Bioconjugate Chemistry最新文献

{"title":"Mechanistic Characterization of the Potency of THIOMAB Antibody-Drug Conjugates Targeting <i>Staphylococcus aureus</i> and ETbR-Expressing Tumor Cells Using Quantitative LC-MS/MS Analysis of Intracellular Drug Accumulation.","authors":"Hilda Hernandez-Barry, Josefa Dela Cruz-Chuh, Kimberly K Kajihara, Jyoti Asundi, Richard Vandlen, Donglu Zhang, Wouter L W Hazenbos, Thomas Pillow, Yichin Liu, Cong Wu, Katherine R Kozak, Kelly M Loyet","doi":"10.1021/acs.bioconjchem.4c00533","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00533","url":null,"abstract":"<p><p>THIOMAB drug conjugate (TDC) technology provides site-specific conjugation of linker drugs to antibodies, allowing for targeted delivery of the payload. While a direct measurement of TDC cytotoxic potency allows efficient screening and confirmation that new drugs conjugated to antibodies result in proper processing in cells, additional mechanistic characterization is often needed to provide information-rich data to guide further optimization of TDC design. For example, a quantitative understanding of how TDCs are processed intracellularly can help determine which processing step is impacting payload delivery and thereby inform the basis of the TDC efficacy. Here, we measure the cellular accumulation of two different TDC drug payloads: MAPK (mitogen-activated protein kinase) pathway inhibitor targeting ETbR-expressing tumor cells and an antibiotic active against <i>Staphylococcus aureus</i> with an <i>in vitro</i> cell-based drug release LC-MS/MS assay in a 96-well format. This assay allowed us to correlate the cellular potency of each unconjugated molecule with the amount of payload that accumulated inside the cell. In the case of the pathway inhibitor drug, the biochemical characterization of TDC processing by cathepsin B and purified human liver enzyme extract demonstrated a correlation between the efficiency of the linker drug cleavage and intracellular payload accumulation. For the antibody-antibiotic conjugate, kinetic analysis of intracellular free drug retention provided valuable insight into the chemistry modifications needed for an efficient TDC. Taken together, we demonstrated the utility of quantitative LC-MS/MS assays as one tool in guiding the design of more effective TDCs via the mechanistic release characterization of two distinct payloads.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143778586","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Fc Multisite Conjugation and Prolonged Delivery of the Folate-Targeted Drug Conjugate EC140.","authors":"Yan Zheng, Hong Cheng, Sibo Jiang, Wanyi Tai","doi":"10.1021/acs.bioconjchem.5c00037","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00037","url":null,"abstract":"<p><p>Small molecule-drug conjugate (SMDC) is a targeted drug delivery technology that develops in parallel with the antibody-drug conjugate. However, the clinical translation of SMDC faces challenges due to its limited circulating half-life in vivo. The drawback in pharmacokinetics is that it restricts the exposure time of SMDC to tumor tissues and ultimately reduces the therapeutic efficacy. In this study, we chemically conjugated a folate-targeted SMDC EC140 to the long-circulating Fc protein at multiple sites, yielding a stable and high-DAR Fc-SMDC conjugate (Fc-EC140). Fc-EC140 can bear approximately 4 molecules of EC140 per Fc protein (drug-antibody ratio = 4.1) and display enhanced potency in folate receptor (FR)-positive tumor cells compared to the SMDC comparator. In addition, Fc-EC140 retains the FcRn-mediated recycling function and displays an extended half-life of 28 h in the mice. In vivo, antitumor experiments demonstrate that intravenous administration of Fc-EC140 (Q7D × 3 at a dose of 15 mg/kg) nearly cures the KB tumors, which is far more effective than the comparator EC140 administrated at equivalent doses. This study presents a new strategy for the targeted delivery of SMDC.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770720","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Self-Assembly of Toll-Like Receptor (TLR2/6) Agonist Lipidated Amino Acid or Peptide Conjugates: Distinct Morphologies and Bioactivities.","authors":"Valeria Castelletto, Lucas R de Mello, Juliane Pelin, Ian W Hamley","doi":"10.1021/acs.bioconjchem.5c00051","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00051","url":null,"abstract":"<p><p>Toll-like receptor (TLR) agonists are of interest in immunotherapy and cancer vaccines. The most common agonists of TLR2 are based on Pam₂Cys or Pam₃Cys. In the former, two palmitoyl (Pam) fatty acids are linked to a glycerylcysteine motif by ester linkages. Pam₃Cys is analogous but contains an extra Pam group on the