The characterization showed that the surface morphology of five graphene-like magnetic sawdust biochars prepared with different preparation conditions were quite different. The specific surface area and pore structure increased with the increment of K2FeO4 addition. The results have shown that graphene-like magnetic sawdust biochar (1:1/900 °C) had the best removal on E2. The experimental results indicated that pseudo-first-order kinetic model and the Langmuir model could describe the adsorption process well, in which the equilibrium adsorption capacity  of 1:1/900 °C were 59.18 mg·g−1 obtained from pseudo-first-order kinetic model and the maximum adsorption capacity (qmax) of 1:1/900 °C were 133.45 mg·g−1 obtained from Langmuir model at 298K. At the same time, lower temperatures, the presence of humic acid (HA), and the presence of NaCl could be regulated to change the adsorption reaction in order to remove E2. Adsorption capacity was decreased with the increase of solution pH because pH value not only changed the surface charge of graphene-like magnetic sawdust biochar, but also affected the E2 in the water. The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction. To sum up, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water.The occurrence of environmental endocrine disrupting chemicals (EDCs) in aquatic environments has caused extensive concern. Graphene-like magnetic sawdust biochar was synthesized using potassium ferrate (K AgriMohammad ,17. Furt2FeO4 as an activator, which possessed a positive electrochemical performance as electrode materials for supercapacitors. Zhou et al. stated that K2FeO4 was a promising reagent because it could not only improve the pore structure of the material, but also generate new active sites, such as FeO42− and Fe2O3 [2FeO4 could load the iron oxide compound on the surface of the material, which provided convenience for the magnetic ionization technology, and at the same time overcame the disadvantages of the difficulty of collecting biochar after adsorption, making the recovery of biochar a reality.There are several methods for the modification of the biochar surface, including physical or chemical activation, steam activation, and coating for the contaminants’ removal from wastewater . Among tnd Fe2O3 . In addi2FeO4 could fulfil the synchronous carbonization and graphitization for converting biomass to porous graphitic carbon biochar, which have been used as advanced electrode materials [2FeO4 to produce graphitic biomass carbon (GBC) by the simultaneous carbonization and graphitization of sawdust biochar. K2FeO4 was utilized as graphitization catalyst (Fe), activating agent (KOH) and magnetizing agent (Fe) [Sawdust is an abundant biomass waste resource everywhere and sawdust biochar has great application potential in the removal of water environmental pollutants ,25. Prepaterials ,26, cataaterials , and adsaterials . Based oent (Fe) . The syn2FeO4 dosage; (2) to analyze the surface characteristics of the adsorbent with multiple characterization methods, such as scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS); (3) to explore the adsorption performance of graphene-like sawdust biochar on E2; (4) to discuss the possible adsorption mechanism of graphene-like sawdust biochar removal E2 from water.The main research objectives of this study are: (1) to produce graphene-like sawdust biochar with different pyrolysis temperatures and different K18H24O2) was purchased from Sigma–Aldrich. K2FeO4, HA, and NaCl were obtained from Shanghai chemical corporation. Other chemicals including sodium hydroxide (NaOH), hydrochloric acid (HCl) and pH buffer solution , used to adjust the solution pH, were purchased from Shanghai Chemical Corp. Deionized water was obtained from Millipore Milli-Q water purification system. All reagents used were of analytical grade or higher and prepared with ultra-pure water.E2  and annealed temperature  were prepared to explore the excellent preparation conditions. 2.0 g BC was dispersed in aqueous 0.1 M K2FeO4 solution whose volume was 20 mL, 50 mL, and 100 mL, respectively with continuous stirring for 12 h, and then dried at 65 °C to obtain. The mass ratios of BC and K2FeO4 were 1:0.2, 1:0.5 and 1:1 respectively. Subsequently, the solid mixture  were heat-treated in a tube furnace at 900 °C for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The mass ratio of 1:1 was heat-treated at a constant annealed temperature  for 2 h under a nitrogen atmosphere with a heating rate of 7 °C min−1. The obtained samples were washed with deionized water, followed by drying at 65 °C. These resultant samples were notated as 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C, and 1:1/1000 °C (mass ratio (BC: K2FeO4)/ annealed temperature). The mass ratios (BC: K2FeO4) and annealed temperature are also in Several samples with different mass ratios . The graphitic structure was analyzed by high-resolution transmission electron microscopy . The surface chemical composition was determined by X-ray photoelectron spectroscopy . Raman spectrometer was performed on a Raman Microscope  with YAG solid-state laser under wavelength of 532 nm. An X-Ray Diffractometer  was used to study the crystallinity. The magnetic properties of samples were examined using a vibratory probe sample magnetometer . The specific surface areas were determined using the Brunauer–Emmett–Teller .2FeO4) and annealed temperature on adsorbents properties were studied. Subsequently, the sample 1:1/900 °C was used for batch adsorption experiments based on the superbly adsorptive properties. In the beginning, three mass ratios  and different annealed temperature  have been used for adsorption experiments. The experiments were performed in a 100 mL conical flask with 50 mL aqueous solution containing the fixed concentration of E2 and adsorbents . More details on the selection of E2 and adsorbents concentrations were presented in The effects of mass ratios  were used to investigate the effect of salinity on adsorption behavior [−1) [Batch adsorption experiments included kinetics adsorption experiments, isothermal adsorption experiments, pH effect, ionic strength effect, and OM effect on E2 adsorption which were conducted at 25 °C with a shaking speed of 160 rpm for 24 h in the vibrational chamber excluding kinetic adsorption experiments . The soll flasks . The pH ior [−1) . The desx/Em = 280 nm/310 nm [E2 concentration was determined using an F-4500 fluorescence spectrophotometer . The emission source is a 450 w xenon lamp which collected the emission matrix spectra of 5 nm in the excitation range of 200–400 nm and 300–500 nm, respectively. Fluorescence intensity of E2 was measured at Em/310 nm . The dat2FeO4, the surface of GBC became more rough and uneven  [2 and K2O could further react with carbon over 700 °C to K. During physical and chemical activations, the carbon lattices expanded irreversibly and resulted in many micropores. Since these chemical reactions occurred at over 700 °C, the specific surface area was mainly related to the addition ratio of the K2FeO4 [2FeO4 made the microporous structure more powerful.Surface morphology and microporous structure of carbonaceous materials were characterized by SEM. The surface of the BC was relatively smooth a. With td uneven b–d. Moreon sites . During  700 °C) . Additioe K2FeO4 . More mi2·g−1. This indicated high BET surface area and total pore volume of 1:1/900 °C in the 2·g−1 and a tiny total pore volume of 0.020 cc/g. Nevertheless, the BET surface area was corresponding increase with K2FeO4 activation of the materials (1:0.2/900 °C and 1:0.5/900 °C). Definitively, the sample 1:1/900 °C demonstrated a powerfully abundant porous structure. The large accessible surface area and its suitable pore size distribution are beneficial for adsorption [The N2 adsorption-desorption isotherms and mesoporous size distribution of carbonaceous materials were presented in sorption .2FeO4 and annealed with high temperature. The graphitization was testified by Raman spectra  is generally used to reflect graphitization of samples. The ID/IG value of BC, 1:0.2/900 °C, 1:0.5/900 °C, 1:1/900 °C, 1:1/800 °C and 1:1/1000 °C were 0.81, 1.01, 1.08, 1.02, 0.98, and 1.01, respectively. The increasing of the ID/IG value indicated that the graphitization of the samples has been shown via the K2FeO4 excitation and high-temperature calcination. The 2D band of the samples 1:1/900 °C and 1:1/1000 °C also verified the formation of few-layered graphene rather than single-layer graphene [2FeO4 and annealed with high temperature. Moreover, the higher graphitization temperature was also beneficial to graphitization [The crystallization and graphitization of the samples were observed by XRD and Raman. The XRD patterns were shown in  spectra b. All saon atoms . The 2D graphene ,34,35. Ttization .The TEM images of BC and 1:1/900 °C have been compared in order to further understand the properties of the materials before and after modification. In 2FeO4. The XPS peak deconvolution for C1s, O1s, and Fe2p spectra of samples was shown in 3+ species on the surface of 1:1/900 °C [0 and Fe2+, respectively [0 was only 9.2%, while the proportion of Fe3+ and Fe2+ was 79.9% and 10.9%, respectively. The 1:1/900 °C become magnetic biochar with the presence of Fe species and produced more oxygen-containing functional groups accompanied by the oxidation of Fe3+ species. From the above characterization results, it can be confirmed that the 1:1/900 °C sample was successfully synthesized by one-step process [−1 which suggested that the adsorbent was enough to achieve solid-liquid separation by a permanent magnet. These results reflected that biochar after modification had excellent magnetic properties and could be easily separated and recycled.To get an insight into the surface chemical composition, the full survey scan XPS a spectra1/900 °C . While tectively . The pro process . SupplemAs presented in R2)  , E2 adsoace area . Moreove process ,41. In t , E2 adsIn −1, while the maximum adsorption capacity of BC was 107.78 mg·g−1 at 298 K. The results indicated that low temperature was more conducive to the removal of E2 by graphene-like sawdust biochar. The relative parameters for two models were summarized in R2), which suggested that the adsorption of E2 by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process [n of the Freundlich adsorption isotherm model  [Kf value related to the adsorption capacity of the Freundlich adsorption isotherm model  [However, the adsorption capacity of 1:1/900 °C was obviously better than that of BC. In the  process ,39,42. Rfficult) , we coulsorbent) , it can The pH value of the solution was crucial to the adsorption process, which not only changed the surface charge of biochar, but also affected the E2 in the water. In Furthermore, to investigate the effect of ionic strength on adsorption of E2 on graphene-like sawdust biochar, different concentrations of NaCl (0.0 M–0.6 M) were added to the E2 solution, owing to the popular existence of this ionic compound in wastewater. In Natural organic matters (NOMs) have been regarded as an important factor affecting the adsorption process since they widely existed in the natural environment . They miAdsorbent recovery is an important consideration as it relates to the operating cost and the feasibility of its practical application. In Different characterizations and experiments were carried out to explore the possible E2 adsorption mechanisms onto 1:1/900 °C. In this work, the graphene-like magnetic structure of 1:1/900 °C have been observed by characterization. On the one hand, the sample 1:1/900 °C had a high surface area, abundant micropores, and high crystallinity properties. These surface structures were beneficial to E2 adsorption, while similar conclusions were also reported in ,43,48. M(1)2FeO4 was 1:1 and the second pyrolysis temperature was 900 °C, the graphene-like magnetic carbon sawdust biochar had the best adsorption capacity on E2.During the carbonization and graphitization process, when the mass ratio of BC to K(2)2FeO4 dosages and pyrolysis temperatures had an important impact on the morphology and structure of the graphene-like magnetic carbon sawdust. The surface area of graphene-like magnetic sawdust biochar was affected by the K2FeO4 dosage. The more addition, the larger the specific surface area. However, under the condition that BC and K2FeO4 was 1:1, the surface area of graphene-like magnetic sawdust biochar showed a positively obvious result.Different K(3)−1 obtained from Langmuir model. Adsorption kinetics and adsorption isotherm indicated that the E2 adsorption by graphene-like sawdust biochar occurred on a homogeneous surface were a monolayer adsorption process and controlled by multiple processes.The maximum adsorption capacity at 298 K was 133.45 mg·g(4)The pH, NaCl, and HA were crucial to the adsorption process because the adsorption capacity was affected by physical-chemical properties caused by the change of surface charge and the salting-out effect.(5)The possible adsorption mechanism for E2 adsorption on graphene-like magnetic sawdust biochar was multifaceted, involving chemical adsorption and physical absorption, such as H-bonding, π-π interactions, micropore filling effects, and electrostatic interaction.Based on the above conclusion, graphene-like magnetic sawdust biochar was found to be a promising absorbent for E2 removal from water as well as wastewater treatment, and the preparation method of graphene-like magnetic sawdust biochar was a green and feasible process. However, as a green technology, the economic viability might be a consideration and there should be some industrial viability of that process/product to be considered strictly.


