Strategies to control NP size, composition, and Mn2+ dissolution rates are essential to improve diagnostic performance of pH-responsive MnO NPs. We are the first to demonstrate that MnO NP size and composition can be tuned by the temperature ramping rate and aging time used during thermal decomposition of manganese(II) acetylacetonate. Two different temperature ramping rates (10°C/min and 20°C/min) were applied to reach 300°C and NPs were aged at that temperature for 5, 15, or 30 min. A faster ramping rate and shorter aging time produced the smallest NPs of ~23 nm. Shorter aging times created a mixture of MnO and Mn3O4 NPs, whereas longer aging times formed MnO. Our results indicate that a 20°C/min ramp rate with an aging time of 30 min was the ideal temperature condition to form the smallest pure MnO NPs of ~32 nm. However, Mn2+ dissolution rates at low pH were unaffected by synthesis conditions. Although Mn2+ production was high at pH 5 mimicking endosomes inside cells, minimal Mn2+ was released at pH 6.5 and 7.4, which mimic the tumor extracellular space and blood, respectively. To further elucidate the effects of NP composition and size on Mn2+ release and MRI contrast, the ideal MnO NP formulation (~32 nm) was compared with smaller MnO and Mn3O4 NPs. Small MnO NPs produced the highest amount of Mn2+ at acidic pH with maximum T1 MRI signal; Mn3O4 NPs generated the lowest MRI signal. MnO NPs encapsulated within poly(lactide-co-glycolide) (PLGA) retained significantly higher Mn2+ release and MRI signal compared to PLGA Mn3O4 NPs. Therefore, MnO instead of Mn3O4 should be targeted intracellularly to maximize MRI contrast.Manganese oxide (MnO) nanoparticles (NPs) can serve as robust pH-sensitive contrast agents for magnetic resonance imaging (MRI) due to Mn The negative contrast of iron oxide NPs is so robust that even single cells can be visualized on MRI if each cell accumulates at least 1 pg of iron [1 MRI due to the paramagnetic properties of the Mn2+ ion. Our group and other studies have shown that intact MnO NPs are in an “OFF” state and create minimal T1 MRI signal due to the Mn2+ ions being tightly bound and inaccessible to the surrounding water molecules [2+, which coordinates with water molecules to decrease T1 and produce a positive MRI signal, thus turning “ON” MRI contrast [The use of metal oxide nanoparticles (NPs) has been increasing over the past decades due to their magnetic, electric, and catalytic properties. Of particular interest to biomedical applications is the ability of metal oxide NPs, such as iron oxide and manganese oxide (MnO), to serve as contrast agents for magnetic resonance imaging (MRI) . Typical of iron . Howeverolecules –10. In acontrast –10.1 MRI contrast agents, MnO NPs have superior MRI properties. Clinically used gadolinium chelates are not pH-sensitive, and are always in an “ON” state, which highlights any well vascularized structure and can lead to false positive diagnoses in which a benign tumor can be mistaken for a malignant tumor [-1s-1 at 1.5T-4.7T [2+ has a higher relaxivity of ~ 7–8 mM-1s-1 at the same field strengths [1 relaxivity changes only ~2–4 times [1 (0.12–0.21 mM-1s-1) [2+ at pH 5 to increase relaxivity to 7 mM-1s-1. MnO NPs with targeting agents can be utilized for enhanced specificity for detection of cancerous tumors through NP dissolution inside tumor cells within low pH endosomes or lysosomes.Compared to gold standard and pH-sensitive gadolinium Tnt tumor –13. In a.5T-4.7T ; Mn2+ hatrengths , 16. Fur–4 times , 18 overmM-1s-1)  at pH 7.2+ to generate higher MRI signal under acidic conditions and allow for more efficient packing of MnO NPs into polymeric or liposomal delivery systems. MnO NPs are commonly synthesized by thermal decomposition of a manganese-based compound such as manganese acetylacetonate (Mn(II) ACAC) [1 signal enhancement of Mn2+ ions released from NPs with different sizes and compositions to determine which formulation maximized MRI contrast at pH 5, 6.5 and 7.4.To enhance MRI signal generation, it is necessary to fine-tune synthesis strategies to control and reduce the size of MnO NPs. It was hypothesized that smaller NP diameters will increase the surface area to volume ratio to facilitate faster dissolution of MnO to MnI) ACAC) –22, Mn oI) ACAC) , Mn acetI) ACAC) , 24–26, I) ACAC)  or Mn stI) ACAC) . SeveralI) ACAC) –22, peakI) ACAC) –23, 26, I) ACAC) , and typI) ACAC)  utilizedMn(II) ACAC, oleylamine , poly (PVA), and rhodamine 6G were obtained from Sigma-Aldrich. Dibenzyl ether , hexane , dichloromethane , Dulbecco's phosphate buffered saline (PBS), sodium citrate dihydrate , citric acid , and manganese(II) chloride tetrahydrate  were purchased from VWR. Hydrochloric acid (HCl) TraceMetal™ Grade was acquired from Fisher Scientific. Ethanol (Decon Laboratories Inc.) was obtained internally from West Virginia University’s Environmental Health and Safety Services. Ester-terminated 50:50 poly (PLGA) (inherent viscosity: 0.55–0.75 dL/g) was obtained from Lactel Absorbable Polymers.3O4) NPs by Xu et al. [2 gas. A constant N2 flow was needed to successfully remove all oxygen from the reaction and obtain the desired product, MnO NPs. Then, the temperature was quickly raised to 300°C under N2 gas using two different ramp rates of 20°C/min or 10°C/min and aged at 300°C for either 5, 15, or 30 min. To assess variability between synthesized NP batches, each of the 6 different temperature profiles were independently run 3 times, obtaining a total of 18 batches of MnO NPs. All of the 18 batches were utilized for further experiments. Upon completion of the reaction, the heating mantle was removed to allow the solution to cool down to room temperature. The MnO NPs were pelleted in Nalgene® Oak Ridge centrifuge tubes following centrifugation at 17,400 x g for 10 min and washed 4 to 5 times in hexane and ethanol using the same centrifugation procedure. Resulting MnO NPs were resuspended in hexane and left in a fume hood to dry overnight. After overnight drying, the MnO NPs were baked over 24 hr in an oven at 100°C. The resulting MnO NPs synthesized by thermal decomposition were hydrophobic and capped with oleylamine.All work for MnO NP synthesis should be performed under a chemical fume hood with proper PPE including safety glasses, nitrile gloves and a lab coat. MnO NPs were fabricated using a standard thermal decomposition reaction of Mn(II) ACAC dissolved in oleylamine and dibenzyl ether based on the synthesis of magnetite  AFor a separate set of experiments, MnO NPs were encapsulated with PLGA using an oil-in-water emulsion technique mediated by sonication . ApproxiTo prepare samples for transmission electron microscopy (TEM), dried MnO NPs and PLGA MnO NPs were suspended in ethanol and deionized water, respectively, using bath sonication. After NP resuspension, 15 μL of the MnO NP mixture was dropped and air dried on 300 mesh copper PELCO® TEM grid support films of carbon type-B . Images were taken using a JEOL JEM-2100 transmission electron microscope at 200 kV for the MnO NPs and 120 kV for the PLGA MnO NPs.3O4, the program obtained an estimated composition for our samples.X-ray diffraction patterns (XRD) of MnO NPs were obtained using a Panalytical X’Pert Pro X-ray diffractometer equipped with a Cu K-Alpha X-ray source operating at 45 kV and 40 mA in the Bragg-Brentano geometry. The spectra were collected over a 2θ range of 5° to 110° at a step size of 0.017° with a 1D silicon strip X-ray detector. The obtained XRD patterns were analyzed using the X’Pert HighScore Plus program. By comparing the XRD spectra of our synthesized MnO NPs with known spectra for MnO and MnScanning electron microscopy (SEM) with energy dispersive x-ray spectroscopy (EDS) was performed to analyze the elemental composition of the MnO NP samples using a Hitachi Scanning Electron Microscope S4700 operated at 15 kV with the EDAX Team EDS System.-6 Pascal. All measurements were performed using a focused Al K-Alpha X-ray source at a photon energy of 1486 eV and power of 25 W with an X-ray spot size of 100 μm. The take-off angle of the photoelectron was set at 45o. Compositional survey scans were obtained using a pass energy of 117.4 eV and an energy step of 0.5 eV. High-resolution detailed scans of each element were acquired using a pass energy of 23.5 eV and an energy step of 0.1 eV. All XPS spectra were referenced to the C1s peak at a binding energy of 284.8 eV.X-ray photoelectron spectroscopy (XPS) measurements were performed using a PHI VersaProbe 5000 Scanning X-Ray Photoelectron Spectrometer  at room temperature and under vacuum greater than 1eFourier-transform infrared spectroscopy (FTIR) measurements on MnO NP samples, oleylamine, PLGA MnO NP samples, and PLGA were performed using a DIGILAB FTS 7000 FTIR spectrometer equipped with a GladiATR attenuated total reflectance (ATR) module from PIKE Technologies.Size distribution of the PLGA MnO NPs was measured through dynamic light scattering (DLS) using a Nano Powder Sizer Malvern Instrument Zetasizer Nano ZS. Six milligrams of the PLGA MnO NPs were suspended in 10 mL of deionized water and bath sonicated prior to analysis.2+ release under different pH conditions, 10 mg of each MnO NP batch was suspended in 1 mL of PBS pH 7.4, 20 mM citrate buffer pH 6.5, or 20 mM citrate buffer pH 5 to simulate the pH of the blood, the tumor microenvironment, and cellular endosomes/lysosomes, respectively. The same three pH conditions were used to evaluate Mn2+ release from unencapsulated smaller MnO NPs (19 ± 6 nm) and unencapsulated smaller Mn3O4 NPs (17 ± 5 nm) to further observe the effect of size and chemical composition on release rate. Similarly, PLGA MnO NPs were suspended under the same conditions to assess Mn2+ release from hydrophilic NPs. Citrate buffers were made by adding anhydrous citric acid and sodium citrate dihydrate to deionized water. The MnO NP or PLGA MnO NP solutions were incubated in Eppendorf tubes under continuous slow rotation (6 rpm) to ensure gentle mixing over 24 hr at 37°C to simulate body temperature. At 1, 2, 4, 8 and 24 hr, the Eppendorf tubes were centrifuged at 17,400 x g for 10 min and the supernatants were collected and analyzed for released Mn2+ content by inductively coupled plasma-optical emission spectrometry (ICP-OES). The remaining pelleted MnO NPs or PLGA MnO NPs were resuspended in 1 mL of fresh buffer and placed back into the rotating incubator until the next time point was collected. The maximum amount of Mn2+ contained within each NP batch was calculated through measuring the total Mn2+ content of