Unlike the taz1Δ mutant used to date, this model accumulates phosphatidylglycerol, thus better approximating the human BTHS cells. We demonstrate that increased phosphatidylglycerol in this strain leads to more pronounced mitochondrial respiratory defects and an increased incidence of aberrant mitochondria compared to the single taz1Δ mutant. We also show that the mitochondria of the pgc1Δtaz1Δ mutant exhibit a reduced rate of respiration due to decreased cytochrome c oxidase and ATP synthase activities. Finally, we determined that the mood-stabilizing anticonvulsant valproic acid has a positive effect on both lipid composition and mitochondrial function in these yeast BTHS models. Overall, our results show that the pgc1Δtaz1Δ mutant better mimics the cellular phenotype of BTHS patients than taz1Δ cells, both in terms of lipid composition and the degree of disruption of mitochondrial structure and function. This favors the new model for use in future studies.Barth syndrome (BTHS) is an inherited mitochondrial disorder characterized by a decrease in total cardiolipin and the accumulation of its precursor monolysocardiolipin due to the loss of the transacylase enzyme tafazzin. However, the molecular basis of BTHS pathology is still not well understood. Here we characterize the double mutant  Mitochondria represent a highly specialized and dynamic functional organelle comprised of two structurally distinct lipid bilayers, the inner and outer mitochondrial membranes. Two negatively charged phospholipids, phosphatidylglycerol (PG) and cardiolipin (CL) are highly specific for mitochondria. CL is an atypical phospholipid with four acyl chains, which exhibits numerous functions in mitochondrial bioenergetics and biogenesis . PG is aTAZ gene, were identified as the primary cause of the X chromosome-linked recessive disease called Barth syndrome (BTHS). Clinically, BTHS is characterized by abnormal mitochondria, dilated cardiomyopathy, neutropenia, skeletal myopathy, growth delay, exercise intolerance, and increased level of organic acid in the urine , a characteristic acyl chain found in mature mammalian CL  and subsequently reacylated to mature CL by a transacylase or acyltransferase . This wahe urine , 8. As aalian CL .Saccharomyces cerevisiae as a model for studying lipid biosynthesis disorders, including BTHS. Steps of CL synthesis are highly evolutionarily conserved; therefore, the yeast taz1Δ mutant has been used as a simple model to study defects resulting from altered remodeling and deficiency of CL  synthase Pgs1 and PG-specific phospholipase Pgc1 through an effective mechanism capable of fast, wide-range, and bidirectional PG regulation . Deletiopiration . Therefode novo synthesis of inositol from glucose-6-phosphate by indirectly blocking myo-inositol phosphate synthase  treatment on the analyzed yeast strains. VPA is a broad-spectrum antiepileptic drug that has been widely used for more than 60 years and is approved by the Food and Drug Administration (FDA) for the treatment of bipolar disorders and neuralgia. Although the mechanism of its therapeutic effect is not yet clear, it is known that among other effects, VPA inhibits synthase . In yeasentation . Here weTAZ gene disruption, PG levels in human cells further increase  similar to the taz1Δ strain, the defect in CL remodeling led to the MLCL accumulation in the double deletion strain and (ii) the absence of Pgc1 resulted in increased levels of PG in this strain, probably mainly at the expense of PC fraction (A). Besides the PG accumulation and PC depletion, no statistically significant difference between the taz1Δ and pgc1Δtaz1Δ strains was detected.As expected, mitochondria of the fraction A. Besidepgc1Δ and crd1Δ strains (taz1Δ significantly differed from the wild type (B). Specifically, in PG isolated from the mitochondria of taz1Δ cells, we found increased palmitoleic acid  and oleic acid . This increase was fully at the expense of stearic acid , which was similar to the changes detected in pgc1Δ and pgc1Δtaz1Δ strains. Consistent with previously published data  and oleic acid (C18:1) and an increase in saturated palmitic (C16:0) and stearic acid (C18:0) bound to CL molecules. Similarly, the fatty acid composition of MLCL was not affected by the further deletion of PGC1 gene (B).Previously, we reported differences between the fatty acid composition of PG accumulated in  strains . Similarild type B. Specifhed data , 13, theGC1 gene B.Besides defects in CL remodeling, decreased cholesterol synthesis has been observed in lymphoblasts of BTHS patients after serum starvation , 25. Thepgc1Δ, taz1Δ, and pgc1Δtaz1Δ strains. Significantly decreased ergosterol levels in both strains lacking TAZ1 gene were detected. Approx. 20% drop of ergosterol content was detected in these strains independent of the presence of Pgc1 (A). The analysis also revealed an increased fraction of sterol esters (SE) in the neutral lipids of all deletion mutants analyzed. In both single deletion mutants, SE fraction was significantly overrepresented if compared with the wild type. Importantly, this fraction further increased in pgc1Δtaz1Δ cells (B), suggesting different mechanisms of SE elevation in each of the single mutants.We compared the neutral lipid content in wild type,  of Pgc1 A. The an1Δ cells B, suggestaz1Δ mutant, aberrant cristae morphology and swollen mitochondria have been observed . As expected, aberrant mitochondria were occasionally observed in taz1Δ cells. The aberrant mitochondria were of a characteristic ring shape. The frequency of this aberration was almost tripled in the double deletion strain (A and B). As apparent at the ultrastructural level, the aberrant mitochondria contained excessive amounts of juxtaposed extended membranes/cristae. At the same time, the morphology of “normal,” rod-like mitochondria of the same strain did not significantly differ from the wild type , a measure of the coupling between the electron transport and ATP synthesis in the analyzed mitochondria , and after uncoupling of mitochondria with CCCP . In single deletion mutants, both OXPHOS and ETS capacity were increased compared with wild type. In the double deletion strain, the OXPHOS capacity was reduced to almost half of the wild-type value. The ETS capacity did not significantly differ from the wild type. This result indicated severe impairment of respiratory activity in the double mutant mitochondria compared with single mutants taz1Δ and pgc1Δ. Consistent with previously published data .Respiratory capacity was measured in isolated mitochondria of the wild type, hed data , 16, we in vitro activities of cytochrome c reductase, cytochrome c oxidase, and ATP synthase  in isolated mitochondria. Measurements of Complex III activity revealed increased cytochrome c reduction in mitochondria of pgc1Δ and taz1Δ strains compared with the wild type. Complex III activity in double mutant pgc1Δtaz1Δ was comparable with the wild type (A). The increased activity in single deletion strains corresponds with the observed increased OXPHOS capacity and ETS capacity (A). Interestingly, we detected different amounts of Complex III subunit Rip1 in pgc1Δ and taz1Δ strains. While increased Rip1 expression could have stood behind the increased Complex III activity in pgc1Δ strain, taz1Δ mitochondria achieved an even higher activity with wild-type Rip1 levels (B). The activity of Complex IV was reduced to half of the wild-type level in mitochondria of pgc1Δtaz1Δ (A). This was probably the reason for the decrease in OXPHOS capacity in this strain (A). It is noteworthy that we found the normal level of Complex IV subunit Cox4 in mitochondria of the double mutant (B). Finally, a small, but significant decrease in Complex V activity compared with the wild type was detected also in the double deletion mutant (C).Next, we measured individual ild type A. The incapacity A. Intere1 levels B. The acc1Δtaz1Δ A. This ws strain A. It is e mutant B. Finalln mutant C.Figure PGC1 deletion in taz1Δ cells. On the one hand, it was capable to compensate for an increased Complex III activity in taz1Δ cells. On the other hand, it reduced the activity of Complex IV and Complex V.Our results identified significant changes in respiration efficiency resulting from As we documented above, the absence of Taz1 leads, among other effects, to a substantial reduction in the level of mitochondrial CL . It has In vitro measurements on mitochondria isolated from the wild type, pgc1Δ, taz1Δ, and pgc1Δtaz1Δ cells revealed a reduced activity of Pgs1 in taz1Δ strain. In contrast, Pgc1 activity in this strain was higher compared with the wild type. In other words, despite the comparable PG levels in the wild type and taz1Δ cells (A and B). In accordance with our expectations, only residual PG degradation was detected in pgc1Δtaz1Δ cells, similar to the single deletion mutant pgc1Δ (B). Surprisingly, the Pgs1 activity was fully restored in pgc1Δtaz1Δ cells – in fact, we detected even slightly increased activity compared with the wild type (A).1Δ cells , mitocho1Δ cells , A and Bnt pgc1Δ B. Surpriild type A.Figure taz1Δ phenotype.The activity of PGP synthase Pgs1 is inhibited by inositol. Consequently, drop in inositol supply leads, among other effects, to increased PG production . Mood-stpgc1Δ, taz1Δ, and pgc1Δtaz1Δ, were grown in SMDGE medium in the absence of inositol and presence of 0.006, 0.06, and 0.6 mM VPA. In accordance with a previous study  we observed decreased and in those lacking TAZ1 allele increased levels of CL following the VPA treatment. Additionally in taz1Δ strain, PG level was increased (A). High VPA concentrations also attenuated Pgs1 activity (B). Below the limit of statistical significance were VPA-induced changes in in vitro degradation of PG by the phospholipase Pgc1 (not shown). We conclude that under selected conditions, VPA slightly increases CL production in strains with impaired CL remodeling.However, even the application of lower VPA concentrations led to statistically significant alterations of the CL levels in mitochondria of all the analyzed strains. Specifically, in cells containing ncreased A. High Vactivity B. Below A, the rate of O2 consumption in mitochondria isolated from pgc1Δtaz1Δ cells was slowed down to about one-half of the wild-type value. Interestingly, the cultivation of pgc1Δtaz1Δ cells with 0.06 mM VPA increased the OXPHOS capacity in these mitochondria almost by 50% (A). Similarly, a significant increase of RCI could be observed in both strains lacking TAZ1 after treatment with 0.06 mM VPA. In contrast, the application of higher VPA concentration decreased RCI in three of four analyzed strains including the wild type, supportive to the generally