Pub Date : 2024-11-15DOI: 10.1016/j.ab.2024.115718
Xitian Zhu , Huijia Chen , Fang Ke
Hydrogen sulfide (H2S) acts as a messenger molecule and can mediate a variety of physiological functions. Conventional methods are seldom used to detect endogenous H2S and present some difficulties in selective and accurate detection. Reaction-based recognition of endogenous H2S by organic small molecule probes with good specificity and biocompatibility. To address this challenge, we developed a novel H2S fluorescent probe 4-(2-(6-hydroxy-2-naphthyl) ethyl)-1-methylpyridinium (DSNP) that triggers a thiolysis reaction through a strong electron withdrawing group, releasing a fluorescent molecule. The simple probe DSNP not only have good selectivity, large Stokes shifts and biocompatibility, but also demonstrated a detection limit as low as 28.4 nM and reaction times as quick as 30 min. Moreover, it has been successfully applied to imaging intracellular H2S in myeloma cells and zebrafish. This study opens new insights to help push this probe forward for its applicability for detailed H2S localization studies in osteosarcoma.
{"title":"A biocompatible fluorescent probe for endogenous hydrogen sulfide detection and imaging","authors":"Xitian Zhu , Huijia Chen , Fang Ke","doi":"10.1016/j.ab.2024.115718","DOIUrl":"10.1016/j.ab.2024.115718","url":null,"abstract":"<div><div>Hydrogen sulfide (H<sub>2</sub>S) acts as a messenger molecule and can mediate a variety of physiological functions. Conventional methods are seldom used to detect endogenous H<sub>2</sub>S and present some difficulties in selective and accurate detection. Reaction-based recognition of endogenous H<sub>2</sub>S by organic small molecule probes with good specificity and biocompatibility. To address this challenge, we developed a novel H<sub>2</sub>S fluorescent probe 4-(2-(6-hydroxy-2-naphthyl) ethyl)-1-methylpyridinium (<strong>DSNP</strong>) that triggers a thiolysis reaction through a strong electron withdrawing group, releasing a fluorescent molecule. The simple probe <strong>DSNP</strong> not only have good selectivity, large Stokes shifts and biocompatibility, but also demonstrated a detection limit as low as 28.4 nM and reaction times as quick as 30 min. Moreover, it has been successfully applied to imaging intracellular H<sub>2</sub>S in myeloma cells and zebrafish. This study opens new insights to help push this probe forward for its applicability for detailed H<sub>2</sub>S localization studies in osteosarcoma.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115718"},"PeriodicalIF":2.6,"publicationDate":"2024-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142643262","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}
Pub Date : 2024-11-12DOI: 10.1016/j.ab.2024.115717
Dipanjan Bhattacharyya , Marcia A. LeVatte , Upasana Singh , Fleur Issac , Mahmoud Karim , Saira Ali , August Sieben , Suyenna Huang , David S. Wishart
Urinary N1, N12-diacetylspermine (DAS) is a known biomarker for colorectal cancer (CRC). However, DAS levels in both healthy and CRC patients’ urine samples are extremely low and often challenging to quantify. Complex and expensive methods do exist to detect DAS in urine, but simpler, less expensive methods to detect DAS are needed, especially in low resource settings. Here we describe a highly efficient, fast, precise, and inexpensive colorimetric assay to detect low levels of DAS in human urine samples. We used recombinant diacetylspermine oxidase (rDAS Ox), expressed and extracted from E. coli, to oxidize DAS, producing three products including hydrogen peroxide (H2O2). The level of DAS present, which correlates with H2O2 levels, was measured using horseradish peroxidase (HRP), which together with H2O2, oxidized Amplex™ Red to produce the pink-colored resorufin. The concentration of resorufin is directly proportional to H2O2 (and DAS) levels. As urine contains metabolites which interfere with these oxidation reactions, we developed a simple two column-based protocol using ion exchange resins to remove these compounds and concentrate the DAS. With this novel cleaning and concentrating method, DAS was concentrated 15 times (confirmed by nuclear magnetic resonance (NMR) spectroscopy) and <1 μM DAS could be detected. Correlation graphs of urine samples spiked with known DAS concentrations versus assay-determined DAS concentrations had high coefficients of determination (R2) for 0–10 μM DAS (0.94) and for 0–1 μM DAS (0.91), clearly demonstrating the excellent performance of the two-column protocol with the rDAS Ox reaction mixture. To the best of our knowledge, this is first reported colorimetric enzymatic assay that quantitates DAS in urine.
