Biophysical reports最新文献

{"title":"Tryptophan oxidation-induced dynamics highlight why stable γS-crystallin variants aggregate.","authors":"Yeonseong Seo, Amanda Abiad, Micco Iwase, Tsoler K Demerdjian, Linyuting Xing, Minseo Kim, Elizabeth M Wingen, Rachel W Martin","doi":"10.1016/j.bpr.2026.100291","DOIUrl":"https://doi.org/10.1016/j.bpr.2026.100291","url":null,"abstract":"<p><p>Human γS-crystallin (γS) is a highly soluble protein found in the eye lens, where it is densely packed with other proteins to form the refractive tissue required for vision. Mutations in γS can compromise its solubility and cause congenital cataract, while post-translational modifications (PTMs) can lead to age-related cataract. γS is especially susceptible to oxidative PTMs, which accumulate from lifelong exposure to UV light. We previously investigated the effects of side chain oxidation at tryptophan 163 (W163) by incorporating an oxidation mimic, 5-hydroxytryptophan (5HTP), in a site-specific manner using genetic code expansion. The resulting variant, γS-W163(5HTP), retains the overall fold of the wild-type γS (γS-WT) even under thermal and chemical stress, yet readily forms large oligomers at physiological temperature. Here, we probe the mechanism underlying this behavior. We first characterize γS-W163S, a serine variant in which the indole ring at position 163 is effectively removed, to further define the role of W163 in γS-crystallin structure and stability. We also map changes in backbone dynamics caused by W163 oxidation in γS-W163(5HTP) using solution-state NMR. Together, our findings support the established model in which crystallin aggregation is decoupled from global destabilization: although the oxidative modification at W163 is structurally tolerated through rearrangement of the local hydrogen bond network, the resulting changes in backbone flexibility render aggregation-competent conformations accessible. This work provides mechanistic insights into how covalent damage to crystallin monomers promotes aberrant intermolecular interactions that lead to cataract formation.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100291"},"PeriodicalIF":2.6,"publicationDate":"2026-10-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149167151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human salivary extracellular vesicles: a comparative study of isolation techniques.","authors":"Fanni Annamária Boros, Alexandra Gentschev, Jan Van Deun, Witold Szymanski, Johannes Graumann, Martin Regensburger, Philipp Arnold, Friederike Zunke","doi":"10.1016/j.bpr.2026.100292","DOIUrl":"https://doi.org/10.1016/j.bpr.2026.100292","url":null,"abstract":"<p><p>Saliva offers a valuable source of analyte for extracellular vesicle (EV) -based biomarker research, however, due to the complex composition of saliva, the isolation of salivary EVs remains technically challenging. The aim of our study is to systematically compare three salivary EV isolation techniques by assessing the obtained EV sample quality and quantity, and to highlight differences between the three methods with respect to costs, isolation time, and potential for parallel sample handling (i.e. sample throughput). For the enrichment of EVs we used three EV isolation techniques with saliva samples of four healthy donors: polymer precipitation (PP), membrane affinity (MA), and size exclusion chromatography (SEC). The vesicles obtained were characterized via transmission electron microscopy (TEM), SDS-PAGE and Western blot (WB), and conventional nanoparticle tracking analysis (NTA) and interferometric nanoparticle tracking analysis (iNTA). Our results indicate that all isolation methods resulted in successful enrichment of EVs of similar size distribution. However, we observed differences with respect to vesicle yield and detectability of positive EV markers, potentially reflecting differences in vesicle integrity and sample purity. Assessment of particle number via NTA and iNTA approaches revealed prominent differences in EV number of MA samples but not in the case of samples prepared with other enrichment methods, highlighting the importance of the choice of analysis method. Our study highlights advantages and potential shortcomings of three salivary EV isolation approaches, and calls attention to the importance of multimodal analysis of EV samples, combining approaches for EV marker detection, imaging, NTA and iNTA.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100292"},"PeriodicalIF":2.6,"publicationDate":"2026-10-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149149658","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantifying the spatio-temporal image degradation under motion blur in fluorescence microscopy.","authors":"Serafim Korovin, Kutay Ugurlu, Dylan Kalisvaart, Manon Kok, Rainer Heintzmann, Kirti Prakash, Carlas S Smith","doi":"10.1016/j.bpr.2026.100290","DOIUrl":"10.1016/j.bpr.2026.100290","url":null,"abstract":"<p><p>The spatial resolution of optical imaging systems is fundamentally restricted by the diffraction limit. However, in widefield live-cell microscopy, the achievable resolution is further constrained by specimen motion, which indicates the existence of a fundamental spatio-temporal resolution trade-off between signal accumulation during full frame integration and the resulting motion blur. To improve the fidelity with which moving objects can be imaged, a quantitative understanding of this spatio-temporal trade-off is necessary. Here, we present a systematic analysis of motion-induced resolution dynamics measured with the