observed growth defect in 0.6 mM VPA-treated cells , in which the increase was at or even below the verge of statistical significance. In all strains treated with 0.6 mM VPA, we observed large increases in the activity of complex IV, the statistical significance of which was greatly exacerbated by the large variance between the individual samples analyzed .The beneficial effect of 0.06 mM VPA on mitochondrial respiration of cells defective in CL remodeling was further analyzed by comparing the changes in the activity of respiration complexes III and IV following the VPA treatment in all studied strains. A pronounced increase of Complex IV activity has been detected in taz1Δ strain . This imbalance of Complex III and IV activities indicated a reduction in respiratory coupling between electron transfer and ATP synthesis, induced by VPA treatment. It is noteworthy here that the direct addition of VPA to isolated mitochondria of untreated pgc1Δtaz1Δ cells, even at a concentration of 0.6 mM, did not affect their respiratory functions (not shown). Apparently, the effect observed on isolated mitochondria of VPA-treated cells reflected VPA-induced changes in cellular metabolism rather than the direct effect of VPA binding to any of the mitochondrial components.In contrast to Complex IV, the activity of Complex III was not strongly affected by treatment with 0.06 mM VPA. The only significant change was the slight decrease of Complex III activity detected in pgc1Δtaz1Δ, exhibited a phospholipid profile, which combined the effects of both single mutations—decreased CL content with MLCL accumulation caused by the TAZ1 deletion and elevated PG due to PGC1 deletion. The increased amount of PE detected in the pgc1Δtaz1Δ strain (A) can be interpreted as a positive compensatory effect of the missing CL, as both PE and CL tend to incorporate into negatively curved membranes. This way PE in concert with PG could structurally substitute CL in respiration complexes and compensate for some CL functions, as described elsewhere (taz1Δ and pgc1Δtaz1Δ strains (B) as was previously described in BTHS patients . For pgc1Δ strain, we reported this observation earlier . In mixed lipid membranes, sterols function as a solvent of highly ordered lipids  and an increase in palmitoleate C16:1) fractions of mitochondrial PG in all analyzed mutant strains B. For pg:1 fractint cells A. In mixd lipids . Ergostetaz1Δ and pgc1Δtaz1Δ strains cultivated in medium containing nonfermentable carbon source ergosterol are depleted, is in an agreement with earlier studies that reported decreased levels of cholesterol in cells of BTHS patients under conditions of serum starvation , the pgc1Δ cells exhibited, similar to taz1Δ and pgc1Δtaz1Δ mutants, a significant increase in sterol ester fraction (B). The pronounced additive effect of the double PGC1 and TAZ1 genes deletion, observed in this case, suggests that if the defect in ergosterol biosynthesis is somehow related to the (saturated) effect on PG acyl chain composition, then it lies upstream of it. SE, together with triacylglycerols, is stored in lipid droplets. Therefore, increased SE fraction in all the analyzed mutant strains, indicating a connection between PG and CL biosynthesis and lipid droplets, is not surprising. We showed before that Pgc1 localizes predominantly onto lipid droplets although the protein is active in membranes of endoplasmic reticulum and mitochondria , which was consistent with their normal ETS capacity. It seems that the primary reason for the decreased OXPHOS capacity in pgc1Δtaz1Δ mutant was a decreased activity of Complex V (C). This interpretation is supported by the observation of more frequent aberrations of cristae morphology in these cells, indicating affected ATP synthase dimers-containing inner mitochondrial membrane ..taz1Δ mutaz1Δ cells. The potentially negative effect of PG accumulation is prevented through both the downregulation of PG synthesis and increased degradation in this mutant. Interestingly enough, this self-protection failed in pgc1Δtaz1Δ cells. Under conditions of absent PG degradation, the double mutant could not suppress the activity of PGP synthase (We found that PG content is tightly controlled in synthase .A). This observation positively correlates with the increase of RCI in taz1Δ and pgc1Δtaz1Δ mitochondria following the treatment with 0.06 mM VPA . Together these findings indicate that VPA partially recovered respiration in these strains. However, these changes are not accompanied by a significant modulation of Pgs1 activity , suggesting that another mechanism of CL elevation took place here. We can only speculate that, for example, VPA facilitated the use of unremodeled CL in the respiratory chain of these cells.It has been published before that during fermentation, the activity of PGP synthase, an enzyme catalyzing the critical step of CL biosynthesis, can be increased by VPA . In thisFig. 8Candida albicans may provide further support for this interpretation  as described . Stained4 , and 1% water in Leica AFS machine and then embedded in Lowicryl HM20 resin (EMS). Ultrathin sections (70 nm) were cut with Ultracut S ultramicrotome equipped with a diamond knife  and placed on copper formvar-coated grids. Sections were examined in an FEI Morgagni 268(D) transmission electron microscope at 80 kV. Images were captured with Mega View G2 CCD camera (Olympus).Yeast cell culture grown in SMDGE I- medium for 24 h at 30 °C was processed as described previously . Briefly2, 1 mM EGTA, 0.1% fatty-acid-free bovine serum albumin, 10 mM KH2PO4) and used for measurement of O2 consumption, the activity of cytochrome c reductase, the activity of cytochrome c oxidase, and ATP-hydrolase activity as described in , and mitochondrial fraction corresponding to 25 μg of mitochondrial protein in a total volume of 120 μl for 20 min at 30 °C. The reaction was stopped by the addition of 1.5 ml chloroform:methanol:HCl mixture (100:100:0.6), followed by the addition of 1 ml of water to produce two phases. Aliquots of 0.45 ml of the organic layer were transferred into scintillation vials and evaporated under the stream of nitrogen. A scintillation mixture (2 ml) was added, and the radioactivity of each sample was determined with a scintillation counter.PGP synthase activity was determined by quantification of the incorporation of radiolabeled substrate [ribed in , 58 withLipids from homogenate fraction prepared by Zymolyase treatment (corresponding to 3 mg of proteins) were extracted, dried under nitrogen stream, resuspended in chloroform:methanol mixture (2:1), and separated by thin-layer chromatography (TLC) as described . RelativPhospholipid analysis was performed as described previously , fatty aAll data are contained within the manuscript and supporting information.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


A molecular clip combining a doubly substituted fluorescent anion–π donor probe and two flexible arms bearing H-bond motifs constitutes a new generation of anion receptors. Five simultaneous non-covalent interactions are highlighted by theoretical complexation studies with five different anions. A large range of analytical techniques  were deployed to evaluate the stoichiometry and association constants with the selected anions. The photophysical and anion–π donor properties of the tetrazine ring allowed fine characterization of the binding properties of the ligand. Based on previously published results, an anti-cooperativity effect in non-covalent interactions was demonstrated. A new generation of anion acceptor is designed from three key structural fragments and five simultaneous non-covalent interactions. More recently, subtle interactions such as cation–π4 or anion–π5,6 have emerged in the field. However, anion binding is more difficult to achieve than cation binding due to the high solvation energy and more diffuse charges of the former.7Non-covalent interactions have been widely used in the elaboration of new molecular organic architectures. Their use in molecular recognition is now established as a milestone in supramolecular chemistry, defined by Jean-Marie Lehn as the “chemistry beyond the molecule”.8–10 We recently turned our attention to a mix of hydrogen bonding and anion–π interaction. Such combination has been studied theoretically by Liu and coworkers,11–14 showing interesting cooperative effects between interactions. Despite these interesting reports, few studies in solution have been published to date.15–18Thus, the combination of non-covalent interactions is a promising strategy to bind anions, but also to construct supramolecular assemblies like folding structures or polymers.19,20 we focused on the tetrazine ring, which exhibits both excellent photophysical21–24 and anion–π binding properties.25,26 We recently showed that photophysical properties are particularly useful to characterize anion complexes in solution in a new anion receptor (1) combining a tetrazine and an urea subunits linked by a short flexible arm.27Following our interest in non-covalent interactions,NAr) would enable access to a new family of anion receptors. In this context, the following questions arose from this design: will folding really occur in solution? What type of complex will be formed? Which interactions will help maintaining this complex? To address these different points, we report the synthesis and exploration of anion binding properties of a double-armed anion receptor, comprising a theoretical approach and a broad scope of experimental methods, namely electrospray-tandem mass spectrometry, NMR, UV-visible and steady state fluorescence as well as time-resolved fluorescence.Examination of the receptor 1 shows that an extra position is available at the tetrazine core for installing an additional urea motif . We hypo28 1 : 1 host–guest complexes were obtained through geometry optimization and frequencies calculations at the APFD/6-31+G level of theory without any solvent effect. For complexes including heavy atoms, a split basis set was applied, composed of APFD/aug-cc-pvtz for bromide or iodide anions, and APFD/6-31+G for other atoms. Geometry optimization and frequencies calculations were also performed using Polarizable Continuum Model (PCM) for acetonitrile. Only a small influence on geometries was observed by application of the solvent model. Optimized geometries are detailed in the ESI (ESI pp.7–20).29 All the results of these calculations are showed in Prior to the examination of the complexation properties of the new ligand synthesized, we evaluated the binding and conformational preferences of our new ligand with five different anions through density functional theory (DFT) calculation using Gaussian 09 software. In order to take into account the dispersion effects essential in the description of non-covalent interactions, the Austin-Frisch-Petersson functional (APFD) was selected.anti conformation of both N and O atoms with respect to the linear CH2–CH2 fragment  are visible around the center of the tetrazine ring, on the N–H hydrogen bond donors and the aromatic substituent of the urea moiety, thus highlighting possible binding sites for anions  the minimization of repulsive interactions between the tetrazine and each of the two arms, and (ii) the 6 is only set by a weak anion–π interaction with hexafluorophosphate in which only one of the fluorine atoms of the anion is involved.Optimized geometries of 2–anion complexes are characterized by a marked folding of the receptor that wraps around the different anions  anions impacts the 3D-shape of the corresponding complexes. Orientation of aryl-urea planes with respect to tetrazine is characteristic of the molecular architecture of complexes. The A arm is almost orthogonal for 2–Cl and 2–Br. The orientation of this arm then become more flattened as the size of the anion increases phenyl group, compared to Hb and Hc. Distances between anion and centroid of tetrazine ring were also determined (30 These distances are close for Cl− (3.31 Å) and Br− (3.42 Å) but markedly increased for I− (3.79 Å). The orientation of the tetrazine ring towards the halide anions is also impacted by the presence of the two urea units. Indeed, in 2–Br, similar Ne–X, Nf–X, Ng–X and Nh–X distances are observed .