unencapsulated MnO NPs (10 mg) or PLGA MnO NPs (10 mg) fully dissolved in 150 μL of HCl trace metal grade using bath sonication. Mn2+ amounts were analyzed using Agilent 720 ICP-OES (1400 watts) with a plasma flow of 15.0 L/min, auxiliary flow of 1.50 L/min, and nebulizer flow of 0.75 L/min. Each sample was evaluated 5 times with a replicate and stabilization time of 10 and 15 s, respectively, and results were averaged. The % Mn2+ released at each time point was calculated using 2+ cumulative release graph was created by adding together the % Mn2+ released from each of the previous time points.To evaluate Mn2+ content of unencapsulated MnO NPs (10 mg) and PLGA MnO NPs (10 mg) with PLGA MnO NP % loading capacity was calculated using the total Mn1 molar relaxivities of Mn2+ at pH 7.4 (PBS), pH 6.5 (20 mM citrate buffer), and pH 5 (20 mM citrate buffer) were determined. To measure r1, manganese(II) chloride tetrahydrate was dissolved in the three buffers to achieve Mn2+ concentrations of 0.182, 0.102, 0.058, 0.032, and 0.018 mM. MRI of the Mn2+ solutions was performed at 1.0 T on a Bruker ICON MRI. T1 measurements were generated by a RARE sequence using an echo time of 10.68 ms. A total of 10 repetition times  were used to acquire images of the tubes. Using Matlab and the T1 longitudinal relaxation equation  of MnO NPs for each temperature condition was calculated from the TEM images using Approximately 25 to 35 TEM images and 800 to 900 MnO NPs were quantified per temperature condition using the line trace tool in ImageJ to measure the NP diameter. Each temperature condition contained 3 independent batches of synthesized MnO NPs. Statistical significance of mean NP diameters, MnMnO NPs were fabricated using a standard thermal decomposition reaction of Mn(II) ACAC dissolved in oleylamine and dibenzyl ether . PreciseTEM was used to assess MnO NP size and a representative image from each temperature condition is shown in As the ramping rate decreased and the aging time at 300°C increased, the average MnO NP diameter grew by a maximum of nearly 54%. When the fastest ramp (20°C/min) and shortest aging time (5 min) was used, the MnO NPs were the smallest, with an average diameter of 23 ± 9 nm. As the aging time was increased to 15 and 30 min, the average MnO NP size increased to 32 ± 11 nm and 32 ± 12 nm, respectively. When the ramp rate was decreased to 10°C/min, the average size of MnO NPs increased to 27 ± 10 nm, 36 ± 12 nm, and 36 ± 13 nm at 5, 15, and 30 min at 300°C, respectively. As shown in The increase in MnO NP size with a slower temperature ramp and an increase in aging time at 300°C was likely due to a longer total reaction time, leading to more opportunity for NP growth and coalescence . HistogrOur MnO NPs tended to exhibit a variation in size, likely due to several factors. First, MnO NP growth could follow an Ostwald ripening process, whereby smaller NPs begin to dissolve and add onto larger ones to cause polydispersity . Second,3O4 and MnO, respectively. All synthesized NPs  during thermal decomposition of Mn(II) ACAC. As the reaction time and temperature is increased, the Mn3O4 NPs begin to be reduced to MnO NPs through an endothermic reaction: Mn3O4 → 3MnO + ½O2 [3O4 to MnO, and lead to a mixed MnO/Mn3O4 composition. Longer aging times provide more thermal energy needed to obtain a full reduction to a pure MnO composition. Once again, for simplicity, in the rest of the manuscript, we will refer to all synthesized manganese oxide NPs as MnO NPs.To our knowledge, our study is the first to show that the ramping rate and aging time at 300°C impact the composition of the synthesized NPs. Mns et al.  and Seo s et al. , but thenO + ½O2 . Shorter2+), while a ΔE of ≥ 5.4 eV indicates Mn2O3 (Mn3+) [3O4 is a mixture of Mn2+ and Mn3+ oxidation states, the Mn3s peak splitting has an intermediate ΔE of 5.6 eV [3O4. XPS results demonstrate that the surface of the NPs oxidizes after synthesis in the presence of air to form a coating of Mn3O4, consistent with what others have found [3O4/MnO versus MnO), but that all NPs are oxidized to include a layer of Mn3O4 on the surface.To complement the bulk analysis of XRD, SEM/EDS, XPS and FTIR were used to assess the surface chemistry of MnO NPs formed with different temperature profiles. SEM/EDS and XPS confirmed the elemental composition of our NP samples to be mainly manganese and oxygen  can be u3 (Mn3+) . Previouf 5.6 eV . Fig 5B ve found . Togethe-1 (marked by asterisks) due to the symmetric and asymmetric CH2 stretching modes, respectively [-1 and 3300 cm-1 (marked by squares) are attributed to the NH2 bending vibration, and the symmetric and asymmetric stretching vibration of the amine group (NH2), respectively [-1 (marked by a triangle) correspond to the vibration of Mn-O and Mn-O-Mn bonds, confirming the chemistry found through XRD, SEM/EDS and XPS [The NP surface chemistry was further studied with FTIR to corroborate hydrophobic capping with oleylamine. ectively . Additioectively . MnO NP ectively , 39. Las and XPS .2+ in acidic media to create a greater T1 MRI signal. The controlled release profile of Mn2+ from MnO NPs was tested over time in 3 different pH conditions: pH 7.4 to mimic the normal physiological pH of the blood, pH 6.5 to mimic the slightly acidic extracellular pH in cancerous tumors due to increased lactic acid production, and pH 5 to mimic the acidic pH of endosomes and lysosomes inside cells. It is well known that following cell uptake, metallic NPs are shuttled to endosomes inside cells [2+ controlled release curve for MnO NPs formed from the 20°C/min ramp aged at 300°C for 30 min. Similar to what we have shown previously [2+ at physiological pH 7.4 was extremely minimal (faint dotted line in 2+ after 24 hr (dashed line in 2+ over 24 hr of ~46% (~2.9 mg)  with smaller MnO NPs (19 ± 6 nm) and smaller Mn3O4 NPs (17 ± 5 nm). TEM images and XRD spectra of the small MnO and small Mn3O4 NPs are shown in 2+ after 24 hours at pH 5 likely due to their increased surface area to volume ratio; however, the increased Mn2+ production from small MnO NPs was not statistically significant  than small MnO NPs  within PLGA, a clinically approved biocompatible and biodegradable polymer, to confirm the trend observed with hydrophobic NPs. TEM showed dark metal oxide nanocrystals trapped within the polymeric NPs  NPs had the highest Mn2+ release after 24 hr at pH 5; approximately 90% of the encapsulated MnO NPs dissociated to Mn2+ compared to only ~45% of encapsulated Mn3O4 NPs  NPs after 24 hours at pH 5  NPs and PLGA MnO (32 nm) NPs. Altogether, our results support the use of MnO over Mn3O4 due to higher Mn2+ generation and MRI signal with and without PLGA encapsulation.PLGA MnO (19 nm) NPs had a significantly lower T at pH 5 , which i all pHs , which s3O4, another strategy to maximize MRI signal from MnO NPs in vitro and in vivo would be to employ NP targeting to enhance uptake into tumor cells to take advantage of the low acidic conditions of endosomes and lysosomes to aid in increased Mn2+ generation. Several receptors are overexpressed on tumor cells depending on the cancer type such as the folate receptor, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), the transferrin receptor, and the mucin-1 (MUC-1) receptor [In addition to utilizing MnO over Mnreceptor , among oreceptor .Besides PLGA encapsulation, NPs can be made hydrophilic through ligand exchange  or lipid2+ ions, as Mn2+ mimics Ca2+ and can enter neurons and muscles. The ability of Mn2+ to travel down neurons has been used for manganese enhanced MRI (MEMRI) in animals to visualize neuronal activity. Bock et al. [2+, injected every 2 days for 12 days, totaling 180 mg/kg Mn2+. MnO NPs should be better tolerated in vivo, as they carry MnO, not free Mn2+ directly. Through incorporating specific NP targeting to tumor cells and confining Mn2+ release to low pH tumor endosomes  were compared with smaller MnO NPs (19 nm) and smaller Mn3O4 NPs (17 nm), with and without PLGA encapsulation. As predicted, the smallest unencapsulated MnO NPs released the most Mn2+ ions at pH 5 and 6.5 and led to the greatest reduction in T1 longitudinal relaxation time, with the highest MRI signal. PLGA encapsulation of large and small MnO NPs reduced the trends observed with unencapsulated MnO NPs possibly due to variability of metal oxide loading within individual NPs. With and without PLGA coating, Mn3O4 NPs were consistently the least effective MRI contrast agents; therefore, it is recommended to utilize MnO over Mn3O4 for MRI applications to expedite Mn2+ release and the resulting MRI signal produced. Future studies will explore varying the chemical reactant ratios to further decrease NP size and polydispersity, and using novel surface functionalization to enhance MnO NP endocytosis into cancer cells to maximize MRI contrast through Mn2+ generation.In conclusion, we are the first to demonstrate that modification of the temperature ramp rate and aging time at 300°C can be used to fine-tune both the diameter and composition of MnO NPs . The fasS1 FigReactant mixtures were heated from room temperature to 60°C over 30 minutes and then to 300°C using two different temperature ramps of a) 20°C/min or b) 10°C/min. Both temperature profiles show an aging temperature at 300°C for 30 minutes prior to cooling. Note how the temperatures measured during the experiments (red circles) closely match the theoretical programmed settings for the temperature controller (black lines), indicating precise control of MnO NP fabrication conditions.(TIF)Click here for additional data file.S2 FigSize distributions of the diameter of MnO NPs produced using the following temperature profiles: a) 5 min at 300°C with a 20°C/min vs. 10°C/min ramp, b) 15 min at 300°C with a 20°C/min vs. 10°C/min ramp, and c) 30 min at 300°C with a 20°C/min vs. 10°C/min ramp. MnO NP diameter increases as the ramping rate decreases and aging time at 300°C increases. The average size for each distribution is shown in (TIF)Click here for additional data file.S3 FigoC with 20°C/min ramp, and f) 30 min at 300°C with 10°C/min ramp. EDS confirmed the presence of Mn and O elements in NP samples.EDS spectra of the MnO NP samples with the following temperature profiles: a) 5 min at 300°C with 20°C/min ramp, b) 5 min at 300°C with 10°C/min ramp, c) 15 min at 300°C with 20°C/min ramp, d) 15 min at 300°C with 10°C/min ramp, e) 30 min at 300 (TIF)Click here for additional data file.S4 FigAsterisks represent oleyl groups, while squares represent amine groups.