Body condition is an important concept in behaviour, evolution and conservation, commonly used as a proxy of an individual's performance, for example in the assessment of environmental impacts. Although body condition potentially encompasses a wide range of health state dimensions , in practice most studies operationalize body condition using a single (univariate) measure, such as fat storage. One reason for excluding additional axes of variation may be that multivariate descriptors of body condition impose statistical and analytical challenges.Structural equation modelling (SEM) is used in many fields to study questions relating multidimensional concepts, and we here explain how SEM is a useful analytical tool to describe the multivariate nature of body condition. In this ‘Research Methods Guide’ paper, we show how SEM can be used to resolve different challenges in analysing the multivariate nature of body condition, such as (a) variable reduction and conceptualization, (b) specifying the relationship of condition to performance metrics, (c) comparing competing causal hypothesis and (d) including many pathways in a single model to avoid stepwise modelling approaches. We illustrated the use of SEM on a real‐world case study and provided R‐code of worked examples as a learning tool.We compared the predictive power of SEM with conventional statistical approaches that integrate multiple variables into one condition variable: multiple regression and principal component analyses. We show that model performance on our dataset is higher when using SEM and led to more accurate and precise estimates compared to conventional approaches.We encourage researchers to consider SEM as a flexible framework to describe the multivariate nature of body condition and thus understand how it affects biological processes, thereby improving the value of body condition proxies for predicting organismal performance. Finally, we highlight that it can be useful for other multidimensional ecological concepts as well, such as immunocompetence, oxidative stress and environmental conditions. Structural equation modelling (SEM) is a powerful and flexible statistical tool that can lead to models of higher predictive power and with more accurate as well as precise estimates compared to conventional approaches. The authors encourage researchers to consider SEM as a flexible framework to quantify the multivariate nature of body condition and thus understand how it affects biological processes. This suggests that it is important to also consider other measures of the health state beyond energy stores, such as physiological measures Table . A solutAdopting a multivariate description of body condition and identifying which condition index best explains variation in performance or fitness impose statistical and analytical challenges that have thus far not been fully addressed. Studies that aim to describe the multivariate nature of body condition have used a variety of statistical methods , which have limited flexibility and implicitly make specific assumptions that are not always recognized. Principal component analyses (PCA) identify axes of body condition based on the correlation patterns among condition variables  and show how it can be used to overcome the above‐mentioned challenges in quantifying a body condition index. SEM is a flexible conceptual framework, where statistical and mathematical tools, along with analytical principles are used to learn about a system Grace, . SEM buiHaematopus ostralegus. We provide R‐code of all analyses with annotations to facilitate its usage and as a learning tool for those new to SEM.We emphasize that the SEM techniques we describe are not novel from a statistical perspective. Instead, our aim is to demonstrate in a comprehensive and educational way its usefulness for the quantification of body condition indices as well as for testing biological hypotheses concerning condition through worked empirical examples on a shorebird species. Data are from a large‐scale study in which we measured a variety of morphological and physiological variables on Eurasian oystercatchers 22.1The global Eurasian oystercatcher population has declined rapidly which can be caused either by reduced reproduction or by reduced survival, and thus our aim is to identify the impact of potential environmental drivers in winter affecting survival. However, survival probabilities of individuals are not directly observable, as we have to infer them from the stochastic realizations of individuals dying or not, leading to information loss . An additional challenge in our long‐lived study species is that mortality events are rare, with an average annual adult survival rate of ~0.9  and helps to identify how environmental drivers (competitor density) affect body condition Figure .n = 28; Table To quantify body condition, 774 oystercatchers were caught with nets at high‐tide roosts and colour‐banded in the Netherlands during 19 catching events spanning two winters  used in the case study were generated from a multi‐state live and dead recoveries model and therefore are on an area level. See Text We relate the body condition index to survival in the year following capture. The survival estimates . CFA includes two types of variables: observed variables and latent variables which are graphically represented, respectively, by boxes and circles Figure . The ‘coThe use of latent variables (as a variable reduction tool) is common in social sciences and psychology Bollen,  and therWe created the variable ‘energy stores’ as a latent variable construct in the SEM framework using CFA Figure . The varSimilarly, we can create a variable ‘bill colour’ as a latent variable in the SEM framework Figure ; Code S2r = 0.98) and the bill colour model , showing linear relationships over the entire range  and one or more predictor variables  effects on condition Figure . The con5In this section, we combine the two models from Sections 5.1To construct the full model Figure , we connWhereas MR assumes independence of error terms, which may bias the estimates  in the oystercatcher wintering grounds increased during recent decades , but that those assumptions do not always hold true. We stress that the negative contribution of the latent construct ‘energy stores’ to body condition results from a negative correlation between mass (corrected for size) and survival Figure , and is We a priori hypothesized a positive relationship of body mass on the condition index, and therefore called the latent variable ‘energy stores’. We can learn from the outcome of this model that our data are not consistent with the theoretical ideas that led us to model the concept of energy stores in the way we did. However, our latent variable ‘energy stores’ could be consistent with interpreting this latent variable as the concept ‘mass‐related agility’ of an individual, but this hypothesis would require further testing. We note that the concept ‘mass‐related agility’ could still be thought of as a condition variable, as it reflects an aspect of health status (responsiveness to danger) and predicts performance .Our results also confirmed the expected negative relationship between corticosterone and the condition index, meaning that birds at sites with higher survival had lower corticosterone levels. Corticosterone fulfils its main functions by mobilizing stored resources to facilitate upregulating metabolism for coping with increased energetic challenges  negative association with oystercatcher density, whereas body condition positively affected annual survival probability , but formal ways to compare such models and identify whether they are sufficient descriptions in the first place have rarely been considered. In SEM, we obtain one overall model fit and can compare model fits between models, which makes it possible to learn about a system. Various metrics of model fit exist for SEM, but we focus here on the comparative fit index . We applied 10‐fold cross‐validation to the original dataset, with 80% of the dataset being used as training dataset to which the SEM  by 9% compared to the conventional (MR/PCA) approach  and the average empirical standard error (E^=1nsim‐1∑i=1nsimθi^‐θ¯2) of the estimate, with θ being the parameter value used to simulate the data , θ^i being the estimated parameter from the simulated ith repetition, θ¯ being the mean of the estimated parameters and nsim being the number of simulated datasets.In addition, we compared the bias and precision of estimators from both approaches. To this end, we simulated 1,000 datasets with 1,000 observations each, based on the covariance patterns found in the case study population using the ‘simulateData’ function in Rosseel, . Next, wRosseel,  or conveRosseel,  approachSimulation results indicate that the SEM method is practically unbiased while the conventional method can have substantial bias and not consistently in the same direction Figure . For con6We showed how to use structural equation modelling (SEM) to quantify body condition, comparing it with the conventional approaches of multiple regression and/or principal component analysis. We illustrated how latent variables can be used to summarize the multidimensional nature of body condition, yielding variable reduction. Next, we showed that composite variables make it possible to define the body condition index based on associations between observed condition variables and a fitness trait, even if the different condition variables contributing to body condition are uncorrelated . This is advantageous, because the direction of a relationship between a condition measure (e.g. body mass) and fitness is often assumed, but rarely known with certainty. Finally, we showed how it is possible in SEM to combine latent and composite variables in one model, which more realistically reflects complex natural situations, as it allows for summarizing both strongly correlated and less correlated measures of condition. A major advantage of SEM is that, in contrast to conventional methods, it can handle variables that are response and explanatory variables at the same time. As a consequence, SEM allows all the relationships to be estimated in a single model, resulting in one estimate for the model fit and generating the possibility to compare competing models, whereas in conventional methods several steps are required  that provide additional challenges (e.g. biased parameter estimates and carrying over the uncertainty from previous models). In addition, we showed how we can correct for the effect of confounding variables in SEM using MIMIC  modelling. Overall, the high flexibility and integration of SEM make it a powerful tool that might increase model predictability and result in unbiased and precise estimates, as illustrated by our worked examples on a case study, and therefore may also provide novel insights in ecological processes.While we used SEM to quantify body condition, there are numerous other contexts in which it can be used to describe complex concepts. For example, in determining an immune response or experienced stress level of an individual, because usually multiple immunological, physiological or hormonal metrics are measured, which may also reflect different aspects of the immune or stress response, and it is a challenge to derive an integrated immune estimate from these data. Similarly, climatic conditions are highly multivariate, and while most studies have, for example, detailed climatic data on various weather variables, studies tend to focus on a single weather variable such as temperature.6.1A condition index based on predicting survival does not necessarily mean that a high body condition of an individual indicates high fitness, because fitness also depends on the association between condition and other fitness components, such as reproductive success. Quantifying condition based on survival data may result in a different condition index than when it is based on the reproductive success of an individual, or a fitness metric that integrates survival and reproduction, such as reproductive value. This difference highlights that definitions of condition can be context‐dependent and may vary during the life cycle, which may be particularly relevant when investigating species that alternate strategies  and a thorough understanding of the definitions and components of the frameworks as well as of the study system is crucial for making biologically meaningful inferences  to illustrate how it can be modelled in SEM in general, but there may be many more confounding variables, and most will not have been measured . The presence of unmeasured confounders is a general challenge in observational studies  is assigned to the error in predicting Y (response variable), implying that the true effect of X on Y is typically underestimated (i.e. regression dilution/attenuation). Pugesek and Tomer . However, latent variables can also be used to account for measurement errors of observed variables. Conventional statistical approaches like MR assume that variables are measured without error (Pugesek & Tomer, nd Tomer  showed tr‐packages, lavaan (Rosseel, r‐package piecewisesem (Lefcheck, SEM statistical packages are under continuous development, but particularly frequentist statistical approaches based on maximum likelihood estimation still have limitations. Random effects in combination with latent variables are currently challenging to model with one of the most widely used Rosseel, . In the For categorical predictor variables, a multi‐group analysis can be conducted. Multi‐group analysis can also be an alternative way to account for random factors with few levels. The question that drives a multi‐group analysis is whether two or more groups might differ in terms of the relationships among parameters and the whole model is run for each level Grace, . To illuNonlinear relationships are common in ecology and there are different ways to address nonlinearity. The easiest way is through transformation, as we did in our case study, applying logit‐transformation for proportional response variables and squaring mass Code . Multi‐gr‐packages that can be used for such Bayesian approaches are rstan (Stan Development Team, jagsui (Kellner, nimble (de Valpine et al., Bayesian approaches to SEM may offer solutions to limitations of frequentist software in dealing with random effects, non‐Gaussian data and multiple‐level categorical predictors, because Bayesian software uses more convenient numerical algorithms. Furthermore, a Bayesian approach allows for constraining estimates using informative priors, so as to include biological knowledge. Bayesian framework has also been used to combine a mark–recapture model with SEM. Cubaynes et al.  show an Kellner,  and nimbTo conclude, we show that SEM is a powerful and flexible statistical tool that can lead to models of higher predictive power and with more accurate as well as precise estimates compared to conventional approaches. Therefore, we encourage researchers to consider SEM as a flexible framework to describe the multivariate nature of body condition and thus understand how it affects biological processes, thereby improving the value of body condition proxies for predicting organismal performance. We emphasize that SEM can also be a useful tool for other multidimensional ecological concepts as well, such as immunocompetence, oxidative stress and environmental conditions.All authors declare no conflict of interest.M.F. and M.v.d.P. conceptualized the research, with A.M.A., B.J.E., E.J., H.d.K., H.‐J.v.d.K., J.N. and S.V. contributed to its development; M.F. and H.‐J.v.d.K. managed the field data; M.F. performed the analysis and drafted the manuscript. All authors contributed to the final draft and approved the final manuscript.Supplementary MaterialClick here for additional data file.