†Synthesis of receptor 2 was achieved using a two steps procedure involving the condensation of ethanolamine on the commercially available 3,5-bis-(trifluoromethyl)phenylisocyanate previously reported by us to form the intermediate urea S1 .27 Secon−), we performed a series of MS and MSn experiments. To this end, 10−4 M equimolar mixtures of 2/Bu4NX were prepared in a 90/10 acetonitrile (ACN)/purified water solution, and electrosprayed into a 3D ion trap mass spectrometer (Bruker Amazon Speed ETD) with an Apollo II electrospray source –X]− anion. [(2)2–X]− adducts are systematically observed, but their intensity is significantly smaller. Depending on the experimental conditions, [(2)3–X]− adducts could also be observed but generally in very weak abundance. The most intense complex therefore exhibits a 1 : 1 stoichiometry, and the experimental isotopic distributions observed indicate that we do not see [(2)n–Xn]n− complexes with n > 1. On the other hand, the rather long size of the anionic receptor 2 allows the incorporation of a second anion and the concomitant formation of [(2)–X2]2− adducts  as confirmed by the isotopic distribution of peaks separated by 0.5 Da (27In order to determine the structure of the complexes that can arise in the gas phase from the interaction of 2 with the different anions (Xy 0.5 Da . Note thn–X]− anions. Starting with [(2)2–X]− species, their unimolecular dissociation upon collision induced dissociation (CID) is solely characterized by the elimination of the intact receptor 2 , which resulted in the following fragmentation scheme.We then tried to obtain structural information about the − (m/z 709). Like with the smaller receptor 1, the extent of deprotonation of 2 and concomitant formation of HX is correlated to the gas-phase acidity of HX (or basicity of X−).31 It corresponds to the main primary process with and Cl− and SCN− which are more basic in the gas phase than Br− and I−. Its abundance then logically decreased for Br− and is even weaker for I− In addition, MS3 experiments showed that the main fragment ions observed onto the MS/MS spectra of [(2)–SCN]− and [(2)–Cl]− ions, namely m/z 622 and 454, can arise from dissociation of [(2)–H]−, and according to our MS4 data, the former further dissociates to give m/z 324, whereas the latter gives rise to m/z 367, 199 and 156 ions. Consequently, for both Cl− and SCN− anions, most of the fragments ions observed imply preliminary formation of the deprotonated receptor, m/z 709. Reasonably, its formation may imply removal of one of the urea protons, and one may assume that removal of Ha or Hd should be more favorable due to resulting stabilization of the anionic charge through resonance with the aromatic ring. Removal of Ha is also consistent with the greater lengthening of Na–Ha and Nd–Hd bonds with respect to Nb–Hb and Nc–Hc bonds evidenced by the calculations. Observation of deprotonation for all anions but PF6− therefore indicates a strong interaction of the anion with the urea moiety, as evidenced by the geometrical parameters from theoretical study.The overall fragmentation picture strongly depends on the nature of X. Like for the anionic receptor 1, the only fragmentation observed in presence of PF− decreases with the decrease of the basicity of X−. This is illustrated in m/z 534, 491, and 411. Note that the two former ions are shifted to m/z 582 and 539, respectively, with iodide. This means that the fragment ion generated incorporates the anion. Indeed, if one considers this second common fragmentation –H]2]2−. Their fragmentation spectra turned to be not informative, as characterized by a charge separation process leading to X− and [(2)–X]− ions.Finally, we also studied the fragmentation of the doubly-charged complexes observed with the halogens,  and S53 [SCN−]In the case of both iodide and thiocyanate, UV-vis titrations ,27 and others.34 Despite the large excess of salts added, a complete quenching cannot be reached. This behaviour indicates that complexes between 2 and chloride or bromide anions, respectively, exhibit a residual fluorescence. Such phenomenon will be further studied by time-resolved fluorescence.Emission quenching is also anion dependant. As can be seen for both chloride and bromide (−] and S55 [SCN−]),On the other hand, in the case of iodide and thiocyanate anions  and a single wavelength nonlinear least squares analysis , followed by bromide anion (1267 L mol−1). Iodide (514 L mol−1) and thiocyanate (499 L mol−1) anions display similar binding constants. No binding constants could be determined for PF6−.From these data, new values of association constants were extracted  by emplo− anion  geometrical parameters (b) NMR shifts (c) photophysical properties and (d) association constants determined by NMR experiments.Up to date, anion receptors combining on a tetrazine core and a hydrogen bond donor are scarcely described.vs. 1.022 to 1.027 Å for 2–Cl).27 An increase of anion-centroid distances is observed in the case of 2 (3.12 Å for 1–Cl vs. 3.31 Å for 2–Cl). These observations are in line with a decrease of the potent non-covalent interactions. A significant change in interactions involved is observed in the case of 2–SCN. In 1–SCN complex, the nitrogen atom points out towards the tetrazine ring, enabling the establishment of an anion–π interaction. In 2–SCN complex, a loss of the anion–π interaction is observed, as the nitrogen of thiocyanate anion now points towards the second urea , consistent with our previously reported study.23Double substitution led to a significant change in photophysical signature compared to 1. A large drop on fluorescence quantum yield is highlighted  display a preponderant dynamic quenching, larger anions  show a more prominent contribution of static quenching. A marked selectivity for chloride anion was observed in both ground and excited states. An interesting anti-cooperative behavior was highlighted in 2 compared to 1. We believe that this work will help the design of future anion receptors combining hydrogen bonding and anion–π interaction. Further studies using other anion receptors are under way and will be reported in due time.In summary, this paper reports the design of a new anion receptor based on a tetrazine core substituted by two arms bearing hydrogen bond donors. DFT calculations enabled to predict a substantial and adaptable folding of this receptor around different anions like chloride, bromide, iodide, thiocyanate and hexafluorophosphate. Based on these insightful preliminary investigations, we successfully synthetized the novel tetrazine based receptor 2. Application to the complexation of anions is reported in both ground and excited states. First, the mass spectrometry study demonstrates the prominent formation in the gas phase of the 1 : 1 stoichiometry complex [(2)–X]There are no conflicts to declare.RA-011-D1RA00630D-s001


Importantly, Δ'17O is relatively insensitive to key fractionation processes, such as Rayleigh distillation in the water cycle that have hindered previous isotope-based assessments of animal water balance. We evaluated the effects of changes in metabolic rate and water intake on Δ'17O values of captive rufous-collared sparrows (Zonotrichia capensis) and two invertivorous passerine species in the genus Cinclodes from the field. As predicted, colder acclimation temperatures induced increases in metabolic rate, decreases in water intake, and increases in the contribution of metabolic water to the body water pool of Z. capensis, causing a consistent change in Δ'17O. Measurement of Δ'17O also provides an estimate of the δ18O composition of ingested pre-formed (drinking/food) water. Estimated δ18O values of drinking/food water for captive Z. capensis were ~ −11‰, which is consistent with that of tap water in Santiago, Chile. In contrast, δ18O values of drinking/food water ingested by wild-caught Cinclodes were similar to that of seawater, which is consistent with their reliance on marine resources. Our results confirm the utility of this method for quantifying the relative contribution of metabolic versus pre-formed drinking/food water to the body water pool in birds.Understanding physiological traits and ecological conditions that influence a species reliance on metabolic water is critical to creating accurate physiological models that can assess their ability to adapt to environmental perturbations  that impact water availability. However, relatively few studies have examined variation in the sources of water animals use to maintain water balance, and even fewer have focused on the role of metabolic water. A key reason is methodological limitations. Here, we applied a new method that measures the triple oxygen isotopic composition of a single blood sample to estimate the contribution of metabolic water to the body water pool of three passerine species. This approach relies on Δ' Understanding the physiological mechanisms that species use to maintain water balance is becoming more relevant as increases in temperature and drought frequency represent significant ecological shifts that are affecting the behavior, distribution, and abundance of animals . BecauseMost