Cubic Sr₂ScGaO₅ Perovskite: Structural Stability, Oxygen Defect Structure, and Ion Conductivity Explored on Single Crystals

Oxygen-deficient Sr₂ScGaO₅ single crystals with a cubic perovskite structure were grown by the floating-zone technique. The transparent crystals of this pure 3D oxygen electrolyte are metastable at ambient temperature, showing one-sixth of all oxygen positions vacant. While neutron single-crystal diffraction, followed by maximum entropy analysis, revealed a strong anharmonic displacements for the oxygen atoms, a predominant formation of ScO₆ octahedra and GaO₄ tetrahedra is indicated by Raman spectroscopic studies, resulting in a complex oxygen defect structure with short-range order. Temperature-dependent X-ray powder diffraction (XPD) and neutron powder diffraction (NPD) studies reveal the cubic Sr₂ScGaO₅ to be thermodynamically stable only above 1400 °C, while the stable modification below this temperature shows the brownmillerite framework with orthorhombic symmetry. Cubic Sr₂ScGaO₅ remains surprisingly kinetically stable upon heating from ambient temperature to 1300 °C, indicating a huge inertia for the retransformation toward the thermodynamically stable brownmillerite phase. Ionic conductivity investigated by impedance spectroscopy was found to be 10^–4 S/cm at 600 °C, while oxygen ¹⁸O/¹⁶O isotope exchange indicates a free oxygen mobility to set in at around 500 °C

Effect of endocytosis inhibitors on endosomal sorting of cystinosin-LKG.

<p>HK-2 cells stably transfected with RFP-tagged cystinosin-LKG, after 48h serum starvation, were treated 30’ with 5 μg/ml chlorpromazine (CPZ) a clathrin-dependent endocytosis inhibitor or with 30 μg/ml methyl-β-cyclodextrin (MβCD) which affects clathrin-independent pathway. Qualitative analysis shows the presence of RFP-tagged cystinosin-LKG on the plasma membrane stained with WGA green, but the RFP signal accumulated more in CPZ treated cells (A). Scale bar = 20 μm. In the same experimental conditions, the uptake of Alexa Fluor® 488 transferrin (488-Tf) was assayed in order to confirm the inhibition of clathrin-dependent endocytosis (B). Scale bar = 20 μm. Quantitative analysis, achieved by protein surface biotinylation and SDS-PAGE, shows that CPZ treatment induces a significant increase of cystinosin-LKG presence on the plasma membrane (C). Means ± SEM of three experiments are shown.</p

Subcellular distribution of the cystinosin-LKG and ΔSSLKG mutant.

<p>HK-2 cells were stably transfected with RFP-tagged cystinosin-LKG or with its mutated form, deleted in C-terminal tail for the last five amino acids (ΔSSLKG). Cells were immunolabeled with LAMP-2 for lysosomes (A), PDI for endoplasmic reticulum (B), GM130 for Golgi (C). Scale bar = 10 μm. Analysis of ROIs (Regions of Interest) shows a Pearson’s Correlation (R_r) for RFP with LAMP-2 greater than with other organelle markers (<i>p</i> < 0.0005). In particular, the mutant ΔSSLKG shows an R_r for LAMP-2 significantly increased (<i>p</i> < 0.0005) compared to the wild type cystinosin-LKG. The presence of cystinosin-LKG and ΔSSLKG mutant on ER is low, moreover the analysis shows very low expression of cystinosin-LKG in the Golgi apparatus, whereas high R_r for GM130 in ΔSSLKG mutant suggests that it is accumulated significantly (<i>p</i> < 0.00001) on the Golgi apparatus (D). Means ± SEM of three experiments are shown.</p

Colocalization studies of cystinosin-LKG carrying the deletion of lysosomal sorting signal YFPQA.

