bioRxiv : the preprint server for biology最新文献

{"title":"The information bottleneck as a principle underlying multi-area cortical representations during decision-making.","authors":"Michael Kleinman, Tian Wang, Derek Xiao, Ebrahim Feghhi, Kenji Lee, Nicole Carr, Yuke Li, Nima Hadidi, Chandramouli Chandrasekaran, Jonathan C Kao","doi":"10.1101/2023.07.12.548742","DOIUrl":"10.1101/2023.07.12.548742","url":null,"abstract":"<p><p>Decision-making emerges from distributed computations across multiple brain areas, but it is unclear <i>why</i> the brain distributes the computation. In deep learning, artificial neural networks use multiple areas (or layers) and form optimal representations of task inputs. These optimal representations are <i>sufficient</i> to perform the task well, but <i>minimal</i> so they are invariant to other irrelevant variables. We recorded single neurons and multiunits in dorsolateral prefrontal cortex (DLPFC) and dorsal premotor cortex (PMd) in monkeys during a perceptual decision-making task. We found that while DLPFC represents task-related inputs required to compute the choice, the downstream PMd contains a minimal sufficient, or optimal, representation of the choice. To identify a mechanism for how cortex may form these optimal representations, we trained a multi-area recurrent neural network (RNN) to perform the task. Remarkably, DLPFC and PMd resembling representations emerged in the early and late areas of the multi-area RNN, respectively. The DLPFC-resembling area partially orthogonalized choice information and task inputs and this choice information was preferentially propagated to downstream areas through selective alignment with inter-area connections, while remaining task information was not. Our results suggest that cortex uses multi-area computation to form minimal sufficient representations by preferential propagation of relevant information between areas.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10369960/pdf/nihpp-2023.07.12.548742v1.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9884104","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":"Disruption of Synaptic Endoplasmic Reticulum Luminal Protein Containment in <i>Drosophila Atlastin</i> Mutants.","authors":"Mónica C Quiñones-Frías, Dina M Ocken, Avital Rodal","doi":"10.1101/2023.09.01.555994","DOIUrl":"10.1101/2023.09.01.555994","url":null,"abstract":"<p><p>The endoplasmic reticulum (ER) extends throughout neurons and regulates many neuronal functions, including neurite outgrowth, neurotransmission, and synaptic plasticity. Mutations in proteins that control ER shape are linked to the neurodegenerative disorder Hereditary Spastic Paraplegia (HSP), yet the ultrastructure and dynamics of neuronal ER remain largely unexplored, especially at presynaptic terminals. Using super-resolution and live imaging in <i>D. melanogaster</i> larval motor neurons, we investigated ER structure at presynaptic terminals of wild-type animals and null mutants of the ER shaping protein and HSP-linked gene, Atlastin. Previous studies using an ER luminal marker reported diffuse localization at <i>Atlastin</i> mutant presynaptic terminals, which was attributed to ER fragmentation. However, using an ER membrane marker, we discovered that <i>Atlastin</i> mutant ER forms robust networks with only mild defects in structural dynamics, indicating the primary defect is functional rather than architectural. We demonstrate that <i>Atlastin</i> mutants progressively displace overexpressed luminal ER proteins to the cytosol during larval development, specifically at synapses, while these proteins remain correctly localized in cell bodies, axons, and muscles. This synaptic-specific displacement phenotype, previously unreported in non-neuronal cells, emphasizes the importance of studying neurons to understand HSP pathogenesis.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/c6/5c/nihpp-2023.09.01.555994v1.PMC10491308.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10263491","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":"Barcode-free multiplex plasmid sequencing using Bayesian analysis and nanopore sequencing.","authors":"Masaaki Uematsu, Jeremy M Baskin","doi":"10.1101/2023.04.12.536413","DOIUrl":"10.1101/2023.04.12.536413","url":null,"abstract":"<p><p>Plasmid construction is central to life science research, and sequence verification is arguably its costliest step. Long-read sequencing has emerged as a competitor to Sanger sequencing, with the principal benefit that whole plasmids can be sequenced in a single run. Nevertheless, the current cost of nanopore sequencing is still prohibitive for routine sequencing during plasmid construction. We develop a computational approach termed Simple Algorithm for Very Efficient Multiplexing of Oxford Nanopore Experiments for You (SAVEMONEY) that guides researchers to mix multiple plasmids and subsequently computationally de-mixes the resultant sequences. SAVEMONEY defines optimal mixtures in a pre-survey step, and following sequencing, executes a post-analysis workflow involving sequence classification, alignment, and consensus determination. By using Bayesian analysis with prior probability of expected plasmid construction error rate, high-confidence sequences can be obtained for each plasmid in the mixture. Plasmids differing by as little as two bases can be mixed for submission as a single sample for nanopore sequencing, and routine multiplexing of even six plasmids per 180 reads can still maintain high accuracy of consensus sequencing. SAVEMONEY should further democratize whole-plasmid sequencing by nanopore and related technologies, driving down the effective cost of whole-plasmid sequencing to lower than that of a single Sanger sequencing run.