Ines C Kübler, Jenny Kretzschmar, Maria Nieves Arredondo-Lasso, Sean D Keeley, Luca Claudia Rößler, Katharina Ganss, Tatiana Sandoval-Guzmán, Marko Brankatschk
{"title":"Systemic and local lipid adaptations underlie regeneration in Drosophila melanogaster and Ambystoma mexicanum.","authors":"Ines C Kübler, Jenny Kretzschmar, Maria Nieves Arredondo-Lasso, Sean D Keeley, Luca Claudia Rößler, Katharina Ganss, Tatiana Sandoval-Guzmán, Marko Brankatschk","doi":"10.1038/s41536-024-00375-x","DOIUrl":"10.1038/s41536-024-00375-x","url":null,"abstract":"<p><p>In regenerating tissues, synthesis and remodeling of membranes rely on lipid turnover and transport. Our study addresses lipid adaptations in intestinal regeneration of Drosophila melanogaster and limb regeneration of Ambystoma mexicanum. We found changes in lipid profiles at different locations: transport, storage organs and regenerating tissues. We demonstrate that attenuating insulin signaling, exclusively in fat storage, inhibits the regeneration-specific response in both the fat storage and the regenerating tissue in Drosophila. Furthermore, in uninjured axolotls we found sex-specific lipid profiles in both storage and circulation, while in regenerating animals these differences subside. The regenerating limb presents a unique sterol profile, albeit with no sex differences. We postulate that regeneration triggers a systemic response, where organs storing lipids play a significant role in the regulation of systemic lipid traffic. Second, that this response may be an active and well-regulated mechanism, as observed when homeostatic sex-differences disappear in regenerating salamanders.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11522293/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142548922","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Regeneration-specific promoter switching facilitates Mest expression in the mouse digit tip to modulate neutrophil response.","authors":"Vivian Jou, Sophia M Peña, Jessica A Lehoczky","doi":"10.1038/s41536-024-00376-w","DOIUrl":"10.1038/s41536-024-00376-w","url":null,"abstract":"<p><p>The mouse digit tip regenerates following amputation, a process mediated by a cellularly heterogeneous blastema. We previously found the gene Mest to be highly expressed in mesenchymal cells of the blastema and a strong candidate pro-regenerative gene. We now show Mest digit expression is regeneration-specific and not upregulated in post-amputation fibrosing proximal digits. Mest homozygous knockout mice exhibit delayed bone regeneration though no phenotype is found in paternal knockout mice, inconsistent with the defined maternal genomic imprinting of Mest. We demonstrate that promoter switching, not loss of imprinting, regulates biallelic Mest expression in the blastema and does not occur during embryogenesis, indicating a regeneration-specific mechanism. Requirement for Mest expression is tied to modulating neutrophil response, as revealed by scRNAseq and FACS comparing wildtype and knockout blastemas. Collectively, the imprinted gene Mest is required for proper digit tip regeneration and its blastema expression is facilitated by promoter switching for biallelic expression.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11519450/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142523616","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Gregory P Campbell, Dwarkesh Amin, Kristin Hsieh, George S Hussey, Anthony J St Leger, Jeffrey M Gross, Stephen F Badylak, Takaaki Kuwajima
{"title":"Immunomodulation by the combination of statin and matrix-bound nanovesicle enhances optic nerve regeneration.","authors":"Gregory P Campbell, Dwarkesh Amin, Kristin Hsieh, George S Hussey, Anthony J St Leger, Jeffrey M Gross, Stephen F Badylak, Takaaki Kuwajima","doi":"10.1038/s41536-024-00374-y","DOIUrl":"10.1038/s41536-024-00374-y","url":null,"abstract":"<p><p>Modulating inflammation is critical to enhance nerve regeneration after injury. However, clinically applicable regenerative therapies that modulate inflammation have not yet been established. Here, we demonstrate synergistic effects of the combination of an HMG-CoA reductase inhibitor, statin/fluvastatin and critical components of the extracellular matrix, Matrix-Bound Nanovesicles (MBV) to enhance axon regeneration and neuroprotection after mouse optic nerve injury. Mechanistically, co-intravitreal injections of fluvastatin and MBV robustly promote infiltration of monocytes and neutrophils, which lead to RGC protection and axon regeneration. Furthermore, monocyte infiltration is triggered by elevated expression of CCL2, a chemokine, in the superficial layer of the retina after treatment with a combination of fluvastatin and MBV or IL-33, a cytokine contained within MBV. Finally, this therapy can be further combined with AAV-based gene therapy blocking anti-regenerative pathways in RGCs to extend regenerated axons. These data highlight novel molecular insights into the development of immunomodulatory regenerative therapy.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11513974/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142512852","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mor Grinstein, Stephanie L Tsai, Daniel Montoro, Benjamin R Freedman, Heather L Dingwall, Steffany Villaseñor, Ken Zou, Moshe Sade-Feldman, Miho J Tanaka, David J Mooney, Terence D Capellini, Jayaraj Rajagopal, Jenna L Galloway
