Rong He , Yanjie Zhu , Weiping Zhou , Muhammad Azhar Hayat Nawaz , Yunhui Li , Jianwei Zhu , Huimin Feng , Anna Maria Nowicka , Wenzhao Han , Cong Yu
{"title":"Aggregation-induced luminescence probe based lateral flow immunoassay for the simultaneous quantitative detection of IL-6/PCT","authors":"Rong He , Yanjie Zhu , Weiping Zhou , Muhammad Azhar Hayat Nawaz , Yunhui Li , Jianwei Zhu , Huimin Feng , Anna Maria Nowicka , Wenzhao Han , Cong Yu","doi":"10.1016/j.jpba.2026.117393","DOIUrl":"10.1016/j.jpba.2026.117393","url":null,"abstract":"<div><div>The detection of important inflammatory biomarkers possesses substantial advantages in guiding clinical decision-making. In particular, simultaneous detection of interleukin-6 (IL-6) and procalcitonin (PCT) significantly improves the differentiation between bacterial and viral infections, a critical challenge in early-stage diagnostics. To address this need, a reliable lateral flow immunoassay (LFIA) incorporating aggregation-induced luminescent nanoparticles (BTA@PS) was successfully developed. A novel aggregation-induced emission (AIE) probe was designed and encapsulated within polystyrene microspheres, thereby overcoming the limitations of aggregation-caused quenching (ACQ) in conventional fluorescent materials. The resulting BTA@PS nanoparticles, conjugated with specific antibodies against IL-6 and PCT, served as stable and effective immunofluorescent probes for the LFIA platform. Under optimized experimental conditions, the developed BTA@PS-LFIA enabled simultaneous quantification of IL-6 and PCT, demonstrating excellent linearity over the range of 2–8000 pg/mL and 0.04–30 ng/mL, respectively, with coefficient of variation (CV) values of below 5 %. Furthermore, this method demonstrated superior detection capability for PCT and IL-6 in serum, confirming its high potential for rapid clinical diagnostics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117393"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146180304","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shreshtha Dash, Molly T. McDevitt, D. David Smith, Peter S. Steyger
{"title":"Determination of Gentamicin C-subtypes in Inner Ear Perilymph Using Liquid Chromatography with Fluorescence Detection","authors":"Shreshtha Dash, Molly T. McDevitt, D. David Smith, Peter S. Steyger","doi":"10.1016/j.jpba.2026.117394","DOIUrl":"10.1016/j.jpba.2026.117394","url":null,"abstract":"<div><div>Gentamicin is a broad-spectrum aminoglycoside used frequently to treat gram-positive and gram-negative bacterial infections. In this study, a new, simple, fast, and sensitive isocratic reversed-phase high performance liquid chromatography (RP-HPLC) method was developed and validated for the detection and quantification of fluorescent OPA-ethanethiol derivatized gentamicin in very small biological sample volumes. To our knowledge, there is no report of the use of ethanethiol for the derivatization of gentamicin with OPA, and the simultaneous determination of the four major C-subtypes of gentamicin using OPA-derivatives. Optimum chromatographic conditions were achieved on a C<sub>18</sub> column with a mobile phase consisting of methanol, glacial acetic acid, and an aqueous solution of sodium 1-heptanesulfonate at a flow rate of 1.0 mL/min under ambient conditions. The method was successfully validated according to the acceptance criteria of USP guidelines in terms of selectivity, linearity, accuracy, precision, and sensitivity. The linearity of the method was demonstrated with a concentration range of gentamicin (10-400 ng/mL) prepared in artificial perilymph. The limit of detection was 0.2 ng/mL and the limit of quantification was 10–11 ng/mL for all four major C-subtypes of gentamicin. Finally, due to its high sensitivity, this method was successfully applied to quantify gentamicin concentrations in the small volumes of perilymph present in the inner ear of mice. Thus, this RP-HPLC-fluorescence method for detecting derivatized gentamicin in preclinical models is promising in terms of simplicity and high sensitivity.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117394"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146157367","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Burçak Demi̇rbakan , Ahmet Çeti̇nkaya , Evrim Güneş Altuntaş , Mehmet Altay Ünal , Mustafa Kemal Sezgi̇ntürk , Sibel A. Özkan