(TIF)Click here for additional data file.S5 Fig2+ release increased with a decrease in pH. Time points are shown for 1, 2, 4, 8 and 24 hr. Error bars show mean ± standard deviation.Controlled release curves are shown for MnO NPs generated with the following temperature profiles: a) 5 min at 300°C with 20°C/min ramp, b) 5 min at 300°C with 10°C/min ramp, c) 15 min at 300°C with 20°C/min ramp, d) 15 min at 300°C with 10°C/min ramp, and e) 30 min at 300°C with 10°C/min ramp. Mn(TIF)Click here for additional data file.S6 Fig3O4 NPs and b) 19 nm MnO NPs. The standard diffraction peaks for known c) Mn3O4 and d) MnO are shown from X’Pert HighScore. Through comparing with the standard diffraction peaks, Mn3O4 NPs are 73–100% Mn3O4 composition and MnO NPs are 67–73% MnO composition. TEM images of e) 17 nm Mn3O4 and f) 19 nm MnO NPs. NPs are smaller in size compared to XRD spectra of a) 17 nm Mn(TIF)Click here for additional data file.S7 Fig1 values for free Mn2+ in 20 mM citrate buffer pH 5 (black), 20 mM citrate buffer pH 6.5 (blue), and PBS pH 7.4 (red). T1-weighted MRI images shown in b-e) were acquired at 1 T with a 400 ms repetition time. T1 MRI of increasing Mn2+ concentrations in b) 20 mM citrate buffer pH 5, c) 20 mM citrate buffer pH 6.5, d) PBS pH 7.4. e) shows T1 MRI images of supernatants collected from small Mn3O4 (17 nm), small MnO (19 nm) and large MnO (32 nm) NPs suspended in pH 5 citrate buffer for 24 hours. MRI signal enhancement is greatest from small MnO NPs and least from small Mn3O4 NPs.a) r(TIF)Click here for additional data file.S8 Fig3O4, b) 19 nm MnO, and c) 32 nm MnO. Metal oxide NPs can be visualized as dark circles inside of the PLGA. NP loading capacity was ~30%. Scale bars are 100 nm.Three different types of metal oxide NPs were coated with PLGA including a) 17 nm Mn(TIF)Click here for additional data file.S9 Fig3O4 (17 nm), b) PLGA MnO (19 nm), and c) PLGA MnO (32 nm). All NPs possess the characteristic peaks of PLGA, represented by circles, as shown in FTIR spectra of PLGA encapsulated NPs: a) PLGA Mn(TIF)Click here for additional data file.S10 Fig−1 and 2989 cm−1 show the C–H stretch of CH2, and C–H stretch of–C–H–, respectively. The peak at 1751 cm−1 is assigned to the C = O stretching vibration of the ester bond and 1165–1087 cm−1 corresponds to the C–O stretching.Circles represent characteristic peaks of PLGA. The peaks at 2993 cm(DOCX)Click here for additional data file.S11 FigSize distributions of PLGA NP diameters by DLS analysis: a) PLGA Mn3O4 (17 nm), b) PLGA MnO (19 nm), and c) PLGA MnO (32 nm). Highest peak for NP diameters is in the 220 to 255 nm bin size range.(TIF)Click here for additional data file.S12 Fig1 MRI images of supernatants collected from PLGA Mn3O4 (17 nm), PLGA MnO (19 nm) and PLGA MnO (32 nm) NPs suspended in pH 5 citrate buffer, pH 6.5 citrate buffer, and pH 7.4 PBS for 24 hours. MRI signal enhancement is significantly greater from PLGA MnO NPs compared to PLGA Mn3O4 NPs.T(TIF)Click here for additional data file.S1 Table(TIF)Click here for additional data file.S2 Table(TIF)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S4 Table(TIF)Click here for additional data file.


Euglena gracilis. The Euglena large ribosomal subunit is distinct in that it contains 14 discrete rRNA fragments that are assembled non-covalently into the canonical ribosome structure. The rRNA is substantially enriched in post-transcriptional modifications that are spread far beyond the catalytic RNA core, contributing to the stabilization of this highly fragmented ribosome species. A unique cluster of five adenosine base methylations is found in an expansion segment adjacent to the protein exit tunnel, such that it is positioned for interaction with the nascent peptide. As well as featuring distinctive rRNA expansion segments, the Euglena ribosome contains four novel ribosomal proteins, localized to the ribosome surface, three of which do not have orthologs in other eukaryotes.Ribosomal RNA is the central component of the ribosome, mediating its functional and architectural properties. Here, we report the cryo-EM structure of a highly divergent cytoplasmic ribosome from the single-celled eukaryotic alga  Ribosomes are macromolecular ribonucleoprotein (RNP) assemblies that function together with transfer RNA (tRNA) in translating messenger RNA (mRNA) into protein. Throughout the three domains of life, ribosomes are highly conserved in composition, structure and sequence . PhylogeEukaryotic cytoplasmic ribosomes (cytoribosomes) typically contain four rRNA species: an 18S-sized component in the SSU and 25S-28S, 5.8S and 5S rRNAs in the LSU Figure . The 18STrypanosoma, Leishmania and Crithidia), the 26S rRNA is further cleaved into six fragments  is substantially more modified than other characterized LSU rRNAs 2HPO4 1 g/l, KH2PO4 1 g/l, MgSO4 0.2 g/l, sodium citrate 0.8 g/l, CaCl2 0.02 g/l, Fe2(SO4)3.nH2O 3 mg/l, MnCl2.4H2O 1.8 mg/l, Co(NO3)2.6H2O 1.3 mg/l, ZnSO4.7H2O 0.4 mg/l, sodium molybdate 0.2 mg/l, CuSO4.5H2O 0.2 mg/l, thiamine hydrochloride 0.01 mg/l, vitamin B12 0.0005 mg/l, phosphoric acid to pH 3.5 and absolute ethanol 2.8 ml/l] at 25°C. Cells were harvested at OD600 = 1 , washed 3× in resuspension buffer  and resuspended in buffer A . Cell disruption was performed by French press  followed by centrifugation steps designed to remove cell debris, chloroplasts and mitochondria; these include 15 min at 1100 × g followed by 30 min at 26 000 × g, both performed at 4°C. The clarified supernatant was gently loaded onto a 1.1 M sucrose cushion in Buffer B  and centrifuged at 116 00 × g at 4°C for 16.5 h. The ribosome-enriched pellet was resuspended at 4°C in Buffer C  and centrifuged at 10 000 × g for 10 min to eliminate residual chloroplast contamination. The clear supernatant was then subjected to a 15–30% sucrose gradient centrifugation in Buffer C . The 80S peak was collected and centrifuged at 320 000 × g for 12 h at 4°C. The pellet was further resuspended in Buffer D  and centrifuged for 1.5 h at 245 000 × g, to remove excess sucrose. Finally, the ribosome pellet was resuspended in Buffer D and stored at –80°C at a final concentration of 10 mg/ml until further use.CACCAUGUUCAAA, GE Dharmacon) containing a kinetoplastid-specific Kozak sequence , A-site tRNAphe  and a synthetic derivative of paromomycin ] with relaxation times of 30 min after the addition of each complex component. Ribosome final concentration was 250 nM.Ribosome complexes with mRNA and tRNA molecules were assembled by sequential addition of a programmed mRNA fragment , a Pund 3 in ) at 1:102/s. Defocus values ranged from 1.5 to –3.5 μm. Movies were patched-framed-motion-corrected and dose-weighted using Motioncor2 (L. donovani ribosome (EMD-7024 and EMD-7025) (Ribosome samples (3.5 μl) were applied to glow-discharged holey carbon grids (Quantifoil R2/2) coated with a continuous thin carbon film. The grids were blotted and plunge-frozen using a Vitrobot Mark IV (Thermo-Fischer Scientific). Cryo-EM micrographs were collected at liquid nitrogen temperature on a Titan Krios electron microscope (Thermo-Fischer Scientific) operating at 300 kV. Micrographs were recorded on a Falcon 2 direct electron detector (Thermo-Fischer Scientific) at a nominal magnification of 133K, with a pixel size of 1.05 Å/pixel and a dose rate of ∼1.52 electrons/Åtioncor2 . CTFFINDtioncor2  was usedMD-7025)  as initiMD-7025)  using seMD-7025) . Local rMD-7025)  (SupplemMD-7025) .de novo model building in COOT  were usand 6AZ3 . De novoIX.Elbow . Model rIX.Elbow . The finIX.Elbow .Euglena gracilis transcriptomes and verified by publicly available sequence data available through the Marine Microbial Eukaryotic Transcriptome Sequencing Project, MMETSP  sequence to the cleavage products . Approx.10 μg RP sample was applied on the column and eluted with a 30-min linear gradient of 10–40% (v/v) acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 100 μl/min. The RPs sample before LC separation (1 μg) or the eluate containing the RP component (50 μl) was adjusted at pH 8, digested with 0.25 μg trypsin overnight at 37°C, and the resulting digest was analyzed by a direct nanoflow LC–MS/MS system equipped with a hybrid quadrupole-orbitrap mass spectrometer  as described  and passed through a 23-gauge needle 100 times. The sample was further mixed with 200 μl of chloroform, centrifuged at 10 000 × g for 15 min at 4°C, and the resulting upper phase (∼500 μl) was mixed with a glycogen solution  and isopropanol (500 μl) to precipitate the rRNAs. The rRNAs were collected by centrifugation, dissolved in RNase-free water, and stored at −80°C until use.A260. For further analysis, the purified rRNA fragments were digested with RNase T1 or RNase A in 100 mM triethylammonium acetate buffer (pH 7.0) at 37°C for 60 min.Purified rRNA fragments were further separated by the reversed-phase LC on a PLRP-S 4000 Å column  as previously described . In brie∼50 ng) were analyzed by a direct nanoflow LC–MS system as previously described (http://ariadne.riken.jp/) was used for the assignment of the tandem mass spectral data. with the E. gracilis rRNA sequence  as reference. The Ariadne search parameters were: the maximum number of missed cleavages was one; two methylations per RNA fragment at any residue position were allowed; an RNA mass tolerance of ±5 ppm and a tandem spectral tolerance of ±20 ppm were allowed.The RNase T1/A digest of LSU6 and LSU8 rRNA  was analyzed by the nanoflow LC–MS system described above. The LC–MSescribed . A summaE. gracilis ribosome we purified intact 80S ribosomes and assembled in vitro a ternary complex with mRNA and tRNAs. We then acquired cryo-EM data that were primarily processed through several rounds of 2D and 3D classifications. Our initial set of data included 325,727 particles, yielding a 3.23-Å reconstruction of the entire ribosome , three tRNAs, an mRNA and a total of 34 RPs for the SSU and 41 for the LSU  and at the tip of the L1 stalk . Ho. HoEuglek Figure . The obsEuglena ribosome surface is highly enriched with rRNA expansion segments (ESs), some of which are Euglena-specific  are clustered in two surface-exposed regions, localized in close proximity to the major rRNA chain end bundles . The resulting tryptic peptide data combined with a whole transcriptome analysis identified a total of 248 proteins, among which were 76 of the 80 inferred E. gracilis cytRPs : all but eL28 and including two paralogs for eL15 . Euglenas cytRPs .Euglena cytoribosome .Our cryo-EM map allowed the building of the inferred RP structures of the e Figure . Howevers Figure , C. The ishmania  and is cishmania . A wholeo eSEug1 , and theo