to day operations by implementing PERFEQTA across multiple departments including Quality Assurance, Donor Services, Human Resources, and their core lab facilities. Central Jersey Blood Center joins The University of Texas – MD Anderson Cancer Center as an early adopter of Sigma’s PERFEQTA, where the SaaS based platform is currently being utilized for a number of automation, scheduling, and electronic data capturing initiatives. “In today’s competitive environment, CJBC is focusing on creating value for its customers. PERFEQTA and QC Manager will allow our team members to re-direct their attention from tracking paperwork, making calculations, repeating entries on forms and trending data to analyzing early warnings, thinking about root causes and maintain continuous improvement” stated CJBC CEO Pascal George, when asked about his desire to implement PERFEQTA and QC Manager 2.0. As part of CJBC’s mission of “saving lives by providing safe, high-quality blood products and services to patients in need,” the organization is committed to adopting new technology that aids in the continuation of this mission. Technological advancements such as software automation, increased business intelligence through data analytics, and the progression toward paperless environments all aid in overall increases in operational efficiency. Products such as PERFEQTA, with its agile codeless application structure give key personnel within the operation the ability to implement change, contributing to the ISO principle of continual improvement. As a tool to help medical laboratories maintain and achieve the standards set forth by ISO:15189, PERFEQTA has been designed with this and other ISO standards in mind.

News Article | October 28, 2016

Terumo BCT will exhibit apheresis collection and blood processing technologies at the annual AABB 2016 Meeting, a premier event targeting blood center and hospital professionals interested in advancing transfusion and cell therapy. As a key meeting sponsor and global leader in this area, Terumo BCT will participate in CME sessions, host a symposium and demonstrate its technologies at AABB 2016. Innovations on display in the booth #937 include: Trima Accel® Automated Blood Collection System–The world’s leading apheresis collection device and the only system on the market today that can collect any transfusable component, in any combination, helping blood centers better meet the demand for blood in their communities. The device can be used for in-center collections or on mobile drives and is supported by a range of innovative business intelligence and operational software that helps drive efficiencies and quality. Spectra Optia® Apheresis System–The industry's next-generation therapeutic apheresis and cell collection platform designed to help operators spend more time focusing on patient care. Terumo BCT Suite of Software Solutions–Innovative technologies that help customers harness the power of connectivity and real-time data, providing visibility into a blood center’s collection and processing procedures. Terumo BCT is an exclusive authorized distributor of B Medical Systems biomedical refrigeration products in the United States and Canada. Please visit B Medical Systems at booth #1044 for product demonstrations. Other Terumo BCT activities taking place throughout the course of the meeting are as follows: 1:00 p.m. Room W230 A/B Orange County Convention Center     Pre-Conference Workshop: (HEMO) Hemovigilance Workshop: Learn How Hemovigilance is Practiced in the U.S. and How Hospitals Can Participate Palani Palaniappan, Executive Vice President,Innovation & Development - Terumo BCT “Safety Innovations and Hemovigilance: Broad Perspectives on Enabling Technologies” 7:00 a.m. Room W240 Orange County Convention Center     Breakfast Symposium: Bringing Value to Your Blood Center: The Potential of Apheresis Therapies     Richard Smith, Global Clinical Scientist, Terumo BCT "Apheresis Optimization for Immunotherapy Collections” Ross Fasano, MD Assistant Professor, Pathology & Laboratory Medicine, Emory University School of Medicine “Value of Red Blood Cell Exchange for Sickle Cell Disease Patients and the Impact to Blood Centers” About Terumo BCT Terumo BCT, a global leader in blood component, therapeutic apheresis and cellular technologies, is the only company with the unique combination of apheresis collections, manual and automated whole blood processing, and pathogen reduction technologies. We believe in the potential of blood to do even more for patients than it does today. This belief inspires our innovation and strengthens our collaboration with customers.

