Biomolecules最新文献

{"title":"The Regulation of Cellular Senescence in Cancer.","authors":"Xianhong Zhang, Yue Gao, Siyu Zhang, Yixiong Wang, Yitian Du, Shuailin Hao, Ting Ni","doi":"10.3390/biom15030448","DOIUrl":"10.3390/biom15030448","url":null,"abstract":"<p><p>Cellular senescence is a stable state of cell cycle arrest caused by telomere shortening or various stresses. After senescence, cells cease dividing and exhibit many age-related characteristics. Unlike the halted proliferation of senescence cells, cancer cells are considered to have unlimited growth potential. When cells display senescence-related features, such as telomere loss or stem cell failure, they can inhibit tumor development. Therefore, inducing cells to enter a senescence state can serve as a barrier to tumor cell development. However, many recent studies have found that sustained senescence of tumor cells or normal cells under certain circumstances can exert environment-dependent effects of tumor promotion and inhibition by producing various cytokines. In this review, we first introduce the causes and characteristics of induced cellular senescence, analyze the senescence process of immune cells and cancer cells, and then discuss the dual regulatory role of cell senescence on tumor growth and senescence-induced therapies targeting cancer cells. Finally, we discuss the role of senescence in tumor progression and treatment opportunities, and propose further studies on cellular senescence and cancer therapy.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940315/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728330","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":"Associations Between Follicular Fluid Biomarkers and IVF/ICSI Outcomes in Normo-Ovulatory Women-A Systematic Review.","authors":"Soha Albeitawi, Saif-Ulislam Bani-Mousa, Baraa Jarrar, Ibrahim Aloqaily, Nour Al-Shlool, Ghaida Alsheyab, Ahmad Kassab, Baha'a Qawasmi, Abdalrahman Awaisheh","doi":"10.3390/biom15030443","DOIUrl":"10.3390/biom15030443","url":null,"abstract":"<p><p>(1) Background: The follicular fluid (FF) comprises a large portion of ovarian follicles, and serves as both a communication and growth medium for oocytes, and thus should be representative of the metabolomic status of the follicle. This review aims to explore FF biomarkers as well as their effects on fertilization, oocyte, and embryo development, and later on implantation and maintenance of pregnancy. (2) Methods: This review was registered in the PROSPERO database with the ID: CRD42025633101. We parsed PubMed, Scopus, and Google Scholar for research on the effects of different FF biomarkers on IVF/ICSI outcomes in normo-ovulatory women. Included studies were assessed for risk of bias using the NOS scale. Data were extracted and tabulated by two independent researchers. (3) Results: 22 included articles, with a sample size range of 31 to 414 and a median of 60 participants, contained 61 biomarkers, including proteins, growth factors, steroid and polypeptide hormones, inflammation and oxidative stress markers, amino acids, vitamins, lipids of different types, and miRNAs. Most of the biomarkers studied had significant effects on IVF/ICSI outcomes, and seem to have roles in various cellular pathways responsible for oocyte and embryo growth, implantation, placental formation, and maintenance of pregnancy. The FF metabolome also seems to be interconnected, with its various components influencing the levels and activities of each other through feedback loops. (4) Conclusions: FF biomarkers can be utilized for diagnostic and therapeutic purposes in IVF; however, further studies are required for choosing the most promising ones due to heterogeneity of results. Widespread adoption of LC-MS and miRNA microarrays can help quantify a representative FF metabolome, and we see great potential for in vitro supplementation (IVS) of some FF biomarkers in improving IVF/ICSI outcomes.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940193/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728249","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":"Conformational Analyses of the AHD1-UBAN Region of TNIP1 Highlight Key Amino Acids for Interaction with Ubiquitin.","authors":"Michael L Samulevich, Liam E Carman, Rambon Shamilov, Brian J Aneskievich","doi":"10.3390/biom15030453","DOIUrl":"10.3390/biom15030453","url":null,"abstract":"<p><p>Tumor necrosis factor ɑ (TNFɑ)-induced protein 3 (TNFAIP3)-interacting protein 1 (TNIP1) is genetically and functionally linked to limiting auto-immune and inflammatory responses. We have shown that TNIP1 (alias A20-binding inhibitor of NF-κB 1, ABIN1), functioning as a hub location to coordinate other proteins in repressing inflammatory signaling, aligns with biophysical traits indicative of its being an intrinsically disordered protein (IDP). IDPs move through a repertoire of three-dimensional