eSEug1 . The E. bona fide Euglena-specific RPs, which we have named eLEgr1–3.In the LSU, the three unassigned densities were found on the ribosome surface, corresponding to small proteins 60–70 amino acids long. Two of the observed densities were localized to the CP region and one overlapped the location of eL28 at the surface-exposed region of the LSU Figure . Guided E. gracilis transcriptome and sequence similarity between eLEgr1 and eL28 is extremely low. The apparent absence of eL28 was surprising because we readily detected an ortholog in kinetoplastid species such as T. cruzi, as well as in other members of Discoba , the eukaryotic supergroup to which Euglenozoa belongs. eL28 functions in most eukaryotes to stabilize ES7L-A  and pseudouridines (Ψ). However, compared to other organisms, in Euglena these modifications differ both in their unprecedented levels and their overall distribution, which extends far beyond the ribosomal core regions ..E. graciEuglena ribosome, revealing their 3D context ,,81,1A clA Figure  that actA Figure . Of note complex  indicate complex . AssuminEuglena cytoribosome is an unusual RNP, which is distinct from other ribosomes in that it harbours four unique core ribosomal proteins, while its LSU rRNA is highly fragmented. The RNAs in this ribosome species (particularly the LSU rRNA) are also substantially enriched in post-transcriptional modifications, which are spread far beyond the catalytic RNA core, likely contributing to the stabilization of rRNA fragments. A notable feature of this ribosome is a cluster of five m1A residues localized to a distinctive LSU rRNA expansion segment on the ribosomal surface and adjacent to the protein exit tunnel. This extension is uniquely positively charged and might serve to stabilize a negatively-charged pocket enriched with rRNA segment ends, and/or to potentially interact with auxiliary proteins that act on the nascent peptide or aid its localization.In summary, the Euglena cytoribosome have been deposited at the Electron Microscopy Data Bank (EMDB-11232). The atomic model has been deposited in the PDB with accession number 6ZJ3. Annotated Euglena gracilis cytRP sequences (CDS and amino acid) have been deposited in GenBank under accession numbers MT583833–MT583918.The cryo-EM maps of the gkaa893_Supplemental_FileClick here for additional data file.


The nucleocapsid protein N from SARS-CoV-2 is one of the most highly expressed proteins by the virus and plays a number of important roles in the transcription and assembly of the virion within the infected host cell. It is expected to be characterized by a highly dynamic and heterogeneous structure as can be inferred by bioinformatics analyses as well as from the data available for the homologous protein from SARS-CoV. The two globular domains of the protein (NTD and CTD) have been investigated while no high-resolution information is available yet for the flexible regions of the protein. We focus here on the 1–248 construct which comprises two disordered fragments (IDR1 and IDR2) in addition to the N-terminal globular domain (NTD) and report the sequence-specific assignment of the two disordered regions, a step forward towards the complete characterization of the whole protein. Coronaviruses (CoVs) are relatively large viruses containing a single-stranded positive-sense RNA genome encapsulated within a membrane envelope  enabling follow-up applications, such as residue-resolved drug screening and interaction mapping.In this frame, we provide here the backbone assignment of the two disordered regions flanking the NTD, the N-terminal IDR1 and the serine-rich disordered region IDR2, in the 1–248 residue construct (IDR1-NTD-IDR2). These data will contribute to the efforts of the research consortium covid19-nmr  was guided by the SARS-CoV homologue  plasmid was obtained from Twist Bioscience.13C, 15N-labelled IDR1-NTD-IDR2 protein was expressed in E. coli strain BL21 (DE3). The culture was grown in 1 L LB medium at 37 °C until OD600 reached 0.8, then transferred in 250 mL of labelled minimal medium (4x) containing 0.25 g/L 15NH4Cl (Cambridge Isotope Laboratories), 0.75 g/L [U]13C6-d-glucose (Eurisotop). After 1 h of metabolite clearance, the culture was induced with 0.2 mM isopropyl-beta-thiogalactopyranoside (IPTG) at 18 °C for 16/18 h.Uniformly The cell pellet was resuspended in 25 mM 2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIS), 1.0 M sodium chloride, 5% glycerol, DNAse, RNAse and 500 µL of 100 × stock of protease inhibitor cocktail (SIGMA) at pH 8.g, 4 °C), then the cleared supernatant was dialyzed overnight at 4 °C into 25 mM TRIS pH 7.2 (binding buffer).Cells were disrupted by sonication. The supernatant was cleared by centrifugation .3, 5% (v/v) D2O in water.Final NMR samples were 280 µM IDR1-NTD-IDR2, 25 mM TRIS pH 6.5, 450 mM sodium chloride, 0.02% NaN1H, 176.05 MHz 13C, and 70.97 MHz 15N frequencies, equipped with a 5 mm cryogenically cooled probehead optimized for 13C direct detection (TXO). Proton direct detected NMR experiments were acquired on a 28.3 T Bruker AVANCE NEO spectrometer operating at 1200.85 MHz 1H, 301.97 MHz 13C, and 121.70 MHz 15N equipped with a 3 mm cryogenically cooled triple-resonance probehead (TCI).All the NMR experiments were acquired at 298 K. Carbon-13 direct detected NMR experiments were acquired on a 16.4 T Bruker AVANCE NEO spectrometer operating at 700.06 MHz 1H and 13C direct detected experiments. In particular, 2D--HSQC, 2D--BEST-TROSY (BT), 2D-CON, 2D-(H)CACO and 2D-(H)CBCACO experiments were performed. Moreover, a series of 3D experiments were acquired: 3D-(H)CBCACON, 3D-(H)CBCANCO, 3D-BT-HNCACB, and 3D-BT-HN(CO)CACB. To compare the resonance values obtained through the carbon detected spectra with the ones obtained with the proton detected ones, 3D-HNCO and 3D-HN(CA)CO were also collected.Backbone assignment was performed by analyzing 2D and 3D 13C detected experiments were acquired in a version optimized for the detection of the highly flexible regions of the protein CBCACON and 3D-(H)CBCANCO , and for the adiabatic π pulse . Composite pulse decoupling was applied on 1H   CACB) or the sensitivity enhanced approach (3D-HNCO and 3D-HN(CA)CO) for the 3D experiments. The 2D--BEST-TROSY used sensitivity-enhanced gradient echo/antiecho coherence selection . The 1H band-selective pulses on the amide region were Pc9  construct of the SARS-CoV-2 nucleocapsid protein N is shown in Fig. i-1-Ni correlations that fall in a very clean region of the CON spectrum (132 < δ(15N) < 140 ppm). The observation of only 7 well-resolved cross-peaks in this region (out of 17 expected for this construct) indeed confirms that C′ direct detection selectively picks up the signals of the disordered regions CBCACON and the (H)CBCANCO  and the fact that this is correlated to resonances of the preceding amino acid  through the 2D CON and 3D (H)CBCACON spectra constitute two features that allow us to unambiguously identify the type of dipeptide (Xi-1-Proi pair) that gives rise to specific signals as highlighted in Fig. α and Cβ resonances enable us to recognize glycine, alanine, serine, and threonine residues; the remaining X-Pro pairs can then be easily identified as deriving from leucine and arginine residues by comparison with the primary sequence of the protein. Therefore, already at this very early stage of the sequence-specific assignment process, most of the observed resonances in this region could be assigned to specific amino acids uniquely considering the type of X-Pro pairs present in the intrinsically disordered regions . Similarly, inspecting the opposite region of the CON spectrum at low 15N chemical shifts  contributes to an excellent resolution allowing us to count 16 resolved cross peaks in this region in the simple 2D mode. This is in line with the number of glycine residues present in the flexible disordered fragments. The classification of these resonances in Xi-1-Glyi pairs achieved through inspection of the (H)CBCACON provides further input for their identification, as described above for the case of Xi-1-Proi pairs. Complete comparative analysis of the 3D (H)CBCACON and 3D (H)CBCANCO spectra enables the identification of the vast majority of the expected resonances of disordered regions. The excellent resolution obtained in the 2D reference spectra, the CON as well as the (H)CACO and (H)CBCACO, provides valuable support for the analysis of crowded regions of the spectra and to the discrimination between different residue types  (vide infra). It is interesting to note how the combined use of these complementary datasets (13C′- and 1HN-detected 3D experiments) provides information that is particularly useful to achieve sequence-specific assignment of intrinsically disordered regions also within highly heterogeneous proteins. The set of 2D spectra CACO, (H)CBCACO), provided they are acquired with high resolution, then becomes a very useful tool to achieve atomic resolution for the vast majority of the amino acids in the highly flexible disordered regions of complex, heterogeneous proteins.As a result, 98% of the disordered fragment IDR1 (only the first methionine is missing) (BMRB 50619) and 91% of the fragment IDR2 (BMRB 50618) could be assigned in a sequence-specific manner  can now be investigated at atomic resolution providing experimental information regarding the many interaction sites that can be predicted through different approaches CACO, (H)CBCACO) in conjunction with 2D HN correlation experiments provide an excellent tool to monitor at atomic resolution their role in the interactions with RNA, with viral proteins or with proteins of the host, as well as with small molecules as potential drugs, opening the way to radically novel, unexplored approaches in drug discovery.The NMR resonance assignments of the IDR1 and IDR2 domains of the N protein from SARS-CoV-2 open the way to understanding the role of these flexible parts of the nucleocapsid protein in modulating its function. The suite of


Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O2 and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O2 migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH2 to reduce the ferric iron, which forms the stable ferric–superoxide–TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 1010 M−1 s−1 forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O2 and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.The substrates O The data and model allow us to derive kinetic constants and energies for a flavoHb-NOD mechanism that employs O2  supporting a ping-pong mechanism. At higher NO concentrations, NO reversibly inhibits the activity, and NO inhibition appears competitive with respect to O2 with roughly 50% inhibition observed at a 1:100 ratio of NO to O2 as previously reported . The data suggest a more complex ping-pong mechanism in which O2 and NO cooperatively activate NO dioxygenation.The activity , similaractivity , 17, 21,ing [O2] A supportreported , 5. O2 sat 20 °C B. The da2 and NO, the weak reversible competitive inhibition by NO, and the larger-than-expected requirements for O2. Similar to the M. tuberculosis HbN-NOD, the flavoHb-NOD appears to utilize a hydrophobic long tunnel (LT) for O2 entry (3− efflux (A). Six residues from the G and H helices form the outer wall of the ∼20-Å LT with three residues from the A helix and single residues from the B, E, and H helices forming the inner wall (B) . T. T2 and wall (B) . Furtherkcal/mol . For exatructure  the cleaC, top). The S. cerevisiae ferric-nitrite enzyme structure shows a widening of the G8-E15 gate by 4 Å and a shift of the entire E-helix by ∼2 Å toward the putative NO entry site , LeuE11 is displaced toward the NO entry site to form a part of the ST interior presumably guiding NO for reaction with the bound Fe3+O2−. By analogy to bound nitrite, O2 displacement of the LeuE11 may widen the gate for passage of the slightly polar 3.2 Å by 4.1 Å van der Waals' dimension NO molecule. The ST may have a capacity for more than one NO molecule.The putative ST shows a highly conserved outer entry defined by GlyG9, GlyH9, LeuG13, and TyrH12  and appeFig. 2E. coli flavoHb structure shows a CD loop in an unusual random coil with the GlnE2 side chain extended forming contact with the adenine ring of FAD in the reductase domain . A chloride ion is found bound in an anion hole in the E helix and CD loop corner . Displacement of the LeuE11 side chain from the iron by nitrite, and presumably by nitrate, appears to twist the E-helix and exert a right-handed torsion to furl the CD loop  thu thuE. coE. coli and S. cerevisiae ferric-nitrite flavoHb structures also show differences in the positioning of the proximal LysF7 forming the bridge for electron transfer (ET) , the LysF7 epsilon ammonium group and GlnE7 amide oxygen atom hydrogen bond a water molecule near the middle of the ∼6-Å-long line-of-flight for ET between the heme carboxylate and FAD C8 methyl group  in which the GlnE7 amide is positioned with TyrB10 hydrogen bonding and stabilizing the Fe3+O2− intermediate and the LysF7 side chain positioned with the straight line-of-flight for ET passing 0.3 Å beyond the van der Waals' radius of the epsilon ammonium nitrogen, which presumably moves to the position of balance between repulsion by the core and attraction by the annular shell for optimal electron tunneling  , 40, sugtructure D, top, tyl group  that is yl group . In thisme plane D, bottomunneling . Electrounneling , 39, 40.2 and NO activation observed in A–B. In the kinetic model depicted in 2 molecules are driven from the aqueous solvent into the LT with an equilibrium constant of K1, and the leading O2 displaces LeuE11 forcing the movement of the E-helix, docks with the ferric iron, the LeuE11 isobutyl group and the TyrB10 hydroxyl, and weakens the G8-E15 gate with an equilibrium constant of K2. NO then enters and passes through the ST further widening the G8-E15 gate with equilibrium constant K3 and leveraging the movement of LeuE11, the E-helix, GlnE7, the F-helix, and the LysF7 side chain while triggering furling of the CD loop with the first-order rate constant k'T and equilibrium constant (KT), ET (kET), and nitrate release (kp). Reduction of the ferric heme causes rapid univalent O2 reduction forming the Fe3+O2− intermediate, which then reacts rapidly with the internally sequestered NO with the bimolecular rate constant k'4 and equilibrium constant K4 forming the Fe3+NO3− intermediate that rapidly isomerizes (kis)  value. The model also allows for excessive NO to permeate a leaky NO entry gate, the nitrate efflux gate, or other pathways and inhibit catalysis with equilibrium constant Kii(NO). In the model, inhibitory NO binds ferric heme forming Fe2+NO+  . Nitrate Fe2+NO+ , which rKT) v= Vm2 v= V2 to each act as uncompetitive inhibitors of catalysis  and Ki(O2) have been included for situations in which NO and O2 do not compete for LT or ST entry, or iron binding, but nevertheless inhibit catalysis. In addition, we have introduced a factor and equilibrium constant Ka(O2) for rare situations in which O2 causes an additional NO-independent activation of catalysis possibly by occupying unique binding sites or cavities  of ∼2 μM at 20 °C , whereas substituting ValG8 with a smaller Ala decreases K3 as expected for a role for ValG8 in gating NO entry. The critical function of ValG8 in the allosteric mechanism is also suggested by the large changes in the KT values in the mutants, and the apparently large frustration of AlaG8 at 20 °C. The data also reveal a role for ValG8 in determining the susceptibility to NO inhibition. AlaG8 shows smaller Ki(NO) and Kii(NO) values, whereas the bulkier LeuG8 shows larger values. The effect is more discernible at 37 than 20 °C, again suggesting greater frustration at the lower temperature. A greater resistance of LeuG8 to NO inhibition also confers a higher Vmax at both temperatures; however, both the NO sensitivity, as indicated by small Ki(NO) and Kii(NO) values, and the impaired allosteric activation, as indicated by large KT values, lower the Vmax for AlaG8. The data support the roles for G8 shown in the kinetic model in Figure 2C. G8 substitutions also cause relatively smaller effects on the O2 dependence (K2) suggesting effects on E-helix and LeuE11 movement.Similar steady-state kinetic data for G8 and E11 substitution mutants are shown in K3 and K2 and a large decrease in KT  values of the E2 variants suggest a CD loop structure dysfunction that opens a migration pathway for NO.GlnE2 within the CD loop was tested for its proposed role in the allosteric mechanism. Data for E2 substitution flavoHbs are shown in 3+O2− intermediate in (flavo)Hbs  and Kii(NO) values, and an inefficiency of NO dioxygenation and larger K4 values. In addition, GlnE7 mutants are expected to show impaired ET switching and altered kET and KT values. Data for B10 and E7 flavoHb variants measured at 20 °C are shown in Ki(NO) and Kii(NO) values, which also limited the O2 and NO concentrations as well as the temperature that could be tested. PheB10 shows a 4-fold larger K2 value while LeuE7 and HisE7 show more modest two- to threefold increases. HisE7 shows a larger KT value possibly reflecting impaired ET while LeuE7 shows negligible effect on the KT value suggesting normal switching at 20 °C, but not 37 °C. K3 values are unexpectedly increased in the PheB10 and E7 mutant NODs and may reflect a role for TyrB10 and GlnE7 in facilitating O2 binding and the displacement of LeuE11 from the ferric iron. Of interest, the calculated Vmax values are not decreased in the mutants indicating the capacity of elevated O2 concentrations to overcome any defects in O2 binding and Fe3+O2− stabilization. The greater susceptibility to NO inhibition at a higher [NO], a lower [O2], and 37 °C may be explained by Fe2+NO formation.TyrB10 and GlnE7 are important for stabilization of the Felavo)Hbs , 49. In lavo)Hbs , mutantsv) also serves as a measure of the rate of ET as expressed as a first-order time-averaged rate constant for ET, and the minimal kET value  where a smaller Δv/Δ[NADH] value indicates a decreased rate of ET, an inefficient tunneling matrix, and dysfunctional gating mechanism. As shown in v versus 1/[NADH] plots at 37 and 20 °C and smaller Δv/Δ[NADH] values  of 1.5 × 107 M−1 s−1 as detected by the transient decrease in FAD absorbance at 460 nm . Similar FAD reduction rates are observed for the PheB10, LeuE7, and HisE7 variants  with the HisE7 mutant showing a greater extent of FAD reduction. FADH2 rapidly reduces the WT flavoHb ferric heme to the ferrous state with a maximum kET value of 120 s−1 at 20 °C as measured by the transient increase in absorbance at 430 nm , similar to the 136 s−1 value previously reported . It is noteworthy that the HisE7 variant showed a biphasic behavior with an initial slow reduction followed by a more rapid phase with incomplete reduction (line 4) despite achieving a more negative FAD/FADH2 reduction potential . The HisE7 variant appears to favor the off conformation  for the fast and slow phases in the 10- to 200-μs range  rebinding is 86% inhibited by 100 μM CO, whereas the fast phase is only inhibited by ∼10%, (2 (2 migration through the LT as well as O2 binding to the iron. The PheB10 and HisE7 variants show similar k'slow (O2) values, but show only ∼50% inhibition of O2 migration and binding by CO in the slow phase suggesting availability of pathways for O2 migration, such as the ST, CD loop, or E-helix heme interface, in addition to the LT. The mutations, iron redox state, ligand types, and concentration affect the τT for ligand binding and the photolysis fraction (Φdis) in ways that are not readily interpretable. We suppose that the large τT values of >8 μs are due to gases transiently maintaining nonspecific pathways for gas migration initially opened by the quake of photolysis  values, but show larger k(O2) values, and respective 19-, 6-, and 35-fold larger Kd(O2) values than the WT enzyme (3+O2− intermediate (k(O2) value measured for the HisE7 variant relative to that for GlnE7 . In addition, the comparable k′slow (NO) and k′slow (O2) values and the large k(NO) values for the ferric flavoHbs  and Kd(NO) values for the ferrous flavoHb (k(NO) values, we can calculate 90% activity recovery following NO binding in 3.2 h for the ferrous NO species and a mere 0.6 ms for the ferric NO form. With NO elimination via reduction (≤0.1 s−1)  , 5, 90% D). The starting activity of the AsnF7 enzyme is 6-fold less than the LysF7 (WT) enzyme and rapidly inactivated. Ninety percent of the starting activity is lost in less than 1000 turnovers or ∼30 s . In the absence of NO, the AsnF7 enzyme loses 85% of its NOD activity in 90 s with apparent zero order kinetics . Thus, NO is not essential for the inactivation, but NO greatly increases the inactivation. Catalase provides modest protection  suggesting formation of Fe3+-OOH, and H2O2, and oxidative damage to the heme. Indeed, flavoHb-NOD activity is sensitive to H2O2, and the heme ligand miconazole (versus 0.27 s−1), a 2.4-fold greater NO reductase activity (0.45 versus 0.19 s−1), a fourfold higher methyl viologen