Galanakis D.K.,State University of New York at Stony Brook | Neerman-Arbez M.,University Hospitals Geneva Medical Center | Kudryk B.,Blood Center | Henschen A.,UCI
Blood Coagulation and Fibrinolysis | Year: 2010

Reported evidence of a role in fibrinolysis by fibrinopeptide (Fp)B-dependent intermolecular fibrin polymerization contacts and of reversed FpA/FpB release sequence from fibrinogen Kingsport led us to investigate the fibrinolytic properties of Kingsport clots. Clot lysis was induced by either plasmin (pH 7.4) or by a mixture of plasminogen and recombinant tissue plasminogen activator and measured by lysis time and by turbidity (350 nm) time course. Clots were formed by thrombin from plasminogen-free fibrinogen (pH 7.4, 8 mmol/l CaCl2), with or without 40 nmol/l factor XIII or 20% afibrinogenemic plasma. Displaying no differences from corresponding normal controls were (a) lysis of repolymerized fibrin clots, and (b) chromogenic measurements of fibrin-stimulated Glu-plasminogen activation by recombinant tissue plasminogen activator. By contrast, thrombin-induced fine and coarse network clots (n = 7) displayed faster turbidity loss than corresponding normal controls and shorter lysis times ranging 31-55% of controls. Comparison of clots of fibrinogen fractions lacking approximately 90% of their alpha chain carboxyl terminal regions, n = 2, also displayed faster plasmin-induced lysis than corresponding controls. To assess the role of FpB release-dependent intermolecular polymerization contacts, clots were prepared in the presence of three molar excess antiβ 15-42 immunoglobulin G, n = 2, and displayed no differences in plasmin-induced lysis from nonimmune immunoglobulin G controls. The reversed FpA/FpB release sequence from Kingsport fibrinogen resulted in clots with decreased resistance to plasmin. We suggest that both markedly slow polymerization and decreased plasmin resistance played causative roles in the hemorrhagic diathesis associated with this dysfibrinogen. © 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins.

News Article | October 29, 2016

The San Francisco Chocolate Factory has launched an Indiegogo Campaign on October 26th. The San Francisco Chocolate Factory, long a tradition in the South of Market area, has had to relocate due to the high commercial rents in San Francisco from the latest technology boom. “We’ve kicked off this Indiegogo Campaign to re-launch our company at the new location and to stay in San Francisco,” according to company founder and CEO, Mike Litton. “We are surrounded by Uber, Zynga and Twitter. We are an equal opportunity chocolate provider, but these technology giants and others are causing commercial rents to soar. We are asking the local technology, business, foodie and chocolate community to help us out by buying some of our chocolate to help us stay in the City that we’ve known for so many years.” The company’s rent at their previous location increased 300% so they had to relocate. They like their new office on Market but are reaching out to chocolate lovers everywhere to consider donating to their Indiegogo campaign to keep them in SF. Donors to the campaign will receive chocolate and other fun gifts in return. The chocolate is packaged in colorful tins with images of iconic San Francisco scenes and themes based upon pairing chocolate with wine, books, tea and coffee. The business started in 2001 and has loyal customers and a following, but a move is hard on any company’s pocketbook. According to Litton, “Even our new location on Market near Van Ness is seeing changes in the few weeks we have been here. The Goodwill store across the street is slated to be torn down and turned into a 29 story building!” With chocolate lover’s help everywhere, The San Francisco Chocolate Factory thinks their Indiegogo Campaign will help them stay in San Francisco where they belong as well as keep the wonderful employees that make up their amazing business. “All we are trying to do here is say: “Hey neighbors, we welcome you. How about buying some of your chocolate and gifts from us as we’ve been here in the community a long time,” said Litton. The San Francisco Chocolate Factory™ was created to provide chocolate lovers with gourmet-quality, superbly packaged chocolate at a non-gourmet price. Since debuting as The San Francisco Chocolate Factory in 1999, San Francisco Chocolate Factory™ has become a favorite with chocolate lovers nationwide. Chocolate is proven to be good for the heart and good for the soul—it creates a stir of passion and evokes love. Chocolate can erase a bad day or a bad deed in an instant. As we at the San Francisco Chocolate Factory™ like to say: Love thy neighbor, and bring them chocolate, often! Known for our unique, custom packaged gourmet chocolates, The San Francisco Chocolate Factory’s™ many different brands—now bridged together under one great company—offer a chocolate opportunity for everyone. From our Tea, Coffee and Wine Lover’s Chocolates, to the souvenir Landmark Collection, the kid friendly Got Chocolate? series to our easy on-the-go Chocolate by Numbers, you’ll find gourmet chocolates perfect for every day and for special occasions. Give The San Francisco Chocolate Factory™ as a gift to each “special someone” in your life—but make sure to order some extra chocolate for yourself! The San Francisco Chocolate Factory’s™ sales have rapidly expanded beyond the shores of the Bay Area—thanks to the many visitors who bring the beautiful chocolate tins home as souvenirs... the chocolates are so delicious, people call desperate to order more! The San Francisco Chocolate Factory™ is an active participant in the San Francisco Bay Area Community. We often provide donations of chocolate to worthy causes to help with fundraising efforts. A few of our most recent donations have gone to valuable community organizations like: City of Dreams, The UCSF Blood Center, The San Francisco Department of the Environment, The Hungry Owl Project, San Francisco Gay Pride, Support for Families with Disabilities, The Asian & Pacific Islander Wellness Center, Metropolitan Fresh Start House, The Bay Institute, The United Way, The San Francisco Sheriff’s Department, and The California Film Institute. Visit. Press Inquiries: Contact: The San Francisco Chocolate Factory PR Sandra Evans & Associates Tel 415 887 9230 sandra(at)seandassoc(dot)com