The better performing PIDG-BT-C20 was used for the fabrication of water-gated organic field-effect transistors (WGOFETs), which showed excellent stability at ambient conditions. The PIDG-BT-C20-based WGOFETs exhibited rapid response when fluoride ions were introduced to the water gate dielectric, achieving a limit of detection (LOD) of 0.40 mM. On the other hand, the devices showed much lower sensitivities towards other halide ions with the order of relative response: F− ≫ Cl− > Br− > I−. The high sensitivity and selectivity of PIDG-BT-C20 to fluoride over other halides is considered to be realized through the strong interaction of the hydrogen atoms of the N–H groups in the indigo unit with fluoride ions, which alters the intramolecular hydrogen-bonding arrangement, the electronic structures, and thus the charge transport properties of the polymer.We report the design and synthesis of two novel indigo donor–acceptor (D–A) polymers, PIDG-T-C20 and PIDG-BT-C20, comprising an indigo moiety that has intramolecular hydrogen-bonds as the acceptor building block and thiophene (T) and bithiophene (BT) as the donor building block, respectively. PIDG-T-C20 and PIDG-BT-C20 exhibited characteristic p-type semiconductor performance, achieving hole mobilities of up to 0.016 and 0.028 cm A p-type indigo polymer semiconductor is developed for water-gated organic field-effect transistors (WGOFET) for sensing fluoride ions. The uptake of a small amount of fluoride ions in drinking water by humans can strengthen bones and prevent osteoporosis or tooth decay.1–3 However, an excess of fluoride ion uptake can lead to dental and skeletal diseases such as fluorosis, osteosarcoma and nephrolithiasis.4 Therefore, maintaining the fluoride concentration in drinking water within a proper range is very important for public health.5,6 Existing fluoride sensors rely mainly on a potentiometric working principle with ion selective electrodes (ISE) with the aid of a reference electrode for signal  measurement. Other fluoride sensors under development are colorimetric or fluorescence-based optical chemosensors in which the qualitative and quantitative analyses of fluoride ions are achieved with a UV-Vis spectrophotometer or a fluorimeter. The mechanisms of these optical fluoride sensors involve the interactions between the fluoride ion and the sensing chromophore, which affect the intramolecular charge transfer (ICT) or excited state intramolecular proton transfer (ESIPT) of hydrogen-bonds,7–12 cleavage of silicon–oxygen or silicon–carbon bonds and binding of fluoride to amides,13–17 π–π interactions,18 and aggregation of nanoparticles.19–23Fluoride  based on small molecule or polymer semiconductors have drawn much attention in recent years because of their low fabrication cost, excellent substrate conformity, high mechanical robustness and versatile function tunability of the organic semiconductors. Therefore, OFETs have many potential applications such as flexible displays, radio frequency identification tags, chemical or biological sensors and therapeutic medical devices.39–44 and polymer45–48 derivatives have recently demonstrated promising semiconductor properties as channel materials in OFETs. We are particularly interested in the intramolecular hydrogen bonds between the N–H and C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O groups of the two vinylogous amides of indigo, N–H⋯OC, which may preferentially interact with fluoride ions.7–12 In this work, we prepared two novel indigo donor–acceptor (D–A) polymers, PIDG-T-C20 and PIDG-BT-C20  was observed, achieving a limit of detection (LOD) of 0.40 mM for fluoride ions. On the other hand, the devices showed much lower sensitivities towards other halide ions with the order of relative response: F− ≫ Cl− > Br− > I−, indicating the excellent selectivity of these sensors to fluoride ions.Indigo is a stable dyestuff, which has been used for textiles for centuries. Indigo and its small moleculeG-BT-C20 , comprisC, for the fluoride sensing is critical in the design of our indigo-based fluoride OFET sensors. However, indigo monomers without solubilizing groups such as Tyrian purple ((E)-6,6′-dibromo--3,3′-dione) are insoluble, posing challenges to its purification and polymerization. Therefore, we synthesized a soluble indigo monomer IDG-C20-Br  formed 1, which was brominated to afford 2, followed by nitration to give 3. The Baeyer–Drewsen indigo synthesis using 3 was then carried out to give the indigo monomer IDG-C20-Br.50 Stille coupling polymerization was carried out between IDG-C20-Br and 2,5-bis(trimethylstannyl)thiophene or 5,5′-bis(trimethylstannyl)-2,2′-bithiophene to produce two polymers PIDG-T-C20 and PIDG-BT-C20, respectively, which were purified by Soxhlet extraction. The molecular weights of the polymers were measured by high temperature gel permeation chromatography (HT-GPC) at 140 °C using 1,2,4-trichlorobenzene (TCB) as eluent. PIDG-T-C20 and PIDG-BT-C20 have number molecular weights (Mn) of 21.6 and 18.6 kDa with dispersities (Đ) of 2.57 and 2.08, respectively. Thermogravimetric analysis (TGA) were performed on PIDG-T-C20 and PIDG-BT-C20, which showed good thermal stability with a 5% weight loss temperature at 315 and 361 °C, respectively. No noticeable endo- or exothermic transitions were found on their differential scanning calorimetry (DSC) thermograms  when compared to the indigo monomer IDG-C20-Br (λmax = 668 nm). This indicated that the polymers have more extended π-conjugation than the indigo monomer. In thin films, they exhibited broader and further red-shifted absorption spectra (λmax = 762 nm for PIDG-T-C20 and 758 nm for PIDG-BT-C20), which could be attributed to the planarization of polymer backbone and intermolecular interaction in the solid state. Cyclic voltammetry (CV) diagrams of PIDG-T-C20 and PIDG-BT-C20 showed oxidative peaks, which were used to calculate their HOMO energy levels to be −5.48 and −5.27 eV, respectively , indicating the presence of a large number of hole traps. A representative PIDG-BT-C20-based OFET device showed a hysteresis ΔVth (the difference between forward and reverse) of 7.9 V  (Rq = 1.1 nm), which may be accounted for the optimum hole mobility achieved at this annealing temperature. Further increasing the annealing temperature to 200 °C led to the formation of more clearly defined aggregates along with larger and deeper cracks. This might create discontinuous regions, resulting in a decrease in mobility. Compared to PIDG-T-C20, the 50 °C-annealed PIDG-BT-C20 film showed larger spherical and rod-like aggregates with a higher roughness (Rq = 2.9 nm). The film became smoother by increasing the annealing temperature to 100 °C (Rq = 1.4 nm) and 150 °C (Rq = 1.1 nm), which might be accounted in part for the improved carrier mobility of PIDG-BT-C20. When the film was annealed at 200 °C, larger aggregates with clearer grain boundaries formed, which could explain the significant drop in mobility of this polymer at this annealing temperature.The atomic force microscopic (AFM) image of the PIDG-T-C20 film annealed at 50 °C showed some micron-sized particles (bright spots) on the rather smooth film surface ( 1.7 nm) . Upon anθ = 3.91° appeared, which corresponds to a d-spacing of 22.6 Å  peak at 2θ = 3.91° (d = 22.6 Å) was observed at a lower annealing temperature of 50 °C (d = 21.3 Å) upon annealing at 100 °C, which indicates a closer interlamellar packing distance. For both polymers, the (100) peak disappeared at annealing temperatures higher than 150 °C, suggesting that the polymer chains might have undergone re-organization to form more disordered chain packing. Although both polymers exhibited maximum crystallinity at the annealing temperature of 100 °C, their highest hole mobilities in OFETs were obtained at the annealing temperature of 150 °C where both polymers were disordered. It has been reported that the film morphology may play a more significant role in charge transport than the crystallinity of the polymer films.59 Therefore, the observed optimal charge transport performance for the 150 °C-annealed films of both polymers may be due to their lower surface roughness as observed in the AFM images at this annealing temperature.The crystallinity of the polymer thin films was studied by using XRD in the reflection mode. For the PIDG-T-C20 film annealed at 50 °C, no diffraction peak was observed, indicating a quite disordered chain packing of this polymer. Upon annealing at 100 °C, a peak at 2f 22.6 Å . This peof 50 °C . The pea2 substrate having interdigitated source and drain electrodes with channel length (L) of 30 μm and channel width (W) of 15.8 mm  and source-drain (VDS) voltages were kept below an absolute value of 1.23 V. The transfer characteristics of the PIDG-BT-C20 WGOFET devices at VDS = −1 mV and VG = 0 to −1.0 V measured in air are shown in IDS of the PIDG-BT-C20 WGOFET device remained relatively steady over time, indicating the excellent stability of this polymer towards water and air. The baseline was found to remain steady after repeated measurements and washing with water multiple times. A reference WGOFET device using regioregular head-to-tail poly(3-hexylthiophene) (P3HT) as the active layer was also fabricated and characterized as a comparison. However, the P3HT based WGOFET device showed significant deviations in IDS over time .61,62WGOFETs were fabricated on the bare Si/SiO 15.8 mm . PIDG-BTe Fig. S8 and star− over other halide ions , a series of aqueous solutions of various sodium halides  with different concentrations were used as analytes. As shown in VDS = −1 mV and VG = −1 V. After 60 s, 5 μL of DI water or sodium halide aqueous solution was injected into the water-gate dielectric. Injection of DI water did not cause any change in the drain current, while all halide solutions caused an immediate current drop. The device exhibited the largest current drop when the NaF solution was injected, indicating the highest sensitivity of the device towards the F− ions. The differences in drain current before and after halide ion introduction were used to calculate the relative response (S) of the device according to the following I0 is the IDS of the baseline and Ianalye is the IDS after analyte introduction. The relative responses (S) for 24 mM NaF, NaCl, NaBr, and NaI solutions were 87%, 52%, 23%, and 22%  of sensing sodium halides with varying halide concentrations, which could be a demonstration of using the sensor device for quantitative analysis of halide solutions. A response saturation in the higher