The Journal of Physical Chemistry B最新文献

{"title":"Exploring the Local Structure of Molten NaF-ZrF₄ through In Situ XANES/EXAFS and Molecular Dynamics.","authors":"Anubhav Wadehra, Omar Oraby, Rajni Chahal, Alexander Levy, Haoxuan Yan, Qing Ma, Uday Pal, Stephen Lam, Karl Ludwig","doi":"10.1021/acs.jpcb.5c00764","DOIUrl":"10.1021/acs.jpcb.5c00764","url":null,"abstract":"<p><p>Molten salts are critical materials for advanced energy systems, particularly in molten salt reactors (MSRs), due to their exceptional thermophysical and chemical properties. While significant progress has been made in understanding their macroscopic behaviors, detailed knowledge of their atomic structures remains limited, particularly in fluoride-based salts with high zirconium concentrations. This study investigates the atomic structure and thermophysical properties of NaF-ZrF₄ salt mixtures (53-47 and 56-44 mol %) using an integrated experimental and computational approach. X-ray absorption near edge structure (XANES) and extended X-ray absorption fine structure (EXAFS) spectroscopy were employed to probe the local environment of Zr atoms across temperatures from 530 to 700 °C, revealing changes in coordination states and bond distances. Complementary ab initio molecular dynamics (AIMD) and neural network-based molecular dynamics (NNMD) simulations were validated against experimental data to elucidate short- and intermediate-range ordering in the melt. The results highlight a temperature-driven transition toward lower Zr coordination numbers and increased structural distortion, providing insights into the fluoroacidity and potential corrosiveness of these salts. This comprehensive understanding of the NaF-ZrF₄ structure supports the development of more reliable models for molten salts, aiding advancements in next-generation nuclear reactors and energy systems.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4747-4755"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143954841","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Spectroscopic Investigations of Excitation Energy Dissipation in the CP43 Preassembly Complexes of Photosystem II Embedded in Clear Native Gel.","authors":"Dariusz M Niedzwiedzki, Sandeep Biswas, Himadri B Pakrasi","doi":"10.1021/acs.jpcb.5c00565","DOIUrl":"10.1021/acs.jpcb.5c00565","url":null,"abstract":"<p><p>The importance of the spectrochemical properties of the pigment-bound CP43 protein lies in its role as the inner antenna complex of photosystem II. Multiple populations of the CP43-containing protein complexes are known to exist in vivo. These complexes have minor differences in molecular masses, and the conventional separation approach using glycerol-gradient ultracentrifugation lacks the resolution to separate them clearly. This hinders the assessment of the spectrochemical properties of different CP43 protein complexes in solution. Using high-resolution clear native PAGE, we successfully separated two known subclasses of the CP43 preassembly complex, ranging between 70 and 90 kDa, and performed spectroscopic analysis of the gel-embedded pigment proteins. These gel-embedded complexes, named CP43p-H and CP43p-L, were investigated by applying various static and time-resolved optical spectroscopies to elucidate their differences. The studies were performed at room temperature and 77 K. The application of time-resolved transient absorption followed by target analysis of the data sets demonstrated fine-tuning in pigment-pigment interactions within the complex. Time-resolved fluorescence demonstrated a truncated fluorescence emission decay for CP43p-L with respect to CP43p-H at both temperatures. The process contributing to such an accelerated quenching in CP43p-L is intriguing. We suggest that in CP43p-L, the pigments are arranged to facilitate a Chl <i>a</i>-to-carotenoid (β-carotene) singlet-singlet excitation energy transfer.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4641-4650"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143951511","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Theoretical Foundation for Interface-Specific Hyper-Rayleigh Scattering in Uniaxial Chiral Assemblies.","authors":"Kevin Murati, Alexander J Higgins, Carly M Clisham, Alexander W Litts, Matthew R Wilson, Yechan Hwang, Patrizia Polichetti, Caitlin E Dunlap, Ting-Ju Ku, Garth J Simpson","doi":"10.1021/acs.jpcb.4c08140","DOIUrl":"10.1021/acs.jpcb.4c08140","url":null,"abstract":"<p><p>The role of incoherence is considered in polarization-dependent second harmonic generation (SHG) measurements of uniaxially oriented assemblies. SHG microscopy continues to find growing utility for tissue, powders, and materials analysis, all of which exhibit structural heterogeneity over length scales comparable to the optical wavelength. In these cases, the detected SHG signal will generally exhibit partial decoherence, invalidating polarization analyses that implicitly assume purely polarized signal detection. The primary goal of the present study is to develop a mathematical framework for interpreting the incoherent component of the SHG signals produced in such instances. While formulas for describing hyper-Rayleigh