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kon-igi · 4 years ago

Note

in cosa è diverso il vaccino Johnson rispetto agli altri, per non necessitare di una seconda dose?

C’è bisogno di un piccolo preambolo.

La differenza tra la SCIENZA e la RELIGIONE è che alla prima ci credi perché ne comprendi i meccanismi mentre con la seconda sei costretto a farlo in modo fideistico.

Quindi decidi tu i vaccini in quale categoria ricadano.

(mi piacerebbe mettere il pulsante ‘salta tutto il testo citato’ ma non esiste e quindi la ‘spiegazione’ è da scrollare fino in fondo)

Vaccine design

The S protein of SARS-CoV-2 corresponding to positions 21,536–25,384 in SARS-CoV-2 isolate Wuhan-Hu-1 (GenBank accession number: MN908947) was codon-optimized for expression in human cell lines. S designs were either based on the native SP, replacement of the native SP by the tissue plasminogen activator (tPA) SP or a SP upstream of the native signal, resulting in a sequence encoding SARS-CoV-2 amino acids 2-1273 and tPA leader as described for MERS-CoV spike protein13,39. In some constructs the furin cleavage site was abolished by amino acid changes R682S and R685G, or proline substitutions (K986P, V987P) were introduced.

Protein expression and purification

A plasmid encoding the SARS-CoV2 S-2P protein12 with the wt SP and with the wt SP replaced by the tPA SP and with a C-tag for purification were codon-optimized and synthesized at GenScript. The constructs were cloned into pCDNA2004. The expression platform used was the Expi293F cells. The cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 6 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The supernatant was subsequently sterile filtered using a 0.22 µm vacuum filter and stored at 4 °C until use. The C-tagged SARS-CoV2 S trimers were purified using a two-step purification protocol by 5 mL CaptureSelect™ C-tag Affinity Matrix (ThermoFisher Scientific). Proteins were further purified by size-exclusion chromatography using a HiLoad Superdex 200 16/600column (GE Healthcare).

Antibodies and reagents

SAD-S35 was purchased from ACROBiosystems (cat# SAD-S35-100ug). ACE2-Fc (ACE2) was made according to Liu et al. 201840. For CR3022, CR301541, CR3046, and S30934 the heavy and light chain were cloned into a single IgG1 expression vector to express a fully human IgG1 antibody. The antibodies were made by transfecting the IgG1 expression construct using the ExpiFectamine™ 293 Transfection Kit (ThermoFisher) in Expi293F (ThermoFisher) cells according to the manufacturer specifications. Antibodies were purified from serum-free culture supernatants using mAb Select SuRe resin (GE Healthcare) followed by rapid desalting using a HiPrep 26/10 Desalting column (GE Healthcare). The final formulation buffer was 20 mM NaAc, 75 mM NaCl, 5% Sucrose pH 5.5. Convalescent serum (SER-0743-00001) was obtained from Sanquin, the Netherlands.

Cell-based ELISA

HEK293 cells were seeded at 2 × 105 cells/mL in appropriate medium in a flat-bottomed 96-well microtiter plate (Corning). The plate was incubated overnight at 37 °C in 10% CO2. After 24 h, transfection of the cells was performed with 300 ng DNA for each well and the plate was incubated for 48 h at 37 °C in 5% CO2. Two days post transfection, cells were washed with 100 μl/well of blocking buffer containing 1% (w/v) BSA (Sigma), 1 mM MgCl2, 1.8 mM CaCl2, and 5 mM Tris pH 8.0 in 1× PBS (GIBCO). After washing, nonspecific binding was blocked, using 100 μl/well of blocking solution for 20 min at 4 °C. Subsequently, cells were incubated in 50 μl/well blocking buffer containing primary antibodies ACE2-Fc (5 µg/mL, 1 µg/mL and 0.2 µg/mL)(1 µg/mL for radar plot), S309 (1 µg/mL), SAD-S35 (1 µg/mL), CR3015 (5 µg/mL), CR3022 (5 µg/mL), CR3046 (5 µg/mL), and convalescent serum (1:400) for 1 hr at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well of washing buffer containing 1 mM MgCl2, 1.8 mM CaCl2 in 1× PBS and then incubated with 100 μl/well of the blocking buffer for 5 min at 4 °C. After blocking, the cells were incubated with 50 μl/well of secondary antibodies HRP conjugated Mouse Anti-Human IgG (Jackson, 1:2500) or HRP Conjugated goat anti-mouse IgG (Jackson, 1:2500) then incubated 40 min at 4 °C. The plate was washed three times with 100 μl/well of the blocking buffer, three times with 100 μl/well washing buffer. 30 μl/well of BM Chemiluminescence ELISA substrate (Roche, 1:50) was added to the plate, and the luminosity was immediately measured using the Ensight Plate Reader.

Flow cytometry

MRC-5 cells (0.4 × 106 cells/well) were seeded in 6-well plates and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes at 5000 vp/cell for 48 h. Harvested cells were washed with PBS and stained with LIVE/DEADTM Fixable Violet Dead Cell Stain Kit (Invitrogen). For SARS-CoV-2 surface staining, cells were washed twice with PBS and then incubated with ACE2-Fc (1 μg/ml), convalescent serum (1:400), and mAbs S309, SAD-S35, CR3022, CR3015 and CR3046 (1 μg/ml) for 30 min in FACS buffer (PBS with 0.5% BSA). Cells were washed twice with FACS buffer and stained with goat anti-Human IgG Alexa Fluor 647 (Invitrogen) or goat anti-Mouse IgG Alexa Fluor 647 (Invitrogen) secondary antibody for 30 min in FACS buffer. Cells were washed twice with FACS buffer and fixed with 1× BD CellFIX (BD Biosciences) for 15 min. Cells were washed once with FACS buffer and resuspended in FACS buffer before analysis on a FACS Canto instrument (BD Biosciences). Data were analyzed with FlowJoTM Software (Becton, Dickinson and Company) and plotted as median fluorescence intensity of the MRC-5 single, live cell population (Fig. S6).

BioLayer interferometry (BLI)

Expi293F cells were transiently transfected using ExpiFectamine (Life Technologies) according to the manufacturer’s instructions and cultured for 3 days at 37 °C and 10% CO2. The culture supernatant was harvested and spun for 5 min at 300 × g to remove cells and cellular debris. The spun supernatant was subsequently sterile filtered using a 0.22 μm vacuum filter and used as the analyte in the experiment. A solution of ACE2-Fc at a concentration of 10 μg/mL was used to immobilize the ligand on anti-hIgG (AHC) sensors (FortéBio cat#18-5060) in 1× kinetics buffer (FortéBio cat#18-1092) in 96-well black flat bottom polypropylene microplates (FortéBio cat#3694). The experiment was performed on an Octet HTX instrument (Pall-FortéBio) at 30 °C with a shaking speed of 1000 rpm. Activation was 60 s, immobilization of antibodies 600 s, followed by washing for 300 s and then binding the S proteins for 1200 s. Data analysis was performed using the FortéBio Data Analysis 8.1 software (FortéBio). Binding levels were plotted as nm shifts at 1200 s after S protein binding.

Differential scanning fluorometry (DSF)

0.2 mg of purified protein in 50 µl PBS pH 7.4 (Gibco) was mixed with 15 µl of 20 times diluted SYPRO orange fluorescent dye (5000 x stock, Invitrogen S6650) in a 96-well optical qPCR plate. A negative control sample containing the dye only was used for reference subtraction. The measurement was performed in a qPCR instrument (Applied Biosystems ViiA 7) using a temperature ramp from 25–95 °C with a rate of 0.015 °C per second. Data were retrieved continuously. The negative first derivative was plotted as a function of temperature. The melting temperature corresponds to the lowest point in the curve.