<p>In HK-2 cells, transiently transfected with the pCTNS-LKG-RFP construct carrying the deletion of the (ΔFPQA), the deleted cystinosin-LKG-RFP could be seen on the plasma membrane as well as in lysosomes. The intensity profile, obtained with RGB Profiler, an ImageJ plugin, showed the RFP signal in lysosomes of ΔFPQA reduced compared to the wild type cystinosin-LKG. Scale bar = 20 μm.</p

Effect of silencing of AP-2 mu chain on endosomal sorting of cystinosin-LKG and ΔSSLKG mutant.

<p>siRNA to human AP-2 mu chain and a scrambled control were transfected into HK-2 cells overexpressing RFP tagged cystinosin-LKG and ΔSSLKG mutant. The analysis of the AP-2 expression by PCR and western blotting show an efficient silencing with a significant transcript reduction (p < 0.005) (A). After 72h silencing of AP-2 mu chain, expression of cystinosin-LKG on the plasma membrane is significantly reduced. Inhibition of the clathrin-independent pathway by treatment with 30 μg/ml methyl-β-cyclodextrin (MβCD) for 30’ combined to AP-2 silencing, permanently prevents endocytic sorting of cystinosin-LKG from the plasma membrane, triggering the accumulation of the protein on the plasma membrane (B). Scale bar = 20 μm. As previously showed, ΔSSLKG mutant is less expressed on the plasma membrane, and this condition is highlighted by AP-2 silencing. After AP-2 silencing, in fact, ΔSSLKG is very low on plasma membrane and the inhibition of the clathrin-independent pathway with MβCD does not affect significantly the distribution (C). Scale bar = 20 μm. Colocalization analysis between RFP and WGA green (plasma membrane) signals shows the Pearson’s Correlation significantly reduced in ΔSSLKG cells (<i>p</i> < 0.001) and in cystinosin-LKG with AP-2 silencing (<i>p</i> < 0.001). In the latter, after MβCD treatment, the Pearson’s Correlation increases about 24% (<i>p</i> < 0.001), while no significant effects is observed in ΔSSLKG mutant (D). Analysis of transferrin uptake indicates that cystinosin-LKG is expressed on the plasma membrane only in cells where AP-2 was not silenced (transferrin internalized) instead silencing of AP-2 associated to the accumulation of transferrin on the plasma membrane, affects negatively the presence of cystinosin-LKG on the plasma membrane (E). Scale bar = 20 μm. Means ± SEM of four (A, B, C, D) or three (E) experiments are shown.</p

Scheme of the cystinosin isoforms structure.

<p>Cystinosin (367 aa) on the left and the cystinosin-LKG isoform (400 aa) on the right, are the main known isoforms to date, for which has been described the transport of cystine. The open-source tool for visualization of proteoforms, PROTTER [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0154805#pone.0154805.ref032" target="_blank">32</a>], displayed the hypothetical structure of the two isoforms. Red regions are two targeting motifs for the protein sorting to lysosomes: GYDQL located at the C-terminal end, and YFPQA located in the putative fifth inter-transmembrane loop. Cystinosin-LKG differs from the canonical cystinosin in the C-terminal region (orange) while the proposed motif critical for the protein sorting to the plasma membrane (SSLKG) is highlighted in green.</p

¹⁴C-cystine uptake from extracellular milieu in HK-2 transfected with different cystinosin isoforms.

<p>¹⁴C-cystine uptake assay performed in HK-2 cells transfected with vehicle, cystinosin, cystinosin-LKG, or ΔSSLKG mutant, shows low uptake of radiolabelled L-[¹⁴C]-cystine across plasma membrane in absence of a proton gradient (light grey columns). In the presence of a proton gradient (dark grey columns) HK-2 cells overexpressing cystinosin-LKG show a significant ¹⁴C-cystine uptake (p < 0.0005) compared to cells transfected with cystinosin; while HK-2 cells overexpressing ΔSSLKG mutant show lower ¹⁴C-cystine uptake (p < 0.004) compared to cells transfected with cystinosin-LKG. Means ± SEM of three experiments are shown.</p