spectral signal-to-noise ratio (SSNR). We developed a simulation framework that models the image formation of objects undergoing arbitrary motion to evaluate the degradation of the spatial resolution under translational, diffusive, and rotational dynamics. Our results demonstrate that for translating objects, the spatial resolution is anisotropically reduced as a function of the orientation of the object relative to the motion vector, leading to the SSNR degrading by up to 50% and the resolution by up to 40% for a 90° change in the motion direction. This motion-blur-induced spectral degradation was further observed on simulated points under Brownian motion and dynamic Drosophila data. Moreover, we show that for rotational motion, conventional radially averaged metrics such as the Fourier ring correlation cannot quantify the effects of angular blur, which the SSNR quantifies accurately. These findings underscore the necessity of an object-oriented imaging approach, in which acquisition parameters such as exposure time are tuned to specific biological spatio-temporal characteristics to optimize the trade-off between motion blur and spatial fidelity.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100290"},"PeriodicalIF":2.6,"publicationDate":"2026-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149032153","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Nanoscale biomechanical remodeling in human myopic corneal lenticule revealed by quantitative dynamic AFM.","authors":"Hai-Qiong Deng, Shu Yang, Xin Wang, Yan Wang","doi":"10.1016/j.bpr.2026.100289","DOIUrl":"10.1016/j.bpr.2026.100289","url":null,"abstract":"<p><p>Increasing evidence shows that myopia, a prevalent ocular disorder and the leading cause of visual impairment worldwide, is associated with significant alterations in the biomechanical properties of the sclera. However, the role of the anterior ocular segment, particularly the hierarchical collagen structure and biomechanical behavior of the corneal stroma, remains poorly understood in the context of myopia development. There is an urgent need to elucidate the relationship between nanoscale changes in corneal collagen ultrastructure, tissue-level biomechanical properties, and the macroscopic progression of myopia. In this study, we used quantitative dynamic atomic force microscopy and transmission electron microscopy to investigate the ultrastructure and biomechanical properties of the corneal stroma in human myopic eyes. Our results demonstrate that with increasing myopia severity, collagen fibril density progressively decreases, fibril packing becomes sparser, and interfibrillar spacing widens. Nanomechanical characterization further revealed decreased stromal stiffness and reduced mechanical stability, indicating a diminished capacity of the tissue to bear mechanical loads. Collectively, this correlative analysis highlights the pivotal role of corneal stromal nanostructure and biomechanics in the pathological development of myopia, providing new insights that could aid in the characterization, diagnosis, and assessment of the condition.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100289"},"PeriodicalIF":2.6,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13657378/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149003050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Morphometric analysis reveals that the chick cranial neural tube expands as an active shell.","authors":"Nimesh Chahare, Chieko Imamura, Nandan L Nerurkar","doi":"10.1016/j.bpr.2026.100280","DOIUrl":"10.1016/j.bpr.2026.100280","url":null,"abstract":"<p><p>Embryonically, the vertebrate brain begins as an approximately uniform, fluid-filled epithelial tube that undergoes rapid volumetric expansion and regionalization to form the morphologically distinct primary brain vesicles. Hydrostatic pressure from fluid secretion into the inner lumen generates tension in the neural tube that has been implicated as a potential driver of cell proliferation during these early stages of brain development. However, a quantitatively rigorous view of 3D morphology and cellular proliferation has remained elusive. Here, we provide a standardized mapping for the mechanical and biological landscape of the developing neuroepithelium along anatomical axes. Using this 3D morphometric framework in chicken embryos, we show that localized curvature characterizes compartmental boundaries. While rapid inflation would typically be expected to stretch and thin the epithelium, we find the opposite: global expansion is coupled with significant tissue thickening, identifying the early brain as an active shell. Moreover, spatial patterns of thickness remain invariant to local curvature. Our results demonstrate a decoupling of geometry and growth, showing that spatially stable distributions of tissue thickness and mitotic activity are maintained throughout massive volumetric expansion, independent of the dramatic geometric reorganization driven by luminal pressure. We conclude that while tension in the neuroepithelium may contribute to proliferative growth at some level, a biological pre-pattern likely plays a driving role in the regionalized expansion of the early embryonic brain.