Analytical biochemistry最新文献_第8页

Decoding ulcerative colitis: Plasma protein-mediated antibody immune responses influence disease risk 解码溃疡性结肠炎：血浆蛋白介导的抗体免疫反应影响疾病风险

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-15 DOI: 10.1016/j.ab.2025.115946

Xuyong Chen, Haidong Wu, Liudan Wang, Yifan Guo, Xinpu Miao

Objective

Previous Mendelian randomization (MR) studies have explored the role of plasma proteins in ulcerative colitis (UC), but the underlying mechanisms, particularly how plasma proteins influence UC risk via immune-mediated pathways, remain unclear. This study integrates two-sample MR and mediation analysis to investigate whether plasma proteins influence UC risk through antibody-mediated immune responses, aiming to identify novel biomarkers and therapeutic targets.

Methods

We analyzed 4907 plasma proteins from a genome-wide association study, 46 immune antibody responses, and UC data from the FinnGen consortium. Two-sample MR was used to assess causal relationships among plasma proteins, antibody responses, and UC. Mediation MR analysis was conducted to evaluate whether specific antibody responses mediate the effect of plasma proteins on UC risk.

Results

A total of 80 plasma proteins with significant causal associations with UC were identified (P < 0.05). Two antibody responses, Epstein-Barr virus (EBV) EA-D antibody levels and anti-HSV-1 IgG seropositivity, were significantly and inversely associated with UC risk (EBV: OR = 0.794, 95 % CI: 0.646–0.974, P = 0.027; HSV-1 IgG: OR = 0.891, 95 % CI: 0.801–0.992, P = 0.035). Five plasma proteins showed significant causal effects on these antibody responses: UBC, TIMD4, and NEFL (linked to EBV EA-D), and TMEM70 and HIF1A (linked to HSV-1 IgG). Mediation analysis revealed that antibody responses explained 9.2 %–13.2 % of the total effects of these proteins on UC risk. For example, UBC increased UC risk partially through reduced EBV EA-D antibodies (mediated effect = 0.0588), while HIF1A contributed to UC risk via suppressed HSV-1 IgG seropositivity.

Conclusion

This study identifies a novel immuno-genetic pathway in UC pathogenesis, where specific plasma proteins influence disease risk through modulation of viral antibody responses. These findings suggest potential targets, such as UBC, HIF1A, and TIMD4, for biomarker development and immune-focused interventions in UC.

先前的孟德尔随机化（MR）研究已经探索了血浆蛋白在溃疡性结肠炎（UC）中的作用，但其潜在机制，特别是血浆蛋白如何通过免疫介导途径影响UC风险尚不清楚。本研究结合两样本MR和中介分析，探讨血浆蛋白是否通过抗体介导的免疫反应影响UC风险，旨在发现新的生物标志物和治疗靶点。方法我们分析了来自全基因组关联研究的4907种血浆蛋白、46种免疫抗体应答和来自FinnGen联盟的UC数据。双样本MR用于评估血浆蛋白、抗体反应和UC之间的因果关系。进行中介MR分析以评估特异性抗体反应是否介导血浆蛋白对UC风险的影响。结果共鉴定出80种与UC有显著因果关系的血浆蛋白(P <；0.05)。eb病毒（EBV） EA-D抗体水平和抗hsv -1 IgG血清阳性与UC风险呈显著负相关(EBV: OR = 0.794, 95% CI: 0.646-0.974, P = 0.027；1型单纯疱疹病毒免疫球蛋白:= 0.891,95%置信区间CI: 0.801 - -0.992, P = 0.035)。五种血浆蛋白对这些抗体反应有显著的因果影响：UBC、TIMD4和NEFL（与EBV EA-D相关），以及TMEM70和HIF1A（与HSV-1 IgG相关）。中介分析显示，抗体反应解释了这些蛋白质对UC风险的总影响的9.2% - 13.2%。例如，UBC部分通过降低EBV EA-D抗体增加UC风险（介导效应= 0.0588），而HIF1A通过抑制HSV-1 IgG血清阳性增加UC风险。结论本研究确定了UC发病机制中的一种新的免疫遗传途径，其中特异性血浆蛋白通过调节病毒抗体反应影响疾病风险。这些发现提示了UC生物标志物开发和免疫干预的潜在靶点，如UBC、HIF1A和TIMD4。