One-Dimensional Oxygen Diffusion Mechanism in Sr₂ScGaO₅ Electrolyte Explored by Neutron and Synchrotron Diffraction, ¹⁷O NMR, and Density Functional Theory Calculations

We investigated moderate-temperature oxygen diffusion mechanisms in Sr₂ScGaO₅ with Brownmillerite structure type. From oxygen isotope ¹⁸O–¹⁶O exchange experiments we determined that oxygen mobility sets in above 550 °C. Temperature-dependent neutron and X-ray (synchrotron) diffraction experiments allowed us to correlate the oxygen mobility with a subtle phase transition of the orthorhombic room-temperature structure with <i>I</i>2<i>mb</i> space group toward <i>Imma</i>, going along with a disorder of the (GaO₄)_∞-tetrahedral chains. From lattice dynamical simulations we could clearly evidence that dynamic switching of the (GaO₄)_∞-tetrahedral chains from its R to L configuration sets in at 600 °C, thus correlating oxygen diffusion with the dynamic disorder. Oxygen ion diffusion pathways are thus constrained along the one-dimensional oxygen vacancy channels, which is a different diffusion mechanism compared to that of the isostructural CaFeO_2.5, where diffusion of the apical oxygen atoms into the vacant lattice sites are equally involved in the diffusion pathway. The proposed ordered room-temperature structure in <i>I</i>2<i>mb</i> is strongly supported by ¹⁷O, ⁴⁵Sc, and ⁷¹Ga NMR measurements, which indicate the presence of crystallographically unique sites and the absence of local disordering effects below the phase transition. The electric field gradient tensor components measured at the nuclear sites are found to be in excellent agreement with calculated values using the WIEN2k program. The oxygen site assignment has been independently confirmed by ¹⁷O­{⁴⁵Sc} transfer of adiabatic populations double-resonance experiments

Polymorphism in Thermoelectric As₂Te₃

Metastable β-As₂Te₃ (<i>R</i>3̅<i>m</i>, <i>a</i> = 4.047 Å and <i>c</i> = 29.492 Å at 300 K) is isostructural to layered Bi₂Te₃ and is known for similarly displaying good thermoelectric properties around 400 K. Crystallizing glassy-As₂Te₃ leads to multiphase samples, while β-As₂Te₃ could indeed be synthesized with good phase purity (97%) by melt quenching. As expected, β-As₂Te₃ reconstructively transforms into stable α-As₂Te₃ (<i>C</i>2/<i>m</i>, <i>a</i> = 14.337 Å, <i>b</i> = 4.015 Å, <i>c</i> = 9.887 Å, and β = 95.06°) at 480 K. This β → α transformation can be seen as the displacement of part of the As atoms from their As₂Te₃ layers into the van der Waals bonding interspace. Upon cooling, β-As₂Te₃ displacively transforms in two steps below <i>T</i>_S1 = 205–210 K and <i>T</i>_S2 = 193–197 K into a new β′-As₂Te₃ allotrope. These reversible and first-order phase transitions give rise to anomalies in the resistance and in the calorimetry measurements. The new monoclinic β′-As₂Te₃ crystal structure (<i>P</i>2₁/<i>m</i>, <i>a</i> = 6.982 Å, <i>b</i> = 16.187 Å, <i>c</i> = 10.232 Å, β = 103.46° at 20 K) was solved from Rietveld refinements of X-ray and neutron powder patterns collected at low temperatures. These analyses showed that the distortion undergone by β-As₂Te₃ is accompanied by a 4-fold modulation along its <i>b</i> axis. In agreement with our experimental results, electronic structure calculations indicate that all three structures are semiconducting with the α-phase being the most stable one and the β′-phase being more stable than the β-phase. These calculations also confirm the occurrence of a van der Waals interspace between covalently bonded As₂Te₃ layers in all three structures