Mass spectrometry

Liquid chromatography-mass spectrometry (LC-MS/MS) was used to determine the N-terminus on either purified soluble protein or on a pull-down of the full-length S protein from cell membranes using either Mab CR3022 or ACE2-Fc. The purified soluble proteins were subjected to direct digest, whereas the membrane-bound spike protein samples were either subjected to direct digest or in gel digestion. The experiments were performed on C18 nRP-LC connected to a ESI-Q-orbitrap mass spectrometer. Data processing for the different proteins was performed using Biopharma Finder 3.1 (Thermo Scientific). Each data file was compared to its corresponding amino acid sequence. Peptides were filtered by mass accuracy, confidence and structural resolution and reported. In order to pick up low abundant peptides, the thresholds for the MS noise level and the S/N were lowered 100-fold in comparison to the normal processing method.

Cell–cell fusion assay

Cell–cell fusion assays were performed to ascertain the relative fusogenicity of the different S protein variants. For fluorescent readout, full-length wild-type SARS-CoV-2 S protein and variants thereof, human ACE2, human TMPRSS2 and GFP were co-expressed from pcDNA2004 plasmids in HEK293 cells using Trans-IT transfection reagent according to the manufacturer’s instructions. Transfections were performed on 70% confluent cell monolayers in 6-well plates. Transfected cells were incubated at 37 °C and 10% CO2 for 24 h before imaging on an EVOS cell imaging system (Thermofisher). Overlays between brightfield and GFP channels were made in ImageJ.

The fluorescent fusion assay was adapted to allow quantitative measurement of cell–cell fusion by leveraging the NanoBiT complementation system (Promega). Donor HEK293 cells were transfected with full-length S and the 11 S subunit in 96-well white flat bottom TC-treated microtest assay plates. Acceptor HEK293 cells were transfected in 6-well plates (Corning) with ACE2, TMPRSS2 and the PEP86 subunit, or just the PEP86 subunit (‘No spike’) as negative control. Eighteen hour after transfection, the acceptor cells were released by 0.1% trypsin/EDTA and added to the donor cells at a 1:1 ratio for 4 h. Luciferase complementation was measured by incubating with Nano-Glo® Live Cell Reagent for 3 min, followed by readout on an Ensight plate reader (PerkinElmer).

Ad26 vectors

Replication-incompetent, E1/E3-deleted Ad26 vectors were engineered using the AdVac system42, here using a single plasmid technology containing the Ad26 vector genome including a transgene expression cassette. The codon-optimized SARS-COV-2 Spike genes (as described in vaccine design) were inserted into the E1-position of the Ad26 vector genomes under transcriptional control of the human cytomegalovirus promoter and the SV-40 polyadenylation sequence. Rescue and manufacturing of the Ad26 vectors was performed in the complementing cell line PER.C6 TetR43,44. Virus particle (vp) titers in the Ad26 vector preparations were quantified by measurement of optical density at 260 nm45 and infectivity was assessed by quantitative potency assay (QPA)46, using PER.C6 TetR cells. PCR including subsequent sequencing of PCR products has confirmed the identity and integrity of the SARS-COV-2 Spike genes (primers used are listed in Table S1). Ad26 vector-mediated expression of SARS-COV-2 Spike genes was confirmed by western blot analysis of cell-culture lysates from infected MRC-5 cells (Fig. 2d). Bioburden levels and endotoxin levels met the preset release criteria for animal experiments.

SDS-PAGE and western blotting

Twenty-four-well plates were seeded with MRC-5 cells (1.25 × 105 cells/well), and after overnight growth transduced with Ad26 vectors encoding SARS-CoV-2 S transgenes. Cell lysates were harvested 48 h post transduction and, after heating for 5 min at 85 °C, samples were loaded under non-reduced conditions on a precast 3–8% Tris-Acetate SDS-PAGE gel (Invitrogen). Proteins were transferred to a nitrocellulose membrane using an iBlot2 dry blotting system (Invitrogen), and membrane blocking was performed overnight at 4 °C in Tris-buffered saline (TBS) containing 0.2% Tween 20 (V/V) (TBST) and 5% (W/V) Blotting-Grade Blocker (Bio-Rad). Following overnight blocking, the membrane was incubated for 1 h with 2.8 µg/ml CR3046. in TBST-5% Blocker. CR3046 is a human monoclonal antibody directed against SARS-CoV-1 Spike. After incubation, the membrane was washed three times with TBST for 5 min and subsequently incubated for 1 h with 1:10,000 IRDye 800CW-conjugated goat anti-human secondary antibody (Li-COR) in TBST-5% Blocker. Finally, the PVDF membrane was washed three times with TBST for 5 min, and after drying developed using an ODYSSEY® CLx Infrared Imaging System (Li-COR). All samples derived from one experiment and were processed in parallel. The uncropped blot is provided in Fig S7.

Animals

Animal experiments were approved by the Central Authority for Scientific Procedures on Animals (Centrale Commissie Dierproeven) and conducted in accordance with the European guidelines (EU directive on animal testing 86/609/EEC) and local Dutch legislation on animal experiments.

Female BALB/c or C57BL6 mice (specific pathogen-free), aged 8–12 weeks at the start of the study were purchased from Charles River laboratories (Sulzfeld, Germany). Mice were immunized via the intramuscular (i.m.) route with 100 μl vaccine (50 μl per hind leg) under isoflurane anesthesia. Intermediate blood samples were collected via submandibular bleeding route. At the end of the experiment, under anesthesia, animals were exsanguinated by heart puncture and sacrificed by cervical dislocation. Blood was processed for serum isolation and spleens were collected. Spleens were processed into single cell suspensions for cellular assays (when applicable).

Virus neutralization assay

Neutralization assays against live SARS-CoV-2 were performed using the microneutralization assay previously described by Algaissi and Hashem47. Vero-E6 cells [CRL-1580, American Type Culture Collection (ATCC)] were grown in Eagle’s minimal essential medium (EMEM; Lonza) supplemented with 8% fetal calf serum (FCS; Bodinco BV), 1% penicillin-streptomycin (Sigma–Aldrich, P4458) and 2 mM L-glutamine (PAA). Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Clinical isolate SARS-CoV-2/human/NLD/Leiden-0001/2020 (Leiden L-0001) was isolated from a nasopharyngeal sample and its characterization will be described elsewhere (manuscript in preparation). The NGS-derived sequence of this virus isolate is available under GenBank accession number MT705205 and shows 1 mutation in the Leiden-0001 virus compared to the Wuhan sequence resulting in Asp614 > Gly at position 614 of the Spike protein. Isolate Leiden-0001 was propagated and titrated in Vero-E6 cells using the tissue culture infective dose 50 (TCID50) endpoint dilution method and the TCID50 was calculated by the Spearman-Kärber algorithm as described48. All work with live SARS-CoV-2 was performed in a biosafety level 3 facility at Leiden University Medical Center.