concentration range was observed. This is possibly because of the sensor-analyte association-dissociation kinetics had reached an equilibrium state,63 and is a typical observation for chemical sensors.64–67vs. [NaF] in the low NaF concentration range of 0–2.4 mM. The slope and coefficient of determination (R2) of the calibration curve was found to be 15.3 and 0.95, respectively. The limit of detection (LOD) was calculated using the 68σ is the relative standard deviation of the sensitivity plot of the device in the absence of analyte and m is the slope of the analyte calibration curve. The LOD for fluoride (NaF) was found to be 0.40 mM for the PIDG-BT-C20 WGOFET device, which is better than the LOD (0.7 mM) of the previously reported OFET-based fluoride ion sensors.38C to recognize the fluoride ions. Since fluorine has the highest electronegativity among all elements, the N–H⋯F− interaction is expected to be the strongest among all halides. The interaction of the amide hydrogen bonds on PIDG-BT-C20 with fluoride ions may be similar to that of some previously reported host-guest supramolecular fluoride chemosensors using amide receptors.69–721H NMR titration71–73 was adopted as an effective method to verify the N–H⋯F− interactions. The small molecule IDG-C20-Br was used as the model compound since the 1H NMR signals of polymer PIDG-BT-C20 were very broad and weak. 1H NMR titration was performed using tetrabutylammonium fluoride (TBAF) in CDCl3. It was observed that the amide proton peak Ha at 9.01 ppm broadened when the ratio of TBAF/IDG-C20-Br was increased from 0 to 0.3 molar equiv. and disappeared at a 0.5 equiv. , bromide (TBAB), or iodide (TBAI) did not result in any observable changes in the Ha signal . Interestingly, it was found that when the PIDG-BT-C20/TBAF and IDG-C20-Br/TBAF blended film samples were dissolved back into chloroform, the spectra of the obtained solutions became identical to those of pristine PIDG-BT-C20 or IDG-C20-Br  with the B3LYP/3-21G* level under tight convergence. The overall charge was set to be −1 and a singlet spin state was used as the initialization parameters for DFT calculations for the model complexes, IDG-F, IDG-Cl, IDG-Br and IDG-I. The neutral indigo model compound IDG was also simulated as a reference .a⋯N bond. When a fluoride anion was added, the formed complex IDG-F has an increased Ha⋯N distance of 1.66 Å, while the distance between Ha and F−, [Ha⋯F], is very short at 1.00 Å, indicating the formation of a new hydrogen-bond . A summary of simulation results can be found in Table S1 in the ESI.†The simulation results show that IDG has an interatomic distance of 1.02 Å for the Hgen-bond . On the 51,76 which contains amide moieties , but no N–H groups and intramolecular hydrogen bonds, was used as a channel in a WGOFET for halide ion sensing. It was found that the device showed the relative responses in the order of F− < Cl− < Br− < I−  for NaF was calculated to be 0.40 mM, which is better than the previously reported OFET based fluoride sensors. The mechanism of fluoride selectivity of the WGOFET sensor was studied through 1H NMR, UV-Vis and computer simulations, which indicated the much stronger interaction of fluoride with the intramolecular hydrogen bond N–H⋯OC in the indigo unit of the polymer compared with other halides. The disruptive effect of fluoride on the hydrogen bond would alter the electronic structure and thus the charge transport properties of the polymer, leading to the high sensitivity of the device towards fluoride. Our results demonstrated that the intramolecular hydrogen bond-containing indigo polymers are a promising class of the semiconductors for OFET based fluoride sensors, which showed good field-effect transistor performance, excellent stability at ambient conditions, and high sensitivity and selectivity towards fluoride ions. They have the potential to be printed on flexible plastic substrate as a low-cost, portable alternative or replacement to other types of fluoride sensors.In this work, two indigo-based donor–acceptor polymers PIDG-T-C20 and PIDG-BT-C20, where the indigo building block has intramolecular hydrogen bonds, were designed and synthesized for use as channel semiconductors in OFETs for fluoride ion sensing. The charge transport performance of PIDG-T-C20 and PIDG-BT-C20 was evaluated in BGBC OFETs, demonstrating highest hole mobilities of up to 0.016 and 0.028 cmThere are no conflicts to declare.RA-009-C9RA04302K-s001


We tested whether a procedure in a hexapod simulator can cause incorrectassumptions of the bank angle  in airline pilots as wellas incorrect interpretations of the attitude indicator (AI).The effect of the leans on interpretation errors has previously beendemonstrated in nonpilots. In-flight, incorrect assumptions can arise due tomisleading roll cues .n = 18) performed 36 runs, in which they were askedto roll to wings level using only the AI. They received roll cues before theAI was shown, which matched with the AI bank angle direction in most runs,but which were toward the opposite direction in a leans-opposite condition(four runs). In a baseline condition (four runs), they received no rollcues. To test whether pilots responded to the AI, the AI sometimes showedwings level following roll cues in a leans-level condition (four runs).Pilots compared to the baseline (6.9%) or leans-level condition (0.0%). There was apronounced learning effect in the leans-opposite condition, as 38.9% ofpilots made an error in the first exposure to this condition. Experience had no significant effects.The leans procedure was effective in inducing AI misinterpretations andcontrol input errors in pilots.The procedure can be used in spatial disorientation demonstrations. Theresults underline the importance of unambiguous displays that should be ableto quickly correct incorrect assumptions due to spatial disorientation. In modern aircraft, the main task of airline pilots is to monitor the automaticsystems and intervene when the automation fails. Such interventions often require aprompt and correct interpretation of the instruments, which may be difficult afterprolonged periods of eventless flight and distraction . In an aopposite to the required direction, which is also referred toas a roll reversal error. It has been argued that these errors may result from anambiguity of the artificial horizon, or attitude indicator (AI), which is the maindisplay system to determine the aircraft bank angle  with less than5000 flight hours, and high experience (n = 10) with more than10,000 flight hours. Participant characteristics are displayed in Eighteen airline pilots participated in the study. All were familiar with flyingmedium- to large-sized aircraft using a W-shaped control column or yoke (such asin Boeing aircraft), which was used in the experiment. Pilots were divided intotwo experience groups: low experience (The experiment was performed in the Simona Research Simulator (SRS) at thefaculty of Aerospace Engineering of the Delft University of Technology see,. The SRSThe simulation used an aircraft flight dynamics model representative of amedium-size twinjet aircraft (Airbus A320). Participants were only able tocontrol the roll axis using a control-loaded column. The only display that wasprovided was a simplified digital primary flight display PFD; see , showingimmediately when the AI appeared, as “an intuitive responsewas desired and reaction time would be one of the outcome measures.” Pilotsfamiliarized themselves with the simulator and controls by flying a number ofturns for approximately 3 min.The tasks were performed as single-pilot crews using the left-hand seat of thesimulator. Pilots were tested either in the morning (9:00–12:00 AM) or in theafternoon (1:00–4:00 PM). Each pilot was first briefed, provided informedconsent, and filled in a general questionnaire. Pilots were told that theexperiment was about “assuming manual control after a period of automatic flightand distraction.” Following a period of automatic flight, they were to take thecontrol column when the autopilot off alert sounded, wait for the AI to appear,and then roll the aircraft level using the AI. They were instructed to respondA leans-inducing motion profile was developed and tuned with nine nonpilots see are asfL = 2000,V = 200; During the entire procedure, the speed remained fixed at approximately 230 knots,and the altitude at 10,000 feet. There was also a continuous light turbulence for the next 33 s, while theplatform maintained a steady roll angle of 3.5° . This ad2. This was abovethe documented perceptual threshold of the vestibular system . This simulated an in-flightsituation of a subthreshold roll to a bank angle. The error rate wasexpected to be around 4.5%–8.7% based on studies in fixed-basesimulators .Leans-opposite condition runs. This variation is shown in Leans-level condition runs. This variation featured the same leans cuesas the baseline and leans-opposite runs, but the AI at the end ; 98 sprThe pilots performed the runs in two sessions of continuous flight, which lasted40 min each. There was a 10-min break between the sessions. The first sessionstarted with eight filler runs, of which the first two runs were practiceruns.The second session started with six filler runs. In the subsequent 12 runsof each session, there were six more filler runs and two runs of each of thetest conditions . One “level”filler run  was featured in the six fillerruns at the start, and one in the next six filler runs of the session.The test conditions and filler runs all featured an equal number of runs with thefast roll cue toward the left and right. Several sequences of the last 12 runsin each session were created using the Latin Square method, so that none of thetest condition runs were systematically preceded by a certain type of run. Eachsession in all sequences ended with a test condition run, and the sequences werecounterbalanced between the experience groups. The following variables were obtained in the test conditions.An error was defined as a roll input away from level following AIpresentation, which caused the control column to exceed 1° of rolldeflection compare .When an error was detected, the maximum bank angle deviation toward the wrongdirection was measured. The initial aircraft bank angle when starting theresponse was subtracted.This was the time between AI presentation and the first input . This was measured for correct inputs only. Ifthere was an initial incorrect interpretation before a correct response,there may be a moment of hesitation and starting the