Integrative Biology最新文献

{"title":"Mechanistic dissection of spatial organization in NF-κB signaling pathways by hybrid simulations.","authors":"Yinghao Wu,&nbsp;Kalyani Dhusia,&nbsp;Zhaoqian Su","doi":"10.1093/intbio/zyab006","DOIUrl":"https://doi.org/10.1093/intbio/zyab006","url":null,"abstract":"<p><p>The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is one of the most important transcription factors involved in the regulation of inflammatory signaling pathways. Inappropriate activation of these pathways has been linked to autoimmunity and cancers. Emerging experimental evidences have been showing the existence of elaborate spatial organizations for various molecular components in the pathways. One example is the scaffold protein tumor necrosis factor receptor associated factor (TRAF). While most TRAF proteins form trimeric quaternary structure through their coiled-coil regions, the N-terminal region of some members in the family can further be dimerized. This dimerization of TRAF trimers can drive them into higher-order clusters as a response to receptor stimulation, which functions as a spatial platform to mediate the downstream poly-ubiquitination. However, the molecular mechanism underlying the TRAF protein clustering and its functional impacts are not well-understood. In this article, we developed a hybrid simulation method to tackle this problem. The assembly of TRAF-based signaling platform at the membrane-proximal region is modeled with spatial resolution, while the dynamics of downstream signaling network, including the negative feedbacks through various signaling inhibitors, is simulated as stochastic chemical reactions. These two algorithms are further synchronized under a multiscale simulation framework. Using this computational model, we illustrated that the formation of TRAF signaling platform can trigger an oscillatory NF-κB response. We further demonstrated that the temporal patterns of downstream signal oscillations are closely regulated by the spatial factors of TRAF clustering, such as the geometry and energy of dimerization between TRAF trimers. In general, our study sheds light on the basic mechanism of NF-κB signaling pathway and highlights the functional importance of spatial regulation within the pathway. The simulation framework also showcases its potential of application to other signaling pathways in cells.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 5","pages":"109-120"},"PeriodicalIF":2.5,"publicationDate":"2021-05-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8129552/pdf/zyab006.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38903857","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A reconfigurable microscale assay enables insights into cancer-associated fibroblast modulation of immune cell recruitment.","authors":"Jiaquan Yu,&nbsp;Amber Piazza,&nbsp;Sidney Sparks,&nbsp;Laurel E Hind,&nbsp;David J Niles,&nbsp;Patrick N Ingram,&nbsp;Wei Huang,&nbsp;William A Ricke,&nbsp;David F Jarrard,&nbsp;Anna Huttenlocher,&nbsp;Hirak Basu,&nbsp;David J Beebe","doi":"10.1093/intbio/zyab004","DOIUrl":"https://doi.org/10.1093/intbio/zyab004","url":null,"abstract":"<p><p>Innate immune cell infiltration into neoplastic tissue is the first line of defense against cancer and can play a deterministic role in tumor progression. Here, we describe a series of assays, using a reconfigurable microscale assay platform (i.e. Stacks), which allows the study of immune cell infiltration in vitro with spatiotemporal manipulations. We assembled Stacks assays to investigate tumor-monocyte interactions, re-education of activated macrophages, and neutrophil infiltration. For the first time in vitro, the Stacks infiltration assays reveal that primary tumor-associated fibroblasts from specific patients differ from that associated with the benign region of the prostate in their ability to limit neutrophil infiltration as well as facilitate monocyte adhesion and anti-inflammatory monocyte polarization. These results show that fibroblasts play a regulatory role in immune cell infiltration and that Stacks has the potential to predict individual patients' cancer-immune response.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 4","pages":"87-97"},"PeriodicalIF":2.5,"publicationDate":"2021-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8793920/pdf/nihms-1762984.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25564238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Microbial-based magnetic nanoparticles production: a mini-review.","authors":"Victor Chmykhalo,&nbsp;Anna Belanova,&nbsp;Mariya Belousova,&nbsp;Vera Butova,&nbsp;Yuriy Makarenko,&nbsp;Vera Khrenkova,&nbsp;Alexander Soldatov,&nbsp;Peter Zolotukhin","doi":"10.1093/intbio/zyab005","DOIUrl":"https://doi.org/10.1093/intbio/zyab005","url":null,"abstract":"<p><p>The ever-increasing biomedical application of magnetic nanoparticles (MNPs) implies increasing demand in their scalable and high-throughput production, with finely tuned and well-controlled characteristics. One of the options to meet the demand is microbial production by nanoparticles-synthesizing bacteria. This approach has several benefits over the standard chemical synthesis methods, including improved homogeneity of synthesis, cost-effectiveness, safety and eco-friendliness. There are, however, specific challenges emanating from the nature of the approach that are to be accounted and resolved in each manufacturing instance. Most of the challenges can be resolved by proper selection of the producing organism and optimizing cell culture and nanoparticles extraction conditions. Other issues require development of proper continuous production equipment, medium usage optimization and precursor ions recycling. This mini-review focuses on the related topics in microbial synthesis of MNPs: producing organisms, culturing methods, nanoparticles characteristics tuning, nanoparticles yield and synthesis timeframe considerations, nanoparticles isolation as well as on the respective challenges and possible solutions.