Functionalized with citrate ligands they show a good colloidal stability in water making them valuable for dual magnetic resonance and optical imaging or image-guided therapy. They exhibit a relatively high transverse relaxivity r2 = 42.3 mM−1·s−1 in water at 37 °C, for an applied static magnetic field of 1.41 T, close to the field of 1.5 T applied in clinics, as they exhibit a red emission by two-photon excited fluorescence microscopy. Finally, when brought into contact with healthy human foreskin fibroblast cells (BJH), for doses as high as 50 µg·mL−1 and incubation time as long as 72 h, they do not show evidence of any accurate cytotoxicity, highlighting their biomedical applicative potential.Red luminescent and superparamagnetic β-NaY Current biomedical imaging techniques are vital for the diagnosis of various diseases. Each imaging mode has its own merits and disadvantages and uses specific probes with particular physical and chemical properties. As a consequence, a single technique does not encompass all the functionalities required for comprehensive imaging. Therefore, multimodal methods, with enhanced signal sensitivity, better spatial resolution, and ability to relay information about biological systems, at the molecular and cellular levels, are becoming strongly recommended. To achieve such a purpose, it is necessary to design and produce multimodal probes which combine in a single object all these requirements.3+ chelates  (50 mM) solution. The resulting suspension was mechanically stirred and heated up to 100 °C for 30 min. The resulting precipitate was collected using a strong magnet, then washed with an excess of ethanol, to remove the non-grafted organic species, and finally dried in air at 60 °C.Citrate grafting was achieved through a simple ligand exchange method, replacing the residual organic moieties at the surface of the fluoride and/or oxide particles by the freshly introduced multivalent citrate species, taking advantage from the complexing ability of their carboxylate and hydroxyl groups . Typical+, Y3+, Eu3+ and Fe3+ standard solutions.Powder X-ray diffraction (PXRD) was performed on all the produced powders using a X’pertPro diffractometer , equipped with a Co-Kα tube  and configured for a θ-θ Bragg-Brentano reflection geometry. Highscore Plus software  was used for phase identification and peak indexation and MAUD software , based on Rietveld refinement , was empTransmission electron microscopy (TEM) was conducted on a JEM 2010 UHR microscope , operating at 200 kV and micrographs were collected thanks to a Gatan Orius SC1000 4008 × 2672 pixel charge-coupled device CCD camera . Dynamic light scattering (DLS) experiments were also performed in order to estimate the hydrodynamic size distribution of the produced citrate functionalized nanoparticles. They were carried out in water thanks to a Zetasizer NanoZS instrument  equipped with a 5.0 mW He-Ne laser operating at 632.8 nm and detecting scattered light at 90° using an avalanche photodiode detector (APD). To complete these analyses, Fourier transformed infrared (FTIR) spectroscopy was performed at ambient temperature using an Equinox FTIR spectrometer  operating in a transmission scheme (KBr pellet). Thermogravimetry (TG) analysis was also carried out by heating a given mass of pre- and post-functionalized particles in air up to 1000 °C (5 °C/min) thanks to a SETARAM TGA92 apparatus.M as a function of temperature T and as a function of magnetic field H was measured on solid state, using a MPMS-5S SQUID magnetometer . In practice, 20 mg of the dried powder were slightly compacted in a plastic sampling tube to avoid their movement during measurements. Magnetization was recorded versus temperature M(T), under a dc magnetic field of 200 Oe, operating within zero field cooling (ZFC) and field cooling (FC) conditions, between 5 and 330 K. Also, magnetization was recorded versus magnetic field M(H) at 310 K (37 °C) by cycling the magnetic field between 70 and −70 kOe.The variation of the magnetization −1. In practice, 0.7 mL of each solution was introduced in a nuclear magnetic resonance (NMR) tube (7.5 mm outer diameter). The tubes were then inserted in a mq60 relaxometer  equipped with a 60 MHz/1.41 T magnet. The 1T and 2T relaxation times were recorded as a function of iron concentrations [Fe], at 310 K. [Fe] was properly calculated by an accurate titration of iron content for all the samples, averaging the values inferred from XRF and colorimetry  -3-carboxyphenyl]amino]thioxomethyl]amino]-4-hydroxy-L-leucine]phalloidin abbreviated as TRITC (λex = 630 nm). Within these operating conditions, NPs cannot be detected by their own emitted red light, due to its superposition with that of labelled cytoskeleton. To detect them, the same slides were observed in parallel with a two-photon microscope, a LEICA TCS SP8 MP FLIM system . The excitation source of this microscope is a femtosecond pulsed infrared laser (Ti: Sapphire). Two hybrid detectors , in non-descanted position were used to optimize the detection of the NPs’ red fluorescence. The spectral analysis was achieved in the confocal scanning head of the microscope which pilots the grating and mirror in front of it. The spectral resolution was 10 nm, covering the range from 380 nm to 780 nm.Optical microscopy was performed to visualize the morphology of the cells before and after NPs incubation. In practice BJH cells were seeded on glass coverslips 24 h before treatment with the same NP doses than previously for a unique incubation time of 48 h. Fixed cells  were mounted with mounting medium . Their nuclei and their cytoskeleton were counterstained with  abbreviated as DAPI (λ0.8Eu0.2F4 and γ-Fe2O3 NPs prepared separately. The structure and the microstructure of the as-produced composite particles were first checked by PXRD and TEM. The recorded PXRD pattern and the collected TEM micrographs are given in 4 (ICDD No. 98-005-1917) and γ-Fe2O3 (ICDD No. 98-008-7119). The cell parameter values were refined and found to be close to those tabulated for bulk materials and close to those previously reported on β-NaY0.8Eu0.2F4  concentration, typically 0.8, 0.4, 0.2 and 0.1 mM. In a second time, these concentrations were double-checked by titration of the stock suspensions by two different analytical methods, namely colorimetry (ultraviolet-visible (UV-Vis) absorbance) and X-ray fluorescence (XRF). Relaxivities were obtained from the slope of the linear variation with [Fe] of the longitudinal (respectively transverse) decay rate of water proton spins, according to:T1 = 3813 ms and T2 = 650 ms as measured experimentally) are taken into account. The curves obtained are plotted in Finally, proton relaxometry measurements were performed on both the citrated γ-Fer2 value of γ-Fe2O3 from 134.1 to 34.6 s−1·mM−1 in the case of the core-shell structure due to the decrease of its saturation magnetization, not compensated by the increase clustering effect, r2 being expected to vary with the square of both the magnetization and the hydrodynamic diameter within the limits of the “outer sphere” theory of superparamagnetic contrast agents [r1 of the core-shells only slightly decreased compared to γ-Fe2O3 from 21.9 to 12.7 s−1·mM−1, showing a good access of the water molecules to the iron oxide surface of the shell. Nevertheless, β-NaY0.8Eu0.2F4@γ-Fe2O3 nanoparticles still remain good candidates as MRI contrast agents, either negative or positive ones, owing to their relatively low r2/r1 ratio of around 2.7 [According to t agents . On the ound 2.7 .0.8Eu0.2F4 and β-NaY0.8Eu0.2F4@γ-Fe2O3 particles were measured, in particular the photoluminescence (PL) spectra of their aqueous colloidal suspensions, obtained by dispersing their citrated counterparts in deionized water. Under a continuous excitation at 396 nm, which corresponds to the spin-forbidden 7F0–5L6 absorption transition of Eu3+ cations [3+ cations from their 5DJ = 0 electronic state to the 7FJ =1, 2, 3 and 4 ones, the intensity of the line at 616 nm being the highest. Interestingly, by decorating the β-NaY0.8Eu0.2F4 with a layer of γ-Fe2O3 crystals, the whole PL core intensity decreased , focusing on the citrated β-NaYJH cells . Within 0.8Eu0.2F4@γ-Fe2O3 nanoparticles are very promising. They allow us to highlight the advantages offered by these all-inorganic bimodal MR and optical imaging probes compared to others, combining also superparamagnetism and lanthanide-based luminescence. The list of such probes is short in the relevant literature. To the best of our knowledge, in comparison to present system, equivalent MRI relaxivity values were measured on iron oxide nanocrystals doped by lanthanide ions, Eu3+ , but less optimized optical responses were reported on them. Indeed, the red emission of these nanocrystals was obtained only under a UV excitation of 254 nm [4 fluoride allows for decreasing this energy, since less non-radiative de-excitation phenomena may proceed. Quite equivalent MRI relaxivity values were also measured on these alternative probes, based on magnetite cores surrounded by a thin lanthanide doped NaYF4 shell , making them less valuable for negative MRI contrasting than ours.Clearly, all the recorded bio-physicochemical properties on these engineered β-NaYb3+-Er3+  or Yb3+-[3+-Tm3+ ). Howevearticles . But, on0.8Eu0.2F4@γ-Fe2O3 nanoparticles of less than 100 nm in size by the so-called polyol process. These composite particles integrate optical and magnetic dual functions in one single nanoprobe geometry. The γ-Fe2O3 satellites make the built hetero-nanostructures suitable for T2-weighted or T1-weighted MRI, while the β-NaY0.8Eu0.2F4 cores offer the ability to be used as biomarkers. The toxicity of the particles as well as their ability to be used as in vitro biomarkers were tested on healthy BHJ human model cells after citrate functionalization to make them hydrophilic. Interestingly, no cell death was reported from AB viability assays for doses as high as 50 µg·mL−1 and incubation time as prolonged as 72 h. All of these interesting results provide a proof of concept that our engineered citrated composite particles are clearly valuable for dual magnetic resonance and optical medical imaging.We have synthesized core-satellite structured β-NaY