Vero-E6 cells were seeded at 12,000 cells/well in 96-well tissue culture plates 1 day prior to infection. Heat-inactivated (30 min at 56 °C) serum samples were analyzed in duplicate. The panel of sera were two-fold serially diluted in duplicate, with an initial dilution of 1:10 and a final dilution of 1:1280 in 60 μL EMEM medium supplemented with penicillin, streptomycin, 2 mM L-glutamine and 2% FCS. Diluted sera were mixed with equal volumes of 120 TCID50/60 µL Leiden -0001 virus and incubated for 1 h at 37 °C. The virus-serum mixtures were then added onto Vero-E6 cell monolayers and incubated at 37 °C in a humidified atmosphere with 5 % CO2. Cells either unexposed to the virus or mixed with 120 TCID50/60 µL SARS-CoV-2 were used as negative (uninfected) and positive (infected) controls, respectively. At 3 days post-infection, cells were fixed and inactivated with 40 µL 37% formaldehyde/PBS solution/well overnight at 4 °C. The fixative was removed from cells and the clusters were stained with 50 µL/well crystal violet solution, incubated for 10 min and rinsed with water. Dried plates were evaluated for viral cytopathic effect. Neutralization titer was calculated by dividing the number of positive wells with complete inhibition of the virus-induced cytopathogenic effect, by the number of replicates, and adding 2.5 to stabilize the calculated ratio. The neutralizing antibody titer was defined as the log2 reciprocal of this value. A SARS-CoV-2 back-titration was included with each assay run to confirm that the dose of the used inoculum was within the acceptable range of 30 to 300 TCID50.

Pseudotyped virus neutralization assay

MLV pseudotyped with SARS-COV-2 S protein was produced as previously described49 with some minor changes. In short, Platinum-GP cells (cell Biolabs, Inc) were transfected with a plasmid encoding the codon-optimized SARS-COV-2 Spike gene from strain Wuhan-Hu-1 (GenBank: MN908947) carrying a 19-aa cytoplasmic tail truncation, a GAG-Pol packaging vector and an MLV vector encoding a luciferase reporter gene using Lipofectamine 3000 transfection reagent (Life Technologies) according to manufacturer’s protocol. Cells were incubated overnight at 37 °C 10% CO2 with OPTIMEM transfection medium. Next day, medium was replaced by OPTIMEM supplemented with 5% FBS and 1% PenStrep and incubated for 48 h prior to harvest. The harvested pseudotyped MLV particles were stored at −80 °C prior to use. In the neutralization assay, soluble ACE2-Fc and mAbs CR3015, CR3022, CR3046 and SAD-S35 (ACROBiosystems) were two-fold serial diluted (n = 3) in DMEM without phenol red supplemented with 1% FBS and 1% PenStrep and incubated with an equal volume of pseudotyped MLV. After 30 min incubation, the mixture was inoculated onto susceptible Vero-E6 cells in MW96 well plates. Luciferase activity was measured after 40 h of incubation by addition of an equal volume of substrate NeoLite (Perkin Elmer) followed by luminescence readout on the EnSight Multimode Plate Reader (Perkin Elmer). The percentage of infectivity was calculated as ratio of luciferase readout in the presence of mAbs normalized to luciferase readout in the absence of mAb.

Direct coat ELISAs

IgG binding to SARS-CoV-2 Spike antigen was measured by ELISA with the full-length in-house produced Spike protein COR200099. COR200099 is an in-house produced SARS-CoV-2 Spike protein, stabilized by two point mutations in the S1/S2 junction that knocks out the furin cleavage site, and by two newly introduced prolines in the hinge region in S2. In addition, the transmembrane and cytoplasmic regions have been replaced by a foldon domain for trimerization mutations, allowing the protein to be produced as soluble protein (see S.dTM.PP Fig. 3C). Briefly, 96-wells Perkin Elmer white ½ area plates were O/N coated with COR200099 protein. Next day plates were washed, blocked for 1 h and subsequently incubated for 1 h with 3-fold serially diluted serum samples in block buffer in duplicate. After washing, plates were incubated for 1 h with goat anti-mouse IgG-HRP in block buffer, washed again and developed using ECL substrate. Luminescence readout was performed using a BioTek Synergy Neo instrument.

For IgG1 and IgG2a ELISAs, a similar protocol was followed as described above, but respectively using Goat anti-Mouse IgG1-HRP and Goat anti-Mouse IgG2a-HRP as secondary antibodies.

ELISpot assay

IFN-γ ELISpot was performed on splenocytes of mice isolated after sacrifice using mouse IFN-γ ELISpot-plus kit (Mabtech). Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. IFN-γ ELISpot assay was performed by stimulating splenocytes from individual mice for 18 h with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 1 μg/peptide/mL. Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. PMA/ionomycin stimulation was used as a positive control (data not shown); medium was used as negative control and used to calculate the lower limit of detection. Stimulation was done overnight in duplicate wells containing 0.5–2.5 × 105 cells per well.

Multiplex ELISA

The Th1/Th2 multiplex ELISA assay was performed on splenocytes obtained after sacrifice. Splenocytes were stimulated by 48 h culturing in the presence of two Spike 15-mer peptide pools (pool 1 and pool 2). Resulting supernatants were diluted 4-fold and measured for the presence of Th1 (IFN-γ) and Th2 cytokines (IL-10, IL-4, and IL-5) using a 10-plex multiplex ELISA proinflammatory panel (mouse) kit (Meso Scale Discovery, V-PLEX Proinflammatory Panel 1 Mouse Kit cat# K15048D). Results shared in this manuscript are the sum of stimulation with peptide pool 1 and pool 2. Ratios of Th1/Th2 cytokines (IFN-γ/IL-10, IFN-γ/IL-4, and IFN-γ/IL-5) were calculated on basis of cytokine measurements in supernatants by dividing the Th1 cytokine (IFN-γ) with the respective Th2 cytokines. Multiplex ELISA measurements were done on supernatant diluted either 2-fold or 4-fold.

ICS assay

The intracellular cytokine staining assay (ICS) was performed on splenocytes obtained two weeks after sacrifice. Splenocytes were obtained by disaggregation of spleens with the gentleMACS dissociator. ICS assay was performed by stimulating splenocytes from individual mice for 16 h, 106 cells per well, with two different peptide pools (pool 1; peptides 1-156, and pool 2; peptides 157-313) consisting of in total 313 15-mers peptides overlapping by 11 amino acids together covering the full-length Spike protein at a final concentration of 0.2 μg/peptide/well. Stimulation with peptide pools was done at 37 °C, 10% CO2 for 1 h in presence of Rat-anti-Mouse CD49d (1:500, BD; cat #553153) and Hamster-anti-Mouse CD28 (1:500, BD; cat#553294). Protein transport was blocked (1:1000; BD GolgiPlugTM cat#51-230KZ) overnight at 37 °C, 10% CO2. Cells were washed twice with PBS and stained during 20 min at room temperature in the dark, according to manufacturers’ instructions, with a violet viability dye (1:5000; Invitrogen Violet ViD cat#L34955). Cells were washed twice with 0.5% BSA in PBS (PBA) and Fc receptors were blocked during 10 min in the fridge in the dark (1:50; BD Mouse Fc block cat# 553142). Cells were washed once with PBA and stained during 30 min in the fridge in the dark with anti-CD3e FITC (BD, cat#553062), anti-CD4 PerCP-Cy5.5 (BD, cat#550954) and anti-CD8α APC-H7 (BD, cat#557654). Cells were washed twice with PBA and permeabilized/fixated during 20 min in the fridge in the dark (BD Cytofix/CytopermTM cat#51-2090KZ/554722), after which 1× BD Perm/WashTM buffer (BD cat#51-2091KZ/554723) was added. Cells were stained during 30 mins in the fridge, in the dark with anti-IFN-γ PE (BD, cat#554412), anti-TNFa PE-Cy7 (557644) and anti-IL-2 APC (BD, cat#554429). Cells were washed twice with Perm/Wash buffer, resuspended in PBA, and fluorescence was measured on the BD FACSCantoTM II and analyzed with BD FACSDivaTM software: Flow Jo version 10.06.1. Cells were gated on single cells, excluding dead cells, and gated for lymphocytes (Fig S8a). CD8-CD4+ and CD8+ T cells were then gated on IFN-γ, IL-2, and TNF-α (Fig S8b).

