Cells最新文献

{"title":"Inflammasome Proteins Are Reliable Biomarkers of the Inflammatory Response in Aneurysmal Subarachnoid Hemorrhage.","authors":"Ruby R Taylor, Robert W Keane, Begoña Guardiola, Sofía López-Lage, Lesmes Moratinos, W Dalton Dietrich, Jon Perez-Barcena, Juan Pablo de Rivero Vaccari","doi":"10.3390/cells13161370","DOIUrl":"10.3390/cells13161370","url":null,"abstract":"<p><p>Aneurysmal subarachnoid hemorrhage (aSAH) is caused by abnormal blood vessel dilation and subsequent rupture, resulting in blood pooling in the subarachnoid space. This neurological insult results in the activation of the inflammasome, a multiprotein complex that processes pro-inflammatory interleukin (IL)-1 cytokines leading to morbidity and mortality. Moreover, increases in inflammasome proteins are associated with clinical deterioration in many neurological diseases. Limited studies have investigated inflammasome protein expression following aSAH. Reliable markers of the inflammatory response associated with aSAH may allow for earlier detection of patients at risk for complications and aid in the identification of novel pharmacologic targets. Here, we investigated whether inflammasome signaling proteins may serve as potential biomarkers of the inflammatory response in aSAH. Serum and cerebrospinal fluid (CSF) from fifteen aSAH subjects and healthy age-matched controls and hydrocephalus (CSF) no-aneurysm controls were evaluated for levels of inflammasome signaling proteins and downstream pro-inflammatory cytokines. Protein measurements were carried out using Simple Plex and Single-Molecule Array (Simoa) technology. The area under the curve (AUC) was calculated using receiver operating characteristics (ROCs) to obtain information on biomarker reliability, specificity, sensitivity, cut-off points, and likelihood ratio. In addition, a Spearman <i>r</i> correlation matrix was performed to determine the correlation between inflammasome protein levels and clinical outcome measures. aSAH subjects demonstrated elevated caspase-1, apoptosis-associated speck-like protein with a caspase recruiting domain (ASC), IL-18 and IL-1β levels in serum, and CSF when compared to controls. Each of these proteins was found to be a promising biomarker of inflammation in aSAH in the CSF. In addition, ASC, caspase-1, and IL-1β were found to be promising biomarkers of inflammation in aSAH in serum. Furthermore, we found that elevated levels of inflammasome proteins in serum are useful to predict worse functional outcomes following aSAH. Thus, the determination of inflammasome protein levels in CSF and serum in aSAH may be utilized as reliable biomarkers of inflammation in aSAH and used clinically to monitor patient outcomes.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11353247/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079276","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel Small Molecules with Anti-Inflammatory and Anti-Angiogenic Activity in a Mouse Model of Oxygen-Induced Retinopathy.","authors":"Adam S Dayoub, Eesha Acharya, Adnan Dibas, Harlan P Jones, Suchismita Acharya","doi":"10.3390/cells13161371","DOIUrl":"10.3390/cells13161371","url":null,"abstract":"<p><p>Retinopathy of prematurity (ROP) has a dual-phase disease pathology; in phase 1, hyperoxia-induced vaso-obliteration occurs in the retinal vasculature due to increased oxidative stress (OS) and inflammation, followed by phase 2, where hypoxia increases the overproduction of growth factors, inducing retinal neovascularization. Toll-like receptor 2 and -4 (TLR2 and TLR4) overactivation, hyper-inflammation, macrophages, and neutrophil infiltration contribute to the developing ROP. AVR-121 and AVR-123 are novel classes of small-molecule dual inhibitors of TLR2/4 tested in a human leukemia monocytic cell line (THP-1) and cord-blood-derived mononuclear cells (CBMCs). Both compounds inhibited TLR2/4 signaling-related inflammatory cytokines in THP-1 cells and inhibited VEGF-induced neovascularization in human retinal endothelial cells (HRECs), which are hallmarks of ROP. In an oxygen-induced retinopathy (OIR) murine model, the intraperitoneal injection of AVR-123 in the hyperoxia phase (P7-P12) or a nanosuspension eyedrop of AVR-123 in the hypoxic phase (P12-P17) significantly reduced vaso-obliteration, angiogenesis, and inflammatory cytokine profiles while not inhibiting the necessary growth factor VEGF in the juvenile mouse eyes. The results are consistent with our hypothesis that targeting the dual TLR2/4 pathway will reduce inflammation, angiogenesis, and vaso-obliteration in vitro and in vivo and reduce cytotoxic immune cells. AVR-123 has the potential to be developed as a therapy for ROP.