On the other hand, the common instant reduction synthesis method leads to significantly agglomerated nanoparticles (Pd85Ni10Bi5/C(I)). EOR activity and stability of these three different carbon supported PdNiBi anode catalysts with a nominal atomic ratio of 85:10:5 were probed via cyclic voltammetry and chronoamperometry using the rotating disk electrode method. Pd85Ni10Bi5/C(II) showed the highest electrocatalytic activity  with low onset potential (0.207 V) for EOR in alkaline medium, as compared to a commercial Pd/C and to the other synthesized ternary nanocatalysts Pd85Ni10Bi5/C(I) and Pd85Ni10Bi5/C(III). This new synthesis approach provides a new avenue to developing efficient, carbon supported ternary nanocatalysts for future energy conversion devices.Direct ethanol fuel cells (DEFC) still lack active and efficient electrocatalysts for the alkaline ethanol oxidation reaction (EOR). In this work, a new instant reduction synthesis method was developed to prepare carbon supported ternary PdNiBi nanocatalysts with improved EOR activity. Synthesized catalysts were characterized with a variety of structural and compositional analysis techniques in order to correlate their morphology and surface chemistry with electrochemical performance. The modified instant reduction synthesis results in well-dispersed, spherical PdThe online version of this article (10.1007/s12678-019-00577-8) contains supplementary material, which is available to authorized users. Crituel cell , 7. Commuel cell .− reaction that leads to a formation of CO2 and water. State-of-the-art catalyst systems lead to an incomplete, 4e− oxidation reaction resulting in CH3COOH  formation. Pt is the most used catalyst for alcohol oxidation in acidic media, but high cost, low availability, and prevalent poisoning by adsorbed CO-like intermediates formed during ethanol oxidation limit its application. The use of alkaline electrolytes significantly improves the kinetics of the ethanol oxidation reaction (EOR) and of the oxygen reduction reaction (ORR) [Nevertheless, slow kinetics for the EOR remains the major challenge for DEFC development. Complete ethanol oxidation is a 12eon (ORR) , 9 and aon (ORR) , 10. Curon (ORR) . This caon (ORR) , 11, 12.2, leading to a lower degree of detachment of Pd nanoparticles from the carbon carrier Vulcan XC72 compared to PtRu/C and Pt/C catalysts. This means that the corrosion resistance of the carbon carrier Vulcan XC72 in the presence of Pd nanoparticles is greater than that of PtRu and Pt nanoparticles, indicating that the Pd/C catalyst is more stable in alkaline media than the other two.Chatenet et al.  investigThe reaction mechanism of EOR on Pd-based catalysts is still not fully understood. Wang et al. as well as other research groups –14 descr− ions, and the conversion of the acyl intermediates to acetate. Liang et al. [3)ads for the adsorbed hydroxyl is the rate determining step -ion adsorption, which is important in the catalyst synthesis [Based on this understanding, much research has been done in recent years to improve the EOR performance of anode materials and to reduce costs by developing new synthesis methods for catalyst nanostructure preparation, specifically to alloy Pd with other metals like Ru, Pb, Sb, As, Ni, or Bi, as well as to improve the active surface area of the catalyst material on its support , 17–19.  reduced –22. Moreed above  reported for EOR , 24. Caiynthesis . Also imynthesis , which rynthesis . It is wynthesis , 27.xNiyBiz/C anode catalysts with high activity and high by-product tolerance toward alkaline EOR but resulted in nanoparticles with inhomogeneous morphology that tended to agglomerate and provided a nonuniform distribution on the carbon support. The new synthesis method utilized the following modifications of the common instant reduction method: (i) using HCl instead of NaCl for dissolution of the PdCl2, (ii) performing the synthesis process under N2 inert atmosphere and ice-bath cooling, and (iii) addition of solid NaBH4 as a reducing agent. Catalysts prepared by our new synthesis method were fully characterized and compared to catalysts obtained by previously published method [Herein, we introduce a new synthesis approach in order to enhance the EOR activity/stability and to improve the dispersion of ternary PdNiBi/C nanocatalyst on Vulcan XC72R carbon support. This method improves upon the approach developed in our previous study , which yd method . Electro2, anhydrous, 59–60% Pd basis, Aldrich), sodium chloride , nickel(II) nitrate hexahydrate (Ni(NO3)2⋅6H2O, Aldrich), bismuth(III) chloride , hydrochloric acid (HCl (aq), Carl Roth), Vulcan XC72R carbon black , sodium borohydride , 2-propanol , sodium hydroxide , and ultrapure water  were used for the development of Pd85Ni10Bi5 nanoparticles on the Vulcan XC72R carbon support material as anode catalysts. A commercial Pd/C  catalyst was used as reference material.Palladium chloride 2∙6H2O salt is carried out in 10 mL of ultrapure water with additional 1.5 mL of 1 M HCl. The precursor salt solution BiCl3 (aq.) is prepared similarly with a few drops of HCl [The dissolution of Ni(NOs of HCl .2 atmosphere in the ice bath. Vulcan XC72R carbon is dispersed in 60 mL of ultrapure water (without the addition of 2-propanol), and the suspension is sonicated two times for 5 min with a cycle of 0.6 and an amplitude of 40%, using an ultrasonic probe . Afterward, the precursor salt solutions are added to the Vulcan XC72R carbon black dispersion under constant sonication, and the pH of the mixture is adjusted to 10 with 1 M NaOH. The addition of the reducing agent NaBH4 (~ 5 eq.) to the catalyst dispersion takes place in chunks (pure solid form) or dropwise in liquid form (0.6 mL of 1 M NaOH and 6 mL of ultrapure water).The overall synthesis process is performed under N2 purging is switched off after 1 h) to obtain the carbon supported Pd85Ni10Bi5/C(II) (NaBH4(s)) and Pd85Ni10Bi5/C(III) (NaBH4(l)) catalysts. After cooling to room temperature, multiple washing with ultrapure water and centrifugation is performed. Finally, the purified catalyst samples are dried at 40 °C for 24 h [The reaction mixture is vigorously stirred at 60 °C for 4 h  and Pd85Ni10Bi5/C(III)) prepared by the modified instant reduction synthesis method, one ternary Pd85Ni10Bi5 catalyst sample on Vulcan XC72R carbon support (Pd85Ni10Bi5/C(I)) prepared by the common instant reduction synthesis method, and one commercial Pd/C catalyst sample were obtained by ICP-OES. The catalyst powders were pre-treated by microwave-assisted pressurized acid digestion using a Multiwave 3000 microwave system . The catalyst samples (~ 10 mg) were digested for 25 min in 7 mL of concentrated nitric acid, 0.2 mL of HClO4 , and 0.2 mL of hydrofluoric acid 40%  at a temperature of 195 °C (heating rate: 13 °C/min) and a power of 1500 W. The digested catalysts were diluted to 25 mL with deionized water, and the ICP-OES analyses were carried out utilizing an Arcos SOP system by Spectro . Following detection wavelengths were used for each element: Bi 223.061 nm, Ni 231.604 nm, and Pd 340.458 nm. Sample blanks and spikes were included in all preparation procedures.The elemental compositions of two ternary Pd85Ni10Bi5/C nanocatalysts were identically analyzed by TEM-EDX according to [d-spacing determinations and element quantifications) was performed by Digital Micrograph software program. For the particle size distribution and average particle diameter determination, we took 100 spherical-shaped particles of the TEM micrograph areas for Pd85Ni10Bi5/C(II) and Pd85Ni10Bi5/C(III) catalysts and measured the diameter of particles manually by FIJI (ImageJ)-free software program [The morphology, particle size distribution, and the chemical composition (atomic ratio) of the Pdrding to . TEM ima program .