Analytical biochemistry最新文献_第8页

Molecularly imprinted polymers in the analysis of chlorogenic acid: A review 分子印迹聚合物在绿原酸分析中的应用：综述。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-11 DOI: 10.1016/j.ab.2024.115616

Himshweta , Neelam Verma , Nitu Trehan , Minni Singh

{"title":"Molecularly imprinted polymers in the analysis of chlorogenic acid: A review","authors":"Himshweta , Neelam Verma , Nitu Trehan , Minni Singh","doi":"10.1016/j.ab.2024.115616","DOIUrl":"10.1016/j.ab.2024.115616","url":null,"abstract":"<div><p>Chlorogenic acid, a phenolic compound, is prevalent across various plant species and has been known for its pharmacological advantages. Health care experts have identified chlorogenic acid as a potential biomarker for treatment of a wide range of illnesses. Therefore, achieving efficient extraction and analysis of chlorogenic acid from plants and their products has become essential. Molecularly imprinted polymers (MIPs) are highly effective adsorbent for the extraction of chlorogenic acid from complex matrices. Currently, there is a lack of comprehensive review article that consolidate the methods utilized for the purification of chlorogenic acid through molecular imprinting. In this context, we have surveyed the common approaches employed in preparing MIPs specifically designed for the analysis of chlorogenic acid, including both conventional and newly developed. This review discusses the advantages, limitations of polymerization techniques and proposed strategies to produce more efficient MIPs for chlorogenic acid enrichment in complex samples. Additionaly, we present advanced imprinting methods for designing MIPs, which improve the adsorption capacity, sensitivity and selectivity towards chlorogenic acid.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141598159","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application RNA G-quadruplex 与配体的相互作用分析及原位成像应用。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-11 DOI: 10.1016/j.ab.2024.115613

Lanxin Jiang , Jie Teng , Xiaojuan Liu , Lulu Xu , Tiantian Yang , Xingping Hu , Shijia Ding , Jia Li , Yongmei Jiang , Wei Cheng

{"title":"Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application","authors":"Lanxin Jiang , Jie Teng , Xiaojuan Liu , Lulu Xu , Tiantian Yang , Xingping Hu , Shijia Ding , Jia Li , Yongmei Jiang , Wei Cheng","doi":"10.1016/j.ab.2024.115613","DOIUrl":"10.1016/j.ab.2024.115613","url":null,"abstract":"<div><p>RNA G4, as an integral branch of G4 structure, possesses distinct interactions with ligands compared to the common DNA G4, thus the investigation of RNA G4/ligand interactions might be considered as a fresh breakthrough to improve the biosensing performance of G4/ligand system. In this study, we comparatively explored the structural and functional mechanisms of RNA G4 and DNA G4 in the interaction with ligands, hemin and thioflavin T (ThT), utilizing the classical PS2.M sequence as a model. We found that although the catalytic performance of RNA G4/hemin system was lower than DNA G4/hemin, RNA G4/ThT fluorescence system exhibited a significant improvement (2∼3-fold) compared to DNA G4/ThT, and adenine modification could further enhance the signaling. Further, by exploring the interaction between RNA G4 and ThT, we deemed that RNA G4 and ThT were stacked in a bimolecular mode compared to single-molecule binding of DNA G4/ThT, thus more strongly limiting the structural spin in ThT excited state. Further, RNA G4/ThT displayed higher environmental tolerance and lower ion dependence than DNA G4/ThT. Finally, we employed RNA G4/ThT as a highly sensitive label-free fluorescent signal output system for in situ imaging of isoforms BCR-ABL e13a2 and e14a2. Overall, this study successfully screened a high-performance RNA G4 biosensing system through systematic RNA G4/ligands interaction studies, which was expected to provide a promising reference for subsequent G4/ligand research.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141603189","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Derivative spectrophotometry-assisted determination of tryptophan metabolites emerges host and intestinal flora dysregulations during sepsis 衍射分光光度法辅助测定色氨酸代谢物可发现败血症期间宿主和肠道菌群的失调。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-10 DOI: 10.1016/j.ab.2024.115605

Mengyu Jiang , Li Li , Yuan Jin , Liuliu Lu , Zhenchen Lu , Wangjie Lv , Xiaoqun Wang , Lei Di , Zhicheng Liu