correct response maythus require more time.As a check that the distraction task induced sufficiently high workload, thesubjective workload of this task was rated on the NASA Task Load Index in theleans-opposite condition. High experience was expected to lead to fewer and lesssevere errors, as well as shorter reaction times, especially in theleans-opposite condition due to fewer misinterpretations. No differences wereexpected in the workload ratings of the distraction task.Generalized Estimating Equations (GEE) tests were performed for ordinal data(error percentage), with the factors: Group (low and high experience), Condition, and the Group × Conditioninteraction. For linear data (error severity and reaction time), a mixed model(Group × Condition) analysis of variance (ANOVA) was performed. As reaction timewas limited to correct responses, the leans-level condition was not included inthis analysis.t-tests (for linear data).Two comparisons were performed: between the leans-opposite and baselinecondition, and between the leans-opposite and leans-level condition. Wecorrected for two comparisons using Bonferroni correction (requiredp = .025).Post-hoc comparisons between conditions were performed with Wilcoxon signed rank or paired-samples To test whether there was a learning effect, the error frequency in the wholegroup in the first run of each condition was compared with that in the last runof each condition using a McNemar test.t-tests.Workload ratings were compared between the groups using independent-samplesNone of the pilots reported any motion sickness issues when asked halfway into theexperiment and at the end. 2 = 9.16, p = .002, but notfor Group, and there was no significant Condition × Group interaction. Thepost-hoc comparisons showed that there was a significant difference between theleans-opposite (median = 25%) and baseline condition (median = 0%),Z = −2.71, p = .007, and between theleans-opposite and leans-level condition (median = 0%), Z =−3.07, p = .002. An interesting finding was that pilots alsomade 3.9% errors in the filler runs, in which the direction of the fast roll cuematched with the shown AI.The GEE analysis of the error percentage showed that there was a significant maineffect for Condition, χp = .016. The error percentage decreased linearly over thefour runs. In the baseline condition, two pilots (11.1%) made an error in thefirst run and none in the last run, but this difference did not reachsignificance. No errors were made in the leans-level condition.The McNemar test showed that there was a significant learning effect in theleans-opposite condition. Seven pilots, or 38.9%, made an error in the firstrun, whereas none of the pilots made an error in the final run,All errors are displayed in F = 9.20, p = .008. The high experiencedgroup responded on average .46 s slower than the low experience group, which wasin contrast to our hypothesis.On average, pilots responded around 2 s following the AI presentation. There wasno significant main effect of Condition, no significant Condition × Groupinteraction, but there was a significant effect of Group,t = 2.19,p = .043, and frustration t = 2.09,p = .043, than the low experience group.Pilots rated the mental demand of the secondary task at 62.4 points , which iWe presented pilots with a leans protocol, which featured a roll motion cue in theopposite bank direction of a subsequently shown AI. We simulated an in-flightsituation of a subthreshold roll to 33.5° bank, and a super-threshold rollback to30°. In response to these cues, 38.9% of pilots made a roll reversal error duringthe first encounter, and 19.4% errors were made on average in the four runs. Thiserror rate was significantly higher than in the baseline condition (6.9%), whichfeatured no roll motion cues followed by a banked AI. This indicates that the errorswere indeed caused by the leans protocol. The error rate was also significantlyhigher than in the leans-level condition (0.0%), which featured the same roll motioncue but followed by a wings-level AI. This indicates that the leans cues induced aninterpretation error of the AI, and that pilots were not responding based only onwhat they felt. In line with previous experiments , this poThe error rate found in this study for airline pilots is lower than that innonpilots, whose expectation of bank angle was manipulated in a fixed-base simulator compared to nonpilots ina comparable experiment  cues oninterpretation of the attitude indicator was investigated.For this, a new leans procedure and simulator motion profile was developedfor a hexapod simulator.The disorienting motion cues caused an increase in roll reversal errors by afactor of almost 3.The results show that incorrect expectations can have hazardous effects ondisplay interpretation, and underline the importance of intuitive displaydesign.The leans procedure, which we found to be effective, can be used for spatialdisorientation research and training in a cost-effective hexapod motionsimulator.


Herein, we demonstrate the critical role of the metal ion in tuning the electronic ground state of such materials. While VCl2(pyrazine)2 is an electrical insulator, TiCl2(pyrazine)2 displays the highest room-temperature electronic conductivity (5.3 S cm–1) for any metal-organic solid involving octahedrally coordinated metal ions. Notably, TiCl2(pyrazine)2 exhibits Pauli paramagnetism consistent with the specific heat, supporting the existence of a Fermi liquid state . This result widens perspectives for designing molecule-based systems with strong metal-ligand covalency and electronic correlations.Electronic synergy between metal ions and organic linkers is a key to engineering molecule-based materials with a high electrical conductivity and, ultimately, metallicity. To enhance conductivity in metal-organic solids, chemists aim to bring the electrochemical potentials of the constituent metal ions and bridging organic ligands closer in a quest to obtain metal- d-orbitals of the metal and the π-orbitals of the organic molecule. Here, the authors present two coordination solids, VCl2(pyrazine)2 and TiCl2(pyrazine)2, with remarkably different electrical conductivity. While the former is an insulator, the latter displays the highest conductivity of any octahedrally coordinated metal ions based metal-organic solid.Achieving high conductivity in metal-organic solids can be challenging, due to the difficulty of obtaining a good overlap between the  One of the main factors that currently limits the further development of such applications is the electrically insulating nature of almost all coordination solids11. Encouraged by the incomparable possibilities available to modern molecular and coordination chemistries for tuning the physical properties of molecule-based materials, scientists are actively working on lifting this limitation12. While organic metals were discovered already in 197313, there are only very few coordination solids displaying clear metallic conductivity. Among them, layered materials, pillared on transition metal ions with square-planar or distorted tetrahedral coordination geometries, have been reported to exhibit metallic properties as well as superconductivity17. For example, Dincă et al. reported metallicity in the M3(hexaiminobenzene)2 , as suggested from ultraviolet photoelectron spectroscopy and band structure calculations18. Later, Marinescu and coworkers demonstrated that air-exposure of an iron 2,3,6,7,10,11-triphenylenehexathiolate MOF led to a material featuring a semiconducting-to-metallic phase transition around room temperature19. The presence of a metallic state is commonly evidenced experimentally by an increase of the electronic conductivity upon lowering the temperature, as reported for a single crystal of CuI2 16. Unfortunately, this straightforward fingerprint of a metallic state is not easy to obtain when the resistivity measurements are performed on pressed polycrystalline or powdered samples for which grain contacts often dominate the charge transport properties20. Therefore, metallic states in coordination solids may indeed be more widespread than experimentally demonstrated.The realization of strong conjugation between metal-centered 26 is found to be lower than for systems based upon square-planar metal-ion nodes, which puts severe limitations on the materials that could be exploited. A significant orbital overlap between the metal-ion centered d orbitals and the bridging ligand π system is key for engendering high conductivity and critical for designing a metallic state. However, this picture is not theoretically constrained to square-planar coordinated metal ions, but solely relies on orbital symmetries and relative energies26. We recently reported the coordination solid CrCl2(pyz)2 (pyz = pyrazine) to behave as a semiconductor that could be converted into an insulator upon post-synthetic modification28. This material, synthesized from CrII and pyrazine, features octahedrally coordinated Cr centers and exhibits a sizable room-temperature conductivity of 0.032 S cm–1. The electrochemical CrIII/CrII reduction potential allows an electron transfer to a single pyrazine ligand during the synthesis leading to CrIII and half-reduced pyrazines (pyz2)– in the final material. This partially reduced ligand scaffold, in conjunction with close spatial proximity of spin-carriers, proffers high electronic conductivity by a charge hopping mechanism. Naturally, the conductivity is expected to be strongly dependent on the energy of the d orbitals that are involved in the conduction bands at the Fermi level. To this end, the substitution of chromium by other neighboring transition elements (M) provides a route to chemically engineer the electronic conductivity in MCl2(pyz)2 analogs.While coordination solids are commonly built with octahedrally coordinated metal ions, their electronic conductivityVCl2(pyz)2 and TiCl2(pyz)2 coordination solids display contrasting electronic properties. While VCl2(pyz)2 features VII metal centers and a neutral pyrazine scaffold, crystallographic analysis, X-ray absorption spectroscopy, and density functional theory (DFT) studies reveal the presence of TiIII in TiCl2(pyz)2 in conjunction with a one-electron reduced ligand scaffold akin to that of the analoguous CrCl2(pyz)2 material27. However, the magnetic properties and electronic conductivity of VCl2(pyz)2 and TiCl2(pyz)2 are strikingly different to those of CrCl2(pyz)2. While VCl2(pyz)2 is a simple antiferromagnetic insulator, both temperature-independent (Pauli) paramagnetism and a