Statistical analysis

Statistical differences between immunization regimens were evaluated two-sided for S-specific binding antibodies as measured by ELISA, NAb titers as measured by VNA, IFN-γ producing cells as measured by ELISpot and cytokine production by MSD and ICS assays. Comparisons between Ad26.S, Ad26.tPA.S, Ad26.tPA.SS, Ad26.tPA.WT.S, Ad26.S.PP, adjuvanted S protein, tPA.S, tPA.S.PP, S and S.PP groups were made using the exact Wilcoxon rank-sum test, Cochran–Mantel–Haenszel test, Mann–Whitney U test, t-test from ANOVA, or z-test from Tobit ANOVA. Results were corrected for multiple comparisons by Bonferroni correction; 3-fold Bonferroni correction Fig. 5a, b, 2-fold Bonferroni correction Fig. 5c, d.

Lo ammetto... SONO STATO STRONZO ma era per farti capire che la mia è la semplificazione di una semplificazione di una semplificazione.

Molto sinteticamente, il vaccino Johnson & Johnson sfrutta una tecnologia specifica di ancoraggio dell’encoding della proteina S (Ad26) a un vettore virale modificato (Adenovirus) simile a quello del vaccino AstraZeneca ma, evidentemente, questa metodica è più efficace nello stimolare una risposta anticorpale che da subito raggiunge livelli stimati tra il 64% e il 72% senza bisogno di una seconda dose di boost.

Perché non te l’ho detto subito?

Perché è bene non dimenticare che anche dietro a una banale compressa di paracetamolo ci sono una serie di azioni, interazioni e reazioni ‘scientifiche’ che in un mondo ideale sarebbe bello che tutti conoscessero ma che allo stato attuale funzionano come la comunione con l’ostia benedetta...

Credeteci e verrete salvati.

#Anonymous

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his-mother-is-lightning · 8 years ago

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actually tho who even knows the US amendments?

** the classics **

1 - GRAPES. the Man cannot g) stop you from complaining about them r) keep you from your weird-as-fuck spaghetti religion a) keep you from inviting 10000 friends to the national mall for a “party” where you all coincidentally also want to save pandas p) stop you or the national enquirer from publishing that the VP has a foot fetish e) make everyone worship that spaghetti thing s) shut you up

2 - you have the right to bear arms. any kind you like: grizzly, black bear, brother bear. get your bear arms

3 - the Man can’t invite friends and have sleepovers at your house without asking first. especially not if those sleepovers take months and they bring guns but no popcorn

4 - the Man needs a good reason to go through your stuff and must get their permission slip signed by the teacher.

5 - the Man must follow the rules about questioning and reminding you . when they break the rules they do not pass 'Go’ and do not collect 200$. no do-overs

6 - when you are challenged to one of those word fights TV talks about, they have to schedule it soon (so you don’t psych yourself out), you get to see who you’re fighting first (is he too swol for you to handle?), and you get to have a lawyer to back you up (with the words, not the fighting). crowd should be made of people ready to cheer for either side

7 - this is boring and doesn’t apply on the state level. if you get sued, the Man has to prove it’s possible that you did the crime and weren’t three states over at ComicCon

8 - they can’t make bail more than you can handle. also, punishments can’t be so fucked up that the jury wants to barf when they hear about it.

9 - just bc the constitutional convention didn’t write it down, it doesn’t mean they can take it away from you (except they can and will and want to)

10 - anything not in the constitution is none of the Man’s business lol

** the post-Madison mess **

11 - you can’t sue states that you don’t live in bc you don’t even go there bro

12 - TJefferson was mad at Aaron Burr so now Vice Presidents are part of the Presidents ticket and not the runner up

13 - why the fuck did we have slavery

14 - what the fuck guys, we just did this. all us citizens get the same rights, and you can’t take them away without due process [the due process baby is born and we shall rejoice of it for years to come]

15 - OMG GUYz us citizens can vote. you racist assholes

16 - congress is hungry and invented income taxes

17 - did you know senators weren’t voted in by the citizens until 1913?

18 - a bunch of ppl pretend they can stop americans from drinking

19 - women are allowed to vote

20 - changed the date of the inauguration. apparently needed its own amendment.

21 - the americans were still drinking? this whole time? that’s so weird. take backsies

22 - you can’t be president more than twice unless your FDR, who is an exception and should not be counted

23 - did you know that DC didn’t have a representitive until 1960? lol, they still don’t. they have some guy sitting on the side of the room in congress to ‘represent’ DC while Idaho suddenly decides they’re bored and want to take away DCs rights

24 - poll taxes are bullshit plz give up on this racist shit

25 - who’s in charge after the president dies? what if we kill his vp? what if we kill the speaker...? i’m asking for a friend

26 - the voting age is 18 based on nothing at all except it makes congress feel good

27 - congress persons can’t change their own salary - it goes into effect after they leave. clearly this is the thing that keeps them under control.

#american history #history #founding fathers #politics #lookitme

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handwork-art · 7 years ago

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CROCHET

Cardigan with wavy stripes

The original sweater wavy stripes pattern is not so complicated as it may seem.

The Magazine “Sabrina” No. 5/2017

DIMENSIONS

36/38 (44/46)

YOU'LL NEED

Yarn (100% cotton, 120 m/50 g) - 200 (250) g of white, 150 (200) g black and 50 (100) g emerald, coral, orange and turquoise; hook №3,5.

PATTERNS AND DIAGRAMS

SAWTOOTH PATTERN

The number of loops multiple of 25 + 24 = knit approx. scheme. Each row start with a given number of VP lift instead of the 1st loop and loops before rapport, rapport constantly repeat, end loops after rapport. To run 1 times 1 - 9th rows, then repeating 2 - 9 ranks, following the instructions of thread colors next to the numbers series.

Attention!

To black-and-white sections do not cut the thread after each row, after the 1st of the series of continuously to perform 2 purl row 2 front row 2 purl row alternately.

THE SEQUENCE OF STRIPS

* The 1st rapport in height: C = emerald

2nd rapport in height: C = cyan,

The 3rd rapport in height: C = coral,

The 4th rapport in height: C = orange,

* constantly repeat.

DENSITY KNITTING

P. X10 is 27.5 p. = 10 x 10 cm; 1 rapport 25. = 9 cm.

ATTENTION!

All items are knit from the top down. The arrows on the pattern = direction of knitting.

PATTERN

THE EXECUTION OF THE WORK

BACK

Black thread to perform a chain of 124 (149) VP + 1 VP lifting and knit pattern scalloped = 4 (5) rapport + start and end loop.

Through 47 cm = 47 rows (55 cm = 55 rows) from the start number to finish the job.

BEFORE

Knit as back.

SLEEVES

Black thread to execute for each sleeve a chain of 74 (99) VP + 1 VP lifting and knit scalloped pattern = 2 (3) rapport + start and end loop.

Through 39 cm = 39 rows from the starting row to finish the job.

ASSEMBLY

Perform shoulder seams on both sides of 11 (14) refer to the Initial row of the sleeve to stretch so that it became straight, and sew the sleeves according to. pattern. To run side seams and seams of sleeves.

#crochet

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kgs03 · 4 years ago

Text

New Post has been published on KG Sinclair

New Post has been published on https://www.kgsinclair.com/2020/06/on1-announces-availability-of-the-new-on1-360-new-on1-photo-raw-2020-5-and-the-new-on1-photo-mobile-for-ios-ipados-and-android/

ON1 Announces Availability of the New ON1 360, New ON1 Photo RAW 2020.5, and the New ON1 Photo Mobile for iOS, iPadOS, and Android.