{"title":"Derivative spectrophotometry-assisted determination of tryptophan metabolites emerges host and intestinal flora dysregulations during sepsis","authors":"Mengyu Jiang , Li Li , Yuan Jin , Liuliu Lu , Zhenchen Lu , Wangjie Lv , Xiaoqun Wang , Lei Di , Zhicheng Liu","doi":"10.1016/j.ab.2024.115605","DOIUrl":"10.1016/j.ab.2024.115605","url":null,"abstract":"<div><p>Sepsis is a life-threatening condition characterized by organ dysfunction resulting from a dysregulated host response to infection. Dysregulated tryptophan (TRP) metabolites serve as significant indicators for endogenous immune turnovers and abnormal metabolism in the intestinal microbiota during sepsis. Therefore, a high coverage determination of TRP and its metabolites in sepsis is beneficial for the diagnosis and prognosis of sepsis, as well as for understanding the underlying mechanism of sepsis development. However, similar structures in TRP metabolites make it challenging for separation and metabolite identification. Here, high-performance liquid chromatography coupled with a diode array detector (HPLC-DAD) was developed to determine TRP metabolites in rat serum. The first-order derivative spectrophotometry of targeted metabolites in the serum was investigated and proved to be promising for chromatographic peak annotation across different columns and systems. The established method separating the targeted metabolites was optimized and validated to be sensitive and accurate. Application of the method revealed dysregulated TRP metabolites, associated with immune disorders and NAD + metabolism in both the host and gut flora in septic rats. Our findings indicate that the derivative spectrophotometry-assisted method enhances metabolite identifications for the chromatographic systems based on DAD detectors and holds promise for precision medicine in sepsis.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141589482","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

PreDBP-PLMs: Prediction of DNA-binding proteins based on pre-trained protein language models and convolutional neural networks PreDBP-PLMs：基于预训练蛋白质语言模型和卷积神经网络的 DNA 结合蛋白预测。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-08 DOI: 10.1016/j.ab.2024.115603

Dawei Qi, Chen Song, Taigang Liu

{"title":"PreDBP-PLMs: Prediction of DNA-binding proteins based on pre-trained protein language models and convolutional neural networks","authors":"Dawei Qi, Chen Song, Taigang Liu","doi":"10.1016/j.ab.2024.115603","DOIUrl":"10.1016/j.ab.2024.115603","url":null,"abstract":"<div><p>The recognition of DNA-binding proteins (DBPs) is the crucial step to understanding their roles in various biological processes such as genetic regulation, gene expression, cell cycle control, DNA repair, and replication within cells. However, conventional experimental methods for identifying DBPs are usually time-consuming and expensive. Therefore, there is an urgent need to develop rapid and efficient computational methods for the prediction of DBPs. In this study, we proposed a novel predictor named PreDBP-PLMs to further improve the identification accuracy of DBPs by fusing the pre-trained protein language model (PLM) ProtT5 embedding with evolutionary features as input to the classic convolutional neural network (CNN) model. Firstly, the ProtT5 embedding was combined with different evolutionary features derived from the position-specific scoring matrix (PSSM) to represent protein sequences. Then, the optimal feature combination was selected and input to the CNN classifier for the prediction of DBPs. Finally, the 5-fold cross-validation (CV), the leave-one-out CV (LOOCV), and the independent set test were adopted to examine the performance of PreDBP-PLMs on the benchmark datasets. Compared to the existing state-of-the-art predictors, PreDBP-PLMs exhibits an accuracy improvement of 0.5 % and 5.2 % on the PDB186 and PDB2272 datasets, respectively. It demonstrated that the proposed method could serve as a useful tool for the recognition of DBPs.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141578729","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High quality RNA extraction protocol for polyphenolics-rich Cotton tissue 针对富含多酚的棉花组织的高质量 RNA 提取方案。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-08 DOI: 10.1016/j.ab.2024.115604

Viralkumar B. Mandaliya , Pritesh Bhatt , Vrinda S. Thaker

引用次数: 0

Calibrating ITC instruments: Problems with weak base neutralization 校准 ITC 仪器：弱碱中和的问题。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-06 DOI: 10.1016/j.ab.2024.115602