residual electronic specific heat term are observed in TiCl2(pyz)2, revealing an unprecedented Fermi liquid metallic state in a coordination solid.In this work, we demonstrate that the novel 2 and VCl2 with a large excess of pyrazine at 200 °C produces deep black TiCl2(pyz)2 and dark purple VCl2(pyz)2, respectively, as microcrystalline powders (see Methods). Scanning electron microscopy 2 over TiCl2(pyz)2, with micrometer and sub-micrometer size crystallites, respectively. Synchrotron powder X-ray diffraction data at room temperature suggest that TiCl2(pyz)2 and VCl2(pyz)2 are isostructural, and structurally similar to the Cr analog 2 (space group Immm), TiCl2(pyz)2 and VCl2(pyz)2 are isomorphous to the previously reported CoCl2(pyz)2 and NiCl2(pyz)2 compounds, crystallizing in the tetragonal I4/mmm space group30. Their structures consist of a perfectly square lattice of pyrazine-bridged metal centers with two trans-coordinated and non-bridging chloride ligands oriented along the crystallographic c direction. Single-crystals were obtained only for VCl2(pyz)2 by optimizing the thermal process during the synthesis (see Methods). X-ray crystallographic analysis on a single crystal of VCl2(pyz)2 at 120 K further corroborates the above findings on microcrystalline powders 2 amount to 2.14 and 2.37 Å, respectively, which are similar to the V–N and V–Cl bond lengths in VCl2(pyz)2 of 2.15 and 2.42 Å (2.14 and 2.42 Å at 120 K), respectively. The molecular compound trans-[VIICl2(py)4] (py = pyridine) closely mimics the local coordination geometry of VCl2(pyz)2 with V–N and V–Cl bond lengths of 2.18 and 2.46 Å, respectively 4]32 complex with 2.17 Å Ti–N and 2.44 Å Ti–Cl bond lengths (at 153 K) with the slightly shorter bonds observed in TiCl2(pyz)2.The reaction of TiClTiCl2(pyz)2 and VCl2(pyz)2, X-ray absorption near edge structure (XANES) spectra were recorded at the Ti and V K-edges , which were used as references. The energy position of the K-edge is a function of effective metal oxidation states or, in the context of the K-edge XANES, a function of the effective nuclear charge (Zeff) seen by the metal core 1s electrons. As the oxidation state increases, the position of the edge shifts up in energy owing to the increase in Zeff. This effect is convincingly illustrated by the spectral changes shown in Fig. II to a MIII formal oxidation state, the edge positions shift by ∼3 and 2.5 eV to higher energy for the titanium and vanadium chlorides, respectively. In the case of the MCl2(pyz)2 coordination solids, the energy position of the Ti K-edge in TiCl2(pyz)2 differs from that of the TiCl3 reference by 0.3 eV, whereas the difference between the V K-edge position in VCl2(pyz)2 and VCl2 is only 0.2 eV. These experimental results confirm the expected different oxidation states for the metal ions in the two MCl2(pyz)2 analogs, TiIII in TiCl2(pyz)2 and VII in VCl2(pyz)2. This conclusion is further supported by the XANES measurements at the Cl K-edge (Supplementary Information Section TiCl2(pyz)2 –) but not in VCl2(pyz)2. These results suggest the presence of a Robin-Day34 Class II or III mixed-valence state between the pyrazine ligands in TiCl2(pyz)2, which is commonly accompanied by an intense absorption in the near-IR region resulting from inter-valence charge transfer 2 spectrum displays an absorption maximum at ∼12,000 cm–1, which may be assigned as the VII-localized 4Tg2 ← 4Ag2 (Oh) transition35.To determine the oxidation state of the metal ions in TiCl2(pyz)2, the high-temperature value of the magnetic susceptibility (χ) - temperature product, χT . The temperature dependence of the magnetic susceptibility shown in Fig. –4 cm3 mol–1) and a residual Curie paramagnetic contribution estimated at 1% of S = 1/2 impurities. This observed temperature-independent paramagnetism suggests the presence of itinerant electrons in TiCl2(pyz)236.In the case of VCl2(pyz)2, the 400-K χT value of 1.03 cm3 K mol–1 is significantly lower than expected for a S = 3/2 VII ion 37. The corresponding fit  and J/kB = –28.2(5) K (with the –2J∑ijSiSj Hamiltonian definition). The strength of the superexchange coupling mediated by neutral pyrazine ligands is remarkably strong, and at least one order of magnitude larger than found in any other coordination solid pillared by neutral pyrazine39. Broken-symmetry DFT calculations performed on a dinuclear [(pyz)3Cl2V(μ-pyz)VCl2(pyz)3] model fragment affords a coupling constant of J/kB = –79 K (Supplementary Information Sections VCl2(pyz)2 mediated by pyrazine. Below ∼120 K, the susceptibility becomes strongly magnetic field-dependent 2 and VCl2(pyz)2 in supercells containing 92 atoms. For VCl2(pyz)2, the lowest energy spin configuration corresponds to an AF ground state with VII ions and a non-magnetic ligand scaffold . In TiCl2(pyz)2, DFT calculations confirm the TiIII oxidation state, the singly reduced pyrazine scaffold, and yield a significant density of state (DOS) at the Fermi level (5 states eV–1 per formula unit), in agreement with the observed temperature independent paramagnetic susceptibility (Pauli paramagnetism). In addition to the Ti d and Cl p-states, C and N p-states of the pyrazine ligands are also found to contribute to the DOS at the Fermi level. This hybridization thus suggests a charge delocalization in the pyrazine scaffold 2 measured on a pressed pellet is as low as ∼10–10 S cm–1, which is a drastic reduction from the value of 0.032 S cm–1 observed for CrCl2(pyz)2. In contrast, σRT is two orders of magnitude higher for TiCl2(pyz)2 and reaches 5.3 S cm–1 at 300 K. Such a large σRT is only surpassed by a handful of coordination solids and is larger than the values found in any system with octahedrally coordinated metal-ion nodes 2 is displayed in Fig. σRT value and the Pauli paramagnetism evidenced in Fig. The room-temperature electronic conductivity, σ exhibits a crossover around 30 K between two different conductivity regimes, each following a σ = σ0 exp(–∆/kBT)1/2 law  measurements were performed up to ±9 T, from 1.8 to 100 K 2 data 2 is semiconducting or metallic, the specific heat, Cp, was measured  was estimated experimentally. Importantly, the γ coefficient is directly proportional to the DOS at the Fermi level and is linked to the degree of electronic correlations (Supplementary Information Section γ is thus expected to vanish according to the absence of electronic states at the Fermi level. In agreement with the conductivity data (vide supra), the signature of an insulator is experimentally observed for VCl2(pyz)2 with CpT–1 extrapolating to zero at 0 K  mJ mol–1 K–2). In contrast, a residual specific heat contribution is clearly detected for TiCl2(pyz)2, yielding γ = 18.0(3) mJ mol–1 K–2. This Sommerfeld coefficient is an order of magnitude larger than that of a normal metal and compares well to the γ value of two canonical unconventional superconductors possessing strong electronic correlations: the overdoped cuprate Tl2Ba2CuOδ6+ (γ = 7(2) mJ mol–1 K–2)43 and Sr2RuO4 (γ = 38(2) mJ mol–1 K–2)44. The Sommerfeld coefficient for TiCl2(pyz)2, which is slightly larger than the value inferred from DFT calculations (γDFT = 9–12 mJ mol–1 K–2), is indicative of electronic correlations within Landau’s Fermi liquid theory45. To further confirm this conclusion, the results from the specific heat and susceptibility measurements were combined to calculate the dimensionless Wilson ratio46, RW = π2kB2χPauli/(3μ0μB2γ), which is theoretically expected to be 1 for noninteracting electrons as well as for correlated Fermi liquids. Experimentally, many strongly correlated electron systems display Wilson ratios between 1 and 2, higher values of RW being reported for correlated Fermi liquids close to a ferromagnetic instability48. In the case of TiCl2(pyz)2, the Wilson ratio is 1.2, supporting the presence of itinerant carriers and a strongly correlated metallic ground state 2 and VCl2(pyz)2, have been reported in this work. Belonging to the family of layered MCl2(pyz)2 materials27, these new analogs display distinctively different and unique physical properties. Divalent VII ions and pristine, non-reduced pyrazine ligands are found in VCl2(pyz)2, which possesses an antiferromagnetically ordered ground state below ∼120 K with unprecedently strong intra-layer VII–VII antiferromagnetic interactions (J/kB = –28 K). In contrast and like CrCl2(pyz)227, TiCl2(pyz)2 contains trivalent metal-ion nodes (TiIII) and, formally, a singly reduced pyrazine ligand per formula unit. This peculiar electronic structure in TiCl2(pyz)2 induces not only the highest experimental electronic conductivity at room temperature (5.3 S cm–1) for any coordination solid based on octahedrally coordinated metal ions, but also a large positive magnetoresistance at low temperature. The combined analysis of specific heat, magnetic measurements and DFT calculations suggests the presence of a correlated Fermi liquid state in TiCl2(pyz)2. Nevertheless, the temperature dependence of the electrical resistivity, ρ, does not follow the expected behavior for a Fermi liquid metal (ρ = ρ0 + constant × T2) due to the granular nature of the material as previously observed in coordination solids. Alternatively, the Sommerfeld coefficient, which largely exceeds the typical values for non-magnetic metals, and the dimensionless Wilson ratio (close to unity) were used to firmly establish the correlated metallic ground state of TiCl2(pyz)2. The ability to chemically tune the ground state of the MCl2(pyz)2 coordination solids, from an antiferromagnetic insulator in VCl2(pyz)2, a semiconducting ferrimagnet in CrCl2(pyz)227 to a strongly correlated Fermi liquid in TiCl2(pyz)2 represents a promising path towards exotic quantum phases of matter, such as superconductivity. Research on strongly correlated coordination solids remains an immensely unexplored area, but as shown in this work, the versatility of coordination chemistry combined with reducible ligands offers a powerful entry point for the exploration of new metallic and, hopefully, superconducting metal-organic materials.In summary, the synthesis and physical characterization of two isostructural coordination solids, 2 99.98%, VCl2 85%, pyrazine ≥99%) were purchased from Sigma-Aldrich and used without further purification. Acetonitrile was dried using an Innovative Technologies solvent purification system and subsequently stored over 3 Å molecular sieves. Due to the air-sensitivity of the reagents and the final products, all manipulations were carried out under a dry dinitrogen or argon atmosphere. The