尿液中的 N1、N12-二乙酰精胺（DAS）是已知的结直肠癌（CRC）生物标志物。然而，健康尿样和 CRC 患者尿样中的 DAS 含量都极低，而且通常难以量化。检测尿液中 DAS 的方法复杂且昂贵，但我们需要更简单、更便宜的方法来检测 DAS，尤其是在资源匮乏的环境中。在此，我们介绍了一种高效、快速、精确且廉价的比色法，用于检测人体尿样中低水平的 DAS。我们使用从大肠杆菌中表达和提取的重组双乙酰精胺氧化酶（rDAS Ox）来氧化 DAS，产生包括过氧化氢（H2O2）在内的三种产物。辣根过氧化物酶（HRP）与 H2O2 一起氧化 Amplex™ Red，生成粉红色的 resorufin，DAS 的含量与 H2O2 的含量相关。雷索卢芬的浓度与 H2O2（和 DAS）水平成正比。由于尿液中含有干扰这些氧化反应的代谢物，我们开发了一种简单的双柱方案，使用离子交换树脂去除这些化合物并浓缩 DAS。通过这种新颖的清洁和浓缩方法，DAS 浓缩了 15 倍（经核磁共振 (NMR) 光谱证实），2) 0-10 μM DAS 的浓缩率为 0.94，0-1 μM DAS 的浓缩率为 0.91，这清楚地表明了双柱方案与 rDAS Ox 反应混合物的卓越性能。据我们所知，这是首次报道定量检测尿液中 DAS 的比色酶法。
{"title":"A novel colorimetric assay for the detection of urinary N1, N12-diacetylspermine, a known biomarker for colorectal cancer","authors":"Dipanjan Bhattacharyya , Marcia A. LeVatte , Upasana Singh , Fleur Issac , Mahmoud Karim , Saira Ali , August Sieben , Suyenna Huang , David S. Wishart","doi":"10.1016/j.ab.2024.115717","DOIUrl":"10.1016/j.ab.2024.115717","url":null,"abstract":"<div><div>Urinary N<sup>1</sup>, N<sup>12</sup>-diacetylspermine (DAS) is a known biomarker for colorectal cancer (CRC). However, DAS levels in both healthy and CRC patients’ urine samples are extremely low and often challenging to quantify. Complex and expensive methods do exist to detect DAS in urine, but simpler, less expensive methods to detect DAS are needed, especially in low resource settings. Here we describe a highly efficient, fast, precise, and inexpensive colorimetric assay to detect low levels of DAS in human urine samples. We used recombinant diacetylspermine oxidase (rDAS Ox), expressed and extracted from <em>E. coli</em>, to oxidize DAS, producing three products including hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). The level of DAS present, which correlates with H<sub>2</sub>O<sub>2</sub> levels, was measured using horseradish peroxidase (HRP), which together with H<sub>2</sub>O<sub>2</sub>, oxidized Amplex™ Red to produce the pink-colored resorufin. The concentration of resorufin is directly proportional to H<sub>2</sub>O<sub>2</sub> (and DAS) levels. As urine contains metabolites which interfere with these oxidation reactions, we developed a simple two column-based protocol using ion exchange resins to remove these compounds and concentrate the DAS. With this novel cleaning and concentrating method, DAS was concentrated 15 times (confirmed by nuclear magnetic resonance (NMR) spectroscopy) and <1 μM DAS could be detected. Correlation graphs of urine samples spiked with known DAS concentrations versus assay-determined DAS concentrations had high coefficients of determination (R<sup>2</sup>) for 0–10 μM DAS (0.94) and for 0–1 μM DAS (0.91), clearly demonstrating the excellent performance of the two-column protocol with the rDAS Ox reaction mixture. To the best of our knowledge, this is first reported colorimetric enzymatic assay that quantitates DAS in urine.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115717"},"PeriodicalIF":2.6,"publicationDate":"2024-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612401","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}
Pub Date : 2024-11-09DOI: 10.1016/j.ab.2024.115712
Olivia Shapiro , Clara Woods , Amanda M. Gleixner , Sara Sannino , Marilyn Ngo , Michael D. McDaniels , Peter Wipf , Neil A. Hukriede , Christopher J. Donnelly , Jeffrey L. Brodsky
Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.