Portland, OR. Jun 29, 2020: ON1, creators of ON1 Photo RAW, announces ON1 360, ON1 Photo RAW 2020.5, and the new ON1 Photo Mobile are now available. ON1 360 is an all-new end-to-end photography workflow solution to capture, edit, and sync photos seamlessly between multiple computers and mobile devices using the new ON1 Photo RAW 2020.5, and the new ON1 Photo Mobile for iOS, iPadOS, and Android.

“ON1 360 is the culmination of years of work and feedback from customers around their frustrations with being forced to upload original photos into cloud systems with no way out. ON1 360 solves these problems by letting photographers choose which photos to sync and where their photos are stored while allowing them access to their photos across their computers and devices,” says Dan Harlacher, VP of Product.

In addition to the new ON1 360, a new version of ON1 Photo RAW version 2020.5 is also available for download today. ON1 Photo RAW is a modern photo organizer, raw processor, layered editor, and effects app, all in one affordable application. ON1 Photo RAW 2020.5 includes the new ON1 360 integration, many performance enhancements, support for new cameras and lenses, and additional bug fixes.

The new and free ON1 Photo Mobile app for iOS, iPadOS, and Android devices is like having a pro-grade camera app and raw processor in the palm of your hand. ON1 Photo Mobile uses the same proprietary raw processing engine developed for ON1 Photo RAW and perfectly compliments the desktop application, ON1 Photo RAW 2020.5, when synced together with ON1 360.

Key Features

One Workflow — A complete end-to-end workflow between multiple computers and mobile devices with ON1 360. ON1 360 connects ON1 Photo RAW 2020.5 on macOS and Windows computers with the new free ON1 Photo Mobile on iOS, iPadOS, and Android devices. Multiple options for syncing files, control in file storage methods, and new capabilities for managing and editing across devices no matter where photos are stored are now available in ON1 360.

Complete Control of Your Photos — At the core of the new ON1 360 is the new cloud storage service and sync technology, which allows photographers to sync photos, metadata, and edits, as well as albums, cataloged folders, and more between each of their computers, devices, external hard drives, or even network drives. Photographers can choose which photos to sync, how they organize photos, where they store their photos, and how they back up their photos.

New Editable Previews — Customers can choose to sync their original raw files or sync their photos using the new Editable Previews that utilize ON1’s new compressed-raw file format. This file format allows photographers to store more photos in the same amount of space without noticeable loss in image quality when processing. Photographers can view, edit, and share their photos without having to store the original photo in the cloud using these Editable Previews. The format is 75% smaller than the original raw file and maintains the same tonal and color range. They are perfect for remote editing, sharing, and even printing common sizes, without noticeable loss in quality, saving customers additional storage space and cost.

New ON1 Photo Mobile Camera Mode — The camera mode in ON1 Photo Mobile works a lot like the camera app on your device, but with more advanced features. The new app can capture raw photos with the tonal range and details you expect from an interchangeable lens camera. The pro-level manual controls include the ability to adjust exposure, shutter speed, manual-focus, and white balance.

New ON1 Photo Mobile Editing Controls — ON1 Photo Mobile uses the same proprietary raw processing engine developed for ON1 Photo RAW. Adjustments include Exposure, Contrast, Shadows, Midtones, Highlights, Whites, Blacks, White Balance, Noise, and Sharpening. Built-in filters will start with, black and white, adding film grain, darkening the edges with a vignette, and targeted color adjustments. ON1 Photo Mobile is available for iOS, iPadOS and Android devices and is free for anyone.

Privacy — Privacy is most important, and with ON1 360, all photos will be kept private. ON1 will never analyze any photos using ON1 360 to build ways to market to photographers.

New Supported Cameras in ON1 Photo RAW 2020.5 — Nikon D6, Sony STL-99, Sony ZV-1

New Supported Lenses in ON1 Photo RAW 2020.5 — Apple iPhone XS, Canon EF 24-105mm f/3.5-5.6 IS STM, Pentax-DA 55-300mm f/4.5-6.3 ED PLM WR RE, SAMYANG AF 85mm F1.4, SAMYANG AF 45mm F1.8, Sigma 105mm F1.4 DG HSM | Art 018, Sigma 35mm F1.2 DG DN | Art 019, Sigma 70-200mm F2.8 DG OS HSM | Sports 018, Sony E 16-55mm F2.8 G, Sony E 70-350mm F4.5-6.3 G OSS, Sony FE 200-600mm F5.6-6.3 G OSS, Tamron E 28-75mm F2.8-2.8, Tokina AT-X 24-70mm f/2.8 PRO FX

Availability and Pricing

ON1 360 plans start out with 10x the storage amount for 20% less cost compared to competitive plans available today. ON1 360 plans include the Software and Service option as well as a Service Add-on option in both monthly and annual payment options. The Software and Service plan includes a subscription license to ON1 Photo RAW 2020, any and all software upgrades and updates, the ON1 Photo Mobile connectivity, and the 360 syncing service. The starting plan includes 200GB of storage at the low cost of $7.99 per month or $89.99 for the year. ON1 still offers Photo RAW as a perpetual-license product separate from ON1 360, for those who prefer to own their software. Customers who own Photo RAW can choose the ON1 360 Add-on and those plans start at $5.99 per month or $59.99 for the year and come with 200GB of storage. This also allows customers to upgrade to future versions of ON1 Photo RAW at a time of their choosing, and it allows them to add or cancel any ON1 360 subscription without losing access to ON1 Photo RAW.

What’s Ahead in the ON1 Roadmap

ON1 will continue developing ON1 Photo RAW for the desktop and ON1 Photo Mobile for devices. There is a detailed product roadmap for ON1 Photo Mobile already available from the ON1 website. Future plans include ON1 getting back into developing products that work within other photo editing workflows as well. There will be new integrations and plug-ins for ON1 products no matter what applications photographers are using.

“As we grow, we will continue to deliver a complete line of products for all workflows, whether it be standalone, plug-in, mobile, or video. It’s already in our plans to deliver all-new plug-ins within the year every photographer will want to use to save them time. We’ve always taken pride in that we are a group of photographers creating software applications specifically for the photographer,” says Craig Keudell, ON1 President & CEO.

About ON1

Since 2005, ON1 has provided award-winning software to millions of photographers worldwide. We accomplish this by being different from other photo software companies. Photography is all we do, we put you the photographer first, and this means our customers have complete control over every aspect of their photography. Our software applications are designed by photographers, for photographers.

The ON1 mission is to deliver the tools photographers need to express themselves through photography. The ability to easily organize, edit, and share their photos anywhere and provide professional quality image processing, the creativity they desire, the service they deserve, and to keep photographers in control of their photography.