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 4","pages":"98-107"},"PeriodicalIF":2.5,"publicationDate":"2021-04-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/intbio/zyab005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25572157","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Oxygenation as a driving factor in epithelial differentiation at the air-liquid interface.","authors":"Sonya Kouthouridis, Julie Goepp, Carolina Martini, Elizabeth Matthes, John W Hanrahan, Christopher Moraes","doi":"10.1093/intbio/zyab002","DOIUrl":"10.1093/intbio/zyab002","url":null,"abstract":"<p><p>Culture at the air-liquid interface is broadly accepted as necessary for differentiation of cultured epithelial cells towards an in vivo-like phenotype. However, air-liquid interface cultures are expensive, laborious and challenging to scale for increased throughput applications. Deconstructing the microenvironmental parameters that drive these differentiation processes could circumvent these limitations, and here we hypothesize that reduced oxygenation due to diffusion limitations in liquid media limits differentiation in submerged cultures; and that this phenotype can be rescued by recreating normoxic conditions at the epithelial monolayer, even under submerged conditions. Guided by computational models, hyperoxygenation of atmospheric conditions was applied to manipulate oxygenation at the monolayer surface. The impact of this rescue condition was confirmed by assessing protein expression of hypoxia-sensitive markers. Differentiation of primary human bronchial epithelial cells isolated from healthy patients was then assessed in air-liquid interface, submerged and hyperoxygenated submerged culture conditions. Markers of differentiation, including epithelial layer thickness, tight junction formation, ciliated surface area and functional capacity for mucociliary clearance, were assessed and found to improve significantly in hyperoxygenated submerged cultures, beyond standard air-liquid interface or submerged culture conditions. These results demonstrate that an air-liquid interface is not necessary to produce highly differentiated epithelial structures, and that increased availability of oxygen and nutrient media can be leveraged as important strategies to improve epithelial differentiation for applications in respiratory toxicology and therapeutic development.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 3","pages":"61-72"},"PeriodicalIF":2.5,"publicationDate":"2021-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7965686/pdf/zyab002.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25442115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Interactivity of biochemical and physical stimuli during epigenetic conditioning and cardiomyocytic differentiation of stem and progenitor cells derived from adult hearts.","authors":"Wendy R Zeng,&nbsp;Pauline M Doran","doi":"10.1093/intbio/zyab003","DOIUrl":"https://doi.org/10.1093/intbio/zyab003","url":null,"abstract":"<p><p>Mixed populations of cardiosphere-derived stem and progenitor cells containing proliferative and cardiomyogenically committed cells were obtained from adult rat hearts. The cells were cultured in either static 2D monolayers or dynamic 3D scaffold systems with fluid flow. Cardiomyocyte lineage commitment in terms of GATA4 and Nkx2.5 expression was significantly enhanced in the dynamic 3D cultures compared with static 2D conditions. Treatment of the cells with 5-azacytidine (5-aza) produced different responses in the two culture systems, as activity of this chemical epigenetic conditioning agent depended on the cell attachment and hydrodynamic conditions provided during culture. Cell growth was unaffected by 5-aza in the static 2D cultures but was significantly reduced under dynamic 3D conditions relative to untreated controls. Myogenic differentiation measured as Mef2c expression was markedly upregulated by 5-aza in the dynamic 3D cultures but downregulated in the static 2D cultures. The ability of the physical environment to modulate the cellular cardiomyogenic response to 5-aza underscores the interactivity of biochemical and physical stimuli applied for cell differentiation. Accordingly, observations about the efficacy of 5-aza as a cardiomyocyte induction agent may not be applicable across different culture systems. Overall, use of dynamic 3D rather than static 2D culture was more beneficial for cardio-specific myogenesis than 5-aza treatment, which generated a more ambiguous differentiation response.