Biophysical reports最新文献

{"title":"Electrically stimulated droplet injector for reduced sample consumption in serial crystallography.","authors":"Mukul Sonker, Diandra Doppler, Ana Egatz-Gomez, Sahba Zaare, Mohammad T Rabbani, Abhik Manna, Jorvani Cruz Villarreal, Garrett Nelson, Gihan K Ketawala, Konstantinos Karpos, Roberto C Alvarez, Reza Nazari, Darren Thifault, Rebecca Jernigan, Dominik Oberthür, Huijong Han, Raymond Sierra, Mark S Hunter, Alexander Batyuk, Christopher J Kupitz, Robert E Sublett, Frederic Poitevin, Stella Lisova, Valerio Mariani, Alexandra Tolstikova, Sebastien Boutet, Marc Messerschmidt, J Domingo Meza-Aguilar, Raimund Fromme, Jose M Martin-Garcia, Sabine Botha, Petra Fromme, Thomas D Grant, Richard A Kirian, Alexandra Ros","doi":"10.1016/j.bpr.2022.100081","DOIUrl":"10.1016/j.bpr.2022.100081","url":null,"abstract":"<p><p>With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its \"off-time\" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 4","pages":"100081"},"PeriodicalIF":2.4,"publicationDate":"2022-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/48/eb/main.PMC9680787.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10565696","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":"Deciphering the conformational dynamics of gephyrin-mediated collybistin activation.","authors":"Nasir Imam,&nbsp;Susobhan Choudhury,&nbsp;Katherina Hemmen,&nbsp;Katrin G Heinze,&nbsp;Hermann Schindelin","doi":"10.1016/j.bpr.2022.100079","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100079","url":null,"abstract":"<p><p>Efficient neuronal signaling depends on the proper assembly of the postsynaptic neurotransmitter machinery. The majority of inhibitory synapses feature γ-aminobutyric acid type A (GABA_A) receptors. The function of these GABAergic synapses is controlled by the scaffolding protein gephyrin and collybistin, a Dbl family guanine nucleotide exchange factor and neuronal adaptor protein. Specifically, collybistin interacts with small GTPases, cell adhesion proteins, and phosphoinositides to recruit gephyrin and GABA_A receptors to postsynaptic membrane specializations. Collybistin usually contains an N-terminal SH3 domain and exists in closed/inactive or open/active states. Here, we elucidate the molecular basis of the gephyrin-collybistin interaction with newly designed collybistin Förster resonance energy transfer (FRET) sensors. Using fluorescence lifetime-based FRET measurements, we deduce the affinity of the gephyrin-collybistin complex, thereby confirming that the C-terminal dimer-forming E domain binds collybistin, an interaction that does not require E domain dimerization. Simulations based on fluorescence lifetime and sensor distance distributions reveal at least a two-state equilibrium of the SH3 domain already in the free/unbound collybistin, thereby illustrating the accessible volume of the SH3 domain. Finally, our data provide strong evidence for a tightly regulated collybistin-gephyrin interplay, where, unexpectedly, switching of collybistin from closed/inactive to open/active states is efficiently triggered by gephyrin.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100079"},"PeriodicalIF":0.0,"publicationDate":"2022-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/f8/6c/main.PMC9680708.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40486156","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 comparative study of interfacial environments in lipid nanodiscs and vesicles.","authors":"Xiao You,&nbsp;Naveen Thakur,&nbsp;Arka Prabha Ray,&nbsp;Matthew T Eddy,&nbsp;Carlos R Baiz","doi":"10.1016/j.bpr.2022.100066","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100066","url":null,"abstract":"<p><p>Membrane protein conformations and dynamics are driven by the protein-lipid interactions occurring within the local environment of the membrane. These environments remain challenging to accurately capture in structural and biophysical experiments using bilayers. Consequently, there is an increasing need for realistic cell-membrane mimetics for <i>in vitro</i> studies. Lipid nanodiscs provide certain advantages over vesicles for membrane protein studies. Nanodiscs are increasingly used for structural and spectroscopic characterization of membrane proteins. Despite the common use of nanodiscs, the interfacial environments of lipids confined to a ~10-nm diameter area have remained relatively underexplored. Here, we use ultrafast two-dimensional infrared spectroscopy and temperature-dependent infrared absorption measurements of the ester carbonyls to compare the interfacial hydrogen bond structure and dynamics in lipid nanodiscs of varying lipid compositions and sizes with ~100-nm vesicles. We examine the effects of lipid composition and nanodisc size. We found that nanodiscs and vesicles share largely similar lipid-water H-bond environments and interfacial dynamics. Differences in measured enthalpies of H-bonding suggest that H-bond dynamics in nanodiscs are modulated by the interaction between the annular lipids and the scaffold protein.