structures rather than being in one set conformation. Here we employed bioinformatic analysis and biophysical interventions via amino acid mutations to assess and alter, respectively, conformational flexibility along a crucial region of TNIP1, encompassing the ABIN homology domain 1 and ubiquitin-binding domain in ABIN proteins and NEMO (AHD1-UBAN), by purposeful replacement of key residues. In vitro secondary structure measurements were mostly in line with, but not necessarily to the same degree as, expected results from in silico assessments. Notably, changes in single amino acids outside of the ubiquitin-binding region for gain-of-order effects had consequences along the length of the AHD1-UBAN propagating to that region. This is evidenced by differences in recognition of the partner protein polyubiquitin ≥ 28 residues away, depending on the mutation site, from the previously identified key binding site. These findings serve to demonstrate the role of conformational flexibility in protein partner recognition by TNIP1, thus identifying key amino acids likely to impact the molecular dynamics involved in TNIP1 repression of inflammatory signaling at large.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940065/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728027","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":"Tissue Resident and Infiltrating Immune Cells: Their Influence on the Demise of Beta Cells in Type 1 Diabetes.","authors":"Sophie L Walker, Pia Leete, Joanne Boldison","doi":"10.3390/biom15030441","DOIUrl":"10.3390/biom15030441","url":null,"abstract":"<p><p>Type 1 diabetes (T1D) is an organ-specific autoimmune disease that results in the selective loss of pancreatic beta cells and an eventual deficit in insulin production to maintain glucose homeostasis. It is now increasingly accepted that this dynamic disease process is multifactorial; involves a variety of immune cells which contribute to an inflamed pancreatic microenvironment; and that the condition is heterogenous, resulting in variable rates of subsequent beta cell damage. In this review, we will explore the current understanding of the cellular interactions between both resident and infiltrating immune cells within the pancreatic environment, highlighting key mechanisms which may promote the beta cell destruction and islet damage associated with T1D.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11939886/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728335","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":"Efficient Fabrication of Human Corneal Stromal Cell Spheroids and Promoting Cell Stemness Based on 3D-Printed Derived PDMS Microwell Platform.","authors":"Yuexi Chen, Jianing Gu, Zekai Cui, Xihao Sun, Yuqin Liang, Chunwen Duan, Xiaoxue Li, Zhanyu Su, Bo Zhang, Jiansu Chen, Zheng Wang","doi":"10.3390/biom15030438","DOIUrl":"10.3390/biom15030438","url":null,"abstract":"<p><p>Spherical culture could promote the plasticity and stemness of human corneal stromal cells (hCSCs). Here, we introduce a novel three-dimensional (3D) cell culture system based on a polydimethylsiloxane (PDMS) microwell platform composed of many V-bottom microcavities to generate human corneal stromal cell spheroids and promote cell stemness. We isolated hCSCs from SMILE-derived lenticules and maintained their physiological phenotype by culturing them in a medium supplemented with human corneal stromal extract (hCSE). Utilizing a PDMS microwell platform fabricated through 3D printing technology, we successfully generated 3D corneal stromal cell spheroids (3D-CSC) with uniform size and stable structure, exhibiting increased expression of pluripotency factors, including OCT4, NANOG, SOX2, KLF4, and PAX6. Furthermore, the iPS supernatant of E8-conditioned medium (E8-CM) significantly enhanced the stemness properties of these cells. RNA sequencing and proteomics analyses revealed that 3D-CSCs exhibited superior proliferation, differentiation, cell adhesion, migration, and neurogenesis compared to traditional monolayer cultures, underscoring the role of biophysical cues in promoting hCSCs stemness. In summary, this study presents an effective 3D cell culture platform that mimics the in vivo microenvironment, facilitating the enhancement of stemness properties and providing valuable insights into corneal tissue engineering and regenerative medicine, particularly for treating corneal opacities and diseases.