{"title":"Assays to measure small molecule Hsp70 agonist activity in vitro and in vivo","authors":"Olivia Shapiro , Clara Woods , Amanda M. Gleixner , Sara Sannino , Marilyn Ngo , Michael D. McDaniels , Peter Wipf , Neil A. Hukriede , Christopher J. Donnelly , Jeffrey L. Brodsky","doi":"10.1016/j.ab.2024.115712","DOIUrl":"10.1016/j.ab.2024.115712","url":null,"abstract":"<div><div>Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and <em>in vivo</em> assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115712"},"PeriodicalIF":2.6,"publicationDate":"2024-11-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612421","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}
Pub Date : 2024-11-08DOI: 10.1016/j.ab.2024.115714
Paul Y. Kim , Michelle Vong , Dani Lee , Chengliang Wu
Dysregulations of blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labeled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7 % and 3.0 %, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.
{"title":"Development of an assay to quantify tranexamic acid levels in plasma","authors":"Paul Y. Kim , Michelle Vong , Dani Lee , Chengliang Wu","doi":"10.1016/j.ab.2024.115714","DOIUrl":"10.1016/j.ab.2024.115714","url":null,"abstract":"<div><div>Dysregulations of blood clot breakdown (fibrinolysis) during vascular trauma can lead to excessive blood loss. Tranexamic acid (TXA) is an inhibitor of fibrinolysis that works by blocking the interaction between plasminogen and fibrin degradation products (FDPs) – a key step in fibrinolysis. Despite the widespread usage, there are no tests available in a clinical setting to monitor TXA levels. We developed a fluorescence resonance energy transfer (FRET)-based assay to quantify TXA concentrations in plasma by using 1) fluorescently labeled plasminogen, and 2) FDPs labeled with a fluorescence quencher. Once plasminogen binds the FDPs, the fluorescent signal is quenched. TXA causes plasminogen to dissociate from the FDPs, thus increasing fluorescence signal in a dose-dependent manner. The dose response was sensitive between 1 and 100 μM (0.16 and 15.7 mg/L). The intraassay and interassay variabilities were determined to be 5.7 % and 3.0 %, respectively. Limit of detection was estimated to be 0.28 μM (0.044 mg/L). When tested for measuring known levels of TXA added to plasma samples, the ratio between measured and expected TXA concentration was 1.0151. Our study demonstrates a novel assay that can rapidly quantify TXA concentrations in plasma samples, thus demonstrating its potential as an in-hospital tool.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115714"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612441","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}
Pub Date : 2024-11-08DOI: 10.1016/j.ab.2024.115711
Michael Gamal Fawzy , Soad S. Abd El-Hay , Alaa Ahmed Mostafa , Youstina Mekhail Metias
Hypertension is a leading cause of cardiovascular mortality, often accompanied by complications such as arrhythmia and stroke. This silent killer requires a multifaceted pharmacological approach for effective management. This article presents new, environmentally friendly spectrophotometric methods for simultaneous quantification of telmisartan (TER) and metoprolol succinate (MTR) in laboratory prepared mixtures and pharmaceutical formulations. The suggested methodologies include the following: area under the curve method (AUC) utilizing area at specific wavelength ranges 228–233 nm (λ1 – λ2) and 240–245 nm (λ3 – λ4) for each analyte and Fourier self-deconvolution method (FD) depending on built-in function to address spectral interferences. In addition, the induced dual wavelength method (IDWL) employing equality factors to obtain absorbance differences at designated wavelengths, ratio difference method (RD) utilizing divisor-based ratio spectra where the utilized divisors were TER 40 μg/mL and MTR 90 μg/mL, and ratio derivative method (RDV) generating spectra through first