reductase activity (1.45 versus 0.36 s−1), a 4.6-fold higher cytochrome b5 reductase activity (0.65 versus 0.14 s−1), a 4.5-fold higher Ngb reductase activity (0.90 versus 0.20 s−1), and a twofold higher trimethylamine-N-oxide (TMAO) reductase activity (0.41 versus 0.21 s−1). CO (50 μM) inhibits TMAO reduction by ∼90% suggesting an increased ET and reduction by the heme. Other reductase activities are relatively resistant to 50 μM CO. Increased rates of reduction demonstrate an increased ET from the flavin and a loss of the electron tunneling path directionality  equal to 13.7 kcal/mol, an amount equivalent to that required to break three to four hydrogen bonds. The largest effect on the Ea is observed with the AlaE11 substitution with a calculated Ea of 24.6 kcal/mol and a 5- to 15-fold larger temperature-dependent KT value further suggesting that LeuE11 movements due to NO collisions, and concerted motions of the CD loop, E and F-helices, and LysF7, form the largest energetic barrier, and also the rate-limiting step in the NOD mechanism. Following this analysis, we can also see that the forward rate constant for the trigger and switching mechanism, k′T, limits and equals the Vmax value.Cooperative activation of the NOD function of the ng model , 4 are ritioning . A critiitioning  and hemetrations  reveal okET), on the other hand, has been reported to be ∼40-fold faster by time-resolved pulse radiolysis (6800 s−1) (−1) (B)   by strengthening bonding to the heme propionate in the ET gate structure and stabilizing the off state  and, moreover, by significantly decreasing the nominal −0.125 V heme mid-point potential (C) together with spectral analysis (unpublished results) suggest a relaxed to tense motion in the heme corresponding to a high spin (S = 5/2) to low spin (S = 1/2) ferric iron transition . Furthermore, thermally fluctuating spin state mixtures in the resting state may account for the observed intermediate ET rates (B). Accordingly, the proposed conformational changes of the F-helix in the trigger step (KT) force a low to high spin transition and associated potential increase, besides switching the ET gate on    than the−1) (−1) B 4) ind indkET),−0.15 V) . Further−0.15 V)  and rest−0.15 V) B, line 4ff state D, top anotential . The E7 otential C togetheansition  with a lng force B. FurtheET rates B. Accord gate on D and 3. on state D, bottom bottom) , 39, shoenergies , correctequation , 39, 44.2− forming peroxynitrite is 29 kcal/mol  and efflux are coupled with CD loop unfurling–furling, and the rate constants thus equal Vmax. We suppose that the initial step in the exergonic NO dioxygenation reaction, as represented by k'4, where k'4 equals Vmax/K4 and k'NOD, ultimately provides the energy required to drive protein motions and support the catalytic cycle. Furthermore, by analogy, a 577-nm photon, bearing 49.6 kcal/mol, and absorbed by the flavoHb during photolysis of O2 or other bound ligands, would suffice to trigger large protein motions via LeuE11, TyrB10, and GlnE7 interactions and contribute to the microsecond-scale heterogeneity of ligand migration evidenced by the data in 2+CO, most motion occurs in the nanosecond range, but CO-induced structural changes and hydration shell fluctuations, nevertheless, continue for microseconds  is only roughly approximated by Vmax/k'slow (O2). K1 may also be further broken down to movement of O2 through the strictures between the docking sites S3, S2, and S1 with a relatively large 6 kcal/mol energy barrier estimated for the narrow stricture 2 controlling passage from S2 to S1 (Ki(NO) values of 0.3 to 1.2 μM determined for NO competition with O2 for LT entry (Ki(CO) values of ∼1 μM  values estimated for the WT flavoHb.In the model, S2 to S1 . DisplacS2 to S1 . Indeed,LT entry , and alsof ∼1 μM , argue f2 molecule, with an induced dipole, interacts magnetically with the paramagnetic ferric iron atom in displacing LeuE11 and, moreover, that TyrB10 and GlnE7, as well as the G-helix dipole, affect this interaction. Indeed, the Fe3+ and O2 interactions are relatively weak given the large micromolar K2 values  may be achieved by a discriminator for the inducible lateral O2 dipole. The diametric PheG20 phenyl group and ThrA12 O-atoms within stricture 3 . We suppose that a small KT value, and a strong trigger, will favor the on position and that larger KT values, like that shown by the HisE7 variant, will favor the off position in the resting state. Further exploration of the effects of mutations, and tunneling matrix modifiers or dopants, on ET rates in flavoHbs should be illuminating provided that the fine structure of the switch mechanism can be mirrored, and the FAD/FADH2 potential controlled, in the resting state(s). Furthermore, the model and 3−, instead of chloride, normally binds and stabilizes the unfurled off configuration of the CD loop. Moreover, CD loop furling–unfurling may play an unrecognized role in the channeling of nitrate by ThrE2 in HbN-NOD  was obtained from Roche Applied Science. Recombinant rat cytochrome b5 (soluble form) and murine Ngb were expressed in E. coli and purified essentially as described  unless otherwise stated. DNA primers were synthesized at the University of Cincinnati Medical Center DNA facility. Praxair  was the supplier of 99.993% Oescribed , 70. NADhmp was constructed by ligating the EcoR1-BamH1 fragment from pAlterhmp  (Fraction V), 100 μM NADH, 0.1 μM FAD with NO, Oescribed , 72. NAD2 concentrations. Vmax, K1, K2, KT, and Kii(NO) values were treated as independent variables for a given flavoHb while allowing K3, K4, and Ki(NO) to vary over limited ranges with varying O2 concentration. Kii(NO) was estimated from NO inhibition at saturating O2 concentrations. Ki(O2) and Ka(O2) were included as independent variables in order to model the AlaE11 mutant data.Rate constants were estimated from the model kinetic equation using Python programming with successive approximations to optimize fits with varying NO and O2-purged buffer (2, NO, and CO association rate constants were determined by photolyzing 50 μM of the respective ferrous flavoHb species with a 300-ns pulse from a 577-nm laser and by spectrophotometrically following ligand reassociation in O2-, NO-, or CO-saturated buffer unless otherwise indicated (2 or CO was measured by the change of absorbance at 436 nm by applying the bound versus unbound difference extinction coefficients of 0.046 and 0.053 μM−1 cm−1, respectively. Binding of NO was measured by the change of absorbance at 422 nm by applying a difference extinction coefficient of 0.040 μM−1 cm−1. O2 and CO dissociation rate constants were determined by stopped-flow rapid mixing with either CO or NO displacement (2 to scavenge H2O2 produced by the NADH oxidase activity of flavoHb and to regenerate O2.FAD and heme reduction were monitored by following the changes in flavoHb absorbance at 460 and 430 nm, respectively, with stopped-flow rapid mixing of 50 μM NADH and 10 μM flavoHb in NData are representative of two or more trials. Structures were modeled and analyzed using Jmol version 14.2.15.All data are contained within the manuscript.The authors declare that they have no conflicts of interest with the contents of this article.


After pandemic, A(H1N1)pdm09 is generally known to be associated with younger adults' infection and greater severity than seasonal A(H3N2) but some inconsistences between recent studies exist.We aimed to compare the epidemiology and clinical outcomes of A(H1N1)pdm09 and A(H3N2) to verify and consolidate about the knowledge of known differences of subtypes.Data were retrospectively collected from the hospital‐based influenza morbidity and mortality surveillance in South Korea in nine tertiary care hospitals, from August 31, 2014, to August 25, 2018. Patients with H1N1pdm09 or H3N2 infection admitted in the emergency room or ward were recruited.P = .030), lower influenza vaccination  and pneumococcal vaccination rates , and fewer underlying diseases  than the A(H3N2) group. Influenza A subtypes were not associated with pneumonia risk (adjusted odds ratios [AOR] of A(H1N1)pdm09: 0.7 [95% confidence interval [CI]: 0.4‐1.2, P = .172]) and in‐hospital mortality (hazard ratio (HR) of A(H1N1)pdm09: 1.0 ). Influenza vaccination reduced in‐hospital mortality in hospitalized patients (HR: 0.3 (95% CI: 0.1‐0.7), P = .005).A total of 1747 patients had influenza A and were divided into two groups those with A(H1N1)pdm09 (n = 240) and those with A(H3N2) (n = 1507). A(H1N1)pdm09 group had younger age pdm09 infection was more common in younger patients without significant difference in pneumonia risk and in‐hospital mortality between subtypes. Influenza vaccination was associated with reduced in‐hospital mortality. Major influenza viruses that cause seasonal epidemics worldwide are influenza A and influenza B. Influenza A viruses have specific subtypes, which depend upon the type of surface antigens as follows: hemagglutinins (H) and neuraminidases (N). In 2009, a novel influenza A H1N1 strain emerged and circulated throughout the world, which was named A(H1N1)pdm09. Currently, it is now one of the most important seasonal influenza A strains along with H3N2. Exact forecasting of dominant influenza type is very important for global vaccination strategies. However, it is unpredictable which influenza type will prevail year by year and varies from country to country. Influenza surveillance in South Korea showed that A(H1N1)pdm09 subtype was more frequent than A(H3N2) in 2015‐2016 . In contrast, A(H3N2) subtype was more frequent than A(H1N1)pdm09 in 2014‐2015 , 2016‐2017 , and 2017‐2018 .The difference in clinical manifestation and outcome between A(H1N1)pdm09 and A(H3N2) has not been clearly defined. Before 2009, several observational data analysis described higher influenza‐associated hospitalization rate and mortality in A(H3N2) predominant seasons, but they are generally regarded as insignificant findings.22.1From August 31, 2014, to August 25, 2018, nine tertiary care hospitals  in South Korea participated in the hospital‐based influenza morbidity and mortality (HIMM) surveillance system. The surveillance system is composed of the following two types of schemes: emergency room (ER)‐based and hospitalized patient‐based influenza‐like illness (ILI)/severe acute respiratory infection (SARI) surveillance.TM Real‐Time Polymerase Chain Reaction (PCR) System. When the subtype data were unavailable by fulfilling more than one of the followings: (a) PCR results were negative; (b) sample was inadequate to analyze due to inappropriate storage