{"title":"3-mercaptopropionic acid agent-based biosensor system using gold-screen printed electrode for aflatoxin B1 detection","authors":"Burçak Demi̇rbakan , Ahmet Çeti̇nkaya , Evrim Güneş Altuntaş , Mehmet Altay Ünal , Mustafa Kemal Sezgi̇ntürk , Sibel A. Özkan","doi":"10.1016/j.jpba.2026.117411","DOIUrl":"10.1016/j.jpba.2026.117411","url":null,"abstract":"<div><div>A novel electrochemical biosensor was constructed for the ultrasensitive detection of aflatoxin B1 (AFB1) in food samples. The biosensor design was based on a 3-mercaptopropionic acid (3-MPA)-modified self-assembled monolayer (SAM) constructed on disposable gold screen-printed electrodes (SPE-Au). Covalent attachment of anti-AFB1 antibodies was achieved on the functionalized electrode surface, and the modification process at each stage was analyzed using CV and EIS techniques. 3-MPA concentration was optimized to enhance analytical performance. The biosensor exhibited a wide linear detection range of 0.1–250 pg/mL with a calculated limit of detection (LOD) of 0.94 pg/mL and limit of quantification (LOQ) of 3.14 pg/mL. It also demonstrated high reproducibility and excellent selectivity toward AFB1. Finally, the biosensor was successfully applied to real food samples, including milk, rice, peanuts, and chili pepper, confirming its reliability and potential for practical food safety monitoring.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117411"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192139","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Shuyi Lv , Jiayi Lin , Yu Sun , Mingqin Kang , Xin Li , Lili Song , Yubo Li
{"title":"Metabolomics technology combined with O2PLS analysis reveals the active components of Paeoniae Radix Alba in preventing renal damage in rheumatoid arthritis","authors":"Shuyi Lv , Jiayi Lin , Yu Sun , Mingqin Kang , Xin Li , Lili Song , Yubo Li","doi":"10.1016/j.jpba.2026.117403","DOIUrl":"10.1016/j.jpba.2026.117403","url":null,"abstract":"<div><div>Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by symmetrical, erosive polyarthritis and multi-organ involvement, with renal complications posing a life-threatening risk. <em>Paeoniae Radix Alba</em>, known as BaiShao (BS) in Chinese, is a widely used herbal medicine for clinical RA treatment, yet its potential in preventing RA-associated renal damage and its underlying active ingredients remain elusive. To address this knowledge gap, we employed a type II collagen-induced arthritis (CIA) rat model. Biochemical assays and histopathological analyses confirmed that BS exerted robust renoprotective effects in CIA rats. Serum and urine metabolomics identified 48 renal damage-related biomarkers, 18 of which showed distinct regulatory trends following BS intervention. Meanwhile, ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was used to characterize 34 chemical components in BS. Through an integrative approach that combines two-way orthogonal partial least squares (O2PLS) and Pearson correlation analysis, 18 active ingredients were identified as key mediators of BS-mediated renoprotection. Collectively, this study establishes a novel metabolomics-O2PLS strategy for discovering active ingredients in BS, laying a foundation for understanding the mechanism underlying the prevention of RA-associated renal damage mediated by BS and providing insights for the development of novel preventive therapeutics.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117403"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192140","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Development and validation of an UPLC–MS/MS method for simultaneous determination of meropenem and its open-ring metabolite in human serum and cerebrospinal fluid with application to clinical samples","authors":"XiangLong Chen , Jinhui Xu , Chengliang Wang, Lijuan Yang, Jinwei Fan, Tongtong Li, Qian Zhang, Yanxia Yu, Lian Tang, Shenjia Huang","doi":"10.1016/j.jpba.2026.117389","DOIUrl":"10.1016/j.jpba.2026.117389","url":null,"abstract":"<div><div>Central nervous system (CNS) infections require adequate drug exposure at the site of action, yet antibiotic meropenem (MER) shows limited cerebrospinal fluid (CSF) penetration and easily undergoes non-enzymatic degradation to an inactive open-ring metabolite (ORM). In this study, we developed a simple, sensitive, and fast liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of MER and ORM in human serum and CSF. Chromatographic separation was accomplished on an Agela Venusil MP C18 column, with MER-d6 and ORM-d6 as internal standards. Methanol was found to promote methanolysis, yielding a characteristic product (<em>m/z</em> 416.2). Therefore, acetonitrile was selected as both the organic phase and the protein-precipitation solvent. Method validation was conducted according to the ICH M10 guideline. Follow validation, the method was successfully applied to 57 serum and 16 CSF samples. ORM concentrations in human CSF were reported for the first time. This method provides a valuable tool to support MER monitoring in patients with CNS infections.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117389"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146119315","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Amit Dahal , Teresa Sierra , Colleen M. Hayes , David N. Assis , Amin Rostami-Hodjegan , Nisanne S. Ghonem , Brahim Achour