metabolic and functional processes of terrestrial animals are sensitive to water balance, where the steady state homeostatic water budget (intake = loss) assumes a constant amount of total body water:D is drinking water; WIPF is (preformed) water in food; WIM is metabolic water formed in the aerobic metabolism of dietary macromolecules; WLC is cutaneous loss of water thorough the skin; WLR is the loss of water through respiratory surfaces; and WLU and WLFC are the loss of water through urine and feces, respectively. For most birds, the total evaporative water loss (TEWL), which is the sum of cutaneous and respiratory losses (WLC+WLR), accounts for between 50 and 80% of total losses depending on hydration conditions, while urine and feces (WLU+WLFC) account for only 15–30% of total water losses , the rate and macromolecular substrate  oxidation, and behavioral attributes . Ultimately, an organism’s water balance is a function of the interplay between the physical environment, the physiological and/or behavioral mechanisms for conserving water (minimizing losses), and the production of metabolic water which is directly linked to metabolic rate  play in maintaining water balance during physiological challenges related to thermoregulation? Does reliance on different water sources vary with thermoregulatory demands? Understanding these mechanisms is critical to creating accurate physiological models that can assess the ability of animals to adapt to potential threats caused by anthropogenic and natural environmental perturbations, especially increases in ambient temperature and drought frequency predicted for many regions over the next century (The contribution of metabolic water to the body water pool is highly variable among birds . Because unknown . For ins century . These p century .2H218O) that require multiple captures of the same individual over short periods of time  and food water (WPF), both of which are ultimately derived from meteoric water , and metabolic water (WIM). Metabolic water (WIM) is assumed to have a Δ'17O value of −0.44‰ reflecting that of inhaled atmospheric oxygen  as:A linear mixing model can be used to calculate the proportional contribution from drinking/food versus metabolic water . BecauseM represents the fractional contribution to body water from metabolic water, and (1−FM) represents the contribution from pre-formed (drinking/food) water. 17O (as described above), and trophic enrichment in which food water is increasingly influenced by prey metabolic water for higher-trophic level consumers. Another important need is applying this simplified model  is very distinct from the biological signal of metabolic water (−0.44‰).Assuming a fixed Δ'low −25‰ . Unique s −0.20‰ ; evaporas −0.20‰ . HoweverZonotrichia capensis), with captive experiments of 15-day exposure to cold (15°C) followed by warm (30°C) environmental conditions. We hypothesize that birds acclimated to the cold conditions will have relatively higher resting metabolic rates (RMRs) but will consume less drinking water than when acclimated to warm conditions. We predict that these responses will yield a change in body water Δ17O values that reflect a net increase in the contribution of metabolic water to the body water pool during cold conditions. Unlike previous applications of oxygen isotopes that have focused exclusively on δ18O, our triple isotope approach is much less sensitive to evaporative 18O-enrichment of body water nor does it require isotopic characterization of all potential water sources. A novel contribution of our study is the application of a new analytical method that estimates the relative contributions drinking and food water (WID+WIPF) vs. metabolic water (WIM) to the body water pool , a coastal group of invertivorous passerines that vary in their ability to use marine resources. Our approach combines phenomenological data collected from the field with results from laboratory experiments designed to identify the physiological mechanisms that constrain how animals respond to environmental conditions  container at the bottom of the tube. After the habituation period, birds were maintained at 15±0.5°C for 15days, and then at 30±0.5°C for another 15days (12:12 light:dark photoperiod). This acclimation period was long enough to ensure complete turnover of the body water pool for a 20–30g passerine  at Los Molles (32°14'22'S 71°30'54'W) on the central coast of Chile. Blood samples were obtained with heparinized microcapillary tubes from the humeral vein immediately after capture. Blood was centrifuged at 10,000rpm for 10min and the plasma was separated from red blood cells and stored at −80°C until isotope analysis.Wild 17O, we cryogenically distilled water from 1 to 2 μl blood plasma samples in a vacuum line, then reacted it with BrF5 at ~300°C for 5–10min, quantitatively converting H2O to O2 and other gasses. These other gasses were removed via liquid nitrogen traps and the O2 was further purified by passing it through a zeolite molecular sieve and a gas chromatography column. O2 was then analyzed on a dual-inlet Thermo Scientific 253 isotope ratio mass spectrometer  at the University of New Mexico Center for Stable Isotopes . The measured values of δ17O and δ18O were used to calculate Δ'17O  that had been calibrated against the international water standards VSMOW2 (δ17O=δ18O=0.000‰) and SLAP2  that birds consumed as:In addition to using Δ'18OAir was 19.4‰ because of fractionation that occurs during absorption of inhaled atmospheric oxygen. This fractionation depends on the efficiency of oxygen absorption  and TEWL for sparrows during 3–4h using standard flow-through respirometry and hygrometry methods that we have previously applied to this species (a) of 15.0±0.5°C and 30.0±0.5°C using an infrared O2-CO2 analyzer equipped with a hygrometer . All trials were conducted in metallic metabolic chambers (2000ml). Briefly, birds were placed in metabolic chambers kept at a constant temperature (15°C or 30°C) that received air free of water and CO2 removed via Drierite and CO2 absorbent at a flow of 750ml/min (±1%). O2 concentrations in the chamber were recorded during the active period between 06:00 and 18:00. Oxygen consumption was calculated according to the following equation , we measured rates of oxygen consumption  of air entering and leaving the chamber as=P/(T×Rw), where P is water vapor pressure of the air in Pascal, T is the dewpoint temperature in Kelvin, and Rw is the gas constant for water vapor , where TEWL is in mg/ml, ρin and ρout are the absolute humidity in kg/m3 of the inlet air and the outlet air, respectively, Vi is the flow rate of the air entering the chamber as given by the mass flow controller (750mlmin−1), and Ve is the flow of exiting air. Ve was calculated following (where FR is the flow rate in ml minollowing  as:in and VO2 (mlmin−1) are known, and we assumed a respiratory quotient (RQ) of 0.71 (2O (kPa) analyzer, the oxygen analyzer (%), and the flow meter was digitalized using a Universal Interface II  and recorded on a personal computer using EXPEDATA data acquisition software . To estimate RMR, we averaged O2 concentrations of the excurrent air stream over a 20-min period after steady state was reached (M) of sparrows using the equivalence of 0.567ml H2O per liter O2 consumed (M/TEWL ratio at different temperature treatments (15°C or 30°C). We also used U and WF into WE as water losses via excreta, V of 0.71 . Output  reached . We esticonsumed . We calcBirds were captured with permits from SAG, Chile (No. 10192/2019). All protocols were approved by the institutional Animal Care Committee of the University of Chile, following the recommendation of the ARRIVE guidelines .17O, δ18O, and δ17O) were compared between warm (30°C) and cold (15°C) treatments using non-parametric two-sample paired t-tests. The statistical analyses were performed in nlme package  with body mass as a covariate, acclimation temperature (15°C and 30°C) as fixed factors, and individual identity as a random factor to control for repeated measures. Assumptions of normality and heteroscedasticity in residuals were examined with Q–Q plots and a plot of residuals against fitted values, respectively . Depende package  using th package .2 h−1) and lower daily water intake (0.19±0.05ml H2Oh−1) in comparison with when they were acclimated at 30°C (RMR: 70.8±12.2ml O2 h−1 and daily water intake: 0.26±0.08ml H2Oh−1); however, there was no difference in TEWL between temperature treatments  at 15°C and 30°C respectively, while TEWL represented 80% of the total water loss at 15°C and 67% of the total water loss at 30°C.Sparrows acclimated at 15°C exhibited higher RMR  metabolic water contributions for C. nigrofumosus and C. oustaleti were 23.0±4.8% and 27.7±4.0%, respectively. Intriguingly, two of the three highest measured Δ'17O values were sourced from C. nigrofumosus, suggesting a greater intake of pre-formed drinking/food water than for C. oustaleti. This inference is consistent with the general hypothesis of reduced dependence on WIM for larger body-sized individuals across birds and mammals (C. nigrofumosus (70–80g) is more than twice the body mass of C. oustaleti  estimated δ18O value of the combined pre-formed drinking/food water ingested by captive sparrows was ~ −11±3‰, which is within the range for tap water and groundwater in the Santiago Basin of central Chile  values near seawater  alone is typically insufficient to meet the water requirements of most vertebrates  in deer mice was similar to that observed here in captive sparrows. Our results confirm that WIM in small passerines increases with thermoregulatory demands and highlights the utility of this method for estimating water balance in laboratory conditions.Metabolic water production  if they are below the lower critical temperature (~25°C) when experiencing water deprivation. For the captive sparrows (~20g) in our study, we calculated a mean WIM/TEWL of ~61%  across a range of environmental temperatures  at 15°C but decreased to 14% at 30°C. These values are slightly lower to the percent contribution of metabolic water to the total body water pool that we estimated from Δ'17O data alone, which were 27.5% for cold-acclimated birds and 24.5% for warm-acclimated birds. The similarity in estimates of WIM using the two approaches is notable because the Δ'17O estimates were based on collection of a single sample and did not rely on any measurements of water intake or loss, highlighting the potential accuracy of this new method , may be due to at least two non-exclusive alternatives. First, the calculation for the amount of water consumed per day was obtained under acclimatization conditions when birds were in larger