derivative application that was measured at 266 nm and 246 nm for TER and MTR, respectively. These methods offer green alternatives for the accurate and precise determination of TER and MTR with exceptional linearity of 3–45, and 15–200 μg/mL for TER and MTR, respectively. Furthermore, the methods showed a coefficient of determination exceeding 0.9995 and good detection and quantification levels. A comprehensive greenness assessment, employing five distinct evaluation tools, confirmed the reduced environmental impact of the proposed methods in terms of waste generation, chemical consumption, and instrument safety. Successful analysis of pharmaceutical formulations and laboratory prepared mixtures containing different TER and MTR ratios confirmed the validity of the proposed methods. Standard addition studies further supported these findings, and the statistical results were comparable to those obtained using a reference method.
{"title":"Specialized greenness sustainability tools for evaluation of the spectrophotometric methodologies greenness: Spectral signal manipulation for resolving the interfering telmisartan and metoprolol succinate spectra in their bulk and pharmaceutical formulation","authors":"Michael Gamal Fawzy , Soad S. Abd El-Hay , Alaa Ahmed Mostafa , Youstina Mekhail Metias","doi":"10.1016/j.ab.2024.115711","DOIUrl":"10.1016/j.ab.2024.115711","url":null,"abstract":"<div><div>Hypertension is a leading cause of cardiovascular mortality, often accompanied by complications such as arrhythmia and stroke. This silent killer requires a multifaceted pharmacological approach for effective management. This article presents new, environmentally friendly spectrophotometric methods for simultaneous quantification of telmisartan (TER) and metoprolol succinate (MTR) in laboratory prepared mixtures and pharmaceutical formulations. The suggested methodologies include the following: area under the curve method (AUC) utilizing area at specific wavelength ranges 228–233 nm (<em>λ</em><sub>1</sub> – <em>λ</em><sub>2</sub>) and 240–245 nm (<em>λ</em><sub>3</sub> – <em>λ</em><sub>4</sub>) for each analyte and Fourier self-deconvolution method (FD) depending on built-in function to address spectral interferences. In addition, the induced dual wavelength method (IDWL) employing equality factors to obtain absorbance differences at designated wavelengths, ratio difference method (RD) utilizing divisor-based ratio spectra where the utilized divisors were TER 40 μg/mL and MTR 90 μg/mL, and ratio derivative method (RDV) generating spectra through first derivative application that was measured at 266 nm and 246 nm for TER and MTR, respectively. These methods offer green alternatives for the accurate and precise determination of TER and MTR with exceptional linearity of 3–45, and 15–200 μg/mL for TER and MTR, respectively. Furthermore, the methods showed a coefficient of determination exceeding 0.9995 and good detection and quantification levels. A comprehensive greenness assessment, employing five distinct evaluation tools, confirmed the reduced environmental impact of the proposed methods in terms of waste generation, chemical consumption, and instrument safety. Successful analysis of pharmaceutical formulations and laboratory prepared mixtures containing different TER and MTR ratios confirmed the validity of the proposed methods. Standard addition studies further supported these findings, and the statistical results were comparable to those obtained using a reference method.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115711"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612444","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}
Pub Date : 2024-11-08DOI: 10.1016/j.ab.2024.115713
David N. Frick , Mujidat Shittu , Chase R. Bock , Zoe P. Wardle , Abdullah A. Rauf , Julian N. Ramos , Joshua G. Thomson , Daniel J. Sheibley , Suzanne F. O'Handley