{"title":"Corrigendum to ‘A liquid biopsy-RNAseq method for monitoring the expression of genes involved in drug disposition: proof-of-concept application to cholestatic liver disease’ [J. Pharm. Biomed. Anal. (2025) 269: 117244]","authors":"Amit Dahal , Teresa Sierra , Colleen M. Hayes , David N. Assis , Amin Rostami-Hodjegan , Nisanne S. Ghonem , Brahim Achour","doi":"10.1016/j.jpba.2026.117385","DOIUrl":"10.1016/j.jpba.2026.117385","url":null,"abstract":"","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117385"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146098610","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yanting Wang , Xiaofei Wang , Ningqi Xia , Hao Chen , Linfeng Zhang , Bin Lu , Diya Lv , Yan Cao
{"title":"Construction of bio-layer interferometry biosensors via cell-free synthesized proteins for fishing bioactive compounds from Chinese herbs","authors":"Yanting Wang , Xiaofei Wang , Ningqi Xia , Hao Chen , Linfeng Zhang , Bin Lu , Diya Lv , Yan Cao","doi":"10.1016/j.jpba.2026.117405","DOIUrl":"10.1016/j.jpba.2026.117405","url":null,"abstract":"<div><div>The application of Bio-Layer Interferometry (BLI) is contingent upon the immobilization of highly purified target proteins onto the sensor. The cumbersome and time-consuming nature of traditional protein expression and purification processes restricts the application of BLI in high-throughput screening of traditional Chinese medicine (TCM). This study aims to develop a rapid and efficient BLI-based platform for screening bioactive components in TCM. An integrated platform combining cell-free protein synthesis (CFPS), BLI, and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-QTOF/MS) was established for efficient TCM bioactive compound discovery. Functional C-X-C chemokine receptor 4 (CXCR4) was synthesized <em>in vitro</em> using a CFPS system, which were then validated by surface plasmon resonance (SPR) and western blotting. Immobilized CXCR4 on NTA biosensors enabled BLI-based high-throughput screening of TCM extracts, followed by target-specific compound recovery and characterized via mass spectrometry. Three bioactive TCM constituents were successfully fished and identified as coptisine, ligustilide, and senkyunolide A. All of them exhibited negligible cytotoxicity at concentrations ranging from 6.25 to 100 μM). Furthermore, ligustilide and senkyunolide A demonstrated certain affinity for CXCR4 with K<sub>D</sub> of 69.86 μM and 14.7 μM, respectively, and significantly inhibited cell migration. This study is the first identification of ligustilide and senkyunolide A as functional ligands of CXCR4. The established CFPS-BLI-UHPLC-QTOF/MS platform enables efficient discovery of low-toxicity, high-affinity CXCR4-targeting therapeutics from TCM.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"273 ","pages":"Article 117405"},"PeriodicalIF":3.1,"publicationDate":"2026-06-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146192141","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"LC-MS peptide mapping of monoclonal antibodies using the mirror proteases trypsin and Tryp-N","authors":"Domantas Sargautis, Bernd Thiede","doi":"10.1016/j.jpba.2026.117361","DOIUrl":"10.1016/j.jpba.2026.117361","url":null,"abstract":"<div><div>Accurate characterization of the amino acid sequence and post translational modifications (PTMs) of monoclonal antibodies (mAbs) is essential for evaluating product quality. Peptide mapping through bottom-up LC/MS analysis is a key methodology for this purpose. While trypsin is commonly the first choice for mAb digestion, it typically yields high but incomplete sequence coverage. As a result, supplementary endoproteases such as Asp-N, chymotrypsin, Glu-C or Lys-C are often employed to enhance coverage. In this report, we evaluated another endoprotease, Tryp-N, which serves as an effective alternative to trypsin for mAb analysis. The sequence coverages achieved for bevacizumab, cetuximab, NISTmAb, and trastuzumab with Tryp-N were comparable to that of trypsin, and the combination of both enzymes slightly improved overall sequence coverage. Notably, both trypsin and Tryp-N generated identical peptides beside the N- and C-terminal ends. The presence of a basic amino acid at opposite ends of the peptide often resulted in complementary sequence coverage of the MS2 of the same peptide sequences. These complementary ion series can be leveraged for precise localization of PTMs, as demonstrated in detail for deamidation, and oxidation sites as well as single amino acid variants (SAVs).</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117361"},"PeriodicalIF":3.1,"publicationDate":"2026-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146024128","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