−1. The software program X’Pert HighScore plus  was used for determination of half-width (Bobserved), lattice parameter, and d-spacing using profile fit and Rietveld refinement. The average crystallite size (L) of located crystal structures in different catalyst powders was estimated according to Scherrer , only Kα1 peaks were used.Bobserved) and the device related width (= Bstandard) using the measurement of the standard material LaB6.Difference between half width , in the range of 5° to 100° 2θ, a step size of 0.02°, and a counting time of 3 s step−1 and a total integration time of 0.1 s point−1. Data analysis was performed using CasaXPS software (http://www.casaxps.com/) with a Shirley-type background and 70–30 Gaussian–Lorentzian peak shapes, except for Pd metal, which was fit with an asymmetric line shape.XPS measurements were performed using a SPECS PHOIBOS 150 Hemispherical Energy Analyzer with a monochromated Al Kα X-ray source. Indium foil was used as substrate for the sample preparation. Survey spectra were measured using a pass energy of 40 eV at a resolution of 0.2 eV step85Ni10Bi5/C and commercial Pd/C nanocatalysts were ex situ characterized by means of thin film rotating disk electrode  technique using a standard three electrode setup in an electrochemical glass cell (Metrohm) [The prepared PdMetrohm) , 41. The2) via a suspension. A mixture of 2-propanol, ultrapure water, and Nafion ionomer solution  as binder was prepared to form a homogeneous suspension of the synthesized catalysts. The slurry was sonicated two times for 5 min with a cycle of 0.6 and an amplitude of 20% using an ultrasonic probe. Before each electrochemical measurement, the RDE was polished using an Al2O3 suspension  with a particle size of 0.05 μm and was finally rinsed with ultrapure water. Afterward, the generated catalyst ink was applied onto the GC disk by a micropipette, to achieve a loading of 56 μgPd cm−2 on the surface. The RDE with the catalyst ink was rotated for approx. 1.5 h at 700 rpm to uniformly dry and distribute the catalyst material. The active layer thicknesses (ALT) of the Pd/C  and Pd85Ni10Bi5/C(I)-(III) catalysts on the GC-RDE are approx. 3 μm and 10 μm, respectively. Details for ALT calculations of the catalysts are given in the ESM.For the preparation of the working electrode, the catalyst was transferred to a GC-RDE , purged 1.0 M potassium hydroxide , and in a mixture of 1 M KOH and 1 M EtOH . Three independent CV measurements of each catalyst to each 6 cycles were performed at 30 °C and with a scan rate of 10 mV∙s−1. The last cycle was used for evaluation of their electrochemical properties. The charge of the integrated reduction peak of PdO to Pd (QPd) located between 0.65 V and 0.90 V in Fig. GC = 0.196 cm2), and catalyst loading of Pd on GC-RDE (cL = 0.056 mgPd⋅cm−2). The Bi oxide reduction is in the same region as the Pd oxide reduction; therefore both resulting charges were used in the calculation of EASA for the ternary Pd85Ni10Bi5/C catalysts as explained in [In order to determine the electrochemical active surface area (EASA) and the EOR activity of all synthesized nanocatalysts, cyclic voltammetry (CV) measurements using RDE were recorded in nitrogen  measurements using RDE were carried out at a potential of 0.83 V at 30 °C for 1 h to examine the EOR stability of the catalysts. All electrochemical characterization results are given as mean value plus standard deviation. Further details about the CV measurement process are described in .85Ni10Bi5/C(I), Pd85Ni10Bi5/C(II), and Pd85Ni10Bi5/C(III) nanocatalysts are summarized in the experimental section above but are briefly discussed here again. In all cases, the common instant reduction method was modified as follows to generate Pd85Ni10Bi5/C(II) and Pd85Ni10Bi5/C(III) catalysts: (a) HCl was used instead of NaCl for improved dissolution of the PdCl2, and (b) the synthesis procedure was performed under N2 inert atmosphere and with ice-bath cooling. Furthermore, solid or liquid NaBH4 was used for the synthesis of Pd85Ni10Bi5/C(II) and Pd85Ni10Bi5/C(III), respectively. Pd85Ni10Bi5/C(I) was prepared by the common instant reduction method according to [Differences in synthesis between Pdrding to . The elexNiyBiz nanocatalysts and commercial Pd/C catalyst were determined using ICP-OES  and Pd85Ni10Bi5/C(III) nanoparticles on Vulcan XC72R carbon support was drastically reduced compared to the Pd85Ni10Bi5/C(I) nanoparticles on Fig. xNiyBiz/C catalysts, which were developed by the common instant reduction synthesis method [85Ni10Bi5/C catalysts are homogeneously distributed on carbon support material. The obtained TEM-EDX results of the Pd85Ni10Bi5/C catalysts  are summarized in Tables Results on Fig. s method . EDX spedaverage) were evaluated from TEM images  catalyst exhibiting smaller particles (daverage = 3.7 ± 0.8 nm) as compared to Pd85Ni10Bi5/C(III) (daverage = 4.7 ± 0.7 nm). This difference is attributed to the use of solid NaBH4 during the synthesis of Pd85Ni10Bi5/C(II) as compared to the use of liquid NaBH4 for Pd85Ni10Bi5/C(III). In both cases, the nanoparticles are significantly more evenly dispersed on the carbon support, which we attribute in part to the inert N2 atmosphere used in synthesis. Synthesis of the Pd85Ni10Bi5/C(I) under ambient atmosphere most likely results in the formation of various undesired oxide species, which easily tend to agglomerate with metal ions onParticle size distribution and average particle diameter  and Pd85Ni10Bi5/C(III) is likely also driven by improved solubility of the precursor salt PdCl2 due to the substitution of HCl for the NaCl used to synthesize Pd85Ni10Bi5/C(I). This is supported by the presence of chloride in the Pd85Ni10Bi5/C(I) catalyst identified by TEM-EDX  plane spacing of the face centered cubic (fcc) crystalline structure of Pd.Fast Fourier transforms of catalyst particles in the high-resolution (HR)TEM micrographs in Fig. 85Ni10Bi5 catalysts, as well as the commercial Pd/C catalyst, also show the characteristic diffraction peaks at 2θ values of 40°, 47°, 68°, 82°, and 87°  (Δ 0.221°) < Pd85Ni10Bi5/C(II) (Δ 0.476°) < Pd85Ni10Bi5/C(III) (Δ 0.884°). The precise peak positions of all catalyst samples are given in the Tables d-spacings of all Pd85Ni10Bi5/C nanocatalysts compared to the commercial Pd/C catalyst  < Pd85Ni10Bi5/C(II) < Pd85Ni10Bi5/C(III) < Pd/C comm. Details about the determined XRD parameters for evaluation of crystallite size  Δ 0.46° < Pd85e Tables –S12 and e Tables –S8 of ale Tables .85Ni10Bi5/C catalysts and commercial Pd/C catalyst were determined by XPS. Survey spectra and extracted elemental concentrations for all catalysts are summarized in Fig. 85Ni10Bi5/C(I) catalysts exhibit a slightly higher oxygen content as compared to the others, which is likely due to synthesis in non-inert atmosphere. Core level spectra of Pd 3d, Ni 2p, and Bi 4f were analyzed directly from the survey XPS spectra, and deconvolution of the Pd 3d, Ni 2p, and Bi 4f spectra was performed to understand the speciation of the different alloy species  and PdO (PdII) – similar to observations in our previous study [85Ni10Bi5/C(III) catalyst contains the most metallic Pd on the surface relative to PdO as compared with the other ternary catalysts  catalyst was found to contain the highest overall Ni content of all three ternary alloys, and fitting of the Ni 2p core level  catalyst exhibits a slightly higher surface Bi content compared to the Pd85Ni10Bi5/C(II) and Pd85Ni10Bi5/C(III) catalysts , with the peak position shifted to higher binding energy relative to pure Bi metal due to the strong binding interaction between Bi and platinum group metals [2O3.The surface oxidation states, chemical compositions, and binding energies of Pd, Ni, and Bi in the synthesized Pdsee Fig. –S9. Two us study . Figure sts Fig. , which ists Fig.  and may sts Fig. . The Pd8vel Fig.  reveals ts Table . Deconvop metals , 36. All85Ni10Bi5/C catalysts synthesized via different procedures was characterized by CV-RDE and CA-RDE and compared to the commercial Pd/C as a benchmark for the alkaline EOR in Figs. The EOR performance of the various Pd3 in the presence of KOH to form Bi2O3 [2 to NiOOH in the positive scan and subsequent reduction of NiOOH to Ni(OH)2 in the negative scan for all three catalysts, indicating the presence of Ni(OH)2 on the surface of the catalysts that is consistent with the XPS analysis [85Ni10Bi5/C(II) and Pd85Ni10Bi5/C(III) relative to Pd85Ni10Bi5/C(I) catalyst, which is likely due to the lower overall concentration of Ni on these samples as measured by XPS (Tables 85Ni10Bi5/C(II) catalyst exhibits the highest EASA compared to the other catalysts  in the potential range of 0.05–1.2 V were recorded without rotation in de-aerated 1.0 M KOH, as well as in a mixture of 1 M KOH and 1 M EtOH at 30 °C Fig. , and demrm Bi2O3 , 28, 34.S Tables –S17. The−2 due to electrochemical double layer charging effects, and the maximum current in the forward scan was utilized as an indicator for the EOR activity of each catalyst. In that context, Fig. 85Ni10Bi5/C(II) catalyst shows the highest peak current density and the lowest onset potential for the alkaline EOR compared to the other catalysts, indicating these catalysts exhibit an optimal content and synergy between the Pd, Ni, and Bi species. The Pd85Ni10Bi5/C(III) catalyst exhibits the lowest EOR activity, which is likely due to excess Ni on the catalyst surface as measured by XPS (Table 85Ni10Bi5/C(III) for the alkaline EOR [Cyclic voltammograms measured in the presence of ethanol Fig.  can be uPS Table . Indeed,line EOR , 31.85Ni10Bi5/C catalysts exhibit higher by-product tolerance relative to commercial Pd/C. The higher Bi content on the surface of Pd85Ni10Bi5/C(I) compared