{"title":"A latent Axin2<sup>+</sup>/Scx<sup>+</sup> progenitor pool is the central organizer of tendon healing.","authors":"Mor Grinstein, Stephanie L Tsai, Daniel Montoro, Benjamin R Freedman, Heather L Dingwall, Steffany Villaseñor, Ken Zou, Moshe Sade-Feldman, Miho J Tanaka, David J Mooney, Terence D Capellini, Jayaraj Rajagopal, Jenna L Galloway","doi":"10.1038/s41536-024-00370-2","DOIUrl":"https://doi.org/10.1038/s41536-024-00370-2","url":null,"abstract":"<p><p>A tendon's ordered extracellular matrix (ECM) is essential for transmitting force but is also highly prone to injury. How tendon cells embedded within and surrounding this dense ECM orchestrate healing is not well understood. Here, we identify a specialized quiescent Scx<sup>+</sup>/Axin2<sup>+</sup> population in mouse and human tendons that initiates healing and is a major functional contributor to repair. Axin2<sup>+</sup> cells express stem cell markers, expand in vitro, and have multilineage differentiation potential. Following tendon injury, Axin2<sup>+</sup>-descendants infiltrate the injury site, proliferate, and differentiate into tenocytes. Transplantation assays of Axin2-labeled cells into injured tendons reveal their dual capacity to significantly proliferate and differentiate yet retain their Axin2<sup>+</sup> identity. Specific loss of Wnt secretion in Axin2<sup>+</sup> or Scx<sup>+</sup> cells disrupts their ability to respond to injury, severely compromising healing. Our work highlights an unusual paradigm, wherein specialized Axin2<sup>+</sup>/Scx<sup>+</sup> cells rely on self-regulation to maintain their identity as key organizers of tissue healing.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11487078/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480633","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Anastasia Pacary, Diane Peurichard, Laurence Vaysse, Paul Monsarrat, Clémence Bolut, Adeline Girel, Christophe Guissard, Anne Lorsignol, Valérie Planat-Benard, Jenny Paupert, Marielle Ousset, Louis Casteilla
{"title":"A computational model reveals an early transient decrease in fiber cross-linking that unlocks adult regeneration.","authors":"Anastasia Pacary, Diane Peurichard, Laurence Vaysse, Paul Monsarrat, Clémence Bolut, Adeline Girel, Christophe Guissard, Anne Lorsignol, Valérie Planat-Benard, Jenny Paupert, Marielle Ousset, Louis Casteilla","doi":"10.1038/s41536-024-00373-z","DOIUrl":"https://doi.org/10.1038/s41536-024-00373-z","url":null,"abstract":"<p><p>The decline in regeneration efficiency after birth in mammals is a significant roadblock for regenerative medicine in tissue repair. We previously developed a computational agent based-model (ABM) that recapitulates mechanical interactions between cells and the extracellular-matrix (ECM), to investigate key drivers of tissue repair in adults. Time calibration alongside a parameter sensitivity analysis of the model suggested that an early and transient decrease in ECM cross-linking guides tissue repair toward regeneration. Consistent with the computational model, transient inhibition or stimulation of fiber cross-linking for the first six days after subcutaneous adipose tissue (AT) resection in adult mice led to regenerative or scar healing, respectively. Therefore, this work positions the computational model as a predictive tool for tissue regeneration that with further development will behave as a digital twin of our in vivo model. In addition, it opens new therapeutic approaches targeting ECM cross-linking to induce tissue regeneration in adult mammals.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11480365/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142480632","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Océane Blaise, Constance Duchesne, Elena Capuzzo, Marie-Anne Nahori, Julien Fernandes, Michael G Connor, Mélanie A Hamon, Javier Pizarro-Cerda, Jean-Jacques Lataillade, Colin McGuckin, Antoine Rousseau, Sébastien Banzet, Olivier Dussurget, Nadira Frescaline