{"title":"Decoding ulcerative colitis: Plasma protein-mediated antibody immune responses influence disease risk","authors":"Xuyong Chen, Haidong Wu, Liudan Wang, Yifan Guo, Xinpu Miao","doi":"10.1016/j.ab.2025.115946","DOIUrl":"10.1016/j.ab.2025.115946","url":null,"abstract":"<div><h3>Objective</h3><div>Previous Mendelian randomization (MR) studies have explored the role of plasma proteins in ulcerative colitis (UC), but the underlying mechanisms, particularly how plasma proteins influence UC risk via immune-mediated pathways, remain unclear. This study integrates two-sample MR and mediation analysis to investigate whether plasma proteins influence UC risk through antibody-mediated immune responses, aiming to identify novel biomarkers and therapeutic targets.</div></div><div><h3>Methods</h3><div>We analyzed 4907 plasma proteins from a genome-wide association study, 46 immune antibody responses, and UC data from the FinnGen consortium. Two-sample MR was used to assess causal relationships among plasma proteins, antibody responses, and UC. Mediation MR analysis was conducted to evaluate whether specific antibody responses mediate the effect of plasma proteins on UC risk.</div></div><div><h3>Results</h3><div>A total of 80 plasma proteins with significant causal associations with UC were identified (P < 0.05). Two antibody responses, Epstein-Barr virus (EBV) EA-D antibody levels and anti-HSV-1 IgG seropositivity, were significantly and inversely associated with UC risk (EBV: OR = 0.794, 95 % CI: 0.646–0.974, P = 0.027; HSV-1 IgG: OR = 0.891, 95 % CI: 0.801–0.992, P = 0.035). Five plasma proteins showed significant causal effects on these antibody responses: UBC, TIMD4, and NEFL (linked to EBV EA-D), and TMEM70 and HIF1A (linked to HSV-1 IgG). Mediation analysis revealed that antibody responses explained 9.2 %–13.2 % of the total effects of these proteins on UC risk. For example, UBC increased UC risk partially through reduced EBV EA-D antibodies (mediated effect = 0.0588), while HIF1A contributed to UC risk via suppressed HSV-1 IgG seropositivity.</div></div><div><h3>Conclusion</h3><div>This study identifies a novel immuno-genetic pathway in UC pathogenesis, where specific plasma proteins influence disease risk through modulation of viral antibody responses. These findings suggest potential targets, such as UBC, HIF1A, and TIMD4, for biomarker development and immune-focused interventions in UC.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115946"},"PeriodicalIF":2.6,"publicationDate":"2025-07-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144654331","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

Enhancing lateral flow assay performance: Buffer additives and protein-membrane interactions 增强横向流动分析性能：缓冲剂添加剂和蛋白质膜相互作用

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-12 DOI: 10.1016/j.ab.2025.115943

Alexander Spreinat , Carola Wilczek , Christin Ronsör , Andrea Ernst

Lateral Flow Assays (LFA) have been valuable tools for point-of-care diagnostics for decades. During periods of high testing demand, as recently in the Covid-19-pandemic, it is necessary to deepen the understanding of test and control line signal intensities. This study investigates the interaction between proteins and structurally different nitrocellulose (CN) membranes and the resulting effects on signal intensity in an LFA under the influence of various buffer additives. The experiments focused on quantitative protein adsorption, protein stability, protein line printing and changes in signal intensity in a human chorionic gonadotropin (hCG)-assay. A method was established for detecting the widths and intensities of fluorescent protein lines. We were able to show that changes in signal intensity of LFAs are driven by accessibility of the antibodies, by hydrophilicity of the membrane or assisted adsorption of antibodies onto the membrane. Additionally, the inclusion of sodium chloride, polysorbate 80 and sodium dodecylbenzenesulfonate can enhance signal intensity. The method developed for protein line analysis has proven to be effective and can help to understand protein-membrane-interactions on a macroscopic level. We demonstrate that LFA-manufacturers have a range of options to fine-tune assay performance without major modifications to assay components.