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100280"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13521015/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148680201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Drosophila jump muscle myofibrils: A new tool for investigating activation and relaxation.","authors":"Axel Fenwick, Bernadette M Glasheen, Anthony Cammarato, Douglas M Swank","doi":"10.1016/j.bpr.2026.100268","DOIUrl":"10.1016/j.bpr.2026.100268","url":null,"abstract":"<p><p>Drosophila models have proven invaluable for studying skeletal and cardiac muscle diseases. While permeabilized indirect flight muscle (IFM) and jump muscle fibers from Drosophila yield insightful mechanical data, these preparations cannot resolve the kinetics of activation and relaxation because calcium diffusion into the fiber core is rate limited at this scale. In contrast, myofibrils have a diameter of only 1-3 μm, making them ideal for measuring physiologically relevant activation and relaxation rates. However, previous attempts using IFMs failed to produce myofibrils that generated measurable active force, likely due to the muscle's inherently low force output. Therefore, instead of using IFMs as the source, we developed a method to isolate myofibrils from the Drosophila jump muscle. By applying brief, low-amplitude sonication to permeabilized jump muscles, we isolated myofibrils that produced 19.8 ± 10.5 mN/mm² net active tension, the first active force measurements from an insect myofibril, with an activation rate of 8.2 ± 4.0 s^-1. Jump muscle myofibrils exhibited the typical biphasic relaxation seen in vertebrates: an initial slow, linear phase lasting 75.6 ± 21.1 ms, followed by a fast exponential decay with a rate constant of 19.7 ± 9.6 s^-1. We also characterized myofibrils from jump muscles transgenically expressing a Drosophila larval body wall myosin isoform (EMB), which we previously reported displays slower actin-binding and detachment kinetics. EMB expression caused a 1.6-fold increase in active tension and a 38% slower activation rate compared with controls but did not change relaxation parameters. These characterizations and comparisons highlight the scientific value of expressing customizable sarcomeric proteins in transgenic Drosophila jump muscle myofibrils to elucidate their contributions to muscle activation and relaxation.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100268"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13316580/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148159019","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cortical brain entropy architecture reveals multidimensional structure of schizophrenia.","authors":"Qiang Li, Vince D Calhoun","doi":"10.1016/j.bpr.2026.100276","DOIUrl":"10.1016/j.bpr.2026.100276","url":null,"abstract":"<p><p>Schizophrenia is a multidimensional psychiatric disorder lacking a unifying systems-level framework. We introduce a cortical brain entropy architecture that characterizes the spatial and hierarchical organization of functional entropy across the cerebral cortex. In large-scale neuroimaging data, this architecture differentiates schizophrenia from health and captures multidimensional network variation. Entropy alterations follow structured cortical organization, revealing coordinated disruptions within association networks. These findings identify cortical brain entropy architecture as a compact systems-level marker of schizophrenia heterogeneity and establish a quantitative link between macroscale cortical entropy and clinical symptom dimensions.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100276"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13380793/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148364215","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Information flow in cultured neuronal networks by transfer entropy.","authors":"Wataru Minoshima, Yumi Segawa, Chie Hosokawa","doi":"10.1016/j.bpr.2026.100278","DOIUrl":"10.1016/j.bpr.2026.100278","url":null,"abstract":"<p><p>Neurons form functional connections in neuronal networks of the brain. Neurotransmission refers to the information flow of electrical signals between neurons, and changes in the strength of this flow characterize brain functions. An evaluation method for neuronal information flow strength is necessary to elucidate the basic principles of brain function. To analyze the strength of information flow, identifying information transmission between time series of electrical spikes in neuronal networks, such as spike trains, is required. In this study, we evaluated the information flow strength in neuronal networks using transfer entropy (TE), an analysis method based on information theory, to elucidate causal relationships between two spike trains. Cultured rat hippocampal neuronal networks were used as living brain models, and extracellular monitoring of action potentials (expressed as spikes) was performed using microelectrode arrays. Spike trains of spontaneous activity and stimulus-evoked responses were measured in multiple neurons, and the causal relationship between electrode pairs was evaluated as an index of information flow between neurons using TE. From the results of spontaneous activity and evoked responses, it was suggested that TE values between electrodes increased with culture days and that the information flow strength was enhanced by network maturation. Furthermore, the correlation between TE values and neuronal population distance was evaluated using TE analysis. We found that neurons that received strong inputs slightly overlapped in their spontaneous activity and evoked responses. These findings suggest that spontaneous activity and evoked responses exhibit similar patterns in neuronal networks. In the graph theoretical analysis based on TE values, the network topology exhibited changes that strengthened information flow over 70 days in culture. Therefore, TE analysis is an effective tool for estimating the information flow between neurons based on neuronal electrical activity recorded at multiple sites.