{"title":"Infected wound repair correlates with collagen I induction and NOX2 activation by cold atmospheric plasma.","authors":"Océane Blaise, Constance Duchesne, Elena Capuzzo, Marie-Anne Nahori, Julien Fernandes, Michael G Connor, Mélanie A Hamon, Javier Pizarro-Cerda, Jean-Jacques Lataillade, Colin McGuckin, Antoine Rousseau, Sébastien Banzet, Olivier Dussurget, Nadira Frescaline","doi":"10.1038/s41536-024-00372-0","DOIUrl":"10.1038/s41536-024-00372-0","url":null,"abstract":"<p><p>Cold atmospheric plasma (CAP) is a promising complement to tissue repair and regenerative medicine approaches. CAP has therapeutic potential in infected cutaneous wounds by mechanisms which remain enigmatic. Here, CAP is shown to activate phagocyte NADPH oxidase complex NOX2. CAP induced increased intracellular reactive oxygen species, alleviated by NOX2 inhibitors. Genetic and pharmacological inhibitions of NOX2 in macrophages and bioengineered skin infected with Staphylococcus aureus and treated with CAP reduced intracellular oxidants and increased bacterial survival. CAP triggered Rac activation and phosphorylation of p40<sup>phox</sup> and p47<sup>phox</sup> required for NOX2 assembly and activity. Furthermore, CAP induced collagen I expression by fibroblasts. Infection and healing kinetics showed that murine skin wounds infected with S. aureus and treated with CAP are characterized by decreased bacterial burden, increased length of neoepidermis and extracellular matrix formation. Collectively, our findings identify mechanisms triggered by CAP that subdue infection and result in enhanced repair following skin injury.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-10-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11447178/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142367428","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Candice Ashmore-Harris, Evangelia Antonopoulou, Rhona E Aird, Tak Yung Man, Simon M Finney, Annelijn M Speel, Wei-Yu Lu, Stuart J Forbes, Victoria L Gadd, Sarah L Waters
{"title":"Utilising an in silico model to predict outcomes in senescence-driven acute liver injury.","authors":"Candice Ashmore-Harris, Evangelia Antonopoulou, Rhona E Aird, Tak Yung Man, Simon M Finney, Annelijn M Speel, Wei-Yu Lu, Stuart J Forbes, Victoria L Gadd, Sarah L Waters","doi":"10.1038/s41536-024-00371-1","DOIUrl":"10.1038/s41536-024-00371-1","url":null,"abstract":"<p><p>Currently liver transplantation is the only treatment option for liver disease, but organ availability cannot meet patient demand. Alternative regenerative therapies, including cell transplantation, aim to modulate the injured microenvironment from inflammation and scarring towards regeneration. The complexity of the liver injury response makes it challenging to identify suitable therapeutic targets when relying on experimental approaches alone. Therefore, we adopted a combined in vivo-in silico approach and developed an ordinary differential equation model of acute liver disease able to predict the host response to injury and potential interventions. The Mdm2<sup>fl/fl</sup> mouse model of senescence-driven liver injury was used to generate a quantitative dynamic characterisation of the key cellular players (macrophages, endothelial cells, myofibroblasts) and extra cellular matrix involved in liver injury. This was qualitatively captured by the mathematical model. The mathematical model was then used to predict injury outcomes in response to milder and more severe levels of senescence-induced liver injury and validated with experimental in vivo data. In silico experiments using the validated model were then performed to interrogate potential approaches to enhance regeneration. These predicted that increasing the rate of macrophage phenotypic switch or increasing the number of pro-regenerative macrophages in the system will accelerate the rate of senescent cell clearance and resolution. These results showcase the potential benefits of mechanistic mathematical modelling for capturing the dynamics of complex biological systems and identifying therapeutic interventions that may enhance our understanding of injury-repair mechanisms and reduce translational bottlenecks.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11442582/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332262","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Mehdi Hassanpour, Amankeldi A Salybkov, Shuzo Kobayashi, Takayuki Asahara