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interkomitet · 6 years ago

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European Parliament resolution on the situation in the Sea of Azov

The European Parliament,

– having regard to its previous resolutions on Russia and Ukraine,

– having regard to the statement by the European External Action Service (EEAS) Spokesperson of 15 May 2018 on the partial opening of the Kerch Bridge,

– having regard to the UN Convention on the Law of the Sea, the Treaty on the Non-Proliferation of Nuclear Weapons and the UN Charter,

– having regard to Council Decision (CFSP) 2018/1085 of 30 July 2018 amending Decision 2014/145/CFSP concerning restrictive measures in respect of actions undermining or threatening the territorial integrity, sovereignty and independence of Ukraine(1), adding six entities involved in the construction of the Kerch Bridge to the list of persons, entities and bodies subject to restrictive measures as set out in Annex I to Regulation (EU) No 269/2014,

– having regard to the Agreement between the Russian Federation and Ukraine on cooperation in the use of the Sea of Azov and the strait of Kerch of 2003, the Budapest Memorandum on Security Assurances of 5 December 1994 and the Package of Measures for the Implementation of the Minsk Agreements of 12 February 2015,

– having regard to Rule 123(2) and (4) of its Rules of Procedure,

A. whereas the situation in the Sea of Azov was addressed by the bilateral agreement of 2003 between Ukraine and Russia, which defines these territories as internal waters of the two states and gives both parties the power to inspect suspicious vessels; whereas both the 2003 agreement and UN Convention on the Law of the Sea provide for the freedom of navigation;

B. whereas the construction of the Kerch Bridge and a gas pipeline and the laying of underwater cables to the illegally annexed Crimean peninsula without Ukraine’s consent constitute another violation of Ukraine’s sovereignty and territorial integrity by the Russian Federation;

C. whereas the Kerch Bridge limits the size of ships that can reach the Ukrainian ports on the Sea of Azov to an air draft of less than 33 metres and a length of less than 160 metres, which has made it impossible for Panamax-class vessels, accounting for over 20 % of all ship traffic before the construction, to enter the Sea of Azov; whereas before the opening of the bridge over the Kerch Strait in spring this year, inspections were random and non-intrusive and did not cause disruptions to the free flow of vessels and cargo;

D. whereas Russia frequently and in an abusive manner blocks and inspects ships going through the Kerch Strait sailing to or from Ukrainian ports; whereas these procedures cause delays of up to one week and result in a decrease in cargo flows and tangible financial losses for the local economy in Ukraine and merchants whose vessels are subject to this regime; whereas according to Ukrainian Government sources more than 200 vessels had to undergo this excessive procedure by the end of September 2018, including over 120 ships registered in the EU, while ships under the Russian flag were exempt from such controls;

E. whereas these cities and the wider region are already facing negative economic and social consequences due to the annexation of Crimea and the ongoing Russian-backed conflict in eastern Ukraine; whereas this new act by Russia has already had a significant negative impact on the local economy and led to a sharp decrease in the turnover of cargo of Ukrainian ports; F. whereas the construction of this massive bridge has had a negative impact on the environment, lowering the sea level in the strait and affecting the water exchange between the Sea of Azov and Black Sea;

G. whereas in September 2018 Ukraine decided to repeal the Treaty of Friendship, Cooperation and Partnership signed in 1997 between Ukraine and the Russian Federation, and to create a naval base in the Sea of Azov, further increasing its military presence there by transferring additional Marine Corps forces and coastal artillery to that coastal area;

1. Deplores the excessive actions of the Russian Federation in the Sea of Azov insofar as they breach international maritime law and Russia’s own international commitments; condemns the excessive stopping and inspection of commercial vessels, including both Ukrainian ships and those with flags of third-party states, including ships under flags of various EU Member States; stresses that inspections of vessels, while being allowed at random, should not be abused or carried out for political reasons with the aim of further destabilising the security, integrity and social and economic situation in Ukraine; calls on the Council and the VP/HR to demand that the Russian Federation immediately end the intensive and discriminatory inspections of vessels and to consider, if necessary, appropriate countermeasures;

2. Expresses its very serious concern about the very volatile security situation in the Sea of Azov, which could easily escalate to an open conflict; is gravely concerned about the continued militarisation of the Sea of Azov and Black Sea region, particularly of the illegally occupied and annexed Crimea peninsular, the development of anti-access/area denial (A2/AD) capabilities by the Russian Federation, including new S-400 anti-aircraft systems, and the redeployment of military and patrol vessels from the Caspian Sea; regrets that the Sea of Azov has become a new maritime dimension of belligerent Russian actions against Ukraine;

3. Condemns the construction of the bridge over the Kerch Strait linking the illegally annexed Crimean peninsula with mainland Russia, and the infringement of navigational rights in Ukraine’s territorial waters; points out that Russia is bound by international maritime law and the bilateral cooperation agreement with Ukraine not to hamper or impede transit passage through the Kerch Strait and the Sea of Azov;

4. Reiterates its support for the independence and territorial integrity of Ukraine, reconfirms Ukraine’s sovereignty over the Crimean peninsula and its part of the Sea of Azov and Ukraine’s absolute right to have full access to the Sea of Azov, as enshrined in the UN Convention on the Law of the Sea;

5. Deplores the illegal extraction of oil and gas resources by the Russian Federation from Ukrainian territory; highlights the possible danger of Russia seizing existing Ukrainian oil and gas fields in the Sea of Azov once it achieves its aim of transforming it into an internal lake within the Russian Federation;

6. Underlines that this pattern of violating the territorial waters of European countries or blocking maritime transport has already been exercised by Russia in the Baltic Sea, in particular against the Baltic States and Poland (Vistula Lagoon);

7. Calls on the VP/HR to follow more closely the evolving security situation in the Sea of Azov, given its growing potential for conflict on Europe’s doorstep, which may have wider security implications affecting the EU and its Member States directly; considers, in this regard, that it would be very useful to appoint an EU Special Envoy for Crimea and the Donbass region, whose responsibilities would also cover the Sea of Azov;

8. Calls on the Vice-President of the Commission / High Representative of the Union for Foreign Affairs and Security Policy (VP/HR) to take the necessary steps to propose that the OSCE Special Monitoring Mission to Ukraine (SMM) mandate, which covers the entire territory of Ukraine, including maritime areas, also cover the new area of tensions in the Azov Sea, and stresses that either the mission should be equipped with the necessary means to perform its monitoring role in maritime areas or a separate international monitoring mission should be established for this body of water;

9. Underlines that the Kerch Bridge has been illegally constructed and welcomes the Council’s decision to impose restrictive measures on six companies involved in its construction; urges the VP/HR, together with the EU Member States in the Council, to make it clear that the targeted sanctions against Russia will be reinforced if the conflict in the Azov Sea escalates further;

10. Reiterates its concern at the involvement of European companies in the construction of the Kerch Bridge, which, through this involvement, knowingly or unknowingly undermined the EU sanctions regime; calls on the Commission, in this regard, to assess and verify the application of the EU restrictive measures in force and on the Member States to share information regarding any national customs or criminal investigations into cases of potential violations;

11. Supports the efforts made by the Ukrainian side in all diplomatic actions and legal procedures provided for by international law and relevant conventions, including the ongoing arbitration process under the UN Convention on the Law of the Sea, with a view to countering Russian hostile practices in the Sea of Azov;

12. Calls on the Commission and the EEAS to provide a full assessment of the economic damage caused by the de facto blockade and to consider possible ways to support the carriers and ports that have been negatively affected, in particular by strengthening the EU’s engagement in Mariupol and Berdyansk, enhancing social resilience and promoting the economic development of these cities and the broader south-east region of Ukraine;

13. Is concerned about the adverse environmental impact of the Kerch Bridge, which might affect the interests of all Black Sea basin countries; calls on Ukraine, the Commission and the Member States on the shores of the Black Sea to monitor the situation, exchange relevant information and identify potential remediation needs;

14. Expresses its condolences and sympathy to the families of the victims of the mass murder at the college in Kerch where 20 people were killed and dozens wounded on 17 October 2018;

15. Instructs its President to forward this resolution to the Council, the Commission, the Vice-President of the Commission / High Representative of the Union for Foreign Affairs and Security Policy, the Secretary-General of the Organisation for Security and Cooperation in Europe (OSCE), the Secretary-General of NATO, the President, Government and Parliament of Ukraine, the President, Government and Parliament of the Russian Federation, and the EU Member States.

http://interkomitet.com/news-of-the-day/european-parliament-resolution-on-the-situation-in-the-sea-of-azov/

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bluebookweb · 7 years ago

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Joint-Employer Ruling Update and P.F. Chang’s Joins ezCater

The TGIF edition of MRM’s Daily Bite features the latest on the “joint employer” ruling from the NLRB, ezCater and P.F. Chang’s, Pizza Boli’s, Southern Glazer’s Wine & Spirits of New York and Bite Squad.