reference compound, trans-[VIICl2(pyridine)4], was synthesized through a modified literature method by the direct reaction of VCl2 and pyridine in a soda glass ampule at 200 °C31. Elemental analyses were performed at the Mikroanalytisches Laboratorium Kolbe . Synthesis of TiCl2(pyz)2: A 25 mL teflon-lined stainless-steel autoclave reactor was charged with TiCl2  and pyrazine , and placed in a furnace (200 °C) for 12 h. The reactor was cooled to room temperature and the black powder product of TiCl2(pyz)2 was washed with 20 mL of acetonitrile and dried in vacuo. Yield: 80–85%. Elemental analysis : C , H , N , Cl , Ti . Synthesis of VCl2(pyz)2: VCl2(pyz)2 was synthesized following a similar procedure to that of TiCl2(pyz)2, but using VCl2 instead of TiCl2. Yield: 80-85%. Elemental analysis : C , H , N , Cl , V . In order to obtain single-crystals suitable to X-ray diffraction analysis, 0.060 g of VCl2 (0.49 mmol) and 1.5 g of pyrazine (19 mmol) were introduced into a soda glass ampule (V = 21 mL) that was cooled in liq. N2 and flame-sealed under vacuum. The ampule was placed in a furnace, which was heated to 200 °C over 5 h. After 80 h at 200 °C, the temperature was increased to 210 °C over 30 h, maintained constant for 2 h and decreased to 200 °C over 30 h. After staying at 200 °C for 80 h, the sample returned to room temperature in about 30 h. The resulting single-crystals were isolated as described above. It is worth noting that it was not possible to obtain single-crystals of TiCl2(pyz)2 even after a similar optimization of the thermal process.All chemicals (TiClFurther characterization methods, including scanning electron microscopy, crystallography, X-ray absorption and optical spectroscopies, magnetism, resistivity, specific heat and DFT calculations are described in the Supplementary informationSupplementary Dataset 1Supplementary Dataset 2Supplementary Dataset 3Supplementary Dataset 4Description of Additional Supplementary Files


CNGA3 in three large consanguineous Pakistani families. Fundus examination and optical coherence tomography (OCT) imaging revealed myopia, thin retina, retinal pigment epithelial cells loss at fovea/perifovea, and macular atrophy. Combination of Sanger and whole exome sequencing revealed three known homozygous missense variants ; c.847C>T, p.(Arg283Trp); c.1279C>T, p.(Arg427Cys)) in CNGA3, the α-subunit of the cyclic nucleotide-gated cation channel in cone photoreceptor cells. All three variants are predicted to replace evolutionary conserved amino acids, and to be pathogenic by specific in silico programs, consistent with the observed altered membrane targeting of CNGA3 in heterologous cells. Insights from our study will facilitate counseling regarding the molecular and phenotypic landscape of CNGA3-related cone dystrophies.Cone photoreceptor dysfunction represents a clinically heterogenous group of disorders characterized by nystagmus, photophobia, reduced central or color vision, and macular dystrophy. Here, we described the molecular findings and clinical manifestations of achromatopsia, a partial or total absence of color vision, co-segregating with three known missense variants of  This re cyclase ,7.CNGA3 or CNGB3 have been reported in humans with achromatopsia (ACHM), cone–rod dystrophy or other disorders such as progressive cone dystrophy [CNGA3 associated spectrum of inherited cone disorders, ACHM accounts for 20–30% cases and is characterized by vision impairment, partial or complete color blindness, nystagmus, and photo dysphoria especially during daytime [CNGA3.In cones, CNG channels are hetero-tetramers that are comprised of three CNGA3 subunits and one CNGB3 subunit . Geneticystrophy ,9,10,11. daytime . Impaire daytime . OverallThe current study followed the tenets of the Declaration Helsinki and received approval from the Institutional review Board (IRB) Committees at the Bahauddin Zakariya University, Pakistan, Liaquat University of Medical & Health Sciences, Pakistan, and University of Maryland, USA. Two families  were enrolled from the Punjab province of Pakistan, while the third family (LUSG06) was identified from the Sindh province. Informed written consent was obtained from all participants. Detailed ophthalmic examination including fundoscopy, slit lamp biomicroscopy, optical coherence tomography  and electroretinography  was performed at the local hospitals in Pakistan. Peripheral blood samples were collected from participating individuals for genomic DNA extraction and genetic analysis.CNGA3 was performed using the proband DNA samples from families PKAB149 and PKED06. While whole exome sequencing (WES) was used to identify the disease-associated variants in family LUSG06. For WES, genomic libraries were recovered for exome enrichment using the Agilent Sure Select Human Expanded All Exon V5 (62 Mb) kit . Libraries were sequenced on an Illumina HiSeq2500  with average 100× coverage. Data analysis used the Broad Institute’s Genome Analysis Toolkit [Sanger sequencing of all the coding exons and exon-intron junctions of  Toolkit , and var Toolkit . Sanger CNGA3 protein structural domains, while Clustal omega multiple alignment was used to assess the conservation of the identified variants across several different species. Varsome  was used for pathogenicity predictions, and allele frequencies were obtained from gnomAD  database. The 3-Dimensional (3D) protein structures were generated using Phyre2 server  with “Intensive Mode” option, which is a combination of template-based modeling and ab initio methods.NCBI  conserved domain was used to generate In order to determine the impact of the newly identified mutation on CNG channel functionality, homomeric as well as heteromeric CNG channels were heterologously expressed and surface expression quantified in human embryonic kidney 293 (HEK293) cells, as previously described .A multi-generation family with four affected individuals was ascertained from Punjab province of Pakistan. Only two (III:2 and III:3) of the four affected individuals participated in the study A. AccordCNGA3 (NM_001298), revealed a transition variant c.827A>G, predicted to introduce a missense change in the encoded protein (p.(Asn276Ser)), segregating in a homozygous fashion among the affected individuals , including six affected individuals suffering with pendular nystagmus, photophobia (in early infancy) with myopia , was enrCNGA3 was found segregating with the phenotype in the family PKED06 ) in y PKED06 A. The p.y PKED06 B and prey PKED06 . The argy PKED06 C. Accordy PKED06 D, and heIn family LUSG06, who were enrolled from Sindh province of Pakistan, the funduscopic examination of the affected individuals revealed the presence of bilateral symmetric pallor optic discs C. AffectCNGA3, which segregated with the phenotype in the family LUSG06  revealed a transition variant c.1279C>T (p.(Arg427Cys)) in exon 8 of y LUSG06 A. The p.y LUSG06 . In conty LUSG06 B facing y LUSG06 C. The p.y LUSG06 D.CNGA3 channels harboring these two variants in HEK293 cells and performed immunocytochemistry experiments ) in an autosomal recessive pattern of inheritance [CNGA3 takes part in forming cone’s ion-conduction channel with its modulator called CNGB3.In humans, cGMP (second messenger) mediates G-protein coupled cascade to carry out visual signal transduction. CNGA3 protein is activated by this cGMP and triggers cation channel opening which ultimately lead towards depolarization of cone photoreceptors. In this study, we have reported three large unrelated consanguineous Pakistani families segregating with three known rescence . Along wchromacy . A cone–eritance . CNGA3 tCNGA3 responsible to cause different disease phenotypes, e.g., achromatopsia, cone–rod dystrophy, and retinitis pigmentosa (HGMD) amongst others. Additionally, CNGB3 mutations in human can cause severe reduction in rod function [CNGA3 harboring missense variants after treating the heterologous cells with chemical and pharmacological chaperons [CNGA3 disease causing variants.To date, there are about 197 different variants reported for function ,26. Two haperons , which cCNGA3 patients. Previous studies have shown that loss of CNGA3 function leads to rod dysfunction, potentially following alterations in cone synapses [Cnga3 null mice also exhibit loss of M-cone opsin-expressing photoreceptors, which can be rescued by gene replacement packaged into adeno-associated virus [CNGA3) for nine patients affected with CNGA3-associated achromatopsia [Adeno-associated virus-mediated gene replacement therapy is also being developed as another treatment option for synapses . Cnga3 ned virus . Intrigumatopsia . In thismatopsia . Thus, e


The thermal expansion of NiGNT–epoxy composite was also restricted in the aligned direction of NiGNT nanostructures. Thus, NiGNT–epoxy composites show great potential as future aerospace, aviation, and automobile materials.In this study, graphene oxide–carbon nanotubes nanostructures decorated with nickel nanoparticles (NiGNT) were prepared through the molecular-level-mixing method, followed by a reduction process, and then applied as reinforcements to enhance the epoxy resin matrix. The ferromagnetism of the Ni nanoparticles allowed NiGNT nanostructures to be vertically aligned within the composite with the assistance of a magnetic field. Due to the alignment distribution of the NiGNT, the composites demonstrated enhanced anisotropic thermal and electrical conduction performances, compared with pure epoxy and randomly distributed composites. The aligned distribution of NiGNT–epoxy composites displayed 2.7 times higher thermal conductivity and around 10 With the increasing requirements for lighter materials in aerospace, aviation, and automobile fields, polymer matrix composites (PMCs) have been widely investigated ,2. EpoxyAmong the filler materials, carbon materials, i.e., carbon fibres (CFs) ,9,10, gr3O4, and other ferrites) has become a common method , sulfuric acid  and acetone were purchased from Sigma-Aldrich , while nitric acid (HNO3) was purchased from Fisher Scientific. Epoxy resin was supplied by Aktinium Technologies Limited and was divided into part A and part B (part B was regarded as the hardener), 2 (LN2), pure Ar gas, and Ar/H2 mixed gas (5% H2 + 95% Ar) were supplied by BOC Gas Cylinders.Graphene oxide (GO) dispersions were purchased from William Blythe Limited , carbon nanotubes (CNTs) were purchased from Carbon Nanotubes Plus , and 3, and 5 mL H2SO4 was kept at 130 °C in an oil bath with a reflux system for 25 min. Afterwards, the acid-treated CNTs were