cages that allowed for movement and flight, conditions that yield higher rates of energy consumption than in the more confined conditions when RMR was measured. In addition, the traditional approach of using an equivalency between oxygen consumption and WIM  does not distinguish between (1) H2O that was produced by condensation reactions that occur during the oxidation of food that contains oxygen bound in macromolecules  and (2) H2O that was produced by complex IV of the electron transport chain, and which therefore only contains inhaled atmospheric oxygen  and contains oxygen bound in macromolecules  as well as phosphate groups . We expect that the influence of dissolved CO2 on body water Δ'17O is small, because the isotopic fractionation associated with loss of exhaled CO2  with ad libitum access to drinking water produced only 1ml metabolic H2O per ~1.5–1.8ml of evaporative water lost at temperatures between 15 and 25°C, showing that without drinking water, the birds would have been in negative water balance. However, when birds were dehydrated for 30days, their TEWL declined and WIM/TEWL increased to one  that were provided drinking water ad libitum had smaller contributions of metabolic water to their body water than did wild desert-adapted small mammals (Peromyscus leucopus) of similar body mass  is likely due to changes in metabolic rates. A question that remains unresolved is whether birds, especially species living in arid seasonal environments, modify their metabolic rate strictly for the purpose of WIM. Lastly, another important variable that influences WIM is the type of oxidative substrate used for aerobic metabolism. For instance, Zebra finches predictably lose body mass when fasting; however, when they are simultaneously water deprived, they lose substantially more body fat than lean (protein-rich) tissue (Passer domesticus) accelerate protein catabolism during acute dehydration  tissue . The catydration , presumaydration .17O-based estimates of the fractional contribution to body water from metabolic water (MF) in captive sparrows with similar estimates for arid- and mesic-adapted wild birds based on allometric equations for water influx rate and field metabolic rate  mentioned above  and high elevation (summer) habitats, which may also be why this species has a relatively high BMR in comparison with other Cinclodes  they were subjected to less variation in temperature and humidity in comparison with wild Cinclodes species , and National Research and Development Agency (ANID).PS, SN, and JW designed the research. SP, PS, ZS, and JW performed the research. SP, AG, KM, and PS analyzed the data. PS, SN, RN, JS-H, KM, and JW wrote the paper. All authors contributed to the article and approved the submitted version.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The findings are valuable to solid-state electrochemical energy storage technologies that require high-efficiency charge transport.Porous electrodes with extraordinary capacitances in liquid electrolytes are oftentimes incompetent when gel electrolyte is applied because of the escalating ion diffusion limitations brought by the difficulties of infilling the pores of electrode with gels. As a result, porous electrodes usually exhibit lower capacitance in gel electrolytes than that in liquid electrolytes. Benefiting from the swift ion transport in intrinsic hydrated nanochannels, the electrochemical capacitance of the nanofluidic voidless electrode (5.56% porosity) is nearly equal in gel and liquid electrolytes with a difference of ~1.8%. In gel electrolyte, the areal capacitance reaches 8.94 F cm Enhancing electrode capacitance without compromising one other metrics for solid-state supercapacitors is of high interest yet difficult to achieve. Here the authors demonstrate a strategy of using nanofluidic electrode with very low porosity to increase the electrochemical capacitance of gel-based solid state supercapacitor. Solid-sate SCs with gel polymer electrolytes impart promising advantages over the traditional SCs with liquid electrolytes, such as easy maintenance, better reliability, and improved manufacture flexibility. However, as the developing electronics go more miniature, there is a growing technological desire for electrode materials delivering both high areal and volumetric capacitance, without compromising the gravimetric capacitance.The demands for safe and fast responding solid-state energy sources continue to rise. With short seconds-to-minutes charging time and long lifespan, supercapacitors (SCs) are in principle superior to batteries for high-power energy storage applications. Solid-state SCs have emerged as high-priority energy storage solution for on-chip and flexible electronic devices−2), which results in small areal capacitance. Enhancing gel electrolyte penetration through increasing the proportion of macropores (>100 nm) in the electrode is commonly applied 11, but this approach would impair the volumetric capacitance. Additionally, it has been stressed that the practical gravimetric capacitance of the electrode would drastically diminish when the mass of electrode-loaded electrolyte is counted14. These facts suggest that enhancing the electrode capacitance without compromising one other metric is usually constrained for solid-state SCs.Porous electrodes are widely used in solid-state SCs. In general, embedding uniform ion penetration network in porous electrodes is key to achieving high capacitance in gel electrolytes. However, gel electrolytes are oftentimes poorly penetrative due to the entanglement and stickiness of cross-linked polymer chains. Such gel penetrability dependence of electrode capacitance causes a significant performance drop compared to electrodes in liquid electrolytes. Many reported works chose to alleviate the low gel penetrability by limiting the mass loading and thickness of electrodes , which forms intrinsically hydrated nanofluidic channels that are inherently ionic conductive. The scale of the confined hydrated ion channel in TALP is close to double Debye length (2λ)18, a parameter that is experimentally examined useful for high-density charge storage21. We also showed the robustness of the layered nanofluidic channels of TALP in keeping regular ion pathways under mechanical compaction, which is distinct from the deformable complex carbon networks23. This property allowed compressing powdery TALP particles to compact pellet electrode with a large apparent density of 1.8 g cm−3 and a very low porosity of 5.56%. The primary TALP particles sheared and fused in the pellets, which produced a spreading nanofluidic ion penetration network throughout the electrode even without external electrolyte flooding. The nanofluidic TALP pellet electrode showed almost equal high areal capacitances in liquid and gel electrolytes (9.10 vs. 8.94 F cm−2), as well as gravimetric and volumetric capacitance, which enables exceptional holistic capacitances that largely outstrip the state-of-the-art porous electrodes in solid-state SCs.We propose a different electrode design concept for solid-state SCs with the purpose of achieving universal high capacitance on all metrics . Our strategy is to use nonporous two-dimensional (2D) nanofluidic structure that is intrinsically dual conductive to electrons and ions as the electrode active materials in solid-state SCs. We validate the strategy using tungstate anion-linked polyaniline (TALP), a layered 2D conductive polymer-oxyanion structure16. The unexfoliated original TALP particles consist of stacked nanosheets with high regularity, as demonstrated through transmission electron microscopy (TEM) analyses  measurement16, is close to 2λ and is promising to store more charges and induce high ionic flux during charging26. Ultrasonic agitating the intact as-synthesized TALP particles in water or ethanol can extensively exfoliate the particles into ultrathin monolayer and few-layer nanosheets . This analysis identifies three modes of free (3 kHz), intermediate (33 kHz), and surface-bound (135 kHz) interlayer D2O in the deuterated particles  is static and has hydrogen bonding with the functional groups on the channel surface29. The free and intermediate D2O between the two layers of surface-bound water are more mobile. This result indicates a three-layer model, which corresponds with a thickness of ~1 nm18. Therefore, the TALP with sub-2nm nanochannels and conductive nanosheets resembles a typical 2D mixed ion-electron conductor, where the width of the ion transport path is on the 2λ length scale that is essential to attain high density of charge storage  display the characteristic semicircles at high-medium frequency and straight tails at medium-low frequency  analysis was conducted by using symmetric two-electrode cells with leak-free solid gel electrolyte membrane  Fig. . For liq36.A is the pre-exponential factor, Ea is the apparent activation energy, kB is the Boltzmann constant, σ is the ion conductivity, and T is the absolute temperature. This value is much lower than that of proton diffusion in nanofluidic channel built from exfoliated vermiculite (190 meV)37. The stability of TALP electrode for repeatedly charging and discharging the nanofluidic channels is examined by 88.6% retention after 5000 cycles at 20 mA cm−2  in a symmetric cell with gel and liquid electrolyte are demonstrated in Fig. −1 at 1 mA cm−2 with a negligible capacitance gap between gel and liquid electrolytes at various current densities. In contrast, when using gel electrolyte, the graphene foam electrode exhibits very low specific capacitance of 10.6 F g−1 at 1 mA cm−2, which is 1/15 of that in liquid electrolyte. The stark contrast in the capacitance gap in liquid and gel electrolytes remarks the advantage of the nanofluidic channels of TALP for solid-state SCs, even without gel permeation.The charge−discharge curves of TALP electrode  and electrode thickness (μm) produces areal capacitance (F cm−2). For this reason, increasing areal capacitance simply relies on adding high mass loading or thickness of the electrode. However, the areal capacitance does not always linearly increase with the increase of electrode mass loading due to the accumulating thickness that imposes difficulty for electrolyte penetration, which is common in practice and especially for the electrode using gel electrolyte31. As a result, the capacitance of porous electrode will level off towards a maximum value7. Due to the electrolyte-independent capacity of ion transport, such maximum value for TALP is theoretically higher than that of the porous electrodes. Within the mass loading ranging up to 50 mg cm−2 (278 μm), TALP electrode keeps the linear relationship between the areal