to Pd85Ni10Bi5/C(III)  Table  likely l85Ni10Bi5/C(II) ~ Pd85Ni10Bi5/C(I) < Pd85Ni10Bi5/C(III) < Pd/C comm., consistent with the by-product tolerance results. The use of a teflon cell is preferred over a glass cell for the electrochemical characterization of catalysts in alkaline media due to the glass corrosion that occurs, as demonstrated by Mayrhofer et al. [The EOR stabilities of all catalyst samples were further evaluated using CA-RDE at an applied potential of 0.83 V at 30 °C for 1 h Fig.  and rever et al. . The cat85Ni10Bi5/C nanocatalysts and of the commercial Pd/C catalyst.Table 85Ni10Bi5/C(II) catalyst displays outstanding specific and mass activity  with a low onset potential (0.207 V) for the alkaline EOR. It was found that the synthesis of the Pd85Ni10Bi5/C(II) catalyst in N2 atmosphere, the use of HCl, and the addition of solid NaBH4 result in the highest EOR performance among all catalyst samples, with activity decreasing as: Pd85Ni10Bi5/C(II) > Pd/C comm. > Pd85Ni10Bi5/C(I) > Pd85Ni10Bi5/C(III). We further demonstrate that the addition of Ni and Bi to monometallic Pd improves the EOR stability and by-product tolerance of all ternary Pd85Ni10Bi5/C catalysts compared to commercial Pd/C catalyst. However, the durability of catalysts prepared by our modified instant reduction approach was not enhanced when compared to PdxNiyBiz/C catalysts synthesized via the common instant reduction approach, suggesting there is room for further optimization of this process. Ongoing research is focused on advanced catalyst support materials such as reduced graphene oxide and N-doped graphene that may lead to overall improvements EOR stability and durability. Nevertheless, our synthesis scheme points to a new strategy for optimizing the structure, homogeneity, and overall performance of ternary PdNiBi catalysts on carbon supports.In this work, a new synthesis method for carbon supported PdNiBi alloy nanocatalysts has been successfully developed, with modifications in the synthesis method resulting in catalysts with smaller particle size and a more homogeneous distribution on the carbon support. The PdESM1(DOCX 11.5 mb)


Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family. It is expressed on the surface of Plasmodium falciparum-infected host red blood cells and binds to specific chondroitin-4-sulfate chains of the placental proteoglycan receptor. The functional ∼310 kDa ectodomain of VAR2CSA is a multidomain protein that requires a minimum 12-mer chondroitin-4-sulfate molecule for specific, high affinity receptor binding. However, it is not known how the individual domains are organized and interact to create the receptor-binding surface, limiting efforts to exploit its potential as an effective vaccine or drug target. Using small angle X-ray scattering and single particle reconstruction from negative-stained electron micrographs of the ectodomain and multidomain constructs, we have determined the structural architecture of VAR2CSA. The relative locations of the domains creates two distinct pores that can each accommodate the 12-mer of chondroitin-4-sulfate, suggesting a model for receptor binding. This model has important implications for understanding cytoadherence of infected red blood cells and potentially provides a starting point for developing novel strategies to prevent and/or treat placental malaria.VAR2CSA is the placental-malaria–specific member of the antigenically variant  Plasmodium parasites cause malaria in humans; however, the deadliest forms of malaria, including cerebral and placental malaria, are caused by Plasmodium falciparum (Pf)  , 2, 3. Trum (Pf) , 14, 15.rum (Pf) , 20, 21.As a result of frequent Pf infections, most adults in malaria endemic areas have acquired protective anti-PfEMP1 antibodies to parasites strains expressing various PfEMP1s, except the placental-specific one, VAR2CSA . During PAM), connected to each other by short linker sequences (A). Together, these domains form a high affinity ligand-binding site with specificity for a minimum 12-mer CSA that has a characteristic, low C4 sulfate content (lsCSA) (N-glycosylated and baculovirus-expressed glycosylated ectodomains (Escherichia coli-expressed DBL3x (PDB codes 3BQI and 3CML) (2WAU and 2Y8D), (4P1T)  , 37. To odomains , 39. Hownd 3CML) , 41, DBLd 2Y8D), , and the, (4P1T)  reveal a, (4P1T) , 46, 47;In the present study, we have expressed and characterized, structurally and functionally, the VAR2CSA ectodomain and a set of N- and C-terminal deletion constructs . These mA and Tables S1 and S2) were expressed in HEK Freestyle 293-F (HEK 293F) cells. The proteins are produced as secreted monomers and purified using a combination of ultrafiltration, nickel affinity, cation exchange, and size-exclusion chromatography. After purification and SDS-PAGE under reducing conditions, a single band at the expected apparent molecular mass was observed for each protein . Similarly, under nonreducing conditions a single band was observed for each protein . The purity of each protein construct, as assessed by the SDS-PAGE band profiles, is ∼98%. Yields of the purified recombinant proteins range from 0.5 to 0.7 mg/liter for NTS-DBL6ε to 14 mg/liter for DBL4ε-DBL6ε. Several additional deletion constructs  were tested for protein production in our system, but these either failed to express or to purify as folded monomers and thus were not analyzed further.The codon-optimized synthetic gene encoding the VAR2CSA ectodomain (NTS-DBL6ε) of Pf 3D7 strain and four deletion constructs corresponding to DBL1x-ID2a, ID2b-DBL6ε, DBL3x-DBL6ε, and DBL4ε-DBL6ε  phosphine , indicating that they are monodisperse. The gR value obtained for NTS-DBL6ε is smaller than those reported previously for DBL1x-DBL6ε , this modification alone would not account for the observed differences in gR values. For each construct, assessment of the molecular weight (P(r), function approaches zero smoothly at r = 0 Å and the Dmax and has a single peak consistent with relatively globular molecules (P(r) distributions for DBL3x-6ε and DBL4ε-DBL6ε each contain an asymmetric peak with a maxima at ∼42 Å and a shoulder centered at ∼62 Å, indicative of a molecule that contains spatially distinct modules, with the first peak due to the distribution of scattering centers (chords) within a module and the shoulder reflecting largely the chords between the modules. The dimensionless Kratky plots further support this analysis (gR = ∼1.7 and (qgR)2I(q)/I(0) = 1.1, in agreement with theoretical values of 1.75 and 1.1 for a globular protein (gR ∼5 (gR (1.8-1.95), indicative of a more elongated molecule. In addition, the plots for DBL3x-DBL6ε and DBL4ε-DBL6ε contain a shoulder at qgR ∼ 4.9 and qgR ∼4, respectively, and return to zero at qRg ∼10, characteristic of proteins containing spatially distinct modules . This is in good agreement with the approximate dimensions of crystal structures of DBL domains; for example, the crystal structure of DBL6ε (gR 23 Å) that can be accommodated within either the head or the base.For each construct, an 2 values . For NTS2 values , resulti2 values A. This iof DBL6ε  is an elab initio bead models were calculated as before. For DBL1x-ID2a, the averaged bead model is smaller than NTS-DBL6ε, as expected from its smaller molecular weight, with approximate dimensions of 115 Å × 75 Å × 50 Å (B). For DBL3x-DBL6ε, the averaged model is asymmetric and elongated in shape (135 Å × 100 Å × 75 Å) (C). For DBL4ε-DBL6ε, the consensus bead model is a triangular prism reminiscent of a letter T (165 Å × 100 Å × 75 Å) with the vertical leg ∼50 Å in diameter and the horizontal arm ∼115 Å × 65 Å × 45 Å (D).To identify the location of DBL1x-ID2a, DBL3x-DBL6ε, and DBL4ε-DBL6ε within NTS-DBL6ε, Å × 50 Å B. For DB × 75 Å) C. For DBÅ × 45 Å D.F). DBL4ε-DBL6ε fits inside DBL3x-DBL6ε leaving an additional volume that, presumably, corresponds to DBL3x; however, it could not be placed in a unique orientation. Thus, the SAXS data are consistent with DBL1x-2IDa occupying the base of the molecule and DBL3x-DBL6ε occupying the length of the molecule including a thinner head region.The bead models of the fragments were then fit into NTS-DBL6ε using chimera, placing DBL3x-DBL6ε first because it was the largest fragment. Although individually, each could occupy a range of positions, there was only one arrangement in which DBL3x-DBL6ε and DBL1x-ID2a fit into the full-length envelope simultaneously without a large conformational rearrangement F. DBL4ε-A shows a representative negative-stained micrograph with well-isolated NTS-DBL6ε particles. 