{"title":"Calibrating ITC instruments: Problems with weak base neutralization","authors":"","doi":"10.1016/j.ab.2024.115602","DOIUrl":"10.1016/j.ab.2024.115602","url":null,"abstract":"<div><p>Modern isothermal titration calorimetry instruments give great precision, but for comparable accuracy they require chemical calibration. For the heat factor, one recommended process is HCl into the weak base TRIS. In studying this reaction with a VP-ITC and two Nano-ITCs, we have encountered some problems, most importantly a titrant volume shortfall Δ<em>v</em> ≈ 0.3 μL, which we attribute to diffusive loss of HCl in the syringe tip. This interpretation is supported by a mathematical treatment of the diffusion problem. The effect was discovered through a variable-<em>v</em> protocol, which thus should be used to properly allow for it in any reaction that similarly approaches completion. We also find that the effects from carbonate contamination and from OH<sup>−</sup> from weak base hydrolysis can be more significant that previously thought. To facilitate proper weighting in the least-squares fitting of data, we have estimated data variance functions from replicate data. All three instruments have low-signal precision of <em>σ</em> ≈ 1 μJ; titrant volume uncertainty is a factor of ∼2 larger for the Nano-ITCs than for the VP-ITC. The final heat factors remain uncertain by more than the ∼1 % precision of the instruments and are unduly sensitive to the HCl concentration.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0003269724001465/pdfft?md5=22b25798750792c14f075a6cbdf3fbb7&pid=1-s2.0-S0003269724001465-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141557788","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum 人血清中游离血红蛋白（有别于血红蛋白-aptoglobin 复合物）的定量方法。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-05 DOI: 10.1016/j.ab.2024.115601

{"title":"Method for quantifying free hemoglobin, distinct from the hemoglobin-haptoglobin complex, in human serum","authors":"","doi":"10.1016/j.ab.2024.115601","DOIUrl":"10.1016/j.ab.2024.115601","url":null,"abstract":"<div><p>The measurement of free hemoglobin (free Hb) in blood is crucial for assessing the risk of organ damage in patients with hemolytic diseases. However, the colorimetric method, commonly used in clinical practice, does not distinguish between free Hb and the hemoglobin-haptoglobin complex (Hb-Hp) in the blood, instead reflecting the total Hb level. Although size-exclusion high-performance liquid chromatography (SEC-HPLC) can specifically measure free Hb, its clinical use is limited by long assay times. Here, we developed a novel assay method for the rapid quantification of free Hb in serum, distinguishing it from Hb-Hp, using a latex agglutination immunoturbidimetric assay (LATIA). This method could be used to measure free Hb in sera in the range of 1–100 μg/mL in approximately 15 min using an automatic biochemistry analyzer. Using Hb-spiked serum samples from healthy adults, there was a high correlation with Hb levels determined using the newly developed method and SEC-HPLC, indicating a high specificity for free Hb. This novel assay can be used to monitor levels of free Hb in patients with various hemolytic diseases and to design therapeutic strategies based on measured values. However, further studies are required to assess its clinical performance.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S0003269724001453/pdfft?md5=3b9e547a6e9b572079f717b830f81c51&pid=1-s2.0-S0003269724001453-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141544469","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A pH ultra-sensitive hydrated iridium oxyhydroxide films electrochemical sensor for label-free detection of Vibrio parahaemolyticus 用于无标记检测副溶血性弧菌的 pH 超灵敏水合氢氧化铱薄膜电化学传感器。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-03 DOI: 10.1016/j.ab.2024.115597

Lin Tian , Yang Li , Huiqing Wang , Xinyi Li , Qian Gao , Yaru Liu , Yao Liu , Qing Wang , Cuiping Ma , Chao Shi

{"title":"A pH ultra-sensitive hydrated iridium oxyhydroxide films electrochemical sensor for label-free detection of Vibrio parahaemolyticus","authors":"Lin Tian , Yang Li , Huiqing Wang , Xinyi Li , Qian Gao , Yaru Liu , Yao Liu , Qing Wang , Cuiping Ma , Chao Shi","doi":"10.1016/j.ab.2024.115597","DOIUrl":"10.1016/j.ab.2024.115597","url":null,"abstract":"<div><p><em>Vibrio parahaemolyticus</em> (<em>V. parahaemolyticus</em>) is a major foodborne pathogen, which can cause serious foodborne illnesses like diarrhoea. Rapid on-site detection of foodborne pathogens is an ideal way to respond to foodborne illnesses. Herein, we provide an electrochemical sensor for rapid on-site detection. This sensor utilized a pH-sensitive metal-oxide material for the concurrent isothermal amplification and label-free detection of nucleic acids. Based on a pH-sensitive hydrated iridium oxide oxyhydroxide film (HIROF), the electrode transforms the hydrogen ion compound generated during nucleic acid amplification into potential, so as to achieve a real-time detection. The results can be transmitted to a smartphone via Bluetooth. Moreover, HIROF was applied in nucleic acid device detection, with a super-Nernst sensitivity of 77.6 mV/pH in the pH range of 6.0–8.5, and the sensitivity showed the best results so far. Detection of <em>V. parahaemolyticus</em> by this novel method showed a detection limit of 1.0 × 10<sup>3</sup> CFU/mL, while the time consumption was only 30 min, outperforming real-time fluorescence loop-mediated isothermal amplification (LAMP). Therefore, the characteristics of compact, portable, and fast make the sensor more widely used in on-site detection.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141537439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Surface display provides an efficient expression system for production of recombinant proteins and bacterial whole cell biosensor in E. coli 表面展示为在大肠杆菌中生产重组蛋白和细菌全细胞生物传感器提供了一种高效的表达系统。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-02 DOI: 10.1016/j.ab.2024.115599