几十年来，横向流动测定法（LFA）一直是即时诊断的宝贵工具。在测试需求高的时期，如最近的covid -19大流行期间，有必要加深对测试和控制线信号强度的理解。本研究研究了在不同缓冲添加剂的影响下，蛋白质与结构不同的硝化纤维素（CN）膜之间的相互作用及其对LFA中信号强度的影响。实验重点研究了人绒毛膜促性腺激素（hCG）测定中蛋白的定量吸附、蛋白稳定性、蛋白线打印和信号强度的变化。建立了一种检测荧光蛋白线宽度和强度的方法。我们能够证明LFAs信号强度的变化是由抗体的可及性、膜的亲水性或抗体在膜上的辅助吸附所驱动的。此外，氯化钠、聚山梨酯80和十二烷基苯磺酸钠的包合可以增强信号强度。该方法用于蛋白质系分析已被证明是有效的，可以帮助在宏观水平上理解蛋白质-膜的相互作用。我们证明lfa制造商有一系列的选择来微调分析性能，而不需要对分析成分进行重大修改。

{"title":"Enhancing lateral flow assay performance: Buffer additives and protein-membrane interactions","authors":"Alexander Spreinat , Carola Wilczek , Christin Ronsör , Andrea Ernst","doi":"10.1016/j.ab.2025.115943","DOIUrl":"10.1016/j.ab.2025.115943","url":null,"abstract":"<div><div>Lateral Flow Assays (LFA) have been valuable tools for point-of-care diagnostics for decades. During periods of high testing demand, as recently in the Covid-19-pandemic, it is necessary to deepen the understanding of test and control line signal intensities. This study investigates the interaction between proteins and structurally different nitrocellulose (CN) membranes and the resulting effects on signal intensity in an LFA under the influence of various buffer additives. The experiments focused on quantitative protein adsorption, protein stability, protein line printing and changes in signal intensity in a human chorionic gonadotropin (hCG)-assay. A method was established for detecting the widths and intensities of fluorescent protein lines. We were able to show that changes in signal intensity of LFAs are driven by accessibility of the antibodies, by hydrophilicity of the membrane or assisted adsorption of antibodies onto the membrane. Additionally, the inclusion of sodium chloride, polysorbate 80 and sodium dodecylbenzenesulfonate can enhance signal intensity. The method developed for protein line analysis has proven to be effective and can help to understand protein-membrane-interactions on a macroscopic level. We demonstrate that LFA-manufacturers have a range of options to fine-tune assay performance without major modifications to assay components.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115943"},"PeriodicalIF":2.6,"publicationDate":"2025-07-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144631456","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

Proton-coupled electron transfer in hallachrome, a natural 1,2 anthraquinone: Linking electrochemical properties to biological activity 一种天然1,2蒽醌——汉那色胺的质子耦合电子转移：将电化学性质与生物活性联系起来。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-11 DOI: 10.1016/j.ab.2025.115945

Felice C. Simeone

Hallachrome is a 1,2-anthraquinone secreted by marine polychaete worms whose toxicity and antimicrobial properties have been reported; the origin of this biological activity, however, remains elusive. Voltammetric studies reveal reversible redox behavior in the pH range 1–10, consistent with a two-electron, two-proton (2e⁻/2H⁺) mechanism. The electron-donating substituents in hallachrome (hydroxyl and methyl groups) stabilize the protonated hydroquinone form and result in a substantial cathodic shift of 0.4–0.5 V compared to unsubstituted 1,2-benzoquinone.

The electrochemical analysis reveals that hallachrome (redox potential −0.12 V vs. SHE at pH 7) might be capable, from a thermodynamic point of view, of oxidizing key cellular antioxidants, including NADH (−0.32 V), NADPH (−0.32 V), and glutathione (−0.24 V). These findings provide fundamental insights into the structure-activity relationships governing quinone electrochemistry and establish a foundation for understanding hallachrome's biological activity.