2D classification of particles picked from this and other equivalent images . The smaller lobe (L1) contains a single distinct volume and is referred to hereafter as the head, whereas the larger lobe  is comprised of three distinct regions, hereafter called the body, the tail, and the feet. Furthermore, inspection of these side-on classes suggest that the head is connected to the body via a thin, stain-excluding neck . Particles were picked from this and equivalent images to obtain 2D classes that fit into one of three distinct subsets: classes similar to those observed with NTS-DBL6ε alone ; Y-shaped classes in projection that are characteristic views for the free mAb ; and classes corresponding to side-on images of complexes between NTS-DBL6ε and the Y-shaped mAb . In this final set of 2D classes, the Y-shaped mAb is clearly visible at the end of the head, providing an unambiguous identification of the location of the C-terminal cMyc tag adjacent to the head domain . It follows that DBL6ε is located in the density adjacent to the bound antibody at the free end of the head, furthest away from the neck and body. The relatively short linker between DBL5ε and DBL6ε (∼10-20 amino acids) and the clear connectivity of the density in the head suggests the remaining head density arises from DBL5ε.To generate a higher resolution model than possible by SEC-SAXS analysis, single particle reconstruction of negative-stained images was calculated for all of the constructs. During data collection, examination of regions throughout the grids revealed that all constructs produced both positively and negatively stained images on the same grid and, in some cases, within the same field. Therefore, data were collected from areas of the grids in which negatively stained particles dominated. Fig. 3A and Fig. 4A), which are slightly larger than those estimated from the corresponding SAXS bead model. Consistent with the views in the 2D classes, the 3D volume is comprised of three layers that are together broadly reminiscent of a duck, with a head, body, feet, and tail . The strong concordance between the reprojections from the 3D volumes and their corresponding 2D classes  and had similar characteristics to the head, tail, and body volumes of NTS-DBL6ε. For the C-terminal deletion construct, DBL1x-ID2a, an asymmetric bilobal molecule, was visible in 2D classes . As expected from our analysis, the DBL1x-ID2a volume localizes to the “feet” and ID2b-DBL6ε to the head, body, and tail features of the NTS-DBL6ε volume, respectively. Together, these data demonstrate that L1 contains DBL5ε and DBL6ε, L2 contains DBL3x and DBL4ε, and L3 contains DBL1x, ID1, and DBL2x. Placed in this manner, the tip of the larger density in the DBL1x-ID2a reconstruction extends from the feet into the region in the body near the DBL3x-DBL4ε volume. This suggests that DBL2x and ID2a would be contained in the larger volume and thus DBL1x in the smaller volume of the DBL1x-2IDa reconstruction.To determine whether the particular structural features in layers L1–L3 of NTS-DBL6ε could be assigned to specific regions of the protein, single particle reconstructions of negative-stained EM images of the N- and C-terminal deletion constructs ID2b-DBL6ε and DBL1x-2IDa, respectively, were calculated and the resulting volumes compared with the EM-based reconstruction of NTS-DBL6ε. For the N-terminal deletion construct ID2b-DBL6ε . The DBL3x-DBL6ε construct . The weaker connecting density is likely due to a combination of a lower signal-to-noise ratio of images of these constructs and, potentially, relatively more conformational freedom . Because the distinctive volume of DBL5ε and DBL6ε in L1 is common to all N-terminal deletion constructs, it provided a visual constrain for assessing fit. In each case, applying this constrain, the placement of volumes is unique and reveals that in L2, DBL4ε is located below both DBL5ε and DBL6ε in the body and DBL3x forms the tail . It should be noted that for all constructs containing DBL4ε, DBL5ε, and DBL6ε, the domains occupy equivalent locations in all maps, however, in the DBL3x-DBL6ε map, the volume corresponding to DBL6ε is rotated by ∼45° relative to its location in NTS-DBL6ε and ID2b-DBL6ε. The distinctly different orientation of the terminal domain is also evident when comparing some 2D classes . Furthermore, although the two arms of the S-shape are better defined in the volume of ID2b-DBL6ε , there was no obvious connected density that could be assigned to ID2b. It is not clear whether the presence of ID2b directly improves the reconstruction quality via a stabilization of the structure or it is simply due to the higher signal-to-noise of the negative-stained images for this construct.To further assign volumes in L2, single particle reconstructions of DBL3x-DBL6ε  and DBL4Fig. 6 freedom . The volThe structural analysis of the EM and SAXS data were performed independently and thus a comparison of the models obtained from each technique provides reliable validation for the proposed structure. Adjusted for resolution, the volumes for each of the constructs generated by SAXS and negative-stained EM are in good agreement. To further compare the models, EM density maps were converted to dummy atom models using EM2DAM (ATSAS 3.0) and theoretical SAXS curves were calculated from them and compared with the experimental SAXS curves . There wA. DBL6ε fit into the expected density at the tip of the molecule and DBL5ε fit into the remaining density in the head. Based on the connection determined in the negative strain reconstruction, DBL3x fit into the tail and DBL4ε was constrained to be adjacent to DBL3x and DBL5ε. DBL1x and DBL2x fit into the base, although the arrangement of these two domains is speculative because the folds of ID1 and ID2 are not known. DBL3x and DBL4ε can be accommodated in an arrangement similar to the structure of the DBL3x-DBL4ε tandem domains (B and C).For rigid body fitting of data, models corresponding to DBL1x, DBL2x, DBL4ε, and DBL5ε were generating using Chimera and Modeller , 55. StrFig. 7 domains . The DBL domains . FittingN-glycosylation moieties to nascent polypeptides post-translationally, potential N-glycosylation sites in the protein sequence, which might be modified during export in the HEK 293F cells, were removed by site-directed mutagenesis, as described by Srivastava et al. , and DBL6ε (PDB codes 2WAU and 2Y8D). However, the remaining surface-exposed, free cysteine residues potentially are available to form inter-domain disulfide bonds that could stabilize the overall architecture of VAR2CSA. Although the current studies, due to their limited resolution, do not provide direct evidence for the existence of inter-domain disulfide bonds, at least two discrete volumes of stain-excluding density between the spatially distinct regions of the head and DBL4ε are observed in 2D classes of all DBL4ε-containing reconstructions. This is consistent with the presence of inter disulfide bonds and could account, in part, for the observed rigidity of this region of the structure. It should be noted that only one of these densities, which we term the neck, connecting one end of the head to DBL4ε is routinely observed in the 3D maps at the typical contour levels used. This most likely reflects a combination of conformational heterogeneity between the head and the body and limitations in image quality and number for the minor tilted views compared with the dominant side-on views. Despite being rigid, overall the structure is relatively open, which would allow access of kinases to the various domains that are phosphorylated in the endogenous and recombinant VAR2CSA . Examination of the disulfide bonding pattern observed in the previously determined crystal structures identifies 8 canonical intra-domain disulfide bonds, although only one set (C(8)-C(12)) is observed in all the crystal structures of DBL3x (PDG codes 3CML and 3BQI), DBL4ε (part of PDB code  VAR2CSA . The two VAR2CSA . The rem1C4S) each pore has dimensions that could accommodate CSA 10-12-mers (B and C). In this model, binding of carbohydrate in P1 would involve residues from DBL1x, ID1, DBL2x, DBL4ε, and likely ID2a, and in P2 involve residues from DBL2x, DBL3x, DBL4ε, and possibly ID2b. Enveloping the carbohydrate in a pore provides a larger surface area for binding interactions while minimizing the accessibility of this functionally-essential surface to immune surveillance. CSA and its nonsulfated form, chondroitin, can be described as stiff worm like polymers (persistence lengths ∼90-110 Å (1CSA), a fully extended 12-mer CSA chain has an end-to-end distance of ∼100-115 Å with an estimated minimum root mean square end-to-end distance of ∼90-100 Å, although it is likely to be closer to the fully extended length in solution . Based solution . TherefoThe EM reconstruction of the ectodomain, described here, provides additional insight into how VAR2CSA might function in the context of infected red blood cells binding to CSA. Previous studies have shown that, in the knob-like structures observed on the surface of infected cells, VAR2CSA molecules are present largely at the tips , 60. TheIn conclusion, these studies have revealed for the first time, the structure of VAR2CSA, at moderate resolution, its orientation relative to the IRBC membrane and the relative arrangement of domains. The structure contains two clear pores that transverse the molecule and suggest a possible model for host receptor binding. This model provides plausible explanations for the high affinity CSA interaction and the ability to evade initial immune surveillance.N-linked glycosylation arising from the mammalian machinery  was used as a starting point to generate a synthetic gene (amino acids 59-2641). Serine or threonine residues at 20 sites, identified as sites of potential achinery , were reet al. . In some cases, protease inhibitors  were added to the media after 48 h and the cultures were harvested 72 