{"title":"Anti-inflammatory Prowess of endothelial progenitor cells in the realm of biology and medicine.","authors":"Mehdi Hassanpour, Amankeldi A Salybkov, Shuzo Kobayashi, Takayuki Asahara","doi":"10.1038/s41536-024-00365-z","DOIUrl":"10.1038/s41536-024-00365-z","url":null,"abstract":"<p><p>Endothelial inflammation plays a crucial role in vascular-related diseases, a leading cause of global mortality. Among various cellular players, endothelial progenitor cells (EPCs) emerge as non-differentiated endothelial cells circulating in the bloodstream. Recent evidence highlights the transformative role of EPCs in shifting from an inflammatory/immunosuppressive crisis to an anti-inflammatory/immunomodulatory response. Despite the importance of these functions, the regulatory mechanisms governing EPC activities and their physiological significance in vascular regenerative medicine remain elusive. Surprisingly, the current literature lacks a comprehensive review of EPCs' effects on inflammatory processes. This narrative review aims to fill this gap by exploring the cutting-edge role of EPCs against inflammation, from molecular intricacies to broader medical perspectives. By examining how EPCs modulate inflammatory responses, we aim to unravel their anti-inflammatory significance in vascular regenerative medicine, deepening insights into EPCs' molecular mechanisms and guiding future therapeutic strategies targeting vascular-related diseases.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11442670/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332260","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Jeanne E Barthold, Luyao Cai, Kaitlin P McCreery, Kristine M Fischenich, Kevin N Eckstein, Virginia L Ferguson, Nancy C Emery, Gert Breur, Corey P Neu
{"title":"Integrative cartilage repair using acellular allografts for engineered structure and surface lubrication in vivo.","authors":"Jeanne E Barthold, Luyao Cai, Kaitlin P McCreery, Kristine M Fischenich, Kevin N Eckstein, Virginia L Ferguson, Nancy C Emery, Gert Breur, Corey P Neu","doi":"10.1038/s41536-024-00367-x","DOIUrl":"https://doi.org/10.1038/s41536-024-00367-x","url":null,"abstract":"<p><p>The repair of articular cartilage after damage is challenging, and decellularized tissue offers a possible treatment option to promote regeneration. Here, we show that acellular osteochondral allografts improve integrative cartilage repair compared to untreated defects after 6 months in an ovine model. Functional measures of intratissue strain/structure assessed by MRI demonstrate similar biomechanics of implants and native cartilage. Compared to native tissue and defects, the structure, composition, and tribology of acellular allografts preserve surface roughness and lubrication, material properties under compression and relaxation, compositional ratios of collagen:glycosaminoglycan and collagen:phosphate, and relative composition of types I/II collagen. While high cellularity was observed in bone regions and integration zones between cartilage-allografts, recellularization of chondral implants was inconsistent, with cell migration typically less than ~750 µm into the dense decellularized tissue, possibly limiting long-term cellular maintenance. Our results demonstrate the structural and biomechanical efficacy of acellular allografts for at least six months in vivo.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11438864/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142332261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sihao He, Tianyong Hou, Jiangling Zhou, Bo Yu, Juan Cai, Fei Luo, Jianzhong Xu, Junchao Xing
{"title":"Implication of CXCR2-Src axis in the angiogenic and osteogenic effects of FP-TEB.","authors":"Sihao He, Tianyong Hou, Jiangling Zhou, Bo Yu, Juan Cai, Fei Luo, Jianzhong Xu, Junchao Xing","doi":"10.1038/s41536-024-00364-0","DOIUrl":"https://doi.org/10.1038/s41536-024-00364-0","url":null,"abstract":"<p><p>Application of tissue-engineered bones (TEBs) is hindered by challenges associated with incorporated viable cells. Previously, we employed freeze-drying techniques on TEBs to devitalize mesenchymal stem cells (MSCs) while preserving functional proteins, yielding functional proteins-based TEBs (FP-TEBs). Here, we aimed to elucidate their in vivo angiogenic and osteogenic capabilities and the mechanisms. qPCR arrays were employed to evaluate chemokines and receptors governing EC migration. Identified C-X-C chemokine receptors (CXCRs) were substantiated using shRNAs, and the pivotal role of CXCR2 was validated via conditional knockout mice. Finally, signaling molecules downstream of CXCR2 were identified. Additionally, Src, MAP4K4, and p38 MAPK were identified indispensable for CXCR2 function. Further investigations revealed that regulation of p38 MAPK by Src was mediated by MAP4K4. In conclusion, FP-TEBs promoted EC migration, angiogenesis, and osteogenesis via the CXCR2-Src-Map4k4-p38 MAPK axis.</p>","PeriodicalId":54236,"journal":{"name":"npj Regenerative Medicine","volume":null,"pages":null},"PeriodicalIF":6.4,"publicationDate":"2024-09-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11415383/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142300797","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}