α-amine. Here, we compare the self-assembly in aqueous solution of the parent Pam₂CysOH and Pam₃Cys amino acid conjugates to that of Pam₂CysSK₄ and Pam₃CysSK₄ which are potent TLR2 agonists bearing the CysSK₄ peptide sequence. All four conjugates exhibit a critical aggregation concentration above which self-assembled structures are formed. We find through a combination of small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and confocal fluorescence microscopy remarkable differences in self-assembled nanostructures. Pam₂CysOH and Pam₃CysOH both form unilamellar vesicles, although these are larger for the latter compound, an effect ascribed to enhanced membrane rigidity. This is in contrast to previously reported morphologies for Pam₂CysSK₄ and Pam₃CysSK4, which are spherical micelles or predominantly wormlike micelles, respectively [Hamley, I. W.; et al. <i>Toll-like Receptor Agonist Lipopeptides Self-Assemble into Distinct Nanostructures</i>. Chem. Comm. 2014, 50, 15948-15951]. We also examine the effect of introduction in the bulky <i>N</i>-terminal Fmoc [fluorenylmethoxycarbonyl] group on the self-assembly of Fmoc-Pam₂CysOH. This compound also forms vesicles (above a critical aggregation concentration, determined from dye probe fluorescence experiments) in aqueous solution, larger than those for Pam₂CysOH and with a population of perforated/compound vesicles. The carboxyl-coated (and amino-coated for Pam₂CysOH) vesicles demonstrated here represent a promising system for future development toward bionanotechnology applications such as immune therapies. Conjugates Pam₂CysOH, Pam₂CysSK₄, and Pam₃CysSK₄ show good cytocompatibility at low concentrations, and in fact, the cell compatibility extends over a wider concentration range for Pam₂CysOH. The TLR2/6 agonist activity was assessed using an assay that probes secreted alkaline phosphatase (SEAP) in NF-κB-SEAP reporter HEK293 cells expressing human TLR2 and TLR6, and Pam₂CySOH shows significant activity, although not to the extent of Pam₂CysSK4 or Pam₃CysSK₄. Thus, Pam₂CysOH in particular is of interest as a vesicle-forming TLR2/6 agonist and stimulator of immune response.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143762484","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Choice of an Optimal Modular Strategy for the Synthesis of DOTA-Containing Heterobivalent Agents Targeting PSMA and GRPr.","authors":"Stanislav A Petrov, Gleb P Grigoriev, Grigory A Orlov, Nikolay Y Zyk, Yuri K Grishin, Vitaly A Roznyatovsky, Maria A Beloglazkina, Juliana V Petrova, Aleksei E Machulkin, Mariia S Larkina, Anastasia Prach, Ruslan Varvashenya, Vitalina Bodenko, Evgenii Plotnikov, Mekhman S Yusubov, Elena K Beloglazkina","doi":"10.1021/acs.bioconjchem.5c00033","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00033","url":null,"abstract":"<p><p>Heterodimeric approaches have emerged as a promising method for simultaneously targeting multiple receptors on tumor cells using a single molecule. Simultaneous targeting of the prostate-specific membrane antigen (PSMA) and the gastrin-releasing peptide receptor (GRPr) holds the potential to improve the accuracy of prostate cancer diagnosis. The aim of this study was to develop a convenient and simple modular strategy for the creation of heterobivalent (HBV) conjugates targeting PSMA/GRPr receptors. For this purpose, we developed and compared six alternative routes for the stereoselective synthesis of HBV conjugates designed to deliver the chelating agent DOTA to PSMA/GRPr receptors. The comparison of these alternative synthetic pathways took into account such factors as efficiency, complexity, synthesis, and purification details, as well as yields of the target compounds. Optimal conditions for the stereoselective synthesis of HBV ligands to PSMA and GRPr, which could serve as molecular platforms for the targeted delivery of therapeutic or diagnostic agents to these receptors, were revealed. For synthesized HBV ligand <b>26</b>^<b>x</b> and its HBV conjugate with DOTA <b>27</b>, the complete signal assignment in ¹H, ¹³C, and ¹⁵N NMR spectra was achieved using 2D NMR techniques. Based on these data, comprehensive signal assignments were provided for all final compounds in their NMR spectra. The final HBV conjugate <b>27</b> was labeled with Lu-177, with yields >99%, and the obtained radiotracer was studied in vitro for its binding specificity, with determining of the <i>K</i>_D and <i>B</i>_max using LNCaP (PSMA+) and PC-3 (GRPr+) cell lines.