PubMed | Center of Oncology of Poland, Autonomous University of Barcelona, University of Basel, German Cancer Research Center and 2 more.
Type: | Journal: Scientific reports | Year: 2015

A genome-wide association study (GWAS) performed on a high-incidence Italian population followed by replications on low-incidence cohorts suggested a strong association of differentiated thyroid cancer (DTC) with single nucleotide polymorphisms (SNPs) at 9q22.33, 2q35, 20q11.22-q12 and 14q24.3. Moreover, six additional susceptibility loci were associated with the disease only among Italians. The present study had two aims, first to identify loci involved in DTC risk and then to assess the cumulative effect of the SNPs identified so far in the Italian population. The combined analysis of the previous GWAS and the present Italian study provided evidence of association with rs7935113 (GALNTL4, OR = 1.36, 95%CI 1.20-1.53, p-value = 7.41 10(-7)) and rs1203952 (FOXA2, OR = 1.29, 95%CI 1.16-1.44, p-value = 4.42 10(-6)). Experimental ENCODE and eQTL data suggested that both SNPs may influence the closest genes expression through a differential recruitment of transcription factors. The assessment of the cumulative risk of eleven SNPs showed that DTC risk increases with an increasing number of risk alleles (p-trend = 3.13 10(-47)). Nonetheless, only a small fraction (about 4% on the disease liability scale) of DTC is explained by these SNPs. These data are consistent with a polygenic model of DTC predisposition and highlight the importance of association studies in the discovery of the disease hereditability.

PubMed | German Cancer Research Center, University of Pisa and Blood Center
Type: Journal Article | Journal: Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology | Year: 2016