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"2 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d4/19/main.PMC9518727.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9623325","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":"Identification and quantification of within-burst dynamics in singly labeled single-molecule fluorescence lifetime experiments.","authors":"Paul David Harris,&nbsp;Eitan Lerner","doi":"10.1016/j.bpr.2022.100071","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100071","url":null,"abstract":"<p><p>Single-molecule spectroscopy has revolutionized molecular biophysics and provided means to probe how structural moieties within biomolecules spatially reorganize at different timescales. There are several single-molecule methodologies that probe local structural dynamics in the vicinity of a single dye-labeled residue, which rely on fluorescence lifetimes as readout. Nevertheless, an analytical framework to quantify dynamics in such single-molecule single dye fluorescence bursts, at timescales of microseconds to milliseconds, has not yet been demonstrated. Here, we suggest an analytical framework for identifying and quantifying within-burst lifetime-based dynamics, such as conformational dynamics recorded in single-molecule photo-isomerization-related fluorescence enhancement. After testing the capabilities of the analysis on simulations, we proceed to exhibit within-burst millisecond local structural dynamics in the unbound <i>α</i>-synuclein monomer. The analytical framework provided in this work paves the way for extracting a full picture of the energy landscape for the coordinate probed by fluorescence lifetime-based single-molecule measurements.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/1b/01/main.PMC9534301.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33510528","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":"Heterogeneous migration routes of DNA triplet repeat slip-outs.","authors":"Simona Bianco,&nbsp;Tianyu Hu,&nbsp;Oliver Henrich,&nbsp;Steven W Magennis","doi":"10.1016/j.bpr.2022.100070","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100070","url":null,"abstract":"<p><p>It is unclear how the length of a repetitive DNA tract determines the onset and progression of repeat expansion diseases, but the dynamics of secondary DNA structures formed by repeat sequences are believed to play an important role. It was recently shown that three-way DNA junctions containing slip-out hairpins of CAG or CTG repeats and contiguous triplet repeats in the adjacent duplex displayed single-molecule FRET (smFRET) dynamics that were ascribed to both local conformational motions and longer-range branch migration. Here we explore these so-called \"mobile\" slip-out structures through a detailed kinetic analysis of smFRET trajectories and coarse-grained modeling. Despite the apparent structural simplicity, with six FRET states resolvable, most smFRET states displayed biexponential dwell-time distributions, attributed to structural heterogeneity and overlapping FRET states. Coarse-grained modeling for a (GAC)₁₀ repeat slip-out included trajectories that corresponded to a complete round of branch migration; the structured free energy landscape between slippage events supports the dynamical complexity observed by smFRET. A hairpin slip-out with 40 CAG repeats, which is above the repeat length required for disease in several triplet repeat disorders, displayed smFRET dwell times that were on average double those of 3WJs with 10 repeats. The rate of secondary-structure rearrangement via branch migration, relative to particular DNA processing pathways, may be an important factor in the expansion of triplet repeat expansion diseases.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"None"},"PeriodicalIF":0.0,"publicationDate":"2022-09-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9586884/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40665358","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":"Optimization of cryo-electron microscopy for quantitative analysis of lipid bilayers","authors":"Frederick A. Heberle, Doug Welsch, H. L. Scott, M. Waxham","doi":"10.1101/2022.08.23.505005","DOIUrl":"https://doi.org/10.1101/2022.08.23.505005","url":null,"abstract":"Cryogenic electron microscopy (cryo-EM) is among the most powerful tools available for interrogating nanoscale structure of biological structures. We recently showed that cryo-EM can be used to measure the bilayer thickness of lipid vesicles and biological membranes with sub-angstrom precision, resulting in the direct