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 3","pages":"73-85"},"PeriodicalIF":2.5,"publicationDate":"2021-03-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25463201","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Pericyte migration and proliferation are tightly synchronized to endothelial cell sprouting dynamics.","authors":"Laura Beth Payne,&nbsp;Jordan Darden,&nbsp;Ariana D Suarez-Martinez,&nbsp;Huaning Zhao,&nbsp;Alissa Hendricks,&nbsp;Caitlin Hartland,&nbsp;Diana Chong,&nbsp;Erich J Kushner,&nbsp;Walter L Murfee,&nbsp;John C Chappell","doi":"10.1093/intbio/zyaa027","DOIUrl":"https://doi.org/10.1093/intbio/zyaa027","url":null,"abstract":"<p><p>Pericytes are critical for microvascular stability and maintenance, among other important physiological functions, yet their involvement in vessel formation processes remains poorly understood. To gain insight into pericyte behaviors during vascular remodeling, we developed two complementary tissue explant models utilizing 'double reporter' animals with fluorescently-labeled pericytes and endothelial cells (via Ng2:DsRed and Flk-1:eGFP genes, respectively). Time-lapse confocal imaging of active vessel remodeling within adult connective tissues and embryonic skin revealed a subset of pericytes detaching and migrating away from the vessel wall. Vessel-associated pericytes displayed rapid filopodial sampling near sprouting endothelial cells that emerged from parent vessels to form nascent branches. Pericytes near angiogenic sprouts were also more migratory, initiating persistent and directional movement along newly forming vessels. Pericyte cell divisions coincided more frequently with elongating endothelial sprouts, rather than sprout initiation sites, an observation confirmed with in vivo data from the developing mouse brain. Taken together, these data suggest that (i) pericyte detachment from the vessel wall may represent an important physiological process to enhance endothelial cell plasticity during vascular remodeling, and (ii) pericyte migration and proliferation are highly synchronized with endothelial cell behaviors during the coordinated expansion of a vascular network.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 2","pages":"31-43"},"PeriodicalIF":2.5,"publicationDate":"2021-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919101/pdf/zyaa027.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25311317","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Patterns of virus growth across the diversity of life.","authors":"Tianyi Jin,&nbsp;John Yin","doi":"10.1093/intbio/zyab001","DOIUrl":"https://doi.org/10.1093/intbio/zyab001","url":null,"abstract":"<p><p>Although viruses in their natural habitats add up to less than 10% of the biomass, they contribute more than 90% of the genome sequences [1]. These viral sequences or 'viromes' encode viruses that populate the Earth's oceans [2, 3] and terrestrial environments [4, 5], where their infections impact life across diverse ecological niches and scales [6, 7], including humans [8-10]. Most viruses have yet to be isolated and cultured [11-13], and surprisingly few efforts have explored what analysis of available data might reveal about their nature. Here, we compiled and analyzed seven decades of one-step growth and other data for viruses from six major families, including their infections of archaeal, bacterial and eukaryotic hosts [14-191]. We found that the use of host cell biomass for virus production was highest for archaea at 10%, followed by bacteria at 1% and eukarya at 0.01%, highlighting the degree to which viruses of archaea and bacteria exploit their host cells. For individual host cells, the yield of virus progeny spanned a relatively narrow range (10-1000 infectious particles per cell) compared with the million-fold difference in size between the smallest and largest cells. Furthermore, healthy and infected host cells were remarkably similar in the time they needed to multiply themselves or their virus progeny. Specifically, the doubling time of healthy cells and the delay time for virus release from infected cells were not only correlated (r = 0.71, p < 10-10, n = 101); they also spanned the same range from tens of minutes to about a week. These results have implications for better understanding the growth, spread and persistence of viruses in complex natural habitats that abound with diverse hosts, including humans and their associated microbes.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 2","pages":"44-59"},"PeriodicalIF":2.5,"publicationDate":"2021-02-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/intbio/zyab001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25393291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Growth of tumor emboli within a vessel model reveals dependence on the magnitude of mechanical constraint.","authors":"Jonathan Kulwatno,&nbsp;Jamie Gearhart,&nbsp;Xiangyu Gong,&nbsp;Nora Herzog,&nbsp;Matthew Getzin,&nbsp;Mihaela Skobe,&nbsp;Kristen L Mills","doi":"10.1093/intbio/zyaa024","DOIUrl":"https://doi.org/10.1093/intbio/zyaa024","url":null,"abstract":"<p><p>Tumor emboli-aggregates of tumor cells within vessels-pose a clinical challenge as they are associated with increased metastasis and tumor recurrence. When growing within a vessel, tumor emboli are subject to a unique mechanical constraint provided by the tubular geometry of the vessel. Current models of tumor emboli use unconstrained multicellular tumor spheroids, which neglect this mechanical interplay. Here, we modeled a lymphatic vessel as a 200 μm-diameter channel in either a stiff or soft, bioinert agarose matrix to create a vessel-like constraint model (VLCM), and we modeled colon or breast cancer tumor emboli with aggregates of HCT116 or SUM149PT cells, respectively. The stiff matrix VLCM constrained the tumor emboli to the cylindrical channel, which led to continuous growth of