Linkage analyses and association studies suggested that inherited genetic variations play a role in the development of differentiated thyroid carcinoma (DTC).We combined the results from a genome-wide association study (GWAS) performed by our group and from published studies on DTC. With a first approach, we evaluated whether a SNP published as associated with the risk of DTC could replicate in our GWAS (using FDR as adjustment for multiple comparisons). With the second approach, meta-analyses were performed between literature and GWAS when both sources suggested an association, increasing the statistical power of the analysis.rs1799814 (CYP1A1), rs1121980 (FTO), and 3 SNPs within 9q22 (rs965513, rs7048394, and rs894673) replicated the associations described in the literature. In addition, the meta-analyses between literature and GWAS revealed 10 more SNPs within 9q22, six within FTO, two within SOD1, and single variations within HUS1, WDR3, UGT2B7, ALOX12, TICAM1, ATG16L1, HDAC4, PIK3CA, SULF1, IL11RA, VEGFA, and 1p31.3, 2q35, 8p12, and 14q13.This analysis confirmed several published risk loci that could be involved in DTC predisposition.These findings provide evidence for the role of germline variants in DTC etiology and are consistent with a polygenic model of the disease. Cancer Epidemiol Biomarkers Prev; 25(4); 700-13. 2016 AACR.

PubMed | German Cancer Research Center, University of Pisa, University of Bonn and Blood Center
Type: | Journal: BMC cancer | Year: 2016

Genome-wide association studies (GWASs) have identified several single-nucleotide polymorphisms (SNPs) influencing the risk of thyroid cancer (TC). Most cancer predisposition genes identified through GWASs function in a co-dominant manner, and studies have not found evidence for recessively functioning disease loci in TC. Our study examines whether homozygosity is associated with an increased risk of TC and searches for novel recessively acting disease loci.Data from a previously conducted GWAS were used for the estimation of the proportion of phenotypic variance explained by all common SNPs, the detection of runs of homozygosity (ROH) and the determination of inbreeding to unravel their influence on TC.Inbreeding coefficients were significantly higher among cases than controls. Association on a SNP-by-SNP basis was controlled by using the false discovery rate at a level of q*< 0.05, with 34 SNPs representing true differences in homozygosity between cases and controls. The average size, the number and total length of ROHs per person were significantly higher in cases than in controls. A total of 16 recurrent ROHs of rather short length were identified although their association with TC risk was not significant at a genome-wide level. Several recurrent ROHs harbor genes associated with risk of TC. All of the ROHs showed significant evidence for natural selection (iHS, Fst, Fay and Wus H).Our results support the existence of recessive alleles in TC susceptibility. Although regions of homozygosity were rather small, it might be possible that variants within these ROHs affect TC risk and may function in a recessive manner.

Ates S.C.,Yildiz Technical University | Bagirova M.,Yildiz Technical University | Allahverdiyev A.M.,Yildiz Technical University | Kocazeybek B.,Istanbul University | Kosan E.,Blood Center
Acta Tropica | Year: 2013

In recent years, the role of donor blood has taken an important place in epidemiology of Leishmaniasis. According to the WHO, the numbers of patients considered as symptomatic are only 5-20% of individuals with asymptomatic leishmaniasis. In this study for detection of Leishmania infection in donor blood samples, 343 samples from the Capa Red Crescent Blood Center were obtained and primarily analyzed by microscopic and serological methods. Subsequently, the traditional culture (NNN), Immuno-chromatographic test (ICT) and Polymerase Chain Reaction (PCR) methods were applied to 21 samples which of them were found positive with at least one method. Buffy coat (BC) samples from 343 blood donors were analyzed: 15 (4.3%) were positive by a microculture method (MCM); and 4 (1.1%) by smear. The sera of these 343 samples included 9 (2.6%) determined positive by ELISA and 7 (2%) positive by IFAT. Thus, 21 of (6.1%) the 343 subjects studied by smear, MCM, IFAT and ELISA techniques were identified as positive for leishmaniasis at least one of the techniques and the sensitivity assessed. According to our data, the sensitivity of the methods are identified as MCM (71%), smear (19%), IFAT (33%), ELISA (42%), NNN (4%), PCR (14%) and ICT (4%). Thus, with this study for the first time, the sensitivity of a MCM was examined in blood donors by comparing MCM with the methods used in the diagnosis of leishmaniasis. As a result, MCM was found the most sensitive method for detection of Leishmania parasites in samples obtained from a blood bank. In addition, the presence of Leishmania parasites was detected in donor bloods in Istanbul, a non-endemic region of Turkey, and these results is a vital importance for the health of blood recipients. © 2013 Elsevier B.V.