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940411/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728208","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":"Salivary-Gland-Mediated Nitrate Recirculation as a Modulator for Cardiovascular Diseases.","authors":"Baoxing Pang, Xingyun Qi, Huiliang Zhang","doi":"10.3390/biom15030439","DOIUrl":"10.3390/biom15030439","url":null,"abstract":"<p><p>Cardiovascular diseases (CVDs), which include multiple disorders of the heart and blood vessels, are the leading causes of death. Nitric oxide (NO) is a vasodilator that regulates vascular tension. Endogenous NO is produced via the L-arginine-nitric oxide synthase (NOS) pathway. In conditions of cardiovascular dysfunction, NOS activity is impaired, leading to NO deficiency. In turn, the reduction in NO bioactivity exacerbates the pathogenesis of CVDs. Exogenous intake of inorganic nitrate supplements endogenous production via the nitrate-nitrite-NO pathway to maintain the NO supply. Salivary glands play an essential role in the conversion of nitrate to NO, with approximately 25% of circulating nitrate being absorbed and secreted into saliva. As a result, salivary nitrate concentrations can exceed that in the blood by more than tenfold. This recycled nitrate in saliva serves as a reservoir for NO and performs NO-like functions when endogenous NO production is insufficient. In this review, we summarize the emerging benefits of dietary nitrate in CVDs, with a particular focus on salivary-gland-mediated nitrate recirculation in maintaining NO bioavailability and cardiovascular homeostasis. Salivary-gland-mediated nitrate recirculation provides a novel perspective for potential intervention of CVDs.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940199/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728303","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 Exon-Based Transcriptomic Analysis of Parkinson's Disease.","authors":"Sulev Kõks","doi":"10.3390/biom15030440","DOIUrl":"10.3390/biom15030440","url":null,"abstract":"<p><p>Parkinson's disease (PD) is a neurodegenerative disease with a complicated pathophysiology and diagnostics. Blood-based whole transcriptome analysis of the longitudinal PPMI cohort was performed with a focus on the change in the expression of exons to find potential RNA-based biomarkers. At the moment of diagnosis, the expression of exons was very similar in both control and PD patients. The exon-based analysis identified 27 differentially expressed exons in PD patients three years after the diagnosis compared to the health controls. Moreover, thirteen exons were differentially expressed during the three-year progression of the PD. At the same time, control subjects had only minimal changes that can mostly be attributed to being related to aging. Differentially regulated exons we identified in the PD cohort were mostly related to different aspects of the pathophysiology of PD, such as an innate immune response or lysosomal activity. We also observed a decline in the expression of the <i>OPN1MW3</i> gene that is related to colour vision, which suggests that colour vision analysis could be a practical biomarker to monitor the progression of PD.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940335/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728324","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":"Liver Injury Following Intravenous Methylprednisolone Pulse Therapy in Multiple Sclerosis: The Experience from a Single Academic Liver Center.","authors":"Dimitris Kounatidis, Natalia G Vallianou, Georgios Kontos, Hariklia Kranidioti, Nikolaos Papadopoulos, Alexandros Panagiotopoulos, Krystalia Dimitriou, Vasileios Papadimitropoulos, Melanie Deutsch, Spilios Manolakopoulos, Dimitrios Vassilopoulos, John Koskinas","doi":"10.3390/biom15030437","DOIUrl":"10.3390/biom15030437","url":null,"abstract":"<p><p>Intravenous methylprednisolone (IVMP) pulses, widely used for managing multiple sclerosis (MS) exacerbations, can lead to acute liver injury, presenting a diagnostic challenge in distinguishing between drug-induced autoimmune-like hepatitis (DI-ALH) and idiopathic autoimmune hepatitis (AIH). This study aimed to delineate the clinical and biochemical features of IVMP-induced liver injury, discern its etiology, and evaluate the efficacy of glucocorticoid (GC) therapy in treatment. A retrospective analysis of 13 relapsing MS patients with IVMP-induced liver injury was conducted. Liver injury was classified as hepatocellular, cholestatic, or mixed, with severity assessment guiding liver biopsy in selected cases. Causality was assessed using the Roussel Uclaf Causality Assessment Method (RUCAM) and the Simplified Diagnostic Criteria for AIH. All patients were initially monitored for a minimum of six months, with a mean follow-up period of 4.30 years. The median onset of liver injury was 37.46 days post-IVMP, with a mean peak alanine transaminase (ALT) level of 618.46 U/L. antinuclear antibody (ANA) positivity was observed in 61.53% of cases, with elevated serum immunoglobulin G (IgG) at 15.38%. Hepatocellular injury was universal among patients, and causality assessment predominantly supported DI-ALH. GC therapy was administered in six cases, achieving favorable outcomes in all but one, which necessitated rituximab. Biochemical normalization occurred within a mean of 55.41 days, with GC-treated patients recovering faster (48 days). These findings support the hypothesis that IVMP can induce hepatocellular injury, likely DI-ALH, during MS exacerbations. A tapering GC regimen proved effective in promoting recovery, particularly in severe cases. Additionally, this study introduced a diagnostic and therapeutic algorithm for managing IVMP-induced liver injury, offering a practical framework for clinical application.