Send news items to Barbara Castiglia at [email protected].

Joint-Employer Ruling Applauded

The National Labor Relations Board (NLRB) voted 3-2 to reverse its own 2015 ruling that waste management company Browning Ferris Industries could be considered a joint employer with Leadpoint Business Services, a staffing agency it subcontracted, even if it had only indirect or unexercised control over Leadpoint’s workers. The 2015 ruling contradicted guidelines followed for more than 30 years that said a company had to have direct control over the actions of a subcontractor or franchisee’s employees in order to be a joint employer.

Today’s vote overturned the Browning-Ferris ruling and reinstated the previous standard requiring direct control.

The National Retail Federation welcomed the vote to reverse a controversial expanded definition of a “joint employer” adopted during the Obama administration that has increased businesses’ exposure to lawsuits.

“This is an important move to restore the common-sense definition of what constitutes this type of employment relationship that stood for decades,” NRF Senior Vice President for Government Relations David French said. “The board’s 2015 decision created an impossible scenario where one business could unfairly and improperly be held accountable for the actions of another business. Today’s vote puts an end to those harmful and unnecessary changes that exposed companies to almost limitless liability.”

The action comes just over a month after the House voted 242-181 to pass the Save Local Business Act, legislation supported by NRF that would have overturned the Browning-Ferris decision if the NLRB had not acted.

ezCater Teams with P.F. Chang’s

ezCater is partnering with P.F. Chang’s adding all 212 of its U.S. locations to ezCater’s online platform. P.F. Chang’s joins more than 150 restaurant chains on ezCater’s platform.

Off-premises dining and catering have been a growth area for the restaurant industry, which overall has experienced negative month-to-month same-store sales for the better part of the past two years, according to the Restaurant Industry Snapshot from Black Box Intelligence. In contrast, business spending on catering has grown more than 30 percent in the past three years to become a $22 billion market, according to data from foodservice research firm Technomic.

Restaurant industry veteran Jim Rand has spent more than a decade building catering operations for national brands and recently joined P.F. Chang’s as VP of off-premises dining to lead the company’s catering and off-premises strategy.

“Partnering with ezCater is an easy and effective way to grow catering revenue,” said Rand. “ezCater quickly added all of our U.S. locations to its platform and we began receiving orders immediately. In the last three months we’ve seen meaningful growth in our catering revenue through ezCater. The ezCater platform has allowed us to quickly reach a large corporate customer base over a broad geographic area, which would have taken longer had we done it on our own.”

Over 60,000 restaurants and caterers partner with ezCater to reach a nationwide customer base of businesspeople, including customers from more than 90 percent of the Fortune 500. California Pizza Kitchen, Potbelly Sandwich Shop, and Fazoli’s are among other brands that recently joined ezCater as partners, adding 300 Potbelly Sandwich Shops, 200 California Pizza Kitchen locations, and 100 Fazoli’s restaurants to ezCater’s nationwide network. These restaurants join longtime chain partners like Firehouse Subs, Dickey’s Barbeque Pit, and Honeybaked Ham.

“We’re thrilled to be adding such notable brands like P.F. Chang’s to our partner network,” said Victoria Brady, VP of caterer partnerships at ezCater. “We take pride in building good relationships with all of our restaurant partners and love that we can help them grow their business. ezCater’s unparalleled national reach makes our platform particularly ideal for any brand looking to drive business across all locations.”

Pizza Boli’s Update

Pizza Boli’s, which opened its first store in 1985, now has more than 75 locations in Maryland, Virginia,Pennsylvania, New Jersey, and the District of Columbia to go alongside a new website.

“The focus of everything we’re doing in our stores and online is to satisfy our customers: those who have been part of the Pizza Boli’s family for decades, and those who are just discovering why ‘we deliver more’ isn’t only our slogan, but it’s also our promise,” said marketing director Maroula G. Mavrophilipos, adding that key improvements and additions that customers can look forward to enjoying in the coming weeks and months include:

A colorful and clear new website that displays flawlessly on all screens and devices.

A new and larger menu based on feedback from customers.

A re-invented online e-club that offers more special offers and promotions than ever before.

A new loyalty program called “Boli’s Rewards” that lets customers earn their way to free food with every purchase.

A new app (iOS and Android) for fast, easy and secure mobile ordering.

New gift cards that allow Pizza Boli’s fans to give a gift that they know will be appreciated and enjoyed.

A creative new logo that thoughtfully echoes the past, embraces the present, and points to the future.

An exciting new store design that features modernized esthetics, and a more engaging and stylish decor — but without diminishing Pizza Boli’s signature fun, relaxed and family-friendly atmosphere.

Added Mavrophilipos: “The things that our loyal customers love about Pizza Boli’s — such as our incredible variety, commitment to quality and great service — haven’t changed, and will never change. The many exciting developments underway position us to deliver even more happiness and satisfaction over the next 33 years — and well beyond that!”

Additional details on Pizza Boli’s new offerings will be announced in the coming weeks. For all other information, visit www.pizzabolis.com.

Southern Glazer’s Opens Port Elizabeth Distribution Center

Southern Glazer’s Wine & Spirits of New York, the New York division of the largest North American wine and spirits distribution company, upgraded Port Elizabeth, N.J. facility is now open and serving customers throughout the New York Metro market. The facility was originally a storage center for the company and has been repurposed and updated this year to augment existing distribution operations located in Syosset, N.Y. It will immediately enable Southern Glazer’s to expand its production capability in the market by millions of cases, improving customer service and supporting growing demand throughout the New York Metro area.

“The Southern Glazer’s team has worked tirelessly to transform this facility into a world-class distribution center,” said Martin Crane, EVP and General Manager of Southern Glazer’s Wine & Spirits of New York. “It was an important and much needed investment that will enable us to improve our performance and better deliver on the promises we make to our customers. We have not only increased our capacity by twenty-five percent, but are also now in a better position than ever to support what we expect to be continued growth in this market.”

The 286,000-square foot Port Elizabeth facility features the latest distribution automation technology and equipment, including state-of-the-art storage, conveyor, and sortation systems. It also includes expansion capabilities to handle additional capacity as volumes increase The Port Elizabeth distribution facility will work in coordination with Southern Glazer’s existing 350,000-square foot Syosset distribution center.

“On behalf of the Southern Glazer’s New York leadership team, we want to thank our dedicated employees and the support of both local and NY state officials, who enabled us to complete this major project quickly and efficiently,” added Roy Kohn, VP of Operations for Southern Glazer’s Wine & Spirits of New York. “We’re continuing to assess our long-term needs for continued expansion in New York.”

Bite Squad Unlimited Rolls Out

More customers will have the opportunity to try out Bite Squad Unlimited, the company’s new subscription-based service that gives users free delivery, no matter how many times they order, for one monthly payment. Following an invite-only beta test this fall, enrollment in the service is now being offered to select customers during the checkout process on the Bite Squad website.