{"title":"Analysis of gut microbiota and intestinal secondary bile acids metabolism in rats after short-term antibiotic treatment","authors":"Zhuan Yang , Xiaoping Zhang , Bo Lv , Yunli Yu","doi":"10.1016/j.jpba.2026.117365","DOIUrl":"10.1016/j.jpba.2026.117365","url":null,"abstract":"<div><div>Antibiotics have a profound impact on the overall taxonomic composition of gut microbiota. As gut microbiota influence host bile acids metabolism, changes in the composition of gut microbiota induced by antibiotics are certain to alter host bile acids profile. However, the differences in the effects of various antibiotics on bile acids metabolism and their associations with the impact on intestinal microbiota remain unelucidated. Here, investigation was conducted into how different antibiotics impact host intestinal bile acids metabolism via gut microbiota using in vivo study and multi-omics approaches. Four antibiotics (cefixime, clarithromycin, moxifloxacin and metronidazole) were used to treat normal rats for a short period (3 days), and then gut microbiota, intestinal bile acids profile, BSH and 7α-dehydroxylases activity mediated bile acid metabolism were measured. The results showed that bile acid metabolism in intestine was significantly altered along with the abundance change in bile acid-producing microbiota. It was found that moxifloxacin and metronidazole inhibited the transformation of primary BAs to secondary BAs in the intestine as compared to cefixime and clarithromycin, which was exposed to be associated with its regulation of <em>Ruminococcaceae</em> by Spearman’s correlation analysis (correlation with CDCA/LCA is r = -0.89, FDR<0.001 and CA/DCA is r = -0.708, FDR<0.01). The abundance of <em>Ruminococcaceae</em> in the gut decreased by 87 % after moxifloxacin intervention, while metronidazole completely suppressed the abundance of intestinal <em>Ruminococcaceae</em>. Further analysis revealed that <em>Ruminococcaceae</em> were most strongly associated with the activity of 7α-dehydroxylases (r = 0.701, FDR<0.001). Specifically, we found that <em>Ruminococcus flavefaciens</em> had the closest association with alterations in secondary bile acids (r < -0.68, FDR<0.01). Collectively, our research demonstrated that different antibiotics exerted varying impacts on the production of intestinal secondary bile acids, which was related to their differential effects on the gut microbiota. This work provides novel insights into the interplay between antibiotics and microbial metabolites.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117365"},"PeriodicalIF":3.1,"publicationDate":"2026-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146078999","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Paulo Cezar de Moraes , Alessandra Koehler , Letícia Maria Eidt , Cristiane Almeida Soares Cattani , Valeriano Antonio Corbellini , Maria Lúcia Scroferneker
{"title":"Identification of leprosy reactions using Fourier transform infrared (FTIR) spectroscopy supervised by clinical evaluation","authors":"Paulo Cezar de Moraes , Alessandra Koehler , Letícia Maria Eidt , Cristiane Almeida Soares Cattani , Valeriano Antonio Corbellini , Maria Lúcia Scroferneker","doi":"10.1016/j.jpba.2026.117354","DOIUrl":"10.1016/j.jpba.2026.117354","url":null,"abstract":"<div><div>Leprosy is a chronic granulomatous, infectious and disabling disease, whose etiological agents are <em>Mycobacterium leprae</em> and <em>M. lepromatosis</em>. Leprosy reactions are the main causes of peripheral nerve damage and sequelae of the disease. There is no laboratory test that alone allows the identification of these episodes, causing delays in their diagnosis and treatment. The objective of this study was to propose a methodology based on Fourier transform infrared (FTIR) spectroscopy to detect leprosy reactions in the saliva of leprosy patients, supervised by the clinical diagnosis of these episodes. A total of 131 saliva samples were included and analyzed by attenuated total reflection (ATR)-FTIR; a supervised analysis with partial least squares discriminant analysis (PLS-DA), after orthogonal signal correction (OSC), was used to classify the samples into two groups: with and without leprosy reactions. The PLS-DA model with one latent variable and one OSC component showed 100 % sensitivity and specificity both in the calibration and prediction sets. Thus, all samples were correctly classified, allowing the diagnosis of leprosy reactions in saliva with high accuracy. Therefore, the FTIR-based chemometric model proved promising for the rapid and early identification of these episodes, contributing to clinical management of patients with leprosy.</div></div>","PeriodicalId":16685,"journal":{"name":"Journal of pharmaceutical and biomedical analysis","volume":"272 ","pages":"Article 117354"},"PeriodicalIF":3.1,"publicationDate":"2026-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145981050","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}