When stressed, cells synthesize di-adenosine polyphosphates (ApnA), and cellular organisms also express proteins that degrade these compounds to release ATP. Most of these proteins are members of the nudix hydrolase superfamily, and several are involved in bacterial pathogenesis, neurodevelopment, and cancer. The goal of this project is to assist in the discovery of inhibitors of these enzymes that could be used to study ApnA function and the cellular role of these nudix enzymes. Because these enzymes cleave Ap4A and Ap5A to produce ATP, two standard ATP detection techniques were optimized and compared here for their suitability for high throughput screening. In the first assay, cleavage is monitored by coupling to a reaction catalyzed by firefly luciferase. In the second assay, cleavage is detected by coupling to hexokinase, glucose 6-phosphate dehydrogenase, and diaphorase. Although the former assay was more sensitive, the latter was more reproducible, linear, and suitable for screening and kinetic analyses. The assays were used to characterize the kinetics of reactions catalyzed by various nudix enzymes isolated from E. coli, humans, and Mycobacterium tuberculosis, the bacterium that causes tuberculosis. Results reveal subtle differences between the proteins that might be exploited to identify specific small molecule inhibitors.
当受到压力时，细胞会合成二腺苷多磷酸盐（ApnA），细胞生物体也会表达降解这些化合物以释放 ATP 的蛋白质。这些蛋白质大多属于 nudix 水解酶超家族，其中有几种涉及细菌致病、神经发育和癌症。本项目的目标是协助发现这些酶的抑制剂，用于研究 ApnA 的功能和这些 nudix 酶的细胞作用。由于这些酶会裂解 Ap4A 和 Ap5A 以产生 ATP，因此对两种标准 ATP 检测技术进行了优化和比较，以确定它们是否适用于高通量筛选。在第一种检测方法中，通过与萤火虫荧光素酶催化的反应耦合来监测裂解。在第二种检测方法中，通过与己糖激酶、6-磷酸葡萄糖脱氢酶和二磷酸盐酶偶联来检测裂解。虽然前一种检测方法更灵敏，但后一种检测方法的可重复性和线性更好，适合筛选和动力学分析。这些检测方法被用来描述从大肠杆菌、人类和结核分枝杆菌（导致结核病的细菌）中分离出来的各种 nudix 酶催化反应的动力学特征。研究结果揭示了蛋白质之间的细微差别，可以利用这些差异找出特定的小分子抑制剂。
{"title":"Optimization of a high throughput screening platform to identify inhibitors of asymmetric diadenosine polyphosphatases","authors":"David N. Frick , Mujidat Shittu , Chase R. Bock , Zoe P. Wardle , Abdullah A. Rauf , Julian N. Ramos , Joshua G. Thomson , Daniel J. Sheibley , Suzanne F. O'Handley","doi":"10.1016/j.ab.2024.115713","DOIUrl":"10.1016/j.ab.2024.115713","url":null,"abstract":"<div><div>When stressed, cells synthesize di-adenosine polyphosphates (Ap<sub>n</sub>A), and cellular organisms also express proteins that degrade these compounds to release ATP. Most of these proteins are members of the nudix hydrolase superfamily, and several are involved in bacterial pathogenesis, neurodevelopment, and cancer. The goal of this project is to assist in the discovery of inhibitors of these enzymes that could be used to study Ap<sub>n</sub>A function and the cellular role of these nudix enzymes. Because these enzymes cleave Ap<sub>4</sub>A and Ap<sub>5</sub>A to produce ATP, two standard ATP detection techniques were optimized and compared here for their suitability for high throughput screening. In the first assay, cleavage is monitored by coupling to a reaction catalyzed by firefly luciferase. In the second assay, cleavage is detected by coupling to hexokinase, glucose 6-phosphate dehydrogenase, and diaphorase. Although the former assay was more sensitive, the latter was more reproducible, linear, and suitable for screening and kinetic analyses. The assays were used to characterize the kinetics of reactions catalyzed by various nudix enzymes isolated from <em>E. coli</em>, humans, and <em>Mycobacterium tuberculosis</em>, the bacterium that causes tuberculosis<em>.</em> Results reveal subtle differences between the proteins that might be exploited to identify specific small molecule inhibitors.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115713"},"PeriodicalIF":2.6,"publicationDate":"2024-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612443","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}
Pub Date : 2024-11-07DOI: 10.1016/j.ab.2024.115716
Jia Deng, Ye Yuan, Min Zou, Xudong Liu, Xianxian Zhao, Hongli Liu
There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.