Hallachrome是一种由海洋多毛类蠕虫分泌的1,2-蒽醌，其毒性和抗菌特性已被报道；然而，这种生物活性的起源仍然难以捉摸。伏安研究表明，在pH值1 ~ 10范围内，其氧化还原行为是可逆的，符合双电子、双质子（2e-/2H+）机制。hallachrome中的给电子取代基（羟基和甲基）稳定了质子化对苯二酚的形式，与未取代的1,2-苯醌相比，导致了0.4-0.5 V的阴极位移。电化学分析表明，从热力学角度来看，hallachrome （pH = 7时氧化还原电位-0.12 V vs. SHE）可能能够氧化关键的细胞抗氧化剂，包括NADH (-0.32 V), NADPH （-0.32 V）和谷胱甘肽（-0.24 V）。这些发现为研究醌类电化学的构效关系提供了基础，并为进一步了解hallachrome的生物活性奠定了基础。

引用次数: 0

Simultaneous multi-element determination in whole blood and urine via dual-mode ICP-MS 双模式ICP-MS同时测定全血和尿液中的多元素。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-11 DOI: 10.1016/j.ab.2025.115941

Yan Li , Jian-Yuan Zhong , Ya-qi Mo , Li-mei Qin , Michael Aschner , Yue-ming Jiang

The content of elements in the body has a significant relationship with human health. However, the wide variation in the concentrations of different elements in blood or urine poses challenges for accurate detection. This study presents a dual-mode ICP-MS methodology integrating kinetic energy discrimination (KED) and dynamic reaction cell (DRC) technologies, specifically designed to address the analytical challenge of quantifying 22 physiologically critical elements (Be, V, Cr, Mn, Fe, Ca, Mg, Ba, Co, Cd, Cu, Zn, As, Se, Ti, Sr, Ni, Mo, Sn, Sb, Tl, Pb) spanning six orders of magnitude in concentration (μg/L to g/L) within complex biological matrices. Rejection parameter q (RPq) adjusts the low-mass cutoff, while rejection parameter a (RPa) controls the high-mass cutoff. Optimizing both extends the low-interference dynamic range. The method's core innovation lies in the synchronized optimization of quadrupole parameters RPa and RPq, which enables simultaneous suppression of matrix-derived polyatomic interferences while maintaining linear detector response across ultra-wide dynamic ranges. The method employs a dual-mode analysis approach: (1) In urine analysis, the KED-He mode is suitable for analyzing all elements. (2) In whole blood analysis, DRC-NH₃ mode is used for interference-prone elements (Mn, Cr, Ca), and KED-He mode is used for the remaining elements. Strategic optimization of quadrupole rejection parameters (RPa/RPq) achieved intra-day precision of 0.5–7.2 % (blood) and 1.6–5.5 % (urine), with inter-day variations ≤9.6 %, demonstrating robust performance for multi-element profiling across clinically relevant concentration ranges.

人体中元素的含量与人体健康有着重要的关系。然而，血液或尿液中不同元素的浓度差异很大，这给准确检测带来了挑战。本研究提出了一种双模式ICP-MS方法，集成了动能鉴别（KED）和动态反应池（DRC）技术，专门用于解决复杂生物基质中22种生理关键元素（Be， V, Cr, Mn, Fe, Ca, Mg, Ba, Co, Cd, Cu, Zn, As, Se, Ti, Sr, Ni, Mo, Sn, Sb, Tl, Pb）浓度（μg/L至g/L）跨越6个数量级的分析挑战。拒止参数（Rpq）调节低质量截止，拒止参数（Rpa）控制高质量截止。两者的优化扩展了低干扰动态范围。该方法的核心创新在于同步优化四极参数Rpa和Rpq，从而能够在超宽动态范围内同时抑制矩阵衍生的多原子干扰，同时保持线性探测器响应。该方法采用双模式分析方法：(1)在尿液分析中，KED-He模式适用于分析所有元素。(2)全血分析中，易干扰元素（Mn、Cr、Ca）采用dc - nh3模式，其余元素采用ed - he模式。四极排斥参数（RPa/RPq）策略优化实现了0.5-7.2%（血液）和1.6-5.5%（尿液）的日内精度，日内变化≤9.6%，在临床相关浓度范围内展示了多元素分析的稳健性能。