scattering (HRS) from isotropic systems are reasonably well established, practical systems encountered experimentally in SHG microscopy measurements are often of lower symmetry. The next lowest symmetry below isotropic, and therefore the next most common samples likely to be encountered experimentally, are uniaxial assemblies, in which one spatial axis is unique from the other two. Such systems include surface assemblies, poled films, stretched polymers, lipid bilayers, most collagenous tendons, and numerous other naturally occurring biological structures. In this work, the general theory for HRS of uniaxially oriented assemblies is developed, including both achiral and chiral uniaxial assemblies. Intriguingly, this analysis predicts the possible observation of large electric dipole-allowed chiral-specific observables within just the incoherent component of the SHG response of chiral assemblies exhibiting with polar, uniaxial ensemble symmetry. The incoherent chiral contributions exhibit distinctly different symmetry than the established chiral sensitivity of coherent SHG in uniaxial assemblies, which is independent of polar order. These predictions provide context for the prior reports of chiral-specific SHG microscopy of tissues and suggest new experimental strategies for performing surface-specific and chiral-specific nonlinear optical analysis of chiral assemblies.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4651-4669"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143953185","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluation of Orientation-Dependent Cation-π Pairwise Effects within Collagen Triple Helices.","authors":"Tzu-Jou Yao, Yung-En Ke, Wen-Ling Lin, You-Cheng Lin, Chih-Han Yang, Tsai-Ling Hsu, Jia-Cherng Horng","doi":"10.1021/acs.jpcb.4c08691","DOIUrl":"10.1021/acs.jpcb.4c08691","url":null,"abstract":"<p><p>Various noncovalent interactions have been introduced to explore their impacts in folding a collagen triple helix. Among these interactions, the cation-π interaction represents one of the compelling forces stabilizing the triple helix. Still, the effects depend on the pairwise components and the orientation between the cationic and aromatic moieties. To gain more insights into this interaction within a collagen trimer, we prepared a series of collagen-mimetic peptides (CMPs) with cationic residues and aromatic residues incorporated to examine the contributions of two types of axial cation-π pairs (N → C and C → N cationic-to-aromatic pairwise) and the lateral cation-π pair. Circular dichroism (CD) measurements indicate that the N → C axial pairs have a significant stabilization effect. In contrast, the lateral and the C → N axial pairs destabilize the fold, and the lateral pairs cause the most destabilization consequences. We further designed and prepared the CMPs containing various lateral and axial cation-π pairs to investigate the coupling consequences in homotrimers and heterotrimers. From CD data, we found that the predicted differences in melting temperatures using individual cation-π pairwise contributions were comparable to the observed values for the designed homotrimers. CD and NMR measurements showed favorable cation-π interactions could effectively induce the folding of heterotrimers, in which the CMPs with more N → C axial pairs formed a more stable trimer than those containing a smaller number of N → C axial pairs. In this study, we have disclosed more valuable information about the properties of cation-π pairwise effects within a collagen triple helix, which can be considered in designing collagen-related peptides and materials.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4605-4613"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12086834/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Determining the Role of Electrostatics in the Making and Breaking of the Caprin1-ATP Nanocondensate.","authors":"Maria Tsanai, Teresa Head-Gordon","doi":"10.1021/acs.jpcb.5c01925","DOIUrl":"10.1021/acs.jpcb.5c01925","url":null,"abstract":"<p><p>We employ a multiscale computational approach to investigate the condensation process of the C-terminal low-complexity region of the Caprin1 protein as a function of increasing ATP concentration for three states: the initial mixed state, nanocondensate formation, and dissolution of the droplet as it reenters the mixed state. We show that upon condensation, ATP assembles via pi-pi interactions, resulting in the formation of a large cluster of stacked ATP molecules stabilized by sodium counterions. The surface of the ATP assembly interacts with the arginine-rich regions of the Caprin1 protein, particularly with its N-terminus, to promote the complete phase-separated droplet on a length scale of tens of nanometers. In order to understand droplet stability, we analyzed the near-surface electrostatic potential (NS-ESP) of Caprin1 and estimated the zeta potential of the Caprin1-ATP assemblies. We predict a positive NS-ESP at the Caprin1 surface for low ATP concentrations that defines the early mixed state, in excellent agreement with the NS-ESP obtained from NMR experiments using paramagnetic resonance enhancement. By contrast, the NS-ESP of Caprin1 at the surface of the nanocondensate at moderate levels of ATP is highly negative compared to that at the mixed state, and estimates of a large zeta potential outside the highly dense region of charge further explain the remarkable stability of this phase-separated droplet assembly. As ATP concentrations rise further, the strong electrostatic forces needed for nanocondensate stability are replaced by weaker Caprin1-ATP interactions that drive the re-entry into the mixed state that exhibits a much lower zeta potential.