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100278"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13476481/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148537803","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of E. coli acid resistance systems in proton motive force formation during fermentation.","authors":"Heghine Gevorgyan, Tamara Abaghyan, Stella Hakobyan, Sophie Brameyer, Kirsten Jung, Karen Trchounian","doi":"10.1016/j.bpr.2026.100266","DOIUrl":"10.1016/j.bpr.2026.100266","url":null,"abstract":"<p><p>The regulation of intracellular pH (pH_in) and the maintenance of a proton motive force (Δp) are vital processes for cellular viability. Escherichia coli employs different amino acid decarboxylase acid resistance (AR) systems: specifically, Gad (glutamate-dependent), Cad (lysine-dependent), and Adi (arginine-dependent) systems for pH_in and pH_ex regulation under acidic conditions. This study elucidated the pH-dependent roles of these AR systems in the formation and regulation of the Δp components (membrane potential [ΔΨ] and transmembrane proton gradient [ΔpH]) and proton flux rate (J_H⁺) during fermentation at different levels of acid stress: (no stress [pH 7.6], mild acid stress [pH 6.5], moderate acid stress [pH 5.8], and sublethal acid stress [pH 5.4]). The regulatory role of AR systems in Δp and pH homeostasis demonstrated significant special contingent at levels of acid stress. Moreover, AR systems affected the Δp components and the proton flux rate from the cytoplasm to the external environment. Under no acid stress, the Gad system played a role in the regulation of ΔpH by maintaining a higher extracellular pH (pH_ex). The Adi system demonstrated significance in balancing of Δp components, and the observed compensatory variation in balancing both ΔΨ and ΔpH in ΔadiY mutant emphasized the critical role of Adi in maintaining Δp stability under mild acid stress, pH 6.5. The Cad system was important in regulating ΔpH under moderate acid stress levels by influencing pH_in. Under sublethal acid stress, all AR systems were crucially involved in ΔpH regulation and J_H⁺. Thus, our data show that each AR system possesses a crucial and distinct role in Δp formation that is highly dependent on the acid stress level. Moreover, the role of one AR system was, in most cases, not fully compensated by the others, underscoring the importance of these AR systems individually in pH homeostasis in E. coli during fermentation.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100266"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13273468/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148038067","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A lightweight deep learning framework for fast, real-time super-resolution fluctuation imaging.","authors":"Miyase Tekpınar, Jelle Komen, Hana Valenta, Ran Huo, Klarinda de Zwaan, Peter Dedecker, Nergis Tomen, Kristin Grußmayer","doi":"10.1016/j.bpr.2026.100279","DOIUrl":"10.1016/j.bpr.2026.100279","url":null,"abstract":"<p><p>Capturing dynamic cellular processes in live cells requires fast imaging with very high resolution beyond the diffraction limit. Fluctuation-based super-resolution techniques overcome this limit by exploiting correlations in fluorescence blinking, but they typically require hundreds of frames and computationally intensive post-processing, prohibiting real-time imaging of fast cellular events. Recent deep learning approaches aim to increase temporal resolution; however, many rely on extensive pre-processing or large, complex models that increase training costs and inference latency, preventing real-time deployment. To address this, we employ a lightweight recurrent neural network model that integrates sequential low-resolution frames to extract spatio-temporally correlated signals. It significantly improves temporal resolution by reducing the required number of frames down to as few as 8 frames while doubling the spatial resolution in an inference time under 30 ms. Furthermore, gentle imaging conditions are essential for extracting reliable biologically relevant information, especially in long-term experiments. Our method is suitable for live-cell imaging under extreme signal/noise ratio conditions, allowing imaging under very low laser intensities to prevent photodamage. By combining simulation-based training with an efficient network architecture, we introduce real-time super-resolution fluctuation imaging (RESURF), a deep-learning-based real-time super-resolution fluctuation imaging framework. We demonstrate that RESURF generalizes across different biological structures and can be readily adapted to various microscope setups using a small data set for transfer learning. The accompanying data set, comprising simulations and experiments across multiple subcellular structures and labeling strategies, establishes a benchmarking platform for fluctuation-based super-resolution techniques. RESURF offers a practical, low-latency deep-learning framework for high-throughput imaging and real-time, smart live-cell super-resolution imaging.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100279"},"PeriodicalIF":2.6,"publicationDate":"2026-09-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13524874/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148686770","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}