目前迫切需要一种简单而又极其精确的生物传感器来分析肿瘤的发生。在此，我们提出了一种新型的荧光和无酶方法，用于检测 p53 基因级联近接连接介导的催化发夹组装和 DNA 酶辅助的信号反应。当目标 p53 基因存在时，p53 与 L1 和 L2 链之间的相互作用会启动催化发夹组装，并随后暴露 P3 探针中的 DNA 酶。暴露的 DNA 酶与 P4 探针的环状区域结合，产生一个挑刺位点，从而释放出大量与 P4 茎部结合的 ATMND。这导致荧光反应放大，成为检测 p53 基因的荧光信号。这种方法可以准确灵敏地鉴定 p53 基因，其线性反应范围为 1 fM 至 1 nM，检测限低至 0.23 fM。此外，这种荧光方法还可用于临床样本的检测，并具有良好的回收率。最重要的是，这种多功能平台可以通过改变相应的识别单元来分析不同的靶标，在肿瘤发生分析的床旁检测方面显示出巨大的潜力。
{"title":"A simple and enzyme-free method for sensitive p53 analysis based on DNAzyme-mediated signal amplification.","authors":"Jia Deng, Ye Yuan, Min Zou, Xudong Liu, Xianxian Zhao, Hongli Liu","doi":"10.1016/j.ab.2024.115716","DOIUrl":"https://doi.org/10.1016/j.ab.2024.115716","url":null,"abstract":"<p><p>There is an urgent demand for a simple yet extremely accurate biosensor to analyze tumorigenesis. Herein, we present a novel fluorescent and enzyme-free approach for detecting p53 gene cascading proximity ligation-mediated catalytic hairpin assembly and DNAzyme-assisted signal reaction. When the target p53 gene is present, the interaction between p53 and L1 and L2 chains initiates catalytic hairpin assembly and subsequently exposes DNAzyme in the P3 probe. The exposed DNAzyme binds with the loop region of the P4 probe and generates a nicking site, resulting in the release of a significant amount of ATMND that is conjugated in the stem section of P4. This leads to an amplified fluorescence response, which serves as a fluorescence signal for the detection of the p53 gene. This method allows for the accurate and sensitive identification of the p53 gene, exhibiting a linear reaction range of 1 fM to 1 nM, with a limit of detection as low as 0.23 fM. Furthermore, this fluorescent method has been utilized for the examination of clinical samples with a favorable recovery rate. Crucially, this versatile platform may be expanded to analyze different targets by changing the corresponding recognition unit, showing great potential for point-of-care testing in tumorigenesis analysis.</p>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":" ","pages":"115716"},"PeriodicalIF":2.6,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612419","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}
Pub Date : 2024-11-07DOI: 10.1016/j.ab.2024.115715
Zhe Ju, Qing-Bao Zhang
Lysine β-hydroxybutyrylation (Kbhb) is newly discovered β-hydroxybutyrylate-derived histone modification which has been associated with the pathogenesis of many human diseases. To further elucidate the biological significance and molecular mechanism of Kbhb, it is necessary to accurately identify the Kbhb sites from protein sequences. In this study, a novel computational model named iBhb-Lys is developed for the identification of Kbhb sites. Four types of features are combined to encode each Kbhb site as a 3266-dimensional feature vector. And the autoencoder network is used to reduce the dimensionality of feature space, due to the high dimensionality of the combined features. In addition, to effectively reduce the influence of noise and outlier on classification, a new fuzzy support vector machine algorithm is proposed by incorporating the density around the sample into the fuzzy membership function. As illustrated by independent test, the AUC value of iBhb-Lys has increased by 2.22 % compared to the existing predictor KbhbXG. Feature analysis shows that some amino acid composition features, such as the occurrence frequency of leucine and histidine residues around Kbhb sites, contribute profoundly to the identification of Kbhb sites. The conclusions drawn in this study may provide useful reference for studying the molecular mechanism of Kbhb.