capacitance and electrode mass loading and achieves an ultrahigh areal capacitance of 8.94 (solid line) and 9.10 (dash line) F cm−2 when using gel and liquid electrolyte, respectively  scarcely decreases with the increasing electrode thickness under a low current density of 1 mA cm−2. Under a high current density of 30 mA cm−2, the decrease of specific capacitance is less than 20% in which a considerable portion is brought by the resistance of gel electrolyte. Such thickness-independent capacitive performance of TALP can be attributed to the percolating nanofluidic ion channels. By comparison, some porous electrodes usually demonstrate gradual loss of volumetric capacitance as the electrode thickness (mass loading) increases39  than that of the typical porous electrodes (21.7–230 F cm−3). Electrode with high areal capacitance, yet small thickness, is desirable for compact solid-state SCs. With similar areal capacitance (~1.8 F cm−2), TALP electrode attains a thickness of only 56 μm, which is nearly half of the composite film made of graphene/conductive polymer 5 or about six times less than the carbon cloth 8. Many gel-infilled porous electrodes have a low volumetric capacitance that restricts the areal capacitance (0.174–3.38 F cm−2) despite the large thickness (up to 1500 μm)6. TALP electrode allows extra-large areal capacitances (8.94 F cm−2) in nonporous and relatively thin (278 μm) electrodes because of its nanofluidic ion diffusion network that is uniformly distributed throughout the whole electrode. For solid-state SCs, porous electrodes hardly deliver compact capacitive performance comparable to that of the nanofluidic TALP electrode.When the holistic capacitance metrics are considered Fig. , nanoflu−1 at exceptionally large mass loading of 50 mg cm−2, which is on par with those recorded at low mass loading levels (~10 mg cm−2) and exceeds those at relatively high mass loading (~20 mg cm−2). When the practical gravimetric capacitance is considered, the total mass of gel-infilled porous electrodes needs to include the gels occupied by the pores. Therefore, the values of gravimetric capacitance in Fig. Preserving high gravimetric capacitance at large mass loading is the key to high areal capacitance. The TALP electrodes can acquire high gravimetric capacitance about 180 F g−2). In contrast to the porous electrodes that are restrained by the electrolyte-dependent capability of charge storage, the electrolyte-independent capacitive behavior of the intrinsic nanofluidic TALP electrodes results in a significantly higher areal performance, as well as an extraordinary holistic performance. The uniformly distributed ion paths are critical for achieving the remarkable volumetric (>320 F cm−3) and gravimetric (>170 F g−1) capacitances over a wide range of electrode thickness up to 278 μm and high mass loading up to 50 mg cm−2 in gel electrolyte. Adjustment of the channel size will promisingly extend its use to gel electrolytes based on organic solvents or ionic liquids. This progress demonstrates the advantages of 2D nanofluidic materials as electrode materials for solid-state SCs and promises a universal strategy for designing electrodes used in solid-state electrochemical energy storage devices.The practical significance of percolating nanofluidic channels for solid-state SCs is detailed in this work. TALP electrode with interconnecting intrinsic nanofluidic ionic paths demonstrates great potential for large areal capacitance with both gel and liquid electrolytes /ammonium persulfate (0.3 M) was added dropwise to 50 mL aqueous solution of H2SO4 (0.1 M)/aniline (0.2 M). The mixing was fulfilled under 5 °C and the duration was approximately 30 min. The mixed solution was kept under 5 °C and continuously stirred for 24 h. The product was collected after filtration and washing and subject to drying in a vacuum at 60 °C for 24 h. All chemicals were purchased from Sigma-Aldrich with purity above 98%, and directly used without further purification.The TALP used in this study was synthesized following previous reportsThe TALP powder was mixed and ground with carbon black at a weight ratio of 9:1. The binder-free electrode was made by cold pressing the dry mixture in a cylindrical die with a diameter of 10 mm. The reported capacitance values in this study are based on the total mass, area, or volume of the pellets containing 90% TALP and 10% carbon black. TALP pellet is fabricated from pure TALP powder, and its fabrication process is the same as that of TALP electrode. Note, TALP electrode is used for electrochemical tests and TALP pellet is used for all the other tests.2SO4  were added to the PVA solution at 90 °C under stirring until the solids were completely dissolved. The as-made sol was casted and dried on the glass to get the gel membrane. The peeled gel membrane was saturated in 1 M Na2SO4 aqueous solution. The gel membrane was then fully dried before use.7.5 g polyvinyl alcohol  was dissolved in 100 mL deionized water at 90 °C to obtain a viscous clear solution. Then 7.5 g Polyethylene glycol  and 1.5 g Na−2. Before assembly, the TALP and porous graphene electrodes were fully dried and not soaked with liquid or sol electrolyte.The model solid-state SC cells were made by sandwiching the gel membrane with two symmetric TALP electrodes or porous graphene electrodes with a mass loading of 6 mg cm2SO4 electrolyte was added to the cell container to ensure the electrodes and separators are fully soaked. Before adding electrolyte, the TALP electrodes were kept dry.The flooded SC cells were made by sandwiching the separator between two symmetric TALP electrodes. Excessive aqueous 1 M Na−1. Focused ion beam processing and image collection were performed with Carl Zeiss AURIGA® CrossBeam® Workstation. Atomic force microscopy results were collected from a Bruker Dimension ICON SPM with tapping mode. Theta optical tensiometer was used to measure the contact angle. Micromeritics Tristar 3030 was utilized for porosity analysis.SEM characterization was carried out on the FEI Nova NanoSEM 450 FESEM at 5 kV. XRD was conducted on a Bruker D8 Thin‐Film XRD with Cu Kα radiation (λ = 1.54056 Å) with a step interval of 2° min42. The rotation electron diffraction (RED) software suite was used to process 3D ED data43. Note that the yield of crystalline sample on TEM grid is low and this phenomenon is coherent with the quasi amorphous feature of XRD for TALP.Samples were crushed into fine powders in a mortar. Then the fine powders were transferred to lacy-carbon copper grids by directly spray them onto the grid. TEM images and diffraction patterns were taken by using JEOL JEM-2100 FEG TEM operated at 200 KV. A Gatan Ultrascan CCD camera was used for collecting HRTEM images and a Gatan Orius CCD detector was used for collecting diffraction patterns. The 3D ED data were collected on a JEOL JEM-2010 LaB6 TEM equipped with an Amsterdam Scientific Instrument Timepix detector using Instamatic softwareThe micro-CT imaging was performed using the HeliScan micro-CT system with a GE Phoenix Nanofucus tube with a diamond window and a high-quality flatbed detector . The samples were scanned in a circular trajectory with the following setting: 80 kV, 85 µA (tube current), exposure time 0.47 s, 3 accumulations, 0.75 Al filter, and 2880 projections per revolution. The voxel size obtained from this sample is 1.6 µm. The tomographic reconstruction was performed using QMango software developed by the Australian National University. The porosity is calculated from the ratio of pore volume and the total volume of the electrode analyzed.2O  for 1 week to substitute the interlayer H2O. Prior to ssNMR measurement, the deuterated POAFs were dried overnight under vacuum at 100 °C. The ssNMR was run on a Bruker Advance III 300 MHz spectrometer with a 7 Tesla superconducting magnet operating at frequencies of 300 MHz and 46 MHz for the 1H and 2H nuclei respectively. Approximately 80 mg of sample was packed in a 4 mm zirconia rotor fitted with a Kel-F cap and spun to 6 kHz at the magic angle in a 4 mm HX double-resonance MAS probe head. The 2H NMR signal was acquired with a solid echo sequence using optimized hard 1 μs radiofrequency pulses, with 60 kHz 1H SPINAL decoupling and 1 s recycle delays. A total of 30 k signal transients were co-added to yield sufficient signal to noise and the overall signal was simulated using the Dmfit software to extract the quadrupolar parameters.Deuterated TALP powders were made by soaking normal TALP in D−2. Cyclic voltammetry was run at 1 mV s−1. The cell voltage was 0−0.8 V.Common electrochemical measurements were conducted in two-electrode liquid-state or solid-state cells. EIS at different temperatures, cyclic voltammetry, and galvanostatic charge/discharge were all conducted on a Biologic VSP potentiostat. Impedance was collected from 10 mHz to 200 kHz. The current density for the galvanostatic test ramped from 1 to 20 mA cmThe capacitance of a single electrode in this study was calculated from the data acquired with symmetric two-electrode cells.Ca) was calculated as following:Ia represents current density (A cm−2), t represents discharge period (s) and V is the voltage window of discharge.The areal capacitance (Cg) was calculated as following:meletrode is the mass loading (g cm−2).The gravimetric specific capacitance (Cv) was calculated as following:L is the electrode thickness.The volumetric capacitance (Cins) of each segmental voltage windows (dV) is defined different from the average capacitance for the entire voltage window (V). Therefore, it is necessary to calculate the Cins of the differential voltage window using the following equation:Cins represents the instantaneous capacitance of the differential voltage windows, I represents the current density applied in GCD test, dt represents the segmental discharge period for time t, dV represents the segmental voltage window for time t. The t−V or Q−V curve for dt or dV period is believed linear. The average capacitance (Cave) on the actual voltage window V can be calculated by using the following equation:Cave represents the average capacitance of the whole voltage window, V represents the whole voltage window, Cins represents the instantaneous capacitance of the segmental voltage windows, and dV represents the segmental voltage window. The example calculation is shown in Supplementary Note, which reveals that the average capacitance is very close to that calculated from C = I × t/V that is used in this work to ensure consistency with the literature.As shown in Fig. Supplementary InformationPeer review file