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143770718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Recent Advances in Aggregation-Induced Emission Bioconjugates.","authors":"Guiquan Zhang, Daming Zhou, Rong Hu, Anjun Qin, Ben Zhong Tang","doi":"10.1021/acs.bioconjchem.5c00036","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00036","url":null,"abstract":"<p><p>Fluorescence imaging technology is playing increasing roles in modern personalized and precision medicine. Thanks to their excellent photophysical properties, organic luminogens featuring aggregation-induced emission (AIE) characteristics (AIEgens) have attracted considerable attention over the past two decades. Because of their superior biocompatibility, ease of processing and functionalization, excellent water solubility, high responsiveness, and exceptional signal-to-noise ratio (SNR) for biotargets, AIE bioconjugates, formed by covalently linking AIEgens with biomolecules, have emerged as an ideal candidate for bioapplications. In this review, we summarize the progress in preparation, properties, and application of AIE bioconjugates in the last five years. Moreover, the challenges and opportunities of AIE bioconjugates are also briefly discussed.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-04-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750151","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Advances in the Treatment of Spinal Cord Injury with Nanozymes.","authors":"Zuohong Chen, Yili Wang, Shaofang Zhang, Huanhuan Qiao, Shuquan Zhang, Hao Wang, Xiao-Dong Zhang","doi":"10.1021/acs.bioconjchem.5c00100","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00100","url":null,"abstract":"<p><p>Spinal cord injury (SCI) with increasing incidence can lead to severe disability. The pathological process involves complex mechanisms such as oxidative stress, inflammation, and neuron apoptosis. Current treatment strategies focusing on the relief of oxidative stress and inflammation have achieved good effects, while many problems and challenges remain such as the side effect and short half-life of the therapeutic agents. Nanozymes exhibiting good biocatalytic activities can sustainably scavenge free radicals, inhibit neuroinflammation, and protect the neurons. With high stability in physiological conditions and cost-effectiveness, the nanozymes provide a new strategy for SCI treatment. In this Review, we outline the advances of nanozymes and their enzyme-mimicking activities and highlight the progress in the intervention of SCI-adopting nanozymes. We also propose future directions and clinical translation for the nanozyme strategy against SCI.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750135","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Introducing TAPY as a Versatile Alternative to TPP for Selective Mitochondrial Targeting in Cancer Cells.","authors":"Jean C Neto, Federico Lucantoni, Leydy V González, Eva Falomir, Juan F Miravet, Francisco Galindo","doi":"10.1021/acs.bioconjchem.4c00554","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00554","url":null,"abstract":"<p><p>The understanding of diseases such as cancer and Alzheimer's, along with natural aging processes, heavily relies on the study of mitochondrial function. Optical techniques like fluorescence imaging microscopy are pivotal for this purpose, enabling precise mapping of subcellular structures, including mitochondria. In this study, we explored TAPY (triarylpyridinium) cations, a novel family of mitochondrial carriers resembling the well-known triphenylphosphonium cation (TPP). Six TAPY-bodipy (BDP) dyads were prepared and chemically characterized. Confocal Laser Scanning Microscopy (CLSM) studies demonstrated that the systems were delivered selectively to the mitochondria of cancer cells (MCF-7, A549, HT-29). Remarkably, these dyads did not target the mitochondria of normal cells (HEK-293, HMEC-1), suggesting their potential use in distinguishing cancerous cells from healthy ones. A model compound comprised of the same bodipy cargo but attached to TPP was also synthesized and tested. Notably, in preliminary comparative assays with MCF-7 cells, the dyad TAPY(OMe)-BDP outperformed the TPP derivative in mitochondrial imaging, achieving twice the final fluorescence intensity. The potential chemical diversity achievable with TAPY cations is considerable, with many derivatives being accessible starting from readily available commercial products. This implies that, based on the strategy outlined in this study, carefully optimized TAPY derivatives for targeted mitochondrial delivery could potentially be developed in the future as alternatives or complements to TPP, with the present work acting as a proof of concept.