News Article | December 7, 2016

An article in Blood shows that administering the anti-hypertensive drug amlodipine in conjunction with conventional chelation therapy helps combat health problems caused by the buildup of iron in organs Iron accumulation in myocardial cells, potentially resulting in heart failure or fatal arrhythmia, is one of the complications most feared by patients with thalassemia major, a hereditary disease also known as Mediterranean anemia. An article by Brazilian researchers published in the journal Blood reports that a daily dose of amlodipine combined with chelation resulted in more effective reduction of cardiac iron in a clinical trial involving 62 patients. Amlodipine is an inexpensive drug with few side effects and is already available for the treatment of hypertension. "The drug has been used clinically for decades and is considered safe for adults and children. As an adjunct to standard treatment, it can be greatly beneficial to patients and has few side effects," said Juliano de Lara Fernandes, a researcher at José Michel Kalaf Research Institute in Campinas, São Paulo State, Brazil, and principal investigator for the project. The trial was conducted in partnership with researchers at the University of Campinas Blood Center (Hemocentro UNICAMP), Boldrini Child Cancer Center, and São Paulo Blood Center (CHSP), among others. Thalassemia major, Fernandes explained, is an inherited blood disorder in which the body makes an abnormal form of hemoglobin, the protein in red blood cells that carries oxygen. The disorder results in a low red cell count, which leads to chronic anemia, so patients require blood transfusions every three to four weeks. The downside of this treatment is a buildup of iron in the organism. "The iron in red blood cells is normally reused when new red cells are produced, but transfusions introduce a lot of extra iron into the patient. The concentration of iron can double after ten transfusions," Fernandes said. The body lacks mechanisms to excrete the excess iron, which builds up in the cells of several organs, especially the heart and liver. This accumulation is usually treated with chelating drugs, which bind with the excess iron to produce compounds that can be excreted in urine or feces. "Chelation therapy works well in peripheral organs, but it's hard to remove iron from the heart," Fernandes said. "Myocardial dysfunctions are currently the main cause of death among patients with thalassemia and can emerge in children from the age of ten." The most serious problem of all, he added, is caused by an accumulation of non-transferrin bound iron (NTBI) in myocardial cells. NTBI is toxic and can cause cell death. Normally scarce in the bloodstream, it can increase significantly as a result of successive transfusions. NTBI enters and leaves the liver without causing much damage to the organ, but it enters the heart via a channel whose main role is to carry calcium into cells. "It occurred to us that drugs capable of blocking the calcium channel could also prevent NTBI from entering the heart and therefore increase the efficacy of chelation therapy," Fernandes said. "Calcium-channel blockers are widely used to treat problems such as high blood pressure and irregular heart beat." The hypothesis was tested in 62 patients with thalassemia major. This number was considered sufficiently representative because the disease is rare. The volunteers were divided into two groups. Both were given conventional chelation therapy, but amlodipine was administered to only one. The other received oral placebo. Before the clinical trial began, peripheral venous blood samples were collected for chemistry and hematology analyses, and MRI scans were performed on patients who had not had one within 30 days before enrollment. Depending on the iron concentrations found in their organs, each group was subdivided into those with and without initial cardiac iron overload. MRI scans were repeated a year later. "Myocardial iron concentration fell 21% in patients with initial iron overload who were treated with chelation plus amlodipine, whereas it increased by 2% in those with initial overload who were treated with chelation plus placebo," Fernandes said. A comparison of results for the subgroups without initial iron overload showed no significant difference between those who received amlodipine and those who received placebo. "Perhaps we would have needed to monitor these patients for a longer period to see the benefits of preventive therapy with amlodipine for people who don't have excess iron in their organs," Fernandes said. "For those who do, however, the results show it's worth using amlodipine. There's no need to change the existing therapy. It's enough to administer the anti-hypertensive orally every day."

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