visualization of nanoscopic domains of different thickness in multicomponent lipid mixtures and giant plasma membrane vesicles. Despite the great potential of cryo-EM for revealing the lateral organization of biomembranes, a large parameter space of experimental conditions remains to be optimized. Here, we systematically investigate the influence of instrument parameters and image post-processing steps on the ability to accurately measure bilayer thickness and discriminate regions of different thickness within unilamellar liposomes. We also demonstrate a spatial autocorrelation analysis to extract additional information about lateral heterogeneity. Significance Raft domains in unstimulated cells have proven difficult to directly visualize owing to their nanoscopic size and fleeting existence. The few techniques capable of nanoscopic spatial resolution typically rely on interpretation of indirect spectroscopic or scattering signals or require stabilizing the membrane on a solid support. In contrast, cryo-EM yields direct images of nanoscale domains in probe-free, unsupported membranes. Here, we systematically optimize key steps in the experimental and analysis workflow for this new and specialized application. Our findings represent an important step toward developing cryo-EM into a robust method for investigating phase behavior of membranes at length scales relevant to lipid rafts.","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":"183 12 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2022-08-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"78579660","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":"Is orange carotenoid protein photoactivation a single-photon process?","authors":"Stanisław Niziński,&nbsp;Ilme Schlichting,&nbsp;Jacques-Philippe Colletier,&nbsp;Diana Kirilovsky,&nbsp;Gotard Burdzinski,&nbsp;Michel Sliwa","doi":"10.1016/j.bpr.2022.100072","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100072","url":null,"abstract":"<p><p>In all published photoactivation mechanisms of orange carotenoid protein (OCP), absorption of a single photon by the orange dark state starts a cascade of red-shifted OCP ground-state intermediates that subsequently decay within hundreds of milliseconds, resulting in the formation of the final red form OCP^R, which is the biologically active form that plays a key role in cyanobacteria photoprotection. A major challenge in deducing the photoactivation mechanism is to create a uniform description explaining both single-pulse excitation experiments, involving single-photon absorption, and continuous light irradiation experiments, where the red-shifted OCP intermediate species may undergo re-excitation. We thus investigated photoactivation of <i>Synechocystis</i> OCP using stationary irradiation light with a biologically relevant photon flux density coupled with nanosecond laser pulse excitation. The kinetics of photoactivation upon continuous and nanosecond pulse irradiation light show that the OCP^R formation quantum yield increases with photon flux density; thus, a simple single-photon model cannot describe the data recorded for OCP <i>in vitro</i>. The results strongly suggest a consecutive absorption of two photons involving a red intermediate with ≈100 millisecond lifetime. This intermediate is required in the photoactivation mechanism and formation of the red active form OCP^R.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100072"},"PeriodicalIF":0.0,"publicationDate":"2022-08-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/3a/12/main.PMC9680785.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40704942","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":"Irregular particle morphology and membrane rupture facilitate ion gradients in the lumen of phagosomes.","authors":"Maksim V Baranov,&nbsp;Melina Ioannidis,&nbsp;Sami Balahsioui,&nbsp;Auke Boersma,&nbsp;Rinse de Boer,&nbsp;Manoj Kumar,&nbsp;Masato Niwa,&nbsp;Tasuku Hirayama,&nbsp;Qintian Zhou,&nbsp;Terrence M Hopkins,&nbsp;Pieter Grijpstra,&nbsp;Shashi Thutupalli,&nbsp;Stefano Sacanna,&nbsp;Geert van den Bogaart","doi":"10.1016/j.bpr.2022.100069","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100069","url":null,"abstract":"<p><p>Localized fluxes, production, and/or degradation coupled to limited diffusion are well known to result in stable spatial concentration gradients of biomolecules in the cell. In this study, we demonstrate that this also holds true for small ions, since we found that the close membrane apposition between the membrane of a phagosome and the surface of the cargo particle it encloses, together with localized membrane rupture, suffice for stable gradients of protons and iron cations within the lumen of the phagosome. Our data show that, in phagosomes containing hexapod-shaped silica colloid particles, the phagosomal membrane is ruptured at the positions of the tips of the rods, but not at other positions. This results in the confined leakage at these positions of protons and iron from the lumen of the phagosome into the cytosol. In contrast, acidification and iron accumulation still occur at the positions of the phagosomes nearer to the cores of the particles. Our study strengthens the concept that coupling metabolic and signaling reaction cascades can be spatially confined by localized limited diffusion.