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-03-31","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143750148","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Aptamer- vs Fab-Conjugated Liposomes: A Comparative Study in Targeting Acute Myeloid Leukemia Cells.","authors":"Hyesoo Jin, Wooseong Noh, Kangwuk Kyung, Woon-Seok Yeo, Ye Han Song, Yong-Seok Heo, Dong-Eun Kim","doi":"10.1021/acs.bioconjchem.5c00065","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00065","url":null,"abstract":"<p><p>Acute myeloid leukemia (AML) is a hematologic malignancy characterized by uncontrolled proliferation of abnormal myeloid cells with a generally poor prognosis despite advancements in chemotherapy and stem cell transplantation. To enhance therapeutic efficacy and minimize systemic toxicity, we designed liposomal nanoparticles functionalized with two distinct targeting ligands, a DNA aptamer or fragment-antigen-binding (Fab) antibody, targeting the surface marker transmembrane glycoprotein CD33 antigen (CD33) on AML cells. Aptamer- and Fab-conjugated liposomes (Apt-Lipm and Fab-Lipm, respectively) were prepared and tested for cellular uptake by CD33-positive AML cell lines. Comparative studies revealed that Fab-Lipm exhibited significantly superior binding affinity, targeting efficiency, and cellular uptake compared with Apt-Lipm. Furthermore, we demonstrated the intracellular distribution and endocytic pathways of Fab-Lipm during the cellular uptake. This comparative study of aptamer- and Fab-conjugated liposomes suggests that the Fab-conjugated liposomal system offers enhanced precision in targeting AML cells for the development of effective therapeutic strategies against hematologic malignancies.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727011","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"AIE Photosensitizer with Tuned Membrane Interactions for Effective-Gram-Negative Bacteria Elimination.","authors":"Edward Kamya, Shangzhao Yi, Zhongzhong Lu, Jincong Yan, Hewan Dawit, Shah Mehmood, Yi Cao, Renjun Pei","doi":"10.1021/acs.bioconjchem.5c00132","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.5c00132","url":null,"abstract":"<p><p>Photodynamic antimicrobial therapy (PDAT) for efficient bacterial infection eradication critically relies on photosensitizers (PSs) that can specifically target and disrupt bacterial membranes. However, the complex membrane architecture of Gram-negative bacteria poses a significant challenge to the efficacy of most aggregation-induced emission (AIE) PSs. Herein, we introduce TPQ, an AIE PS meticulously designed to overcome this challenge by incorporating an outer membrane disruption ability, thereby boosting PDAT efficacy against Gram-negative bacteria. TPQ demonstrated excellent microbial imaging and potent PDAT activity against both Gram-positive and Gram-negative bacteria, attributed to its inherent fluorescence, high singlet oxygen generation, and balanced electrostatic and hydrophobic interactions with bacterial membranes. Notably, TPQ achieved exceptional PDAT activity (>97% efficacy) against Gram-negative bacteria while exhibiting minimal cytotoxicity to mammalian cells. Furthermore, TPQ-mediated PDAT effectively healed <i>Escherichia coli</i>-infected wounds on mice models with assured biosafety. This work provides valuable insights into the rational design of AIE PSs and highlights the synergistic effect of membrane disruption for advancing PDAT applications, particularly against recalcitrant Gram-negative bacterial infections.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143717591","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Photochemical-Promoted Cross-Coupling Reaction of Alkyl Boronate Esters with DNA-Conjugated Aryl Bromides for DNA-Encoded Library Synthesis.","authors":"Baiyang Mu, Yiwei Zhang, Xudong Wang, Rui Jin, Weiwei Lu, Nidhal Selmi, Sixiu Liu, Avinash Bhat, Sara Pahlén, Giulia Bergonzini, Zhiqiang Duan, Yinan Song, Xiaojie Lu","doi":"10.1021/acs.bioconjchem.4c00581","DOIUrl":"https://doi.org/10.1021/acs.bioconjchem.4c00581","url":null,"abstract":"<p><p>The C(sp²)-C(sp³) cross-coupling reaction is an effective way to increase the C(sp³) content in compound collections for drug discovery, enhancing molecular diversity and offering a unique chemistry starting point. In this study, we report a mild, DNA-compatible, and off-DNA-inert photochemical cross-coupling reaction inspired by the amino radical transfer strategy. This method demonstrates broad substrate scopes for DNA-encoded library (DEL) constructions, utilizing commonly available structures on DNA and diverse alkyl boronate ester building blocks, which have not been widely applied in the current DEL chemical space.</p>","PeriodicalId":29,"journal":{"name":"Bioconjugate Chemistry","volume":" ","pages":""},"PeriodicalIF":4.0,"publicationDate":"2025-03-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143727012","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"化学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}