“We can see that food delivery is becoming more and more ingrained in our customers’ daily lives,” said Bite Squad co-founder and CEO Kian Salehi. “We’re working to make the ordering process as easy and worry-free as possible. With Unlimited, customers no longer have to deal with delivery fees – they can simply order as many times as they want and enjoy the convenience of the service.”

Bite Squad Unlimited is designed to provide increased value to customers who order delivery more than twice per month. With a monthly payment that varies by city and location (up to $9.99), customers can order as many orders as they like – all day, every day – with no individual delivery fees. All partner restaurants on the Bite Squad platform, within a four mile radius of a customer’s delivery location, are available with an Unlimited subscription.

“This is a big win for everybody,” said Salehi. “It gives us a more predictable and sustainable revenue source, and it gives substantial cost savings to customers who want more frequent deliveries.”

The company expects to roll out broader availability of Unlimited in early 2018. Founded in the summer of 2012 in Minneapolis, Minnesota, Bite Squad has partnered with local restaurants across more than 30 metropolitan areas.

Joint-Employer Ruling Update and P.F. Chang’s Joins ezCater posted first on happyhourspecialsyum.blogspot.com

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helios7media-blog · 7 years ago

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Acer Predator Orion 9000 - World's most Powerful Gaming PC of 2017

With four-card graphics cards and an 18-core processor, the Acer Predator Orion 9000 is an extreme platform for top-of-the-line gaming, with a curved Acer Predator X35 monitor that supports NVIDIA® G-SYNC ™ and Acer HDR Ultra ™ brings spectacular graphics with the top gaming experience. On August 30, 2017, at the IFA Fair in Berlin, Acer introduced novelties in its premium gamers product line. Predator Orion 9000 with Windows 10, the most powerful desktop ever. https://www.youtube.com/watch?v=TD7rKBJ_GAs

Acer Predator Orion 9000: The power you can see

Designed in a silver-black casing, it looks like a spacecraft with adjustable RGB illumination along the front side of the front frame. The massive side window does not hide the interior of a powerful interior, which is very attractive in the water. "The Predator Orion 9000 is the most powerful computer we ever did," said Jeff Lee, general director of computing, IT Products Business, Acer. "With four-card graphics cards and an 18-core processor, this is the platform that can put the most demanding tasks from the gaming world."

Cooling and IceTunnel 2.0 keep the temperature of the hardware in normal

The Predator Orion 9000 Series has a current cooling and Acer IceTunnel 2.0 to keep the temperature within normal limits while burdened with high-end hardware inside the enclosure. IceTunnel 2.0 is an advanced airflow management solution that cleverly separates the system into several heat zones, each having an individual air flow tunnel that cools all parts of the computer. Large metal mesh panels on the front and top of the housing allow for more cool air, while simultaneously ejecting the heated air inside, the fluid cooled CPU. Part of the air flow is diverted to the rear of the motherboard to cool down the storage devices.

Extreme platform for playing

https://www.youtube.com/watch?v=vceY6QlZ3ww As one of the major AMD OEM partners, they bring the latest Radeon ™ RX Vega graphics to your desktop. Acer raises the chart in very high graphics, with the ability to connect up to 4 Radeon RX Vega cards to deliver virtually photorealistic images to your monitor, in real time and at high resolution, with high refresh rates. Players also can embed instead of the AMD GPU, two NVIDIA GeForce GTX 1080Ti cards in SLI, which easily support virtual reality. "We are delighted that Acer has selected the AMD Radeon ™ RX Vega graphics card for the most modern computer ever made. RX Vega is a perfect addition to the predator Orion regarding beauty and power. One RX Vega delivers ultra-high resolution at smooth 60 fps. And with new features like High Bandwidth Cache Controller and Rapid Packed Math, players can expect their system to work better with the latest and upcoming game titles, "said Scott Herkelman, VP, and CEO, Gaming Radeon Technologies Group, AMD. The Predator Orion 9000 will also offer the latest 18-core Intel® Core ™ i9 Extreme Edition processor with up to 128GB four-channel DDR4 memory, which should be more than enough for easy PC performance and the most demanding tasks with exceptional performance. One-punch overclocking allows enthusiasts to select turbo performance at the touch of a button. The excellent connectivity includes two USB 3.1 Gen 2 connectors (one type-C and one type-A), eight USB 3.1 Gen 1 connectors (one type-C and seven type A) and two USB 2.0 connectors (type A). The Predator Orion 9000 supports a total of three M.2 slots to expand the ability to increase speed, power, and PC capabilities, and the four PCIe x16 slots provide enough expansion for video cards.

Monitor Acer Predator X35: Inspiring visualization

This large 35-inch, 21: 9 monitor has a sporty convincing curve of 1800R and a brilliant (3440 x 1440) WQHD resolution. With the NVIDIA G-SYNC, Acer HDR Ultra and quantum dot technology, it also delivers the best possible contrast quality with a high dynamic range. Advanced local LED illumination in 512 individually controlled zones illuminates only when and where needed. The Predator X35 delivers a wider and deeper saturated color spectrum that covers 90 percent of the DCI-P3 color standard and the brightness range a few times higher than the traditional Dynamic Range Monitor. Fast response time of 4 ms and high frequency of 200 Hz in combination with NVIDIA G-SYNC allow you to play smoothly and vividly without flickering the picture. Equipped with Predator GameView, there are eight pre-set image view modes with optimization for different types of action. In addition to Standard, ECO, Graphic and Movie, there are three special modes of action, including Action, Racing, and Sports, which can be accessed easily via hotkeys or on-screen display (OSD) menus. Players can also define their custom profile and program each way according to their wishes. Acer BlueLightShield ™ technology enables users to reduce harmful blue light emissions by selecting from four different filter settings via the OSD menu. Premium panel VA allows wide viewing angles up to 178 degrees horizontally and vertically. Also, Dark Boost technology allows you to display fine detail in less-light environments.

Prices and availability

The Acer Predator Orion 9000 Desktop Gaming Series will be available in North America in December this year, with prices starting at $ 1,999; and in the European Union already in November, starting at 1.999 euros.

The Predator X35 display will be available in Q1 2018.

The exact specifications, pricing, and availability vary depending on the region. To find out more about availability, product specifications, and prices on specific markets, contact your nearest Acer office or visit acer.com for more information. Click to Post

#acer #desktop #gaming pc

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slapthebass · 3 years ago

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Source: music-guitar-instrument.blogspot.com>

#kaoru #dir en grey #y: 2009 #m: GIGS Guitar Book #for reference #g: ESP Ganesa #g: b&purple Ganesa #g: ESP VP-200 #g: shiny black VP #g: ESP VP Custom #g: pink VP #g: PRS Custom24 #g: black PRS #g: Fender Jaguar #g: Gibson Flying V Medallion #g: Gibson Les Paul CUSTOM #g: Jerry Jones Baby Sitar #pic: sourced

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kgs03 · 4 years ago

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New Post has been published on KG Sinclair

New Post has been published on https://www.kgsinclair.com/2020/06/on1-announces-availability-of-the-new-on1-360-new-on1-photo-raw-2020-5-and-the-new-on1-photo-mobile-for-ios-ipados-and-android/

ON1 Announces Availability of the New ON1 360, New ON1 Photo RAW 2020.5, and the New ON1 Photo Mobile for iOS, iPadOS, and Android.

Key Features

Privacy — Privacy is most important, and with ON1 360, all photos will be kept private. ON1 will never analyze any photos using ON1 360 to build ways to market to photographers.

New Supported Cameras in ON1 Photo RAW 2020.5 — Nikon D6, Sony STL-99, Sony ZV-1

Availability and Pricing

What’s Ahead in the ON1 Roadmap

About ON1

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