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/14/e4/nihpp-2023.04.12.536413v2.PMC10120676.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9391431","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":"Normative evidence weighing and accumulation in correlated environments.","authors":"Nathan Tardiff, Jiwon Kang, Joshua I Gold","doi":"10.1101/2024.05.29.596489","DOIUrl":"10.1101/2024.05.29.596489","url":null,"abstract":"<p><p>The brain forms certain deliberative decisions following normative principles related to how sensory observations are weighed and accumulated over time. Previously we showed that these principles can account for how people adapt their decisions to the temporal dynamics of the observations (Glaze et al., 2015). Here we show that this adaptability extends to accounting for correlations in the observations, which can have a dramatic impact on the weight of evidence provided by those observations. We tested online human participants on a novel visual-discrimination task with pairwise-correlated observations. With minimal training, the participants adapted to uncued, trial-by-trial changes in the correlations and produced decisions based on an approximately normative weighing and accumulation of evidence. The results highlight the robustness of our brain's ability to process sensory observations with respect to not just their physical features but also the weight of evidence they provide for a given decision.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11160761/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141297463","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":"Social context and the evolution of delayed reproduction in birds.","authors":"Liam U Taylor, Josef C Uyeda, Richard O Prum","doi":"10.1101/2023.08.02.551693","DOIUrl":"10.1101/2023.08.02.551693","url":null,"abstract":"<p><p>One puzzling feature of avian life histories is that individuals in many different lineages delay reproduction for several years after they finish growing. Intraspecific field studies suggest that various complex social contexts-such as cooperative breeding groups, nesting colonies, and display leks-result in delayed reproduction because they require forms of sociosexual development that extend beyond physical maturation. Here, we explicitly propose this hypothesis and use a full suite of phylogenetic comparative methods to test it, analyzing the evolution of age at first reproduction (AFR) in females and males across 963 species of birds. Phylogenetic regressions support increased AFR in colonial females and males, cooperatively breeding males, and lekking males. Continuous Ornstein-Uhlenbeck models support distinct evolutionary regimes with increased AFR for all of cooperative, colonial, and lekking lineages. Discrete hidden state Markov models suggest a net increase in delayed reproduction for social lineages, even when accounting for hidden state heterogeneity and the potential reverse influence of AFR on sociality. Our results support the hypothesis that the evolution of social contexts reshapes the dynamics of life history evolution in birds. Comparative analyses of even the most broadly generalizable characters, such as AFR, must reckon with unique, heterogeneous, historical events in the evolution of individual lineages.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/cb/cd/nihpp-2023.08.02.551693v1.PMC10418290.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10038219","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":"Investigating tumor-associated macrophages and their polarization in colorectal cancer using Boolean implication networks.","authors":"Ekta Dadlani, Tirtharaj Dash, Debashis Sahoo","doi":"10.1101/2023.08.01.551559","DOIUrl":"10.1101/2023.08.01.551559","url":null,"abstract":"<p><p>Tumor-associated Macrophages (or TAMs) are amongst the most common cells that play a significant role in the initiation and progression of colorectal cancer (CRC). [Ghosh et al., 2023] have built a Boolean-logic dependent model to propose a set of gene signatures capable of identifying macrophage polarization states. The signature, called the Signature of Macrophage Reactivity and Tolerance (SMaRT), comprises of 338 human genes (equivalently, 298 mouse genes). The SMaRT signature was