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11353024/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079359","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Signalling Pathways of Inflammation and Cancer in Human Mononuclear Cells: Effect of Nanoparticle Air Pollutants.","authors":"Agata Niechoda, Maciej Roslan, Katarzyna Milewska, Piotr Szoka, Katarzyna Maciorowska, Adam Holownia","doi":"10.3390/cells13161367","DOIUrl":"10.3390/cells13161367","url":null,"abstract":"<p><p>Fine inhalable particulate matter (PM) triggers an inflammatory response in the airways and activates mononuclear cells, mediators of tissue homeostasis, and tumour-promoting inflammation. We have assessed ex vivo responses of human monocytes and monocyte-derived macrophages to standardised air pollutants: carbon black, urban dust, and nanoparticulate carbon black, focusing on their pro-inflammatory and DNA-damaging properties. None of the PM (100 μg/mL/24 h) was significantly toxic to the cells, aside from inducing oxidative stress, fractional DNA damage, and inhibiting phagocytosis. TNFα was only slightly increased. PM nanoparticles increase the expression and activate DNA-damage-related histone H2A.X as well as pro-inflammatory NF-κB. We have shown that the urban dust stimulates the pathway of DNA damage/repair via the selective post-translational phosphorylation of H2A.X while nanoparticulate carbon black increases inflammation via activation of NF-κB. Moreover, the inflammatory response to lipopolysaccharide was significantly stronger in macrophages pre-exposed to urban dust or nanoparticulate carbon black. Our data show that airborne nanoparticles induce PM-specific, epigenetic alterations in the subsets of cultured mononuclear cells, which may be quantified using binary fluorescence scatterplots. Such changes intercede with inflammatory signalling and highlight important molecular and cell-specific epigenetic mechanisms of tumour-promoting inflammation.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352816/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079364","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The Influence of Betulin and Its Derivatives on Selected Colorectal Cancer Cell Lines' Viability and Their Antioxidant Systems.","authors":"Marcel Madej, Celina Kruszniewska-Rajs, Magdalena Kimsa-Dudek, Agnieszka Synowiec-Wojtarowicz, Elwira Chrobak, Ewa Bębenek, Stanisław Boryczka, Stanisław Głuszek, Jolanta Adamska, Sebastian Kubica, Jarosław Matykiewicz, Joanna Magdalena Gola","doi":"10.3390/cells13161368","DOIUrl":"10.3390/cells13161368","url":null,"abstract":"<p><p>Oxidative stress is considered one of the main reasons for the development of colorectal cancer (CRC). Depending on the stage of the disease, variable activity of the main antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), is observed. Due to limited treatment methods for CRC, new substances with potential antitumor activity targeting pathways related to oxidative stress are currently being sought, with substances of natural origin, including betulin, leading the way. The betulin molecule is chemically modified to obtain new derivatives with improved pharmacokinetic properties and higher biological activity. The aim of this study was to evaluate the effects of betulin and its new derivatives on viability and major antioxidant systems in colorectal cancer cell lines. The study showed that betulin and its derivative EB5 affect the antioxidant enzyme activity to varying degrees at both the protein and mRNA levels. The SW1116 cell line is more resistant to the tested compounds than RKO, which may be due to differences in the genetic and epigenetic profiles of these lines.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352258/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079373","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Direct Binding of Synaptopodin 2-Like Protein to Alpha-Actinin Contributes to Actin Bundle Formation in Cardiomyocytes.","authors":"Hiroshi Yamada, Hirona Osaka, Nanami Tatsumi, Miu Araki, Tadashi Abe, Keiko Kaihara, Ken Takahashi, Eizo Takashima, Takayuki Uchihashi, Keiji Naruse, Kohji Takei","doi":"10.3390/cells13161373","DOIUrl":"10.3390/cells13161373","url":null,"abstract":"<p><p>Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352367/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079245","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Molecular Mechanisms of Autophagy Decline during