The search for artificialtopological superconductivity has beenlimited by the stringent conditions required for its emergence. Asexemplified by the recent discoveries of various correlated electronicstates in twisted van der Waals materials, moiré patterns canact as a powerful knob to create artificial electronic structures.Here, we demonstrate that a moiré pattern between a van derWaals superconductor and a monolayer ferromagnet creates a periodicpotential modulation that enables the realization of a topologicalsuperconducting state that would not be accessible in the absenceof the moiré. The magnetic moiré pattern gives riseto Yu–Shiba–Rusinov minibands and periodic modulationof the Majorana edge modes that we detect using low-temperature scanningtunneling microscopy (STM) and spectroscopy (STS). Moiré patternsand, more broadly, periodic potential modulations are powerful toolsto overcome the conventional constraints for realizing and controllingtopological superconductivity. This system has a complex electronic structure combininga Fermi surface reconstruction of NbSe2 stemming from itscharge density wave,17 together with astrong moiré arising from the lattice mismatch between NbSe2 and CrBr3. It is surprising that such complexelectronic structure with no external control parameters turns outto give rise to a state featuring topological superconductivity. Theemergence of topological superconductivity in a NbSe2/CrBr3 heterostructure13 can be rationalizedas follows. The ferromagnetic state of CrBr3 induces anexchange field on the electronic structure of NbSe2, andthe mirror symmetry breaking of the heterostructure creates a strongRashba spin–orbit coupling in the NbSe2 bands. Whenincluding the intrinsic s-wave superconducting order of NbSe2, the low energy electronic structure harvests the three fundamentalingredients for the emergence of artificial topological superconductivity:10 s-wave superconducting order, Rashba spin–orbit coupling,and exchange fields. Such low-energy effective model has been shownto faithfully capture the phenomenology observed experimentally.13 However, interesting additional microscopiccontributions have been so far unaddressed. First, Majorana edge modesshowed strong regular modulation at the edges of the topological superconductingisland. Second, the mismatch between the NbSe2 and CrBr3 monolayers gives rise to a moiré pattern modulatingall the parameters in space. And finally, the emergence of topologicalsuperconductivity in the minimal model required a delicate fine-tuningof the NbSe2 Fermi level. Here we extend our earlier experimentalresults on the CrBr3/NbSe2 system, demonstratinghow the previous three features are naturally accounted by emergentmoiré phenomena of the heterostructure.There are many routes to realizingtopological superconductivity in artificial structures,3/NbSe2 system can be theultimate driving force of its topological superconducting state. Inparticular, the strongly modulated electrostatic potential and exchangecoupling in the moiré heterostructure give rise to modulatedYu–Shiba–Rusinov (YSR) bands that allow for the emergenceof topological superconductivity in generic regimes where it is otherwiseforbidden. We explain theoretically and demonstrate experimentallythat the moiré modulation of the topological state emergingfrom the YSR bands is also visible in the spatial distribution ofthe one-dimensional topological Majorana modes. Our results put moiréphysics forward as a powerful knob enabling topological superconductivity.Finally, conceptually similar effects can be realized by creatinga periodic potential modulation  insemiconducting devices,25 which offers new ways of controlling topological superconductivitytoward the realization of topological qubits in the future.Here, we showthat the apparent complexity created by the moirépattern in the CrBr26 Specifically, we take a Hamiltonian that includesall the known ingredients for topological superconductivity: s-wavesuperconductivity, Rashba spin–orbit coupling, and ferromagnetism.27 We introduce the moiré modulation through spatial variationof the parameters of the tight-binding model: on-site energies, thehoppings, the exchange coupling, Rashba spin–orbit coupling,and the s-wave superconductivity (details are given in the Supporting Information). Despite the increasingcomplexity of the Hamiltonian H from having spatiallydependent order parameters, their effects on enabling a topologicalsuperconducting state in arbitrary conditions can be easily rationalized.In order to illustrate these possibilities, we first focus on a minimalcase: a one-dimensional moiré system  ∼ cos(Ωx), for a given wavevectorΩ, will cause folding of the band structure and opening of minigapsbetween the folded bands as illustrated in 30 As shown in Figure S1). This idea provides a new direction to explore topological superconductivityin designed one-dimensional systems, such as nanowires grown witha long-range modulation,32 in doping regimes inwhich it would not be allowed otherwise.For a one-dimensional model with uniform order parameters,topologicalsuperconductivity can only appear at the top bottom of the band, asshown in 3 monolayer grown on a bulk NbSe2 substrate ,revealing a well-ordered moiré superstructure with 6.3 nm periodicityarising from the lattice mismatch between the CrBr3 andthe NbSe2 layers.33 The moirépattern matches a structure with 19 NbSe2 unit cells accommodating10 unit cells of CrBr3, thus forming a 6.3 nm × 6.3nm superstructure. This also matches the measured lattice constantsof CrBr3 and NbSe2.This phenomenologycan be extended to two-dimensional systems thatnaturally arise due to the moiré modulation in van der Waalsheterostructures. 3 layer34 with the superconductivity fromthe NbSe2 substrate gives rise to the YSR bands insidethe superconducting gap that are also modulated by the moirépattern. The formation of YSR band is shown in I/dV spectrum taken in the middle of the CrBr3 island has a pair of conductance onsets at ±0.35 mV.This spectroscopic signature can be compared to a dI/dV spectrum of bare NbSe2, where a hardgap with an extended region of zero differential conductance aroundzero bias is observed (t ≈ 0.3Δ (see Supporting Information). In order to visualizethe spatial modulation of the YSR band, we have recorded grid dI/dV spectroscopy maps  rather than energyvariations of the YSR band as further demonstrated in the Supporting Information .The interaction of the magnetism of the CrBropy maps 2c–e over3 and CrBr3 bilayers.36 This feature suggests that themoiré pattern not only modulates the absolute value of theeffective exchange but also can change its sign.36 Second, as a consequence of the modulation of the exchange field,the superconducting order parameter will also be modulated in theopposite way, due to the competition of s-wave superconductivity fromNbSe2 and the proximity induced exchange field.37 Third, the modulation of the onsite energiesstems from an electrostatic effect associated with the stacking. Wecan directly measure the modulation of this electrostatic potentialthrough the spatial modulation of the conduction band edge of CrBr3 . Fourth, modulationsin the hoppings are expected from small relaxation effects, well-knownin other dichalcogenide-based twisted systems.39 Fifth, the charge density wave of NbSe217 will introduce additional short-range modulation in boththe hopping and local onsite energies.41 While allthese effects can be incorporated into the effective model, we canreproduce the experimental results even using a minimal model thatonly incorporates spatially varying exchange interactions and onsiteenergies . This resultsin a topological superconducting band structure  as shown in Above, we have focused on the impact of the moirépatternon the bulk electronic structure, but the moiré electronicstructure also gets imprinted on the topological edge modes. In particular,the emergence of a YSR moiré band structure suggests that topologicaledge modes may inherit the moiré distribution of the bulk YSRstates. We now focus on the edge of a CrBr3/NbSe2. First, the spatial modulation of the edge modesdirectly corresponds to the moiré modulation of the bulk Yu–Shiba–Rusinovbands. Second, there is no need for the fine-tuning of the chemicalpotential as the moiré modulation results in a topologicalphase around charge neutrality over a broad range of the values ofthe chemical potential. And third, the detrimental effect of the Isingspin–orbit interaction is mostly removed by the modificationof the band structure due to the charge-density wave modulation ofthe NbSe2. Concomitant to this moiré-enabled superconductingstate, moiré-modulated YSR bands appear, whose topologicalband structure is ultimately responsible for the topological superconductingstate. We have demonstrated this idea in a CrBr3/NbSe2 twisted heterostructure, showing the emergence of moiréYSR bands and moiré-modulated edge modes, the two paradigmaticexperimental signatures of a moiré-enabled topological state.Moiré-enabled topological phase transitions are especiallypowerful in twisted van der Waals heterostructures, where the twistangle can be used as a knob to push the system to a topological superconductingstate. Our results demonstrate the possibility of using twist engineeringto design topological quantum materials with a high potential forcreating a platform for realizing strongly interacting topologicalsuperconductors. This provides a new paradigmatic direction in thefield of topological twistronics.To summarize, we have demonstrated that moiré modulationsallow realization of topological superconductivity in parameter regimesotherwise forbidden by the electronic structure. In particular, byaccounting for the moire modulation, we have solved three open questionson the emergence of topological superconductivity in CrBr