constructed using datasets that were not specialized towards any particular disease. To specifically investigate macrophage polarization in CRC, in this paper, we (a) perform a comprehensive analysis of the SMaRT signature on single-cell human and mouse colorectal cancer RNA-seq datasets and (b) adopt transfer learning to construct a \"refined\" SMaRT signature that specifically characterizes TAM polarization in the CRC tumor microenvironment. Towards validation of our refined gene signature, we use: (a) 5 RNA-seq datasets derived from single-cell human datasets; and (b) 5 large-cohort microarray datasets from humans. Furthermore, we propose the translational potential of our refined gene signature while investigating microsatellite stability and CpG island methylator phenotype (CIMP) in colorectal cancer. Overall, our refined gene signature and its extensive validation provide a path for its adoption in clinical practice in diagnosing colorectal cancer and associated attributes.</p><p><strong>Availability and implementation: </strong>The data, codes, and software packages used in our research are linked and shared publicly at https://github.com/tirtharajdash/TAMs-CRC .</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10418212/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9993255","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":"Distinct Activation Mechanisms of CXCR4 and ACKR3 Revealed by Single-Molecule Analysis of their Conformational Landscapes.","authors":"Christopher T Schafer, Raymond F Pauszek, Martin Gustavsson, Tracy M Handel, David P Millar","doi":"10.1101/2023.10.31.564925","DOIUrl":"10.1101/2023.10.31.564925","url":null,"abstract":"<p><p>The canonical chemokine receptor CXCR4 and atypical receptor ACKR3 both respond to CXCL12 but induce different effector responses to regulate cell migration. While CXCR4 couples to G proteins and directly promotes cell migration, ACKR3 is G protein-independent and scavenges CXCL12 to regulate extracellular chemokine levels and maintain CXCR4 responsiveness, thereby indirectly influencing migration. The receptors also have distinct activation requirements. CXCR4 only responds to wild-type CXCL12 and is sensitive to mutation of the chemokine. By contrast, ACKR3 recruits GPCR kinases (GRKs) and β-arrestins and promiscuously responds to CXCL12, CXCL12 variants, other peptides and proteins, and is relatively insensitive to mutation. To investigate the role of conformational dynamics in the distinct pharmacological behaviors of CXCR4 and ACKR3, we employed single-molecule FRET to track discrete conformational states of the receptors in real-time. The data revealed that apo-CXCR4 preferentially populates a high-FRET inactive state, while apo-ACKR3 shows little conformational preference and high transition probabilities among multiple inactive, intermediate and active conformations, consistent with its propensity for activation. Multiple active-like ACKR3 conformations are populated in response to agonists, compared to the single CXCR4 active-state. This and the markedly different conformational landscapes of the receptors suggest that activation of ACKR3 may be achieved by a broader distribution of conformational states than CXCR4. Much of the conformational heterogeneity of ACKR3 is linked to a single residue that differs between ACKR3 and CXCR4. The dynamic properties of ACKR3 may underly its inability to form productive interactions with G proteins that would drive canonical GPCR signaling.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10635023/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157687","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":"Gamma-burst cortical activity in awake behaving macaques.","authors":"Benjamin T Acland, Ben Julian A Palanca, Janine Bijsterbosch, Lawrence H Snyder","doi":"10.1101/2023.09.26.559594","DOIUrl":"10.1101/2023.09.26.559594","url":null,"abstract":"<p><p>Electrophysiological recordings during ketamine anesthesia have revealed a slow alternating pattern of high- and low- frequency activity (a \"gamma-burst\" pattern) that develops with the onset of general anesthesia. We examine the role of NMDA receptor antagonism in generating the gamma-burst pattern and the link between gamma-bursts and dissociative anesthesia. We compare the effects of ketamine with those of the highly selective NMDA receptor antagonist CGS 19755 on multi-site intracranial electrophysiology and behavior in rhesus macaques. Remarkably, we find that animals given a moderate dose of CGS 19755 are able to perform a difficult memory task, while at the same time showing electrophysiological activity similar to ketamine anesthesia, with one key difference: a lack of delta-band LFP modulation. This difference demonstrates that ketamine's ability to drive strong delta-band oscillations relies on additional mechanisms beyond NMDA receptor antagonism alone, and points to a key role for the activity underlying delta-band oscillations in causing anesthesia.