Aging.","authors":"Shaun H Y Lim, Malene Hansen, Caroline Kumsta","doi":"10.3390/cells13161364","DOIUrl":"10.3390/cells13161364","url":null,"abstract":"<p><p>Macroautophagy (hereafter autophagy) is a cellular recycling process that degrades cytoplasmic components, such as protein aggregates and mitochondria, and is associated with longevity and health in multiple organisms. While mounting evidence supports that autophagy declines with age, the underlying molecular mechanisms remain unclear. Since autophagy is a complex, multistep process, orchestrated by more than 40 autophagy-related proteins with tissue-specific expression patterns and context-dependent regulation, it is challenging to determine how autophagy fails with age. In this review, we describe the individual steps of the autophagy process and summarize the age-dependent molecular changes reported to occur in specific steps of the pathway that could impact autophagy. Moreover, we describe how genetic manipulations of autophagy-related genes can affect lifespan and healthspan through studies in model organisms and age-related disease models. Understanding the age-related changes in each step of the autophagy process may prove useful in developing approaches to prevent autophagy decline and help combat a number of age-related diseases with dysregulated autophagy.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352966/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Functionalized Congeners of 2<i>H</i>-Chromene P2Y₆ Receptor Antagonists.","authors":"Paola Oliva, Asmita Pramanik, Young-Hwan Jung, Sarah A Lewicki, Jamie M Mwendwa, Jong Hwan Park, Kenneth A Jacobson","doi":"10.3390/cells13161366","DOIUrl":"10.3390/cells13161366","url":null,"abstract":"<p><p>The P2Y₆ receptor (P2Y₆R), a G_q-coupled receptor, is a potential drug discovery target for various inflammatory and degenerative conditions. Antagonists have been shown to attenuate colitis, acute lung injury, etc. In the search for competitive antagonists, we have investigated the SAR of 3-nitro-2-(trifluoromethyl)-2<i>H</i>-chromene derivatives, although high affinity is lacking. We now reveal that long-chain amino-functionalized congeners display greatly enhanced affinity in the antagonism of UDP-induced Ca²⁺ mobilization in <i>human</i> (h) P2Y₆R-transfected 1321N1 astrocytoma cells. A 6-(Boc-amino-<i>n</i>-heptylethynyl) analogue <b>30</b> (MRS4940) had an IC₅₀ of 162 nM, which was a 123-fold greater affinity than the corresponding unprotected primary alkylamine, 107-fold greater than the corresponding pivaloyl derivative <b>30</b>, and 132-fold selective compared to the P2Y₁₄R. However, similar Boc-amino chains attached at the 8-position produced weak µM affinity. Thus, the P2Y₆R affinity depended on the chain length, attachment point, and terminal functionality. Off-target activities, at 45 sites, were tested for acylamino derivatives <b>20</b>, <b>24</b>, <b>26</b>, <b>30</b>, <b>31</b>, and <b>37</b>, which showed multiple interactions, particularly at the biogenic amine receptors. The more potent analogues may be suitable for evaluation in inflammation and cancer models, which will be performed in future studies.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352859/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079268","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Implication of the LRR Domain in the Regulation and Activation of the NLRP3 Inflammasome.","authors":"Margaux Cescato, Yixiang Y J Zhu, Laurent Le Corre, Bénédicte F Py, Sophie Georgin-Lavialle, Mathieu P Rodero","doi":"10.3390/cells13161365","DOIUrl":"10.3390/cells13161365","url":null,"abstract":"<p><p>The NLRP3 inflammasome is a critical component of the innate immune response. NLRP3 activation is a tightly controlled process involving an initial priming to express NLRP3, pro-IL-1 β, and pro-IL-18, followed by an activation signal. The precise mechanism of activation is not fully understood due to the diverse range of activators, yet it effectively orchestrates the activation of caspase-1, which subsequently triggers the release of proinflammatory cytokines IL-1β and IL-18. NLRP3 dysregulation can lead to a variety of inflammatory diseases, highlighting its significant role in immune response and disease pathogenesis. NLRP3 is divided into three domains: the PYD, the NACHT, and the LRR domains. This review focuses on the LRR domain of NLRP3, detailing its structural characteristics, its function in pathogen sensing, its role in the degradation process, and its involvement in inflammasome auto-inhibition and activation. Additionally, we discuss the impact of mutations within the LRR domain found in atypical Cryopyrin-Associated Periodic Syndromes (CAPS), highlighting the clinical relevance of this domain.