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4705-4714"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955199","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Two Relaxation Mechanisms for Rejuvenation of Stable Polystyrene Glass.","authors":"Saba Karimi, Junjie Yin, James A Forrest","doi":"10.1021/acs.jpcb.5c00413","DOIUrl":"10.1021/acs.jpcb.5c00413","url":null,"abstract":"<p><p>We report on the rejuvenation of thin films of polystyrene (PS) as they are heated from stable glassy states, prepared either through vapor deposition or physical aging. For films with thicknesses <i>h</i> ≳ 200 nm and <i>h</i> ≲ 50 nm, the rejuvenation of vapor-deposited stable PS glass films is quantitatively described with zero free parameters by simulations using parameters previously obtained from isothermal rejuvenation measurements in the same material. For films with thickness <i>h</i> ∼ 140 nm, the behavior of the vapor-deposited films becomes more complicated and exhibits significant deviations from model predictions. After rejuvenation and physical aging, such films are then described by a single free parameter. The results of these studies suggest a new distinct mechanism that can result in rejuvenation of glassy PS, and hence two distinct relaxation processes that can couple to the material density.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4811-4817"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955860","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Noncovalent Lasso Entanglements are Common in Experimentally Derived Intrinsically Disordered Protein Ensembles and Strongly Influenced by Protein Length and Charge.","authors":"Quyen V Vu, Ian Sitarik, Mai Suan Li, Edward P O'Brien","doi":"10.1021/acs.jpcb.5c01260","DOIUrl":"10.1021/acs.jpcb.5c01260","url":null,"abstract":"<p><p>Noncovalent lasso entanglements are conformations in which a protein backbone segment forms a loop closed by noncovalent interactions and that loop is threaded one or more times by either the N- or C-terminal segment of the backbone or both. While these entanglements are common in globular proteins, their presence in intrinsically disordered proteins or regions (IDPs/IDRs) remains largely unexplored. Here, we examine whether IDPs/IDRs in their monomeric form populate these conformations and how sequence length and charge composition influence entanglement prevalence. Using experimentally derived IDP/IDR ensembles from the Protein Ensemble Database, we find that 48% (199 of 416) of its entries contain subpopulations with entangled conformations, with 25% of entries having conformational ensembles in which 50% or more are entangled. This includes IDPs such as nuclear pore complex protein Nup153, nonstructural protein V of Hendra virus, and Eukaryotic initiation factor 4F subunit p150. Using molecular simulations, we find that (i) entanglements are most prevalent in weak polyampholytes and polyelectrolytes, and strong polyampholytes but rare in strong polyelectrolytes; (ii) entanglement populations increase with IDP length; (iii) entanglement probability positively correlates with chain compaction; and (iv) most IDPs/IDRs in the human proteome exhibit entangled conformations. A GO enrichment analysis reveals that the entanglement probability correlates with IDP/IDR function and subcellular localization. Thus, these findings indicate that noncovalent lasso entanglements are a widespread structural feature of IDPs/IDRs and have the potential to be biologically relevant.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4682-4691"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143956702","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Transport Properties of Self-Assembling G-Hydrogels: Evidence for a Tunable Fickian Diffusivity","authors":"Alessia Pepe*,&nbsp;Paolo Moretti,&nbsp;Paolo Mariani*,&nbsp;Valentina Notarstefano and Francesca Ripanti,&nbsp;","doi":"10.1021/acs.jpcb.5c0056410.1021/acs.jpcb.5c00564","DOIUrl":"https://doi.org/10.1021/acs.jpcb.5c00564https://doi.org/10.1021/acs.jpcb.5c00564","url":null,"abstract":"<p >The mixing of Guanosine (Gua) and Guanosine 5′-monophosphate (GMP) in water in selected compositions yields highly hydrated, transparent, and self-healing self-assembled supramolecular G-hydrogels, attractive for biomedical applications. This work investigates how hydrogel composition affects solute transport, including diffusion, binding, loading, and release properties, using a set of fluorescent probes with varying size and polarity. Although small/wide-angle X-ray scattering techniques showed that no structural changes are induced by probe addition, even