{"title":"iBhb-Lys: Identify lysine β-hydroxybutyrylation sites using autoencoder feature representation and fuzzy SVM algorithm","authors":"Zhe Ju, Qing-Bao Zhang","doi":"10.1016/j.ab.2024.115715","DOIUrl":"10.1016/j.ab.2024.115715","url":null,"abstract":"<div><div>Lysine β-hydroxybutyrylation (Kbhb) is newly discovered β-hydroxybutyrylate-derived histone modification which has been associated with the pathogenesis of many human diseases. To further elucidate the biological significance and molecular mechanism of Kbhb, it is necessary to accurately identify the Kbhb sites from protein sequences. In this study, a novel computational model named iBhb-Lys is developed for the identification of Kbhb sites. Four types of features are combined to encode each Kbhb site as a 3266-dimensional feature vector. And the autoencoder network is used to reduce the dimensionality of feature space, due to the high dimensionality of the combined features. In addition, to effectively reduce the influence of noise and outlier on classification, a new fuzzy support vector machine algorithm is proposed by incorporating the density around the sample into the fuzzy membership function. As illustrated by independent test, the AUC value of iBhb-Lys has increased by 2.22 % compared to the existing predictor KbhbXG. Feature analysis shows that some amino acid composition features, such as the occurrence frequency of leucine and histidine residues around Kbhb sites, contribute profoundly to the identification of Kbhb sites. The conclusions drawn in this study may provide useful reference for studying the molecular mechanism of Kbhb.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115715"},"PeriodicalIF":2.6,"publicationDate":"2024-11-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142612442","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}
Pub Date : 2024-10-29DOI: 10.1016/j.ab.2024.115701
Xiang Hu , Jingyi Li , Taigang Liu
The escalating global incidence of allergy patients illustrates the growing impact of allergic issues on global health. Allergens are small molecule antigens that trigger allergic reactions. A widely recognized strategy for allergy prevention involves identifying allergens and avoiding re-exposure. However, the laboratory methods to identify allergenic proteins are often time-consuming and resource-intensive. There is a crucial need to establish efficient and reliable computational approaches for the identification of allergenic proteins. In this study, we developed a novel allergenic proteins predictor named Alg-MFDL, which integrates pre-trained protein language models (PLMs) and traditional handcrafted features to achieve a more complete protein representation. First, we compared the performance of eight pre-trained PLMs from ProtTrans and ESM-2 and selected the best-performing one from each of the two groups. In addition, we evaluated the performance of three handcrafted features and different combinations of them to select the optimal feature or feature combination. Then, these three protein representations were fused and used as inputs to train the convolutional neural network (CNN). Finally, the independent validation was performed on benchmark datasets to evaluate the performance of Alg-MFDL. As a result, Alg-MFDL achieved an accuracy of 0.973, a precision of 0.996, a sensitivity of 0.951, and an F1 value of 0.973, outperforming the most of current state-of-the-art (SOTA) methods across all key metrics. We anticipated that the proposed model could be considered a useful tool for predicting allergen proteins.