filtrated, rinsed with distilled water, and dried in a vacuum oven overnight.CNTs from Carbon Nanotubes Plus were firstly acid-treated via a simple method . Specifi3COO)2 was dissolved in 50 mL H2O through magnetic stirring, while acid-treated CNTs and GOs were mixed with a ratio of 1:1 and dispersed under ultrasonication for half an hour to obtain a well-dispersed mixture. The obtained carbon mixture was then added to the nickel solution to obtain a weight ratio of Ni:C of 9:1 and stirred for another 2 h. After stirring, the suspension was quickly frozen under liquid nitrogen (LN2) atmosphere and was then freeze-dried via a Labconco FreeZone 76705 freeze dry system at −55 °C and 0.024 mBar. The obtained precursor powders were then reduced under H2/Ar atmosphere at 450 °C for 2 h in a tube furnace. The pure GO–CNT (GNT) sample was prepared in the same way as the NiGNT sample without the addition of Ni(CH3COO)2.As shown in To prepare the epoxy-based composites, NiGNT nanostructures were uniformly mixed with epoxy part A and then cured after adding epoxy part B, as shown in −1. Thermal–mechanical analysis (TMA) was adopted to analyse the coefficient of thermal expansion (CTE) of the epoxy composites with TMA Q400 from room temperature to 100 °C with 5 °C min−1 under a N2 atmosphere. The thermal diffusivity (α) of all the epoxy-based composites was measured using a NETZSCH Light Flash Apparatus (LFA) 467 Hyper Flash instrument at room temperature. The density (ρ) of the as-prepared pellet was obtained through Archimedes’ principle. The specific heat capacities (pC) of all epoxy-based pellets were recorded with TA DSC2500. The thermal conductivity (X-ray diffraction (XRD) results were obtained through the Bruker D8 Advance system (operated at 40 kV and 35 mA) using Cu K-α radiation as the X-ray source, between 5 and 80° 2θ range at a scanning step of 0.01°, and the time per step is 0.12 s. The morphologies of obtained powders and the cryogenic fracture surfaces of epoxy-based composites which were subsequently coated with 10 nm carbon using a sputtering device were recorded through a JEOL 7100 field-emission gun scanning electron microscope (SEM) with the working voltage of 15 kV. A JEOL 2100+ transmission electron microscope (TEM) equipped with a Gatan Ultrascan 1000XP CCD camera was used at a working voltage of 200 kV to acquire TEM images of NiGNT nanostructures, and images of epoxy-based composites at 80 kV. The TEM sample of NiGNT nanostructures was prepared by dispersing the as-prepared powders in ethanol under sonication and then dropping them onto holey carbon TEM grids, while all composite samples were cut using an ultramicrotome with a fine diamond blade into thin films of ca. 100 nm for observation. X-ray photoelectron spectroscopy (XPS) was carried out on a VG ESCALab Mark II spectrometer machine using Al K-α X-ray as an excitation source, with a wavelength of 1486.6 eV. It was performed under a condition of anode voltage of 12 kV and an emission current of 20 mA. A thermogravimetric (TGA) experiment was performed using TA Q600 SDT. The NiGNT sample was measured from 50 to 700 °C in air, while the epoxy-based composites were measured from 50 °C to 800 °C in air, and both with a ramp rate of 10 °C min2 atmosphere. TGA was introduced to investigate the composition of the as-prepared samples [ samples , and theThe morphology of the NiGNT nanostructures was investigated using SEM and TEM techniques, and illustrative images are shown in 2-hybridised C located at 284.6 eV, the C−O (ether bonds) which corresponded to the hydroxyl groups at 286.3 eV, the C=O groups (ketone bonds) at 288.9 eV, and the sp2 hybrid orbit of benzene plane at 290.8 eV [2-hybridised C (at around 284.0 eV) and defect-containing sp2-hybridised C (at around 285.0 eV), indicating that more C defects were obtained as a result of the reduction process in the preparation procedures [2 hybrid orbit of the benzene plane at 290.8 eV—could still be observed, but the intensity of such functional groups  decreased dramatically in the NiGNT sample. This indicates that the C−O groups, in particular, were reduced during the reduction process.To further investigate the chemical composition of GNT and NiGNT nanostructures, XPS was introduced, and the results of both samples are shown in 290.8 eV . The exiocedures . MeanwhiThe distribution of the NiGNT nanostructures in the epoxy matrix, with and without magnetic field assistance, was analysed using SEM. The cross-sectional back-scattered electron images of all NiGNT–epoxy composites are shown in XRD results of the NiGNT-reinforced epoxy composites and their reference samples are shown in To further explore the distribution of NiGNT nanostructures in the epoxy matrix, TEM images of the M-NiGNT-10 sample cut using an ultramicrotome are displayed in 10 Ω m, which indicated the insulation instinct of the pure epoxy sample. After the addition of NiGNT nanostructures, a decreasing trend of electrical resistivity could be observed. Firstly, a minor decrease was found in the NiGNT-10 sample, around 2 × 109 Ω m, and it further decreased to around 1.6 × 108 Ω m in the M-NiGNT-10 sample. With the growth of the NiGNT filler amount, NiGNT-20 composites showed lower electrical resistivity. The electrical resistivity of the NiGNT-30 composite continued to decrease, and the value for M-NiGNT-30 sample reached 4.6 × 105 Ω m. When compared with previous studies, the as-prepared M-NiGNT-30 composite displayed better electrical conduction performance, such as the aligned functionalised CNTs in epoxy via an electric field (~106 Ω m) [6 Ω m) [3O4-coated graphene nanoplatelets in epoxy via a weak magnetic field (~106 Ω m) [5 times higher than the pure epoxy sample.To investigate the electrical conduction property of the composite samples, the electrical resistivities were obtained through ModuLab XM Materials Test System, and the results are shown in 106 Ω m) , the ali [6 Ω m) , and ali2 s−1, and this value increased to 0.119 mm2 s−1 in the NiGNT-10 composites, which indicated that the addition of NiGNT nanostructures was beneficial to thermal conduction. Meanwhile, the M-NiGNT-10 composites showed further improvement in thermal diffusivity (0.141 mm2 s−1) after the magnetic field assistance in the preparation method. When the ratio of NiGNT nanostructures grew, the thermal diffusivities of NiGNT-20 and NiGNT-30 composites were 0.138 and 0.154 mm2 s−1, respectively, while these values for M- NiGNT-20 and M- NiGNT-30 composites were 0.159 and 0.184 mm2 s−1. This indicated the enhancement of NiGNT nanostructures in the epoxy matrix, as well as the further reinforcement of fillers’ alignment with the assistance of the magnetic field. The obtained thermal conductivities of all the epoxy-based composites are shown in −1 K−1 which is close to most reported values [−1 K−1 for the NiGNT-10 sample and 0.242 W m−1 K−1 for the M-NiGNT-10 sample. Meanwhile, this increasing trend continued with an increase in filler ratios. When the weight ratio became 20 wt.%, the NiGNT-20 sample showed a thermal conductivity value of 0.271 W m−1 K−1, whereas this value was 0.326 W m−1 K−1 for the M-NiGNT-20 sample. With the increase in filler amounts, the obtained thermal conductivities demonstrated further increase, with thermal conductivity reaching 0.362 W m−1 K−1 for NiGNT-30 and 0.462 W m−1 K−1 for M-NiGNT-30, respectively. The obtained thermal conductivity of the M-NiGNT-30 composite was around 2.67 times higher than that of pure epoxy. It was found that the thermal conduction performance was effectively enhanced with the addition of NiGNT samples. Meanwhile, the alignment distribution of the NiGNT fillers which was confirmed by the SEM results , and the CTEs of each sample are displayed in −1 °C−1, and the NiGNT–epoxy reference samples demonstrated a similar CTE when compared with the pure epoxy sample. This indicated that the random distribution of the NiGNT nanostructures did not obviously change the CTE of the epoxy matrix, nor did the increasing amount of NiGNT nanostructures effectively decrease the CTE. Furthermore, the M-NiGNT composites displayed additional enhancement in addition to that from increasing the filler ratios. The obtained CTE of the M-NiGNT-10 composites was 9.15 µm m−1 °C−1 which was lower than the NiGNT-10 sample and even lower than the NiGNT-30 sample. With the increase in the NiGNT nanostructures contents, the CTE of M-NiGNT-20 composites decreased to 8.74µm m−1 °C−1, and this value further dropped to 7.87µm m−1 °C−1 for M-NiGNT-30. Therefore, with the assistance of the magnetic field, the vertical distribution of NiGNT nanostructures demonstrated an obvious effect in restricting the expansion of the epoxy matrix along the vertical direction, and the increase in fillers could further enhance the restriction.The thermal expansion of NiGNT–epoxy composites alongside Z direction was measured under glass transition temperature . Meanwhile, the CTE of the M-NiGNT composite also improved, and the aligned NiGNT fillers could effectively prevent the expansion of the epoxy matrix alongside the Z direction when compared with the random distribution ones. Therefore, the aligned NiGNT nanostructures in epoxy matrix with enhanced performances inspire the fabrication of high-performance epoxy-based materials, especially where anisotropic properties are desirable, and make them potential candidates to be applied in fields such as aerospace, aviation, and automobile manufacturing.In summary, uniform Ni-nanoparticle-decorated GNT (NiGNT) nanostructures were successfully prepared through the MLM process with a subsequent reduction method. The extensive characterisation confirmed the well-dispersed structure and uniform decoration of Ni nanoparticles on the GNTs. With the assistance of a magnetic field, NiGNT nanostructures were uniformly distributed in the epoxy matrix, and the SEM results demonstrated that NiGNT fillers were distributed alongside the direction of the magnetic field. Such a 3D alignment network effectively connected the cross-plane direction of the as-prepared NiGNT–epoxy composites. Due to this 3D alignment network, the NiGNT–epoxy composites showed enhanced thermal conductivity (around 2.67 times that of pure epoxy) and lower electrical resistivity (from around 10