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10557640/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41173593","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":"ATP-release pannexin channels are gated by lysophospholipids.","authors":"Erik Henze, Russell N Burkhardt, Bennett W Fox, Tyler J Schwertfeger, Eric Gelsleichter, Kevin Michalski, Lydia Kramer, Margret Lenfest, Jordyn M Boesch, Hening Lin, Frank C Schroeder, Toshimitsu Kawate","doi":"10.1101/2023.10.23.563601","DOIUrl":"10.1101/2023.10.23.563601","url":null,"abstract":"<p><p>In addition to its role as cellular energy currency, adenosine triphosphate (ATP) serves as an extracellular messenger that mediates diverse cell-to-cell communication. Compelling evidence supports that ATP is released from cells through pannexins, a family of membrane proteins that form heptameric large-pore channels. However, the activation mechanisms that trigger ATP release by pannexins remain poorly understood. Here, we discover lysophospholipids as endogenous pannexin activators, using activity-guided fractionation of mouse tissue extracts combined with untargeted metabolomics and electrophysiology. We show that lysophospholipids directly and reversibly activate pannexins in the absence of other proteins. Secretomics experiments reveal that lysophospholipid-activated pannexin 1 leads to the release of not only ATP but also other signaling metabolites, such as 5'-methylthioadenosine, which is important for immunomodulation. We also demonstrate that lysophospholipids activate endogenous pannexin 1 in human monocytes, leading to the release of IL-1β through inflammasome activation. Our results provide a connection between lipid metabolism and purinergic signaling, both of which play major roles in immune responses.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10634739/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"92157625","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":"H3.3-G34R Mutation-Mediated Epigenetic Reprogramming Leads to Enhanced Efficacy of Immune Stimulatory Gene Therapy in Diffuse Hemispheric Gliomas.","authors":"Maria B Garcia-Fabiani, Santiago Haase, Kaushik Banerjee, Ziwen Zhu, Brandon L McClellan, Anzar A Mujeeb, Yingxiang Li, Claire E Tronrud, Maria L Varela, Molly E J West, Jin Yu, Padma Kadiyala, Ayman W Taher, Felipe J Núñez, Mahmoud S Alghamri, Andrea Comba, Flor M Mendez, Alejandro J Nicola Candia, Brittany Salazar, Fernando M Nunez, Marta B Edwards, Tingting Qin, Rodrigo T Cartaxo, Michael Niculcea, Carl Koschmann, Sriram Venneti, Montserrat Puigdelloses Vallcorba, Emon Nasajpour, Giulia Pericoli, Maria Vinci, Claudia L Kleinman, Nada Jabado, James P Chandler, Adam M Sonabend, Michael DeCuypere, Dolores Hambardzumyan, Laura M Prolo, Kelly B Mahaney, Gerald A Grant, Claudia K Petritsch, Joshua D Welch, Maureen A Sartor, Pedro R Lowenstein, Maria G Castro","doi":"10.1101/2023.06.13.544658","DOIUrl":"10.1101/2023.06.13.544658","url":null,"abstract":"<p><p>Diffuse hemispheric glioma (DHG), H3 G34-mutant, representing 9-15% of cases, are aggressive Central Nervous System (CNS) tumors with poor prognosis. This study examines the role of epigenetic reprogramming of the immune microenvironment and the response to immune-mediated therapies in G34-mutant DHG. To this end, we utilized human G34-mutant DHG biopsies, primary G34-mutant DHG cultures, and genetically engineered G34-mutant mouse models (GEMMs). Our findings show that the G34 mutation alters histone marks' deposition at promoter and enhancer regions, leading to the activation of the JAK/STAT pathway, which in turn results in an immune-permissive tumor microenvironment. The implementation of Ad-TK/Ad-Flt3L immunostimulatory gene therapy significantly improved median survival, and lead to over 50% long term survivors. Upon tumor rechallenge in the contralateral hemisphere without any additional treatment, the long-term survivors exhibited robust anti-tumor immunity and immunological memory. These results indicate that immune-mediated therapies hold significant potential for clinical translation in treating patients harboring H3.3-G34 mutant DHGs, offering a promising strategy for improving outcomes in this challenging cancer subtype affecting adolescents and young adults (AYA).</p><p><strong>Statement of significance: </strong>This study uncovers the role of the H3.3-G34 mutation in reprogramming the tumor immune microenvironment in diffuse hemispheric gliomas. Our findings support the implementation of precision medicine informed immunotherapies, aiming at improving enhanced therapeutic outcomes in adolescents and young adults harboring H3.3-G34 mutant DHGs.</p>","PeriodicalId":72407,"journal":{"name":"bioRxiv : the preprint server for biology","volume":" ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2025-02-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/11/20/nihpp-2023.06.13.544658v1.PMC10312611.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10149707","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}