or missing during transportation; (c) PCR analysis was impossible because of insufficient laboratory capacity; and (d) more than two subtypes of influenza were identified in one sample, we introduced the subtype results from commercial respiratory virus real‐time PCR kit or RAT kits performed in each contributing hospital if possible. The case with unavailable subtype data had been excluded from the analysis.Each season of influenza was divided by 1 year: from August 31, 2014, to August 29, 2015 (2014‐2015 season); from August 30, 2015, to August 27, 2016 (2015‐2016 season); from August 28, 2016, to August 26, 2017 (2016‐2017 season); and from August 27, 2017, to August 25, 2018 (2017‐2018 season). ILI was defined as (a) history or sudden onset of fever ≥38.0°C and (b) presence of ≥1 respiratory symptoms such as cough, sore throat, or rhinorrhea/nasal obstruction which onset within 7 days. SARI was defined (a) history or sudden onset of fever ≥38.0°C; (b) presence of ≥1 respiratory symptoms such as cough, sore throat, or rhinorrhea/nasal obstruction which onset within 7 days; (c) shortness of breath; and (d) admission.The clinical data of subtype‐confirmed patients were collected by retrospectively reviewing medical records. Data on demographic characteristics, histories of vaccination, hospitalization, underlying diseases, clinical manifestations, complications, and in‐hospital mortality were collected using a case report form. Pneumonia was defined as respiratory symptoms with pulmonary infiltrations on chest radiography, which was confirmed by a radiologist. All hospitalized patients were followed up to 90 days along the hospital stay and checked the date of death or the date of discharged alive.The HIMM surveillance including participant enrollment, sample collection, and data analysis was approved by the institutional review board at each contributing hospital and the center hospital, Korea University Guro Hospital .2.2U test was used to compare the mean age and length of hospital stay between two groups, and the results were expressed as median (interquartile range). A P value of <.05 was considered to significant. Potential variables that might be associated to the development of pneumonia during influenza A infection were analyzed by multivariable linear model logistic regression as adjusted odds ratios (ORs). Variables which were significantly associated with pneumonia in the univariable analysis (P < .05) and generally known risk factors of pneumonia  were selected and included in multivariable regression analysis. The association between A(H1N1)pdm09 and A(H3N2) in relation to the overall mortality of hospitalized patients was investigated using a time‐dependent Cox regression analysis to obtain the hazard ratios (HRs). All variables were validated by the graphical analysis of log‐minus‐log plot for the proportional hazards assumption. All statistical analyses were performed using Statistical Package for the Social Sciences version 20.0. The post hoc power analysis of Cox regression was conducted by Power Analysis & Sample Size 2020, NCSS Statistical Software.All categorical clinical data were calculated and analyzed using chi‐square and Fisher's exact test. Mann‐Whitney's 33.1A total of 4760 patients diagnosed with influenza infection were enrolled in the study. A total of 2741 were enrolled at the time of ER visit with 2066 were discharged from ER and 675 were admitted to the wards or ICUs, whereas 2019 were enrolled at wards after admission Figure . At the P = .030), and the median age difference was 7 years. The proportions of patients aged 19‐49 years and 50‐64 years were higher in the A(H1N1)pdm09 group , while the proportion of patients aged above 65 years was higher in the A(H3N2) group . Age group distribution by 10 years showed a gradual decrease in the proportion of patients with A(H1N1)pdm09 with aging and bimodal curve of A(H3N2) . The A(H3N2) group had a higher proportion of patients with more than one underlying disease than the A(H1N1)pdm09 group . Patients in the A(H1N1)pdm09 group more frequently significantly experienced fever, chills, cough, nausea/vomiting, headache, and wheezing than those in the A(H3N2) group.The baseline characteristics of the AH1N1)pdm09 and AH3N2) groups are shown in Table  groups aN1pdm09 a3.2P < .001), had lower vaccination rate , and had considerably more underlying diseases  and lower respiratory symptoms such as sputum  and dyspnea  (Table P = .021). No significant difference was observed in the overall mortality between the two groups (4 deaths (2.7%) vs 22 deaths (1.5%), P = .774).Clinical outcomes of subtype‐confirmed influenza A infection are displayed in Table 1) Table . The preP < .001) in those aged 50‐64 years; AOR: 19.2  in those aged ≥65 years) (Table P = .070), underlying illness , P = .115), and vaccination history , P = .421) were not statistically significant. The influenza A subtype also did not show a meaningful OR (compared to A(H3N2), A(H1N1)pdm09 AOR: 0.7 .All pneumonia patients (n = 183) were hospitalized in the surveillance. In the analysis of logistic regression, older age was a risk factor for pneumonia : 6.9 : 3.2‐14.8, P = .005). Other factors including the influenza A subtype showed no significant difference pdm09 and A(H3N2), did not show a significant influence on the development of pneumonia and in‐hospital mortality. Old age, high rate of vaccination, underlying disease, and pneumonia were associated with A(H3N2) infection. However, when the age, sex, underlying disease, and vaccination were adjusted in the logistic regression among hospitalized pneumonia and Cox regression among hospitalized participants, no significant difference was observed between the A(H1N1)pdm09 and A(H3N2) groups.In 2009, the A(H1N1)pdm09 pandemic strain caused more severe infections in young patients than previous A(H3N2) strains.Streptococcus pneumoniae, and pneumococcal vaccination.Influenza vaccination rate was higher in the A(H3N2) group than in the A(H1N1)pdm09 group, which possibly indicates that the efficacy of this vaccine against A(H3N2) strain is lower than that against A(H1N1)pdm09 strain. The meta‐analysis from 2004 to 2015 showed poor protection rate of seasonal influenza vaccine against A(H3N2) strain.The high susceptibility of A(H1N1)pdm09 infection among younger patients could be explained by the different possession rate of cross‐reactive antibody and birth cohort effect, which also known as immunologic imprinting caused by lack of previous childhood exposure of similar A(H1N1) viruses.In the Cox regression analysis, hospitalized patients who received influenza vaccination had low risk of in‐hospital mortality, and the age group, sex, subtypes, and pneumococcal vaccination did not show any significant effect on in‐hospital mortality. High hazards ratio in older age groups (≥65 years) without statistical significance might be associated with insufficient number of participants and deaths in the analysis. The effectiveness of influenza vaccine is well known. However, there is a controversy regarding whether the vaccine can certainly reduce mortality.P = .474; A(H3N2)group, HR: 0.3 (95% CI: 0.1‐0.7), P = .006, data not shown in the table). This result might be because of the size of the A(H1N1)pdm09 group but also might be influenced by lack of power and multiple‐comparison bias.In addition, we also conducted Cox analysis by subgroup, showed that the difference in HR based on influenza vaccination was only significant in the A(H3N2) group but not in the A(H1N1)pdm09 group (A(H1N1)pdm09 group, HR: 0.3 (95% CI: 0.0‐8.4), The study has a number of limitations, including its retrospective nature. First, the study populations might have more underlying diseases than the general population because most of the enrolled participants were recruited from the emergency rooms and wards of tertiary care hospitals. It is possible that a considerable number of mild influenza infections treated in outpatient clinics or even unrecognized by clinicians were ignored by the surveillance system. This selection bias might be associated with the incidence of pneumonia and death than the virtual influenza burden. Therefore, conducting a cohort surveillance including patients with mild infections would be helpful in determining the definite characteristics of influenza. Second, prescription of antiviral agents, use of adjunctive glucocorticoids, exact timing and type of pneumococcal vaccine, and influenza vaccination in previous seasons were not included in the analysis, which can be confounders of the study results. Furthermore, differences in regional demography and the treatment strategy used by each clinician from participating hospitals should be considered. Third, we did not perform real‐time PCR as subtype confirmation for all influenza A samples because of limited resources and laboratory capacity. Despite there were no factors influenced whether or not the PCR was conducted except laboratory situation, potential selection bias might have been involved. Finally, the low effectiveness of conventional influenza vaccines in older adults has been reported; hence, it is difficult to determine the direct link between influenza vaccination history and reduction of in‐hospital mortality.In conclusion, there was no significant difference in pneumonia and in‐hospital mortality rates between A(H1N1)pdm09 and A(H3N2) infections when adjusted with age, sex, underlying diseases and vaccination. The younger age group was more susceptible to A(H1N1)pdm09 but had fewer risk of pneumonia development than the older age group. Reduced in‐hospital mortality in vaccinated hospitalized patients suggests importance of seasonal influenza vaccination. Prospective large controlled cohort studies would be necessary to clearly describe the differences in clinical features of the influenza subtypes.The authors have no potential conflicts of interest to disclose.Jin Gu Yoon: Formal analysis (lead); Writing‐original draft (lead). Ji Yoon Noh: Conceptualization (supporting); Writing‐review & editing (supporting). Won Suk Choi: Resources . Jacob Lee: Resources . Jin Soo Lee: Resources . Seong‐Heon Wie: Resources . Young Keun Kim: Resources . Hye Won Jeong: Resources . Shin Woo Kim: Resources . Kyung‐Hwa Park: Resources . Joon Young Song: Conceptualization (supporting); Writing‐review & editing (supporting). Hee Jin Cheong: Conceptualization (supporting); Writing‐review & editing (supporting). Woo Joo Kim: Conceptualization (lead); Writing‐review & editing (lead).App S1Click here for additional data file.