CTNND1 encodes a cellular adhesion protein p120-catenin (p120), which is essential for vascularization with unclear function in postnatal physiological angiogenesis. Here, we applied whole-exome sequencing to 140 probands of FEVR families and identified 3 candidate variants in the human CTNND1 gene. We performed inducible deletion of Ctnnd1 in the postnatal mouse endothelial cells (ECs) and observed typical phenotypes of FEVR with reactive gliosis. Using unbiased proteomics analysis combined with experimental approaches, we conclude that p120 is critical for the integrity of adherens junctions (AJs) and that p120 activates Wnt signaling activity by protecting β-catenin from glycogen synthase kinase 3 beta–ubiqutin–guided (Gsk3β-ubiquitin–guided) degradation. Treatment of CTNND1-depleted human retinal microvascular ECs with Gsk3β inhibitors LiCl or CHIR-99021 enhanced cell proliferation. Moreover, LiCl treatment increased vessel density in Ctnnd1-deficient mouse retinas. Variants in CTNND1 caused FEVR by compromising the expression of AJs and Wnt signaling activity. Genetic interactions between p120 and β-catenin or α-catenin revealed by double-heterozygous deletion in mice showed that p120 regulates vascular development through the Wnt/cadherin axis. In conclusion, variants in CTNND1 can cause FEVR through the Wnt/cadherin axis.Familial exudative vitreoretinopathy (FEVR) is a hereditary disorder that can cause vision loss.   FZD4 (frizzled 4) (LRP5 (low-density lipoprotein receptor-related protein 5) (TSPAN12 (tetraspanin 12)  (LRP6 (low-density lipoprotein receptor-related protein 6) (CTNNB1 (catenin beta 1) (ILK (integrin-linked kinase) (DLG1 (discs large MAGUK scaffold protein 1)   , LRP5 (lotein 5) , 9, TSPAanin 12) –12, NDP oglioma) , LRP6 (lotein 6) , and CTN beta 1) –17. Addi kinase) , DLG1 (dotein 1) , and CTNcatenin)  were recCDH5) serving as a central element that joins adjacent endothelial cells through calcium-dependent trans-interactions in the extracellular domain, whereas the cytoplasmic domain is associated with catenins including β-catenin (CTNNB1), γ-catenin (JUP), and p120-catenin (The integrity of the blood vessel barrier is mainly regulated by the adherens junctions (AJs), indispensable cell-cell adhesion molecules at the plasma membrane where adjacent endothelial cells attach to each other with transmembrane linkers . The AJsnin CTNNB, γ-caten-catenin , 23. β-C-catenin , 24.CTNND1 gene) is a core stabilizer of the cadherin/catenin complex that binds to the juxtamembrane domain (JMD) of VE-cadherin in the endothelium  syndrome and nonsyndromic cleft palate  on probands of 140 FEVR families and identified 3 candidate variants in the CTNND1 gene. We further generated an inducible endothelial cell–specific (EC-specific) Ctnnd1-knockout mouse line to show that murine Ctnnd1 is indispensable for postnatal retinal vascularization, which largely phenocopied FEVR-like vascular defects. Using in vivo and in vitro approaches combined with proteomic analysis and rescue assays, we concluded that p120 regulates Wnt signaling through direct stabilization of the cadherin/catenin complex and protection of β-catenin from glycogen synthase kinase 3 beta–ubiquitin–guided (Gsk3β-ubiquitin–guided) degradation. Variants in p120 caused disruption of the AJ integrity, as well as downregulation of Wnt signaling, by disturbing p120/VE-cadherin or p120/β-catenin interaction, which might ultimately cause FEVR. A double-heterozygous deletion study revealed distinct genetic interactions between p120 and β-catenin or α-catenin. These findings reveal a fundamental role for p120 in postnatal vascular development, which provides insights into the pathogenesis of FEVR and other vascular diseases.t palate –32. HoweCTNND1 gene (NM_001085458) were identified in 3 Chinese families: a heterozygous variant c.949C>T (p.R317C) in Family-441, a heterozygous nonsense variant c.1867A>T (p.K623*) in Family-506, and a heterozygous missense variant c.2099G>A (p.R700Q) in Family-3313  variant in Family-441, the proband was diagnosed with FEVR at age 6, manifesting severe retinal exudates with a characteristic peripheral avascular retina in the left eye . His fatThe second heterozygous variant, c.1867A>T (p.K623*), was found in a 1-month-old boy with macular ectopia directed toward the temporal periphery in the right eye and a peripheral avascular area in the left eye . The proThe third heterozygous variant, c.2099G>A (p.R700Q), was found in an 8-year-old boy with peripheral avascularization and severe retinal exudates in the right eye, whereas the vasculature of his left eye was undetectable due to band keratopathy . The youCTNND1 cosegregated with the disease phenotype in their families . The fl/flCtnnd1Pdgfb-iCre-ER (iECKO/iECKOCtnnd1) mice, fl/+Ctnnd1Pdgfb-iCre-ER (iECKO/+Ctnnd1) mice, and their control littermates (fl/flCtnnd1 mice or +/+CtnndlPdgfb-iCre-ER mice) were treated with tamoxifen for 3 consecutive days, from postnatal day 1 (P1) to P3  to P3 . Westernular ECs , D–F.Ctnnd1 in mice  extend numerous filopodia and radially sprout to form the superficial vasculature , 34. The in mice . Interesg plexus , whereasg plexus , demonstg plexus , 34. Aftg plexus , 36. At CKO mice .iECKO/iECKOCtnnd1 mouse retinas, as compared with their littermate controls  is critical for selective vascular permeability, and disruption of BRB integrity might lead to severe leakage, which is a typical phenotype of FEVR , 39, 40.CKO mice , indicatisrupted . We therMR at P6 , indicatMR at P6 . To evalntinuity . Meanwhintinuity , confirmFzd4, Norrin, Tspan12, or Ctnna1 mutant mouse retinas  has previously been reported to indicate active astrocytes in  retinas , 34, 37,trocytes . Astrocytrocytes , 44. We CKO mice , which aiECKO/iECKOCtnnd1 mouse retinas . An unbiased proteomic analysis was performed on the control and CTNND1-knockdown HRECs. A large number of differentially expressed proteins were detected, as shown in the volcano plot, and the abundance of the p120 protein was confirmed to be substantially decreased  in ECs results in reduced membrane localization of the cadherin/catenin complex , c-Myc (MYC), c-Jun (JUN), DKK1, CD44, and claudin5 (CLDN5), were significantly reduced in the absence of p120 , a transcriptional mediator of Norrin/β-catenin signaling  domains (1–5 and 7–10 ARM domains) , 53. TheCtnna1 (α-catenin) in mice causes FEVR-like phenotypes through disruption of the cadherin/catenin complex and abnormal overactivation of β-catenin signaling , LiCl failed to rescue compromised vascular progression and BRB integrity, suggesting that disrupted cadherin/catenin complexes play nonnegligible roles in the progress of FEVR-like phenotypes in iECKO/iECKOCtnnd1 mice. These results further highlighted the role of normal AJ integrity in retinal vascularization, which might be a therapeutic target for FEVR.Interestingly, treatment with LiCl or CHIR-99021, which are inhibitors of Gsk3β, considerably restored Wnt/β-catenin signaling activity and promoted the in vitro proliferation of p120-depleted HRECs . This inCTNND1 gene caused FEVR in a presumed autosomal dominant manner of inheritance, consistent with the fact that most FZD4, LRP5, and TSPAN12 variants are inherited as autosomal dominant FEVR . Th. ThCtnnaCtnnd1 and Ctnna1 prominently contributes to compromised AJ integrity, leading to severe vascular leakage and associated immune responses that induce hyperplasia without affecting vascular progression, whereas double-heterozygous deletion of Ctnnd1 and Ctnnb1 impairs vascular development prominently through inactivation of Wnt signaling, thus leading to more typical FEVR-like phenotypes.It has been reported that α-catenin inhibits Wnt signaling by segregating the available pool of β-catenin in cytosol, and that variants of α-catenin disrupt the cadherin/catenin complex that releases β-catenin from cytosol for nuclear translocation and activation of β-catenin signaling, which may contribute to the pathogenesis of FEVR , 67–70. CTNND1 gene cause FEVR through the combined contributions of AJ disruption and Wnt signaling inactivation  (Pdgfb-iCre-ER mice (iECKO/iECKOCtnna1 model was constructed as previously described (loxP/loxPCtnnb1 mice were obtained from The Jackson Laboratory (stock number 004152).r/Mmucd)  were pur-ER mice . The Ctnescribed . The CtnPatients and their family members were enrolled and evaluated by ophthalmologists at Zhongshan Ophthalmic Center, Sun Yat-sen University, and Xinhua Hospital, Shanghai Jiaotong University.CTNND1 coding sequence was subcloned into a mammalian expression vector with N-terminal FLAG-tagged pCDNA3.1 vector (both from Youbio Biotechnology). CTNND1 variants were generated by site-directed mutagenesis using a Q5 Site-Directed Mutagenesis Kit  and confirmed by Sanger sequencing.Wild-type The microscope  was used to capture confocal images. The Spline Contour2 and line tools in Zen 2.1 software (Zeiss) were used to evaluate intensity mean value of cell junction proteins and vascular progression of whole mounts, respectively. Angiotool software was used to measure the percentage of vessel area as previously described . The immAll the data and materials are available within the article and the supplemental materials. t test with Welch’s correction was applied to comparisons between 2 experimental groups, while 1-way ANOVA or 2-way ANOVA with Dunnett’s or Tukey’s multiple comparison tests were performed in multiple comparisons among more than 2 experimental groups. All the experiments were conducted at least 3 times independently. P < 0.05 was considered statistically significant.Statistical analysis was performed with GraphPad Prism 8.0. To assess statistical significance with a 95% confidence level, unpaired 2-tailed Student’s The clinical study was conducted in accordance with the declaration of Helsinki and approved by the ethical oversight committee of Sichuan Provincial People’s Hospital, Zhongshan Ophthalmic Center, Sun Yat-sen University, and Xinhua Hospital, Shanghai Jiaotong University. Written informed consent was obtained from all participants or legal guardians for minors. The Institutional Animal Care and Use Committee at the Sichuan Provincial People’s Hospital approved all animal study protocols. All experimental procedures were carried out according to the approved protocols.XZ, ZY, and XD conceived the research. MY, SL, and RZ performed the animal model study and cell biology, immunohistochemistry, and gene expression studies. LH, XJ, YH, JL, LP, ED, WL, ZZ, DJ, YZ, ZJ, and YY performed the construction of plasmids and animal breeding. XD, LH, and PZ recruited the participants and performed the WES study and Sanger sequencing analysis. MY, SL, XD, and XZ analyzed data. MY, SL, and XZ wrote the manuscript, with input from other authors.