the emboli, in contrast to the growth rate reduction that unconstrained spheroids exhibit. Emboli morphology in the soft matrix VLCM, however, was dependent on the magnitude of mechanical mismatch between the matrix and the cell aggregates. In general, when the elastic modulus of the matrix of the VLCM was greater than the emboli (EVLCM/Eemb > 1), the emboli were constrained to grow within the channel, and when the elastic modulus of the matrix was less than the emboli (0 < EVLCM/Eemb < 1), the emboli bulged into the matrix. Due to a large difference in myosin II expression between the cell lines, we hypothesized that tumor cell aggregate stiffness is an indicator of cellular force-generating capability. Inhibitors of myosin-related force generation decreased the elastic modulus and/or increased the stress relaxation of the tumor cell aggregates, effectively increasing the mechanical mismatch. The increased mechanical mismatch after drug treatment was correlated with increased confinement of tumor emboli growth along the channel, which may translate to increased tumor burden due to the increased tumor volume within the diffusion distance of nutrients and oxygen.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 1","pages":"1-16"},"PeriodicalIF":2.5,"publicationDate":"2021-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/intbio/zyaa024","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38817709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Matrix degradation and cell proliferation are coupled to promote invasion and escape from an engineered human breast microtumor.","authors":"Emann M Rabie, Sherry X Zhang, Andreas P Kourouklis, A Nihan Kilinc, Allison K Simi, Derek C Radisky, Joe Tien, Celeste M Nelson","doi":"10.1093/intbio/zyaa026","DOIUrl":"10.1093/intbio/zyaa026","url":null,"abstract":"<p><p>Metastasis, the leading cause of mortality in cancer patients, depends upon the ability of cancer cells to invade into the extracellular matrix that surrounds the primary tumor and to escape into the vasculature. To investigate the features of the microenvironment that regulate invasion and escape, we generated solid microtumors of MDA-MB-231 human breast carcinoma cells within gels of type I collagen. The microtumors were formed at defined distances adjacent to an empty cavity, which served as an artificial vessel into which the constituent tumor cells could escape. To define the relative contributions of matrix degradation and cell proliferation on invasion and escape, we used pharmacological approaches to block the activity of matrix metalloproteinases (MMPs) or to arrest the cell cycle. We found that blocking MMP activity prevents both invasion and escape of the breast cancer cells. Surprisingly, blocking proliferation increases the rate of invasion but has no effect on that of escape. We found that arresting the cell cycle increases the expression of MMPs, consistent with the increased rate of invasion. To gain additional insight into the role of cell proliferation in the invasion process, we generated microtumors from cells that express the fluorescent ubiquitination-based cell cycle indicator. We found that the cells that initiate invasions are preferentially quiescent, whereas cell proliferation is associated with the extension of invasions. These data suggest that matrix degradation and cell proliferation are coupled during the invasion and escape of human breast cancer cells and highlight the critical role of matrix proteolysis in governing tumor phenotype.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"13 1","pages":"17-29"},"PeriodicalIF":2.5,"publicationDate":"2021-02-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7856634/pdf/zyaa026.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38795533","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Tumor-macrophage crosstalk: how to listen.","authors":"Tuli Dey","doi":"10.1093/intbio/zyaa023","DOIUrl":"https://doi.org/10.1093/intbio/zyaa023","url":null,"abstract":"<p><p>The tumor microenvironment contains many cellular components influencing tumor behaviors, such as metastasis, angiogenesis and chemo-resistance. Tumor-associated macrophages (TAMs) are one of such components that can also manipulate the overall prognosis and patient survival. Analysis of tumor-macrophage crosstalk is crucial as tumor cells can polarize circulatory monocytes into TAMs. Such trans-polarization of macrophages support tumor mediated evasion and suppression of immune response. Additionally, such TAMs significantly influence tumor growth and proliferation, making them a potential candidate for precision therapeutics. However, the failure of macrophage-dependent therapies at clinical trials emphasizes the fault in current perception and research modality. This review discussed this field's progress regarding emerging model systems with a focused view on the in vitro platforms. The inadequacy of currently available models and their implications on existing studies also analyzed. The need for a conceptual and experimental leap toward a human-relevant in vitro custom-built platform for studying tumor-macrophage crosstalk is acknowledged.</p>","PeriodicalId":80,"journal":{"name":"Integrative Biology","volume":"12 12","pages":"291-302"},"PeriodicalIF":2.5,"publicationDate":"2020-12-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/intbio/zyaa023","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38619297","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}