Background: Several studies have reported the beneficial effects of anti-platelet drugs in cardioprotection against ischaemia–reperfusion injuries. To date, no studies have focused on the indirect cytoprotective effects of ticagrelor via adenosine receptor on the endothelium. Method: By evaluating cell viability and cleaved caspase 3 expression, we validated a model of endothelial cell apoptosis induced by hypoxia. In hypoxic endothelial cells treated with ticagrelor, we quantified the extracellular concentration of adenosine, and then we studied the involvement of adenosine pathways in the cytoprotective effect of ticagrelor. Results: Our results showed that 10 µM ticagrelor induced an anti-apoptotic effect in our model associated with an increase of extracellular adenosine concentration. Similar experiments were conducted with cangrelor but did not demonstrate an anti-apoptotic effect. We also found that A2B and A3 adenosine receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress. Conclusion: we described an endothelial cytoprotective mechanism of ticagrelor against hypoxia stress, independent of blood elements. We highlighted a mechanism triggered mainly by the increased extracellular bioavailability of adenosine, which activates A2B and A3 receptors on the endothelium. Vascular endothelial lesions are associated with the death of apoptotic cells and dysfunction, leading to the development of atherosclerosis ,2,3,4. E−ΔΔCT. HUVECs expressed A2A, A2B, and A3 receptors  for A2AAR (T2h: 6.44 ± 1.99) and A2BAR (T2h: 1.94 ± 0.54)  for A2AAR after 2 h of reoxygenation  at different times of hypoxia (T1h and T2h of hypoxia stress). The relative expression to the control group (normoxic control) was estimated as normalized mRNA levels using the following formula: 2eceptors . As prev ± 0.54) . Over-exn = 6, p < 0.05, n > 6, p > 0.05, HUVECs exposed to 2 h of simulated hypoxia were harvested to determine the expression of cleaved caspase 3. Stress-related to hypoxia resulted in a significant increase in apoptosis. Relative expression of cleaved caspase 3 increased after 2 h of hypoxia: T2h: 360.20 ± 63.50% versus normoxic control group 9.03 ± 4.80%  in the relative expression of cleaved caspase 3 was observed in cells treated with 1 µM (44.80 ± 7.92%) and 10 µM ticagrelor (14.67 ± 3.81%), versus control group 100% (untreated group after 2 h of hypoxia stress) (Anti-apoptotic effect of 1 µM and 10 µM ticagrelor was assessed in HUVECs after 2 h of hypoxia stress. A significant decrease ( stress) .n = 6, p > 0.05) in the relative expression of cleaved caspase 3 was observed in cells treated with 1 µM (102.80 ± 19.57%), nor with 10 µM (81.33 ± 21.22%) and 50 µM cangrelor (90.00 ± 16.53%) versus control group 100% (untreated group after 2 h of hypoxia stress)  .n = 6, p < 0.05) in cells treated with ticagrelor (The release of adenosine from HUVECs exposed to 1 µM and 10 µM ticagrelor in the extracellular medium (in 500 µL of supernatant) was studied using liquid chromatography coupled with a high-resolution mass spectrometer (LC-HRMS). Ticagrelor was added 30 min before hypoxia. Extracellular adenosine concentration increased significantly .The role of adenosine receptors, involved in the anti-apoptotic effect of ticagrelor, was investigated using a panel of selective antagonists . Cells wn = 6, p < 0.05)  . The antp < 0.05). The anti-apoptotic effect of 10 µM ticagrelor was also significantly and partially abolished by blocking COX (64.00 ± 3.14%), and NOS (62.83 ± 6.05%)   . This suIn this study, we evaluated the anti-apoptotic effect of ticagrelor against hypoxia stress in HUVECs free from blood elements. We used a model reported to induce apoptosis without affecting cell cytolysis ,30.Numerous experimental and clinical studies have suggested that P2Y12 receptor antagonists can directly reduce the size of the myocardial infarction ,26,31,32Although this protective effect has been well described in in vivo models ,11, it hMany studies have been conducted to understand the mechanisms of ticagrelor protection. Although the association of the increase in adenosine concentration with the protective effects of ticagrelor was reported ,17, onlyWe reported the expression of A2AAR, A2BAR, and A3AR in endothelial cells in accordance with previous studies and literature ,52,53. IAs described in the literature, our work reported the involvement of A2BAR. Yang et al. studied the cardioprotective effect of another P2Y12 receptor antagonist, cangrelor, during reperfusion in different in vivo animal models ,11,12. CAlthough widely described in cardioprotection ,27,62,63As in previous studies, in a complementary experiment, we aimed to test the effect of cangrelor in our model. As ectoenzymes hydrolyze cangrelor, we consider that the effect of cangrelor on P2Y12 receptors is transient. However, at the platelet level, the duration of its effect is estimated to 60 min. This enzymatic hydrolysis can limit the effect of cangrelor via the P2Y12 receptors. Relative expression of P2Y12 receptor was described in the endothelium . The invOn the other hand, we can, in part, rule out the hypothesis that blocking P2Y12 ticagrelor triggers an intracellular mechanism responsible for the release of ATP/adenosine. We measured extracellular nucleotides concentrations with and without ticagrelor. As described above, we found that, in normoxic controls, 2 h of hypoxia did not increase ATP, or the sum of ATP, ADP, and AMP . ConcentThe involvement of multiple signalling pathways has been described in ticagrelor-mediated cardioprotection against ischaemia. Our results also confirm the involvement of PI3K pathways, NOS, and COX pathways. Previous studies have reported multiple signalling pathways involved in the cardioprotection of ticagrelor, such as Akt ,17,31 anThere are some limitations in the present study that may be considered in future projects. First, the monolayer cell culture model we used does not take into account the environment. Other models, such co-cultures with smooth muscle cells, or 3D models could be developed to study the effect of ticagrelor in a more integrative model. Second, the ticagrelor-induced plasma membrane modifications already demonstrated on the platelet membrane could be explored at the endothelial level. The effects of ticagrelor on intracellular and extracellular nucleotide and nucleoside charge as well as the expression of purinergic receptors could also be explored. Finally, in order to evaluate, the percentage of viable (double negative), early apoptotic (A+/PI−), late apoptotic (A+/PI+), and necrotic (A−/PI+) cells, flow cytometry analysis could be performed in addition to caspase-3 experiments and more extensive apoptosis times may be investigated to study late apoptosis.v/v) fetal calf serum (FCS), 0.4% (v/v) endothelial growth supplement: 0.1 ng/mL human EGF, 1.0 μg/mL hydrocortisone, 1 ng/mL human bFGF, 90 µg/mL heparin, and 1% (v/v) penicillin/streptomycin  were used in a completely humid atmosphere at 37 °C and 5% CO2. The confluent cells were detached using the PromoCell detachment kit containing 30 mM Hepes, trypsin/EDTA solution (0.04%/0.03%), and trypsin neutralizing solution. The FCS was reduced to 1% 24 h before the experiment. All experiments were performed on subconfluent monolayer endothelial cells (80%) after the third passage.As validated and described ,30, HUVEv/v) N2 and 5% (v/v) CO2 for 2 h at 37 °C. PO2 in the hypoxic chamber was reduced to less than 1.5% during the experiments. After 2 h of hypoxia stress, the cells were recovered for analysis or underwent a two-hour reoxygenation phase (5% CO2 at 37 °C). At the same time, control cells were cultured under normoxic conditions. During the experiments, cells and media were harvested at different times to quantify nucleotides and to evaluate cleaved caspase 3 by immunoblotting.The cells were placed in a custom hypoxic chamber  and exposed to 95%  or Cangrelor  were added 30 min before hypoxia. The concentrations used in our experiments were similar to the concentrations described in humans at the recommended dosages ,46, or i−ΔΔCT, according to Livak et al. [During each experiment, cells were collected at during normoxia without hypoxia stress (normoxic control), after 2 h of hypoxia (T2h) and after 2 h of reoxygenation (T2h-2h). Total RNA was extracted using RNeasy Mini-KitTM . cDNA synthesis was obtained from 1 μg RNA using the iScript cDNA synthesis kit . Real-time PCR was performed with Sybr Green PCR reagents and analyzed on an ABI Prism 7500 Fast Real- Time PCR System . PCR was carried out in duplicate for each sample. EEF2 was used as the housekeeping gene. PCR conditions were as follows: 2 min at 50 °C, 2 min at 95 °C, followed by 40 cycles each consisting of 15 s at 95 °C (denaturation) and 1 min at 60 °C . The specificity of PCR amplification was checked using a melting curve step from 65 °C up to 95 °C following the final cycle. Results are presented as normalized mRNA levels using the following formula: 2k et al. . mRNA exv/v) of methanol were added and the extracts were frozen at −80 °C [−1) was also added. Mixtures were evaporated under nitrogen at 40 °C and then reconstituted with 100 μL of ice-cold water (LC-MS hypergrade). Liquid chromatography  coupled with high-resolution mass spectrometer (LC-HRMS) was used for the quantification . High-resolution masses for adenosine  and for nicotinamide D4  were used for quantification. TraceFinder Forensic 3.3  was used for LC-MS, library management, acquisition and processing (Culture medium (50 µL) was collected at different times during the course of each experiment . In order to inhibit ectonucleotidase activity, 75 µL  were boiled in sample loading buffer for 5 min before loading on 12.5% SDS–PAGE for electrophoretic separation and subsequently transferred into polyvinylidene difluoride membranes . After blocking with skimmed milk 5% (m/v) in TBS-T ), membranes were incubated with the primary monoclonal antibodies anti-cleaved caspase 3 at a dilution of 1:1000  or anti-β-actin at a dilution of 1:5000  overnight at 4 °C. After washing with TBS-T, membranes were incubated with appropriate horseradish peroxidase-labelled secondary antibody  for 90 min at room temperature. Immunoreactivity was detected with the Chemiluminescent HRP detection reagent . Quantification was performed by densitometric analysis using the Quantity One software of ChemiDoc XRS (BioRad). ImageLab was used for reprocessing image and quantification [The samples for Western blot were collected as follows: after treatment, the cell culture medium was collected, and cells were washed in fresh Phosphate Buffered Saline (PBS). Protein extraction was performed by adding 100 µL of RIPA Buffer (Sigma-Aldrich) supplemented with protease and phosphatase inhibitor cocktails, per well. Samples were sonicated thrice for ten seconds then centrifuged 30 min at 16,000× fication . Densitoex 560 nm and λem 590 nm using the above-mentioned microplate reader. The fluorescence of the T0 group represented 100% cell viability.The cell viability reagent PrestoBlue is a membrane-permeable solution based on resazurin. The reducing power of living cells forms the red fluorescent compound resorufin, via the mitochondrial enzymes of viable cells. As a result, this change can be detected by measuring fluorescence and absorbance. During the course of each experiment, PrestoBlue  was added to cells 30 min before the studied time . After incubation, fluorescence was measured at λhttp://www.r-project.org). All data are expressed as mean ± sem (Standard Error of the Mean). Before applying the parametric unpaired t-test, the Gaussian distribution of data was assessed by the Shapiro–Wilk normality test and the Kolmogorov–Smirnov test. Differences between groups for data describing Gaussian distribution were evaluated using one-way analysis of variance (ANOVA) followed with a t-test (multiple comparison). When the normality of the distribution was rejected, a non-parametric Kruskal–Wallis test was used for comparisons among groups, followed by post-hoc testing using un-paired Mann–Whitney U tests. For all significant differences concerning primary endpoints, a posteriori powers higher than 80% were checked. Bonferroni correction was applied for multiple comparisons. All p-values were two-tailed with statistical significance indicated by a value of p < 0.05 [Statistical analyses were performed using the Prism 4.00 GraphPad Software, San Diego, CA, USA and R 3.1.4  ticagrelor induced an anti-apoptotic effect in our model, and (2) ticagrelor induced an increase in extracellular adenosine concentration. This effect was dependent on ticagrelor concentration and duration of exposure. In addition, (3) A2BAR and A3AR receptors were involved in the anti-apoptotic effect of ticagrelor in endothelial cells exposed to 2 h of hypoxia stress.