h post-transfection, by centrifugation at 6000 × g, 4 °C. The clarified media, which contained the exported VAR2CSA proteins, was filtered through a 0.22-μm filter, protease inhibitor mixture  added to a final concentration of 1× and the media either frozen at −20 °C or used directly for protein purification. Proteins were purified from fresh or thawed media by a concentration to 50 ml using a VivaFlow 50 cross-flow cassette MWCO 100 k , diluted 1:1 with 20 mm imidazole in Ni buffer , then reconcentrated to a final volume of 50 ml. The expressed proteins were isolated via a three-step procedure. Processed medium was applied to a HisTrap Fast Flow HPLC column , pre-equilibrated with Ni Buffer containing 20 mm imidazole and washed with 5 column volumes of Ni Buffer containing 35 mm imidazole. The bound protein was eluted in a step gradient with Ni Buffer containing 300 mm imidazole. The peak fractions containing the VAR2CSA protein were concentrated using a Centricon 100-kDa concentrator and exchanged to CPX Buffer A  prior to ion-exchange chromatography. The concentrated protein was loaded on an Eshmuno CPX column  pre-equilibrated with CPX Buffer A. The column was washed with 5 column volumes of 95% 50 mm CPX Buffer A:5% CPX Buffer B  and the bound protein was eluted in a 10-column volume linear gradient from 40 to 80% CPX Buffer B. The peak fractions were analyzed by SDS-PAGE and Coomassie Blue staining. Protein-containing fractions were pooled and concentrated  to ∼5 mg/ml and applied to a TSK-G3000SWxL column  pre-equilibrated with SEC Buffer . The protein was eluted with SEC Buffer and protein-containing fractions were analyzed by SDS-PAGE and Coomassie Blue and/or silver staining. Fractions containing purified proteins (>95% pure) were concentrated to 2-8 mg/ml. The proteins were either used for experiments immediately or snap frozen in liquid nitrogen and stored at −80 °C for future use.The expression and purification of proteins is based on Srivatsava et al.  with minm NaKHPO4, pH 6.8, 100 mm NaF were recorded on a Jasco J-1500 spectrophotometer at 25 and 90 °C using a 0.1-cm path length cuvette. Spectra were accumulated at 0.5-nm data intervals at a scan rate of 50 nm/min. For each sample, three scans were collected, averaged, and corrected for base line rotation. Data were converted to molar ellipticity. For thermal denaturation, spectra of proteins (0.2 mg/ml) in 0.1 m NaCl and 10 mm NaKHPO4, pH 6.8, were recorded using a Jasco J-720 spectrophotometer at 222 nm over the temperature range 25 to 85 °C at 1 °C intervals. Data were processed using GraphPad Prism, version 5.CD spectra (190-260 nm) of proteins (0.2 mg/ml) in 10 mm ammonium bicarbonate, pH 8.3. The cysteine sulfhydryl groups were reduced by addition of 5 μl of 10 mm DTT and incubation at 56 °C for 30 min followed by addition of 5 μl of 55 mm iodoacetamide and further incubation for 20 min at room temperature in the dark. The protein was digested using 60 ng of trypsin  for 12 h at 37 ˚C  and eluted in 50% acetonitrile containing 0.5% formic acid and lyophilized. The lyophilized peptides were reconstituted in 10 μl of 3% acetonitrile containing 0.1% formic acid and analyzed by LC–MS/MS. Approximately 0.5 μg of the digested peptides were injected from a NanoLC AS-2 autosampler  into a NanoLC-Ultra-2D Plus HPLC  using a 10-μl injector loop. Trap and elute mode was used to separate each SCX fraction using the microfluidics on a cHiPLC Nanoflex system equipped with a Trap column (200 μm × 0.5-mm ChromXP 3C18-CL 120 Å resin) and a separation column (75 μm × 15 cm ChromXP 3C18-CL 120 Å resin). The column was subsequently washed with mobile phase A  for 1 min at a flow rate of 0.5 μl/min. The peptides were eluted in a linear gradient from 95% A:5% B (100% acetonitrile: 0.1% formic acid) to 70% A:30% B at 0.3 μl/min over 50 min. The eluted peptides were analyzed by in-line detection with an AB Sciex 5600+ Triple TOF MS/MS  mass spectrometer operated in positive and high sensitivity modes. For data acquisition, the detector was set to a mass range of 400 to 1250 (m/z), charge state range of 2+ to 6+, an accumulation time of 250 ms, a collision energy of 10 V, a declustering potential of 80 V, an ion spray potential of 25500 V, and an interface heater temperature of 150 °C. Peptide fragmentation was accomplished by collision-induced dissociation using nitrogen at a pressure of 3.6 × 105 Torr. A maximum of 50 parent ions were subjected to fragmentation per cycle. Fragmentation spectra were collected over a mass range of 50-6000 Da with an accumulation time of 25 ms and rolling collision energy, as determined automatically by the embedded software, and collision energy spread of 3 V. The peptides were identified using Protein Pilot version 5.0.1 with embedded Paragon algorithm version 5.0.1.0, 4874 , CSC , and HA . Microtiter plates were coated with bovine corneal CSPG-II, described above. Separately, NTS-DBL6ε and DBL1x-ID2a proteins  were incubated at room temperature with GAGs at concentrations ranging from 0.03 to 5 μg/ml. After 40 min, 50-μl solutions of protein:GAG mixtures were added to the CSPG-coated plates and incubated for 1 h. The extent of protein binding to the CSPG-coated wells was measured as described above.m NaKHPO4, pH 6.8, 150 mm NaCl. Scattering curves were collected continuously throughout the elution to obtain buffer and protein scattering curves. Data were initially processed using beamline software py4XS (NSLSII) and RAW (APS) and subsequently programs in the ATSAS Suite (versions 2.8 and 3.0) , Brookhaven National Laboratory, Upton, NY or 18ID of the Advanced Photon Source (APS), Argonne National Laboratory, Argonne, IL . For datand 3.0) . Ab initlgorithm , 55, 74.m NaCl, 10 mm NaKHPO4, pH 6.8, at a flow rate of 0.5 ml/min. Fractions containing protein–antibody complexes were identified by the absorbance profile and SDS-PAGE of individual fractions, pooled, concentrated to 0.5 mg/ml, and used for negative staining and EM.cMyc mAb  and NTS-DBL6ε or DBL1x-ID2a proteins were mixed at a ratio of 1:2 molar ratio and incubated for 30 min on ice. Solutions were loaded onto a TGX3000S size-exclusion column (Tosho Biosciences) and eluted with 150 mm mesh; EMS, PA) were prepared by plasma cleaning (air) for 60 s using an Emitech K590X plasma cleaner  immediately prior to use. For NTS-DBL6ε, or DBL1x-2IDa, a 4-μl aliquot of the protein alone, or bound to cMyc mAb  was applied to the grids and incubated at room temperature for 10 s. Excess protein solution was removed from the edge of the grid using thin strips of Whatman No. 1 paper. The grids were washed 3 times with distilled water by placing 35-μl drop and each time touching the surface with filter paper and excess water removed as before. For staining, the grids were floated sequentially on the top of three 35-μl drops of 1% uranyl acetate in water for 10 s for the first two drops and 1 min for the final drop, air dried at room temperature for 1 h, and stored under anhydrous conditions until needed. EM data were collected using a JEOL 2100 transmission electron microscope equipped with a 4K CCD camera. All images were collected at 200 eV at a pixel size of 2.3 Å/pixel using Digital Micrograph.Formvar-coated carbon grids  and visualized using Chimera. Basic homology modeling was performed using the MODELLER interface within Chimera. For each domain, models were evaluated by visual inspection, and GA341 and zDOPE scores . ResiduePXD021873. All remaining data are contained within the document and is available upon request from Dr. John Flanagan, Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine. E-mail: jflanagan@psu.edu.The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE  with the


This increase has led to a decrease in seawater pH that has changed inorganic carbon chemical speciation, increasing the dissolved Posidonia oceanica is a marine angiosperm that uses − (Cl−c) in mesophyll leaf cells due to the efflux through anion channels and, probably, to intracellular compartmentalization. Since anion channels are also permeable to 2, would also promote a decrease of cytosolic −-selective microelectrodes for the continuous monitoring of the cytosolic concentration of both anions in P. oceanica leaf cells. Under light conditions, mesophyll leaf cells showed a 3 caused both a 2 also produced a diminution of the 2) in NSW would have an effect on the cytosolic anion homeostasis mechanisms in P. oceanica leaf cells. In the presence of 0.1 mM ethoxyzolamide, the plasma membrane-permeable carbonic anhydrase inhibitor, the CO2-induced cytosolic P. oceanica leaf pieces in 3 mM P. oceanica and, probably, in other aquatic plants.The concentration of CO Zostera marina in which the uptake of +  in the mesophyll cells of P. oceanica  Delile plants were sampled in Punta de Calaburras (36°30′23.4′′N 4°38′37.6′′W) Málaga, southern Spain, at 2 m depth. Plants with 6 to 12 leaves attached to a piece of the rhizome were collected and transported to the laboratory in a thermos container in less than 30 min. Then, plants were placed in an aquarium filled with continuously aerated natural seawater (NSW). The air used for this purpose was obtained from the compressed air supply of the Faculty of Sciences building. Concentration of