{"title":"Surface display provides an efficient expression system for production of recombinant proteins and bacterial whole cell biosensor in E. coli","authors":"","doi":"10.1016/j.ab.2024.115599","DOIUrl":"10.1016/j.ab.2024.115599","url":null,"abstract":"<div><p>A novel bacterial display vector based on <em>Escherichia coli</em> has been engineered for recombinant protein production and purification. Accordingly, a construct harboring the enhanced green ﬂuorescent protein (EGFP) and the ice nucleation protein (INP) was designed to produce EGFP <em>via</em> the surface display in <em>E. coli</em> cells. The fusion EGFP-expressed cells were then investigated using fluorescence measurement, SDS- and native-PAGE before and after TEV protease digestion. The displayed EGFP was obtained with a recovery of 57.7 % as a single band on SDS-PAGE. Next, the efficiency of the cell surface display for mutant EGFP (EGFP S202H/Q204H) was examined in sensing copper ions. Under optimal conditions, a satisfactorily linear range for copper ions concentrations up to 10 nM with a detection limit of 0.073 nM was obtained for cell-displayed mutant EGFP (mEGFP). In the presence of bacterial cell lysates and purified mEGFP, response to copper was linear in the 2–10 nM and 0.1–2 μM concentration range, respectively, with a 1.3 nM and 0.14 μM limit of detection. The sensitivity of bacterial cell lysates and surface-displayed mEGFP in the detection of copper ions is higher than the purified mEGFP.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141533417","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Detection of foodborne pathogens in contaminated food using nanomaterial-based electrochemical biosensors 利用基于纳米材料的电化学生物传感器检测受污染食品中的食源性病原体。

IF 2.6 4区生物学

Analytical biochemistry Pub Date : 2024-07-02 DOI: 10.1016/j.ab.2024.115600

Ana Yareli Flores-Ramírez , Ramsés Ramón González-Estrada , Martina Alejandra Chacón-López , María de Lourdes García-Magaña , Efigenia Montalvo-González , Alejandra Álvarez-López , Aarón Rodríguez-López , Ulises Miguel López-García

{"title":"Detection of foodborne pathogens in contaminated food using nanomaterial-based electrochemical biosensors","authors":"Ana Yareli Flores-Ramírez , Ramsés Ramón González-Estrada , Martina Alejandra Chacón-López , María de Lourdes García-Magaña , Efigenia Montalvo-González , Alejandra Álvarez-López , Aarón Rodríguez-López , Ulises Miguel López-García","doi":"10.1016/j.ab.2024.115600","DOIUrl":"10.1016/j.ab.2024.115600","url":null,"abstract":"<div><p>Foodborne pathogens are a grave concern for the for food, medical, environmental, and economic sectors. Their ease of transmission and resistance to treatments, such as antimicrobial agents, make them an important challenge. Food tainted with these pathogens is swiftly rejected, and if ingested, can result in severe illnesses and even fatalities. This review provides and overview of the current status of various pathogens and their metabolites transmitted through food. Despite a plethora of studies on treatments to eradicate and inhibit these pathogens, their indiscriminate use can compromise the sensory properties of food and lead to contamination. Therefore, the study of detection methods such as electrochemical biosensors has been proposed, which are devices with advantages such as simplicity, fast response, and sensitivity. However, these biosensors may also present some limitations. In this regard, it has been reported that nanomaterials with high conductivity, surface-to-volume ratio, and robustness have been observed to improve the detection of foodborne pathogens or their metabolites. Therefore, in this work, we analyze the detection of pathogens transmitted through food and their metabolites using electrochemical biosensors based on nanomaterials.</p></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":null,"pages":null},"PeriodicalIF":2.6,"publicationDate":"2024-07-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141533415","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0