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352923/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079273","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Role of Piezo1 in Terminal Density Reversal of Red Blood Cells.","authors":"Kuntal Dey, Ankie M van Cromvoirt, Inga Hegemann, Jeroen S Goede, Anna Bogdanova","doi":"10.3390/cells13161363","DOIUrl":"10.3390/cells13161363","url":null,"abstract":"<p><p>Density reversal of senescent red blood cells has been known for a long time, yet the identity of the candidate ion transporter(s) causing the senescent cells to swell is still elusive. While performing fractionation of RBCs from healthy individuals in Percoll density gradient and characterization of the separated fractions, we identified a subpopulation of cells in low-density fraction (1.02% ± 0.47) showing signs of senescence such as loss of membrane surface area associated with a reduction in band 3 protein abundance, and Phosphatidylserine (PS) exposure to the outer membrane. In addition, we found that these cells are overloaded with Na⁺ and Ca²⁺. Using a combination of blockers and activators of ion pumps and channels, we revealed reduced activity of Plasma membrane Ca²⁺ ATPase and an increase in Ca²⁺ and Na⁺ leaks through ion channels in senescent-like cells. Our data revealed that Ca²⁺ overload in these cells is a result of reduced PMCA activity and facilitated Ca²⁺ uptake via a hyperactive Piezo1 channel. However, we could not exclude the contribution of other Ca²⁺-permeable ion channels in this scenario. In addition, we found, as a universal mechanism, that an increase in intracellular Ca²⁺ reduced the initially high selectivity of Piezo1 channel for Ca²⁺ and allowed higher Na⁺ uptake, Na⁺ accumulation, and swelling.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352946/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Multiscale Modeling of Bistability in the Yeast Polarity Circuit.","authors":"Siarhei Hladyshau, Kaiyun Guan, Nivedita Nivedita, Beverly Errede, Denis Tsygankov, Timothy C Elston","doi":"10.3390/cells13161358","DOIUrl":"10.3390/cells13161358","url":null,"abstract":"<p><p>Cell polarity refers to the asymmetric distribution of proteins and other molecules along a specified axis within a cell. Polarity establishment is the first step in many cellular processes. For example, directed growth or migration requires the formation of a cell front and back. In many cases, polarity occurs in the absence of spatial cues. That is, the cell undergoes symmetry breaking. Understanding the molecular mechanisms that allow cells to break symmetry and polarize requires computational models that span multiple spatial and temporal scales. Here, we apply a multiscale modeling approach to examine the polarity circuit of yeast. In addition to symmetry breaking, experiments revealed two key features of the yeast polarity circuit: bistability and rapid dismantling of the polarity site following a loss of signal. We used modeling based on ordinary differential equations (ODEs) to investigate mechanisms that generate these behaviors. Our analysis revealed that a model involving positive and negative feedback acting on different time scales captured both features. We then extend our ODE model into a coarse-grained reaction-diffusion equation (RDE) model to capture the spatial profiles of polarity factors. After establishing that the coarse-grained RDE model qualitatively captures key features of the polarity circuit, we expand it to more accurately capture the biochemical reactions involved in the system. We convert the expanded model to a particle-based model that resolves individual molecules and captures fluctuations that arise from the stochastic nature of biochemical reactions. Our models assume that negative regulation results from negative feedback. However, experimental observations do not rule out the possibility that negative regulation occurs through an incoherent feedforward loop. Therefore, we conclude by using our RDE model to suggest how negative feedback might be distinguished from incoherent feedforward regulation.</p>","PeriodicalId":9743,"journal":{"name":"Cells","volume":null,"pages":null},"PeriodicalIF":5.1,"publicationDate":"2024-08-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11352540/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142079321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}