CO2 in the air was regularly monitored (390 ± 10 ppm) using an IRGA, LICOR LI-820, Li, Nebraska (USA). Temperature was held at 15°C, and illumination was at a light intensity of 150 µmol photons·m−2·s−1 with a photoperiod 16L/8D. Renewing the seawater every three days, plants were used for experiments within two weeks after sampling.Cytosolic nitrate and chloride were measured by electrophysiological techniques using double-barreled intracellular microelectrodes. Glass capillary preparation details have been previously described . In shor−-microelectrodes, the ionophore I , dissolved in a mixture of polyvinylchloride dissolved in tetrahydrofuran  was used and backfilled into the silanized barrel. Once THF evaporated, the remainder was filled with 0.5 M KCl  that contained 5 mM NaNO3, the putative cytosolic − and For Cl.5 M KCl . As desc.5 M KCl , Cl−-micntration , to mini cytosol . Calibra−, which means any interference will not be important in a physiological situation, as described in 3 and 0.1 M KCl. Then, 3K solutions (0.1–20 mM) containing 10 mM KCl, to saturate the presumed interference using the Cl− concentration reported previously in P. oceanica mesophyll leaf cells (2PO4 (from 15 to 50 mM), to give a constant background ionic strength in the calibration solutions  in 0.5 M KCl) were fixed to Ag/AgCl electrode holders and connected to a high-impedance differential amplifier . Amplifier signals were continuously monitored on a double pen chart recorder (Linseis L250E).For measurements, the microelectrode voltage barrel was backfilled with 0.5 M KCl . Then, t−1, which renewed chamber volume approximately 10 times every minute. This system kept the temperature, the ionic concentration, and gases constant during the experiments. Measurements were made under a microscope light of 150 µmol photons·m−2·s−1.Measurements were performed on leaf pieces (≈ 1 cm long), longitudinally peeled to remove part of the epidermis and fixed with paraffin wax on a Plexiglas transparent chamber (1.1 ml volume). A gravity-based flow-through system permitted controlled changes of the assay medium at a rate of 10 ml·min3 during 2 days. Then, excised leaves (2–3 g fresh weight) were placed separately in 250 ml flasks and incubated in 100 ml NSW (control) or NSW supplemented with 3 mM NaHCO3. The assay was carried out at 25°C with gentle and constant agitation. For each treatment, samples of assay medium were taken at 0, 1, 2, 3, 5, 7, 10, 15, 20, and 30 min. The highly sensitive method for 2− (In order to monitor the net efflux and/or uptake of of NO2−  was used3 stock solution (pH 8.2). CO2-enriched NSW was obtained by bubbling with artificial air . A stock solution of the plasma membrane-permeable carbonic anhydrase inhibitor ethoxyzolamide  was prepared in 0.05 M NaOH. The addition of equivalent volumes of NaOH without the inhibitor had no effect on measurements. All chemicals were analytical grade and were purchased from Sigma-Aldrich.Natural seawater (NSW) supplemented with 3 mM P < 0.05.Time-course measurements are shown as single traces, representative of a number of equivalent experiments carried out under the same conditions, as stated in the figure legends. Data are presented as means, and error bars are standard deviations. Number of repetitions (n) is indicated in every experiment. Data were analyzed using SPSS Statistics, version 21. The significance level was set at P. oceanica showed a stable plasma membrane potential of −174 ± 8 mV  after 25 min in the dark. With a delay of a few minutes, light-dark transition promoted the gradual increase of cytosolic P = 0.02, Student t test) after 30 min in the dark  or P = 0.012, Student t test), followed by a prolonged depolarization that stabilized within approximately 40 min at a membrane potential of −168 ± 3 (n = 5). Also in agreement with the previous study, the addition of 3 mM − (after approximately 4 min) which continued progressively from 9.7 ± 0.2 mM to 6.3 ± 0.3 mM  20 min after P. oceanica mesophyll leaf cell cytosolic −, the diminution of cytosolic P = 0.03, Student t test). In both cases, time courses of cytosolic − decreases aligned with the recovery of the transient membrane hyperpolarization and profound membrane depolarization, supporting a cytosolic leak of negative charge induced by the We have previously reported that in ed value . The simed value . The par to NO3−  allows uP. oceanica mesophyll leaf cells the direct uptake of 2 for photosynthesis has consequences for anion homeostasis  increases and the cytosolic pH (pHc) is alkaline  after 7 min of 1000 ppm CO2 treatment. This maximum hyperpolarization was lower and almost 2 min delayed compared to that induced by the treatment with 3 mM 2; however, in these conditions cytosolic 2, cytosolic P = 0.37, Student t test). Furthermore, in the presence of 0.1 mM EZ, the enrichment of NSW with 1000 ppm CO2 evoked a marked plasma membrane hyperpolarization that reached a steady maximum value of −178 ± 2 mV after 12 min treatment. In the presence of EZ, cytosolic P = 0.53, Student t test). That neither the long-term depolarization nor the drop in cytosolic 2 in the presence of EZ (thus restricting 2 source) points to the need for substantial cytosolic 2) increase.As we have previously reported, in eostasis . In thislic NO3− Figure 2polarize . In terralkaline . These agh HCO3− . In ordeP. oceanica leaves, the time course of external P <0.001, Student t test) within the first minute of incubation. Maximum net efflux rate was 18 ± 2 nmol FW−1·min−1. Then, external FW−1·min−1 until 5 min of incubation to stabilize at a concentration of 9.2 ± 0.1 µM FW−1·min−1 during the first 10 min of incubation to reach a steady lower value of 7.6 ± 0.2 µM P = 0.002, Student t test). External P. oceanica leaves under these conditions.The activation of S-type anion channels may allow the efflux of 2+ for example (for discussion see The concentration of P. oceanica mesophyll leaf cells was 5.7 ± 0.2 mM (n = 10), almost double the value reported for both epidermal and mesophyll leaf cells of A. thaliana  of 9.7 mM Cl−, yielding a −c of 0.6. This ratio is higher than that calculated for C. conicum,−c ≈ 0.1, taking 7.4 mM as the Cl−c (−c ≈ 0.9) or A. thaliana mesophyll leaf cells (−c ≈ 0.3). Those values have been calculated considering Cl−c as 6 mM for barley root cells  mutant (nia1nia2) leaf cells, the A. thaliana nia1nia2 mesophyll leaf cells and a similar time course of cytosolic increase to the rate of NR activity change in response to illumination transitions  and hydroxyl anions, promoting membrane potential and cytosolic pH and Cl− variations  are present  and in contrast to SLAC1, SLAH3 also requires extracellular A. thaliana roots has been proposed for SLAH3 , one SLAH1 homolog (Zosma91g00860.1) and one SLAC1 homolog (Zosma76g00610.1). The presumed homologs of these channels in P. oceanica should be good candidates for the proposed channels for the leak of − ions from the cytosol, but further molecular analyses are needed to characterize the specific role of these anion channels to mediate anion efflux in response to high Although seagrasses have lost stomata differentiation genes  the presseagrass , contain2 is mediated by the activation of S-type (SLAC1/SLAH3) anion channels . However, in response to high −c decrease observed in P. oceanica mesophyll leaf cells (ΔCl−c = 3.4 mM) was much higher than for −c could be explained by a different membrane permeability for both anions and/or different capability of compartmentalization. Nevertheless, even the observed decrease of P. oceanica leaf cells in natural seawater containing high inorganic carbon.As we showed previously, direct af cells . The shoh anions Figure 52 rise. Oceans have been the sink for 30% of CO2 released during the industrial era, at a higher rate (3.8 GTons·year−1) than the 1.8 GTons·year−1 fixed by photosynthesis or the 2 GTons·year−1 removed by abiotic absorption (2 is exchanged into aquatic environments rendering the dissolved inorganic carbon (DIC) equilibrium. Controlled by pH, this equilibrium generates the distribution of DIC species: dissolved CO2, bicarbonate (32−) ions. Consequently, elevated atmospheric CO2 concentration increases the total DIC and lowers the pH, shifting the relative proportion of each DIC species. Under current ocean pH (~8.04) and atmospheric CO2 (~ 410 ppm), the smallest pool of DIC is dissolved CO2, but this will have the greatest increase (> 250%) among the DIC constituents as the pH drops (~ 0.3–0.4 pH units) under the predicted rise in atmospheric CO2 (1000 ppm) for 2100. In contrast, the −1) 2  shows that the long-term exposure (9 months) to acidified seawater (pH 7.82) under constant DIC conditions promotes the diminution of the C/N molar ratio, due to the increase of N content by 21% and 70% in leaves and rhizome, respectively, whereas C content of those organs is not affected by external pH acidification  conditions the expression of nitrate transporter genes in P. oceanica leaves is altered; while NRT1_6.3 and NRT1_2.13 , seagrasses show C3 photosynthetic pathways and are not saturated at the current ocean DIC concentration  awarded to JF and LR and the University of Cambridge's Central Chest (JD).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.