Calculation of extracellular volume fraction (ECV) currently receives increasing interest as a potential biomarker for non-invasive assessment of liver fibrosis. ECV calculation requires hematocrit (Hct) sampling, which might be difficult to obtain in a high-throughput radiology department. The aim of this study was to generate synthetic ECV for hepatic applications without the need for Hct sampling.blood) could be calibrated and used to generate the equation for synthetic Htc and ECV calculation. Conventional and synthetic ECV were calculated. Pearson correlation, linear regression and Bland–Altman method were used for statistical analysis.In this prospective study participants underwent liver MRI. T1 mapping was performed before and after contrast administration. Blood Hct was obtained prior to MRI. We hypothesized that the relationship between Hct and longitudinal relaxation rate of blood (R1 = 1/T1n = 90) and validation (n = 90) cohorts. In the derivation cohort, native R1blood and Hct showed a linear relationship (HctMOLLI = 98.04 × (1/T1blood) − 33.17, R2 = 0.75, P < 0.001), which was used to calculate synthetic ECV in the validation and whole study cohorts. Synthetic and conventional ECV showed significant correlations in the derivation, validation and in the whole study cohorts  with minimal bias according to the Bland–Altman analysis.180 consecutive patients were divided into derivation (Synthetic ECV seems to offer an alternative method for non-invasive quantification of the hepatic ECV. It may potentially overcome an important barrier to clinical implementation of ECV and thus, enable broader use of hepatic ECV in routine clinical practice. Chronic liver disease is a global public health concern and accounts for approximately 2 million deaths per year worldwide . Liver cblood), is feasible [In differentiating between normal and diseased liver parenchyma, the concept of evaluating the T1 relaxation times was first mentioned in the 1980s. Hepatic fibrosis increases the T1 relaxation time of liver parenchyma due to an increase of extracellular matrix and protein concentration. T1 mapping techniques also allow the estimation of extracellular volume fraction (ECV) from native and post-contrast T1. ECV values are calculated from the change in relaxation rate (R1 = 1/T1) of blood and parenchyma corrected for the hematocrit (Hct) . Therefofeasible , 23. Butblood) could be used for synthetic Hct estimation, which permits synthetic ECV calculation without Hct sampling. The aim of this study was (1) to create a synthetic Hct regression model and (2) to investigate whether synthetic Hct can be used for reliable and valid calculation of synthetic ECV compared to conventional ECV.The hypothesis of our study was that a linear relationship between blood Hct and longitudinal T1 relaxation times  and MRI (including MR-elastography). When necessary, the presence of significant fibrosis at MRI was assessed by MR-elastography as a reference standard using previous published cutoffs , 4. ExclAll participants underwent MRI examination on a clinical whole-body 1.5-T system  equipped with 32-channel abdominal coil with digital interface for signal reception. In addition to morphological sequences, patients underwent hepatic T1 mapping with a heart rate independent 10-(2)-7-(2)-5-(2)-3-(2) modified Look-Locker inversion recovery (MOLLI) acquisition scheme with internal triggering . Technic2), drawn centrally in the hepatic segments II, IVa and VII, were calculated  performed image analyses, blinded to the clinical data. For the assessment of T1 relaxation times, the mean relaxation time of three representative regions of interest (ROI) (≥ 1 cmP) and the erythrocyte cytoplasm (R1RBC) [blood = R1p × (1 − Hct) + R1RBC × Hct. Hence, synthetic Hct was derived from the linear relationship between Hct and R1blood and used to calculate synthetic ECV. Synthetic ECV was normalized for synthetic Hct and calculated using the same equation as conventional ECV. Synthetic and conventional ECV were then compared.The longitudinal relaxivity of blood (R1 = 1/T1) demonstrate a linear relationship with blood Htc, and is determined by the relaxivity of the water fractions of plasma (R1 (R1RBC) : R1bloodχ2 test (with the cell count > 5) and Fisher test (with a cell count ≤ 5). A locally derived synthetic ECV was created from the longitudinal relaxivity of blood . This model was created using linear regression, where R1 is the predictor variable and the measured Hct is the outcome. The bivariate Pearson correlation coefficient (r) was used for a correlation analysis between synthetic and blood Hct as well as synthetic and conventional ECV. Agreement between individual sets of blood and synthetic Hct as well as conventional and synthetic ECV was analyzed and represented graphically using the Bland–Altman method. The level of statistical significance was set to P < 0.05.Statistical analysis was performed using software . Patient characteristics are presented as mean ± standard deviation or as absolute frequency, as appropriate. Student t test was used for comparison of continuous variables between two different groups. Dichotomous variables were compared using the n = 27, 15.0%); autoimmune liver diseases, including autoimmune hepatitis, primary sclerosing cholangitis, and primary biliary cirrhosis ; non-alcoholic fatty liver disease ; viral hepatitis  and other rare etiologies such as portal sinusoidal disease, Budd–Chiari syndrome and Fontan-associated hepatopathy  as well as cryptogenic hepatopathy . All patients were randomly divided into the derivation (n = 90) and validation (n = 90) cohorts. The derivation cohort was used to establish the linear regression equation for calculation of synthetic Htc and ECV. The clinical characteristics of the derivation and validation cohorts are presented in Table A total of 180 consecutive patients were included. In the whole study cohort, 87.8% (158/180) of patients had diffuse liver disease and 12.2% (22/180) of patients did not have diffuse liver disease based on past medical history, clinical and laboratory examinations as well as MR-elastography. The mean MR-elastography derived liver stiffness in the group of patients without chronic and/or fibrotic liver disease was 2.1 ± 0.5 kPa. This group of patients consisted of patients with indications for liver MRI examinations as follows: non-specific abdominal symptoms, e.g., non-specific abdominal pain  or liver lesions detection or/and characterization . Indications for all MRI examinations in patients with diffuse liver disease were follow-up and/or malignancy exclusion by known chronic liver disease. Etiologies of liver diseases included: alcoholic liver disease  − 33.17, where Hct is hematocrit (1 to 100%) and R1blood = 1/T1blood in 10−3 s  as well as between conventional and synthetic ECV  were found using the above-mentioned equation. Moreover, we found significant correlations between synthetic and blood Htc (r = 0.87) as well as synthetic and conventional ECV (r = 0.99), in each case P < 0.001  as well as conventional and synthetic ECV . Moreover, synthetic and conventional ECV were highly correlated . Synthetic and blood Hct also correlated well . Bland–Altman analysis demonstrated minimal bias for both Hct  as well as ECV  .Moreover, we found also strong correlation between conventional and synthetic ECV in patients with chronic liver disease in the whole study cohort with a Pearson’s correlation coefficient of 0.98 (The purpose of our study was to (1) create locally derived synthetic Hct values from the linear relationship between blood Hct and the longitudinal relaxivity (R1) of the blood and (2) investigate whether synthetic Hct can be used for reliable and valid calculation of synthetic hepatic ECV compared to conventional hepatic ECV. The main findings of our study are that: (1) synthetic Hct derived from linear regression modeling showed a strong correlation with blood Hct and, (2) synthetic ECV showed a strong correlation with conventional ECV and minimal bias according to the Bland–Altman analysis and, therefore, has a potential to be used as a reliable valid biomarker in routine clinical practice alternatively to conventional ECV.blood). However, the clinical validity of this approach for abdominal applications has not been established yet. A few recent studies in cardiac MRI already demonstrated that synthetic ECV quantification without blood sampling might be a reliable valid tool compared with conventional ECV [Liver fibrogenesis in patients with chronic liver disease is a consequence of cellular damage and following regeneration processes, leading to increased production of connective tissue with extracellular matrix components. This process leads to the extension of extracellular space and an increased accumulation of extracellular contrast agents, which is reflected by prolonged native T1 relaxation times and increased ECV of the liver. Therefore, with a growing body of evidence, calculation of ECV is considered a new promising potential biomarker for non-invasive assessment of liver fibrosis –9. Thereonal ECV , 23, 30.blood has been sufficiently investigated [P < 0.001 in each case). There were also strong correlations between conventional and synthetic ECV in the validation as well as the whole study cohort with r = 0.97 and 0.99, respectively (P < 0.001 in each case). As far as the results of this study can be compared with the results of previous cardiac studies, these findings support previous data, demonstrating higher correlations between synthetic and conventional ECV compared to synthetic and blood Hct [In our study we implemented a simple to obtain synthetic ECV measurement using Hct derived from pre-contrast blood T1. The linear relationship between Hct and R1stigated , 31, 32,lood Hct , 23. On lood Hct , 34. Thelood Hct , 36. Howlood Hct , 23. TheThere are several limitations in our study. The main limitation was that the sample size was modest and all examinations were performed in a single center. Furthermore, the fact that T1 mapping techniques vary across the institutions can additionally limit the applicability of our study results. Furthermore, synthetic Hct requires local calibration, unless MRI scanner, used T1 mapping parameters, and machine for Hct laboratory are the same. Moreover, as the accuracy of current T1 measurements method remains to be established, this study does not claim to report an accurate measure of T1, but that synthetic calculation of hepatic Hct derived from used T1 MOLLI sequence is a stable and reliable approach for routine clinical practice. Another significant limitation for clinical application of synthetic measurements is that equations for synthetic Hct calculation should be derived individually on each MRI scanner using the same acquisition scheme. Therefore, if synthetic ECV is to be used in routine clinical practice where blood Hct cannot be obtained, using a locally derived synthetic Hct regression model for the used T1 mapping sequence is preferred.In conclusion, this is the first study investigating the applicability of synthetic hepatic Hct derived from a regression model for ECV calculation without Htc sampling. Our findings suggest that ECV calculated from synthetic Hct may be a useful, valid and reliable tool compared with conventional ECV. Further multi-centric prospective studies on a larger population are needed to validate these findings across the centers, using different T1 mapping sequences to enable the further clinical implementation of ECV by liver examinations. The use of synthetic ECV may potentially overcome an important barrier for clinical implementation of hepatic ECV measurements.