when intercalation into G-quadruplexes is expected, the internal mesh structure of the hydrogel, modulated by the Gua:GMP ratio, directly impacts probe diffusivity and loading. Tighter networks (e.g., 1:1) slow diffusion and enhance retention compared to looser configurations (e.g., 1:4). Moreover, UV–visible titrations revealed markedly different binding affinities (<i>K</i>_b ≈ 5.7 × 10⁴ M^–1 for DAPI, 8.0 × 10³ M^–1 for ThT, and 1.4 × 10² M^–1 for RhB), which are expected to result in lower diffusion coefficients and slower release, especially for DAPI and ThT. Indeed, diffusion coefficients, obtained via fluorescence recovery after photobleaching and time-resolved fluorescence spectroscopy, reach 90, 20, and 60 μm²/s for FITC-dextran, ThT, and RhB, respectively. Probe release kinetics, modeled via Weibull fitting, indicated sustained release with characteristic times (τ) between 9.6 and 23.2 h and β ≈ 1 in 1× PBS, consistent with predominantly Fickian diffusion. Remarkably, switching to 10× PBS significantly accelerated release (τ reduced by ≈ 40–50%), suggesting that ionic strength and/or pH changes critically affect not only probe-hydrogel interactions but also the internal gel architecture, altering porosity, mesh size, and network tortuosity, thus enhancing molecular mobility. Overall, the G-hydrogel system offers a structurally tunable and composition-dependent platform capable of finely regulating molecular transport and release profiles, making it highly suitable for controlled drug delivery and adaptive biomaterial applications.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":"129 21","pages":"5136–5149 5136–5149"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://pubs.acs.org/doi/epdf/10.1021/acs.jpcb.5c00564","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144166034","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Luminescence and Nanoheterogeneity in Manganese-Containing Ionic Liquid Mixtures.","authors":"Hiroshi Abe, Shusei Maruyama, Fumiya Nemoto, Akihisa Aimi, Hiroaki Kishimura, Daisuke Okuyama, Hajime Sagayama","doi":"10.1021/acs.jpcb.5c01792","DOIUrl":"10.1021/acs.jpcb.5c01792","url":null,"abstract":"<p><p>The luminescence properties of single-component and binary ionic liquids (ILs) were investigated in relation to their nanoheterogeneities. The cations of the ILs were 1-alkyl-3-methylimidazolium and trihexyl(tetradecyl)phosphonium ([P666,14]⁺). The anions were [Cl]^-, [Br]^-, [MnCl₄]^2-, and [MnBr₄]^2-. Even in the liquid state, the binary mixtures of η[P666,14][Br] + [P666,14]₂[MnBr₄] (η = 0.5, 1, 2, and 4) emitted green light at room temperature. In the mixed liquids, the prepeak derived from the nanoheterogeneity was observed at a low <i>Q</i> position on the small- and wide-angle X-ray scattering patterns. With increasing temperature, the luminescent intensities of the IL mixtures increased, contrary to the almost constant prepeak intensity. The luminescence intensity maximum of the mixture (η = 0.5) was observed at 403 K.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4756-4764"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143953272","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Thermodynamics of Mobile Ion in Ion Exchange Membranes: Water-Swollen-Membrane Reference State and Quasi-Regular Solution Model.","authors":"Dongdong Li","doi":"10.1021/acs.jpcb.4c08514","DOIUrl":"10.1021/acs.jpcb.4c08514","url":null,"abstract":"<p><p>Thermodynamics of mobile ions in swollen ion exchange membranes (IEM) are essential for understanding their permselectivity. The theoretical modeling of the ion activity coefficient and the ion partitioning of the IEM is challenging. Based on a water-swollen membrane reference state and a quasi-regular solution model, we successfully correlated the ion activity coefficient in various IEMs. For all the studied 59 systems, the correlation coefficient <i>r</i> and determination coefficient <i>R</i>² are 0.973 and 0.947, respectively. As expected, the water activity in the IEM and the ion partitioning between the IEM and the external salt solution are represented. In the new theory framework, the standard chemical potential of the counterion is different from that of the aqueous solution. Moreover, the ion activity coefficient in the IEM reaches unity for the infinite dilution external salt concentration for all systems. Our quasi-regular solution model only considered the short-range interactions between the co-ions and the \"effective free counterion\", but it showed excellent correlation ability with the activity coefficient data of extensive systems. A power-law relation between the effective concentration of the free counterion and the apparent concentration of the free counterion was suggested. This indicates that the long-range electrostatic interactions in the swollen IEM are negligible due to abundant counterion condensation shielding the charge of the polymer chain.</p>","PeriodicalId":60,"journal":{"name":"The Journal of Physical Chemistry B","volume":" ","pages":"4794-4810"},"PeriodicalIF":2.8,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143955830","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}