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100069"},"PeriodicalIF":0.0,"publicationDate":"2022-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9680789/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40705902","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":"Viro-fluidics: Real-time analysis of virus production kinetics at the single-cell level.","authors":"Joëlle Eid,&nbsp;Marius Socol,&nbsp;Antoine Naillon,&nbsp;Jérôme Feuillard,&nbsp;Luca Ciandrini,&nbsp;Emmanuel Margeat,&nbsp;Benoit Charlot,&nbsp;Marylène Mougel","doi":"10.1016/j.bpr.2022.100068","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100068","url":null,"abstract":"<p><p>Real-time visualization and quantification of viruses released by a cell are crucial to further decipher infection processes. Kinetics studies at the single-cell level will circumvent the limitations of bulk assays with asynchronous virus replication. We have implemented a \"viro-fluidic\" method, which combines microfluidics and virology at single-cell and single-virus resolutions. As an experimental model, we used standard cell lines producing fluorescent HIV-like particles (VLPs). First, to scale the strategy to the single-cell level, we validated a sensitive flow virometry system to detect VLPs in low concentration samples (≥10⁴ VLPs/mL). Then, this system was coupled to a single-cell trapping device to monitor in real-time the VLPs released, one at a time, from single cells under cell culture conditions. Our results revealed an average production rate of 50 VLPs/h/cell similar to the rate estimated for the same cells grown in population. Thus, the virus-producing capacities of the trapped cells were preserved and its real-time monitoring was accurate. Moreover, single-cell analysis revealed a release of VLPs with stochastic bursts with typical time intervals of few minutes, revealing the existence of limiting step(s) in the virus biogenesis process. Our tools can be applied to other pathogens or to extracellular vesicles to elucidate the dissemination mechanisms of these biological nanoparticles.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100068"},"PeriodicalIF":0.0,"publicationDate":"2022-08-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/32/b5/main.PMC9680794.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40704941","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":"Single-chain models illustrate the 3D RNA folding shape during translation.","authors":"Tianze Guo,&nbsp;Olivia L Modi,&nbsp;Jillian Hirano,&nbsp;Horacio V Guzman,&nbsp;Tatsuhisa Tsuboi","doi":"10.1016/j.bpr.2022.100065","DOIUrl":"https://doi.org/10.1016/j.bpr.2022.100065","url":null,"abstract":"<p><p>The three-dimensional conformation of RNA is important in the function and fate of the molecule. The common conformation of mRNA is formed based on the closed-loop structure and internal base pairings with the activity of the ribosome movements. However, recent reports suggest that the closed-loop structure might not be formed in many mRNAs. This implies that mRNA can be considered as a single polymer in the cell. Here, we introduce the Three-dimensional RNA Illustration Program (TRIP) to model the three-dimensional RNA folding shape based on single-chain models and angle restriction of each bead component from previously reported single-molecule fluorescence in situ hybridization (smFISH) experimental data. This simulation method was able to recapitulate the mRNA conformation change of the translation activity and three-dimensional positional interaction between an organelle and its localized mRNAs as end-to-end distances. Within the analyzed cases, base-pairing interactions only have minor effects on the three-dimensional mRNA conformation, and instead single-chain polymer characteristics have a more significant impact on the conformation. This top-down method will be used to interpret the aggregation mechanism of mRNA under different cellular conditions such as nucleolus and phase-separated granules.</p>","PeriodicalId":72402,"journal":{"name":"Biophysical reports","volume":" ","pages":"100065"},"PeriodicalIF":0.0,"publicationDate":"2022-08-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/b2/e4/main.PMC9680788.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40705901","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}