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940579/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727840","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":"Topical Instillation of N-Acetylcysteine and N-Acetylcysteine Amide Impedes Age-Related Lens Opacity in Mice.","authors":"Hidetoshi Ishida, Yu Sasaki, Teppei Shibata, Hiroshi Sasaki, Bhavana Chhunchha, Dhirendra P Singh, Eri Kubo","doi":"10.3390/biom15030442","DOIUrl":"10.3390/biom15030442","url":null,"abstract":"<p><p>Cataracts, the leading cause of blindness globally, are caused by oxidative stress and inflammation, which disrupt lens transparency due to increased accumulation of reactive oxygen species (ROS) as well as protein and DNA damage during aging. Using in vitro, ex vivo, and in vivo models, we determined the protective efficacy of N-acetylcysteine amide (NACA) against oxidative stress-induced and aging-induced cataractogenesis. We found that lens epithelial cells exposed to the oxidative stress inducers hydrogen peroxide (H₂O₂) or tert-butyl hydroperoxide showed significant ROS accumulation and reduced cellular viability. These effects were inhibited by NACA via the suppression of ROS and thioredoxin-interacting protein (<i>Txnip</i>) expression, a regulator of oxidative stress-related cellular damage and inflammation. In ex vivo lens experiments, NACA significantly reduced H₂O₂-induced lens opacity and preserved lens integrity. Similarly to NACA-treated lenses ex vivo, the integrity and opacity of aged mouse lenses, when topically instilled with NACA, were preserved and reduced, respectively, and are directly related to reduced <i>Txnip</i> and increased thioredoxin (<i>Trx</i>) expression levels. Overall, our findings demonstrated the protective ability of NACA to abate aberrant redox-active pathways, particularly the ROS/TRX/TXNIP axis, thereby preventing cataractogenesis and preserving eye lens integrity and ultimately impeding aging-related cataracts.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940285/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728336","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":"Vitamin D Decreases Susceptibility of CD4⁺ T Cells to HIV Infection by Reducing AKT Phosphorylation and Glucose Uptake: A Bioinformatic and In Vitro Approach.","authors":"John D Loaiza, Jose Fernando Gómez, Daniel Muñoz-Escudero, Sandra M Gonzalez, Timothy Kyle Eubank, Maria T Rugeles, Ana Lucía Rodríguez-Perea, Wbeimar Aguilar-Jimenez","doi":"10.3390/biom15030432","DOIUrl":"10.3390/biom15030432","url":null,"abstract":"<p><p>Activated immune cells are highly susceptible to human immunodeficiency virus (HIV) infection. Vitamin D (VitD) induces antimicrobial responses and reduces cellular activation. We investigated VitD effects on HIV-1 replication, glucose uptake, and gene regulation using computational and in vitro approaches. CD4⁺ T cells from healthy male donors were treated with VitD and infected with HIV-1. After 72 h, p24 protein was measured to assess viral replication. VitD effects on anti- and pro-HIV genes were analyzed by a Boolean network model based on curated databases and the literature. CCR5 and CXCR4 coreceptor expression, AKT phosphorylation, and glucose uptake were evaluated by flow cytometry, and expression of some model-identified genes was quantified by qPCR. VitD reduced p24 by 53.2% (<i>p</i> = 0.0078). Boolean network modeling predicted that VitD upregulates antiviral, migration, and cell-differentiation related genes, while downregulating genes related to cellular activation, proliferation, glucose metabolism, and HIV replication, notably <i>AKT1, CCNT1, SLC2A1, HIF1A,</i> and <i>PFKL</i>. In vitro, VitD reduced AKT phosphorylation by 26.6% (<i>p</i> = 0.0156), transcription of <i>CCNT1</i> by 22.7% (<i>p</i> = 0.0391), and glucose uptake by 22.8% (<i>p</i> = 0.0039) without affecting classic antiviral genes or coreceptor expression. These findings suggest an anti-HIV effect of VitD, mediated through AKT and glucose metabolism downmodulation, both involved in cell activation and HIV-1 replication.</p>","PeriodicalId":8943,"journal":{"name":"Biomolecules","volume":"15 3","pages":""},"PeriodicalIF":4.8,"publicationDate":"2025-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11940553/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143728342","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}