{"title":"Alg-MFDL: A multi-feature deep learning framework for allergenic proteins prediction","authors":"Xiang Hu , Jingyi Li , Taigang Liu","doi":"10.1016/j.ab.2024.115701","DOIUrl":"10.1016/j.ab.2024.115701","url":null,"abstract":"<div><div>The escalating global incidence of allergy patients illustrates the growing impact of allergic issues on global health. Allergens are small molecule antigens that trigger allergic reactions. A widely recognized strategy for allergy prevention involves identifying allergens and avoiding re-exposure. However, the laboratory methods to identify allergenic proteins are often time-consuming and resource-intensive. There is a crucial need to establish efficient and reliable computational approaches for the identification of allergenic proteins. In this study, we developed a novel allergenic proteins predictor named Alg-MFDL, which integrates pre-trained protein language models (PLMs) and traditional handcrafted features to achieve a more complete protein representation. First, we compared the performance of eight pre-trained PLMs from ProtTrans and ESM-2 and selected the best-performing one from each of the two groups. In addition, we evaluated the performance of three handcrafted features and different combinations of them to select the optimal feature or feature combination. Then, these three protein representations were fused and used as inputs to train the convolutional neural network (CNN). Finally, the independent validation was performed on benchmark datasets to evaluate the performance of Alg-MFDL. As a result, Alg-MFDL achieved an accuracy of 0.973, a precision of 0.996, a sensitivity of 0.951, and an F1 value of 0.973, outperforming the most of current state-of-the-art (SOTA) methods across all key metrics. We anticipated that the proposed model could be considered a useful tool for predicting allergen proteins.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115701"},"PeriodicalIF":2.6,"publicationDate":"2024-10-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142557025","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}
The identification of Drug-Target Interaction (DTI) is an important step in drug discovery and drug repositioning, and has high application value in multiple fields such as drug discovery, drug repositioning, and repurposing. However, the high cost of experimental validation limits its identification. In contrast, computation-based approaches are both economical and efficient. This review first synthesizes existing chemical genomic approaches, provides a comprehensive summary of prevalent databases for predicting DTIs, and categorizes the feature encodings from recent years. This is followed by an overview and brief description of the methods currently in use for predicting DTIs. The strengths and weaknesses of newly proposed prediction methods in the last five years (2020–2024), including those based on network representation learning and graph neural networks, are then discussed in detail, evaluating the performance of the different methods on a wide range of datasets. Finally, this review explores potential directions for future DTI research, emphasizing how to improve prediction accuracy and efficiency by combining big data and emerging computing technologies.
{"title":"Research progress on Drug-Target Interactions in the last five years","authors":"Yun Zuo, Xubin Wu, Fei Ge, Hongjin Yan, Sirui Fei, Jingwen Liang, Zhaohong Deng","doi":"10.1016/j.ab.2024.115691","DOIUrl":"10.1016/j.ab.2024.115691","url":null,"abstract":"<div><div>The identification of Drug-Target Interaction (DTI) is an important step in drug discovery and drug repositioning, and has high application value in multiple fields such as drug discovery, drug repositioning, and repurposing. However, the high cost of experimental validation limits its identification. In contrast, computation-based approaches are both economical and efficient. This review first synthesizes existing chemical genomic approaches, provides a comprehensive summary of prevalent databases for predicting DTIs, and categorizes the feature encodings from recent years. This is followed by an overview and brief description of the methods currently in use for predicting DTIs. The strengths and weaknesses of newly proposed prediction methods in the last five years (2020–2024), including those based on network representation learning and graph neural networks, are then discussed in detail, evaluating the performance of the different methods on a wide range of datasets. Finally, this review explores potential directions for future DTI research, emphasizing how to improve prediction accuracy and efficiency by combining big data and emerging computing technologies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"697 ","pages":"Article 115691"},"PeriodicalIF":2.6,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142493137","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}
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