{"title":"Simultaneous multi-element determination in whole blood and urine via dual-mode ICP-MS","authors":"Yan Li , Jian-Yuan Zhong , Ya-qi Mo , Li-mei Qin , Michael Aschner , Yue-ming Jiang","doi":"10.1016/j.ab.2025.115941","DOIUrl":"10.1016/j.ab.2025.115941","url":null,"abstract":"<div><div>The content of elements in the body has a significant relationship with human health. However, the wide variation in the concentrations of different elements in blood or urine poses challenges for accurate detection. This study presents a dual-mode ICP-MS methodology integrating kinetic energy discrimination (KED) and dynamic reaction cell (DRC) technologies, specifically designed to address the analytical challenge of quantifying 22 physiologically critical elements (Be, V, Cr, Mn, Fe, Ca, Mg, Ba, Co, Cd, Cu, Zn, As, Se, Ti, Sr, Ni, Mo, Sn, Sb, Tl, Pb) spanning six orders of magnitude in concentration (μg/L to g/L) within complex biological matrices. Rejection parameter q (RPq) adjusts the low-mass cutoff, while rejection parameter a (RPa) controls the high-mass cutoff. Optimizing both extends the low-interference dynamic range. The method's core innovation lies in the synchronized optimization of quadrupole parameters RPa and RPq, which enables simultaneous suppression of matrix-derived polyatomic interferences while maintaining linear detector response across ultra-wide dynamic ranges. The method employs a dual-mode analysis approach: (1) In urine analysis, the KED-He mode is suitable for analyzing all elements. (2) In whole blood analysis, DRC-NH<sub>3</sub> mode is used for interference-prone elements (Mn, Cr, Ca), and KED-He mode is used for the remaining elements. Strategic optimization of quadrupole rejection parameters (RPa/RPq) achieved intra-day precision of 0.5–7.2 % (blood) and 1.6–5.5 % (urine), with inter-day variations ≤9.6 %, demonstrating robust performance for multi-element profiling across clinically relevant concentration ranges.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115941"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625291","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

Spectroscopic method for measuring activity of cis-aconitate decarboxylase, an important metabolic regulator of immune responses 测定免疫反应重要代谢调节因子顺式乌头酸脱羧酶活性的光谱方法

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-11 DOI: 10.1016/j.ab.2025.115944

Kevin Knowlan, Cody L. Hoop, Nadya I. Tarasova

Cis-aconitate decarboxylase (ACOD1) is a key enzyme converting cis-aconitate to itaconate, which has therapeutic potential for inflammatory diseases. Existing methods to measure ACOD1 activity and itaconate are often expensive and complex. We developed a novel, high-throughput spectrophotometric assay using the Fürth-Herrmann reaction. Our method quantifies ACOD1-catalyzed itaconate production by leveraging distinct absorbance ratios of cis-aconitate and itaconate at 386 nm and 440 nm. We optimized parameters, characterized human ACOD1 kinetics, and determined an IC₅₀ for citraconate consistent with previous reports. This simple, fast, and reliable assay, requiring only a UV–Vis spectrophotometer, will accelerate screening for ACOD1 modulators, speeding up therapeutic development.

顺式乌头酸脱羧酶（ACOD1）是将顺式乌头酸转化为衣康酸的关键酶，具有治疗炎性疾病的潜力。现有的测量ACOD1活性和衣康酸的方法通常既昂贵又复杂。我们利用 rth- herrmann反应开发了一种新的高通量分光光度法。我们的方法量化acod1催化衣康酸的生产，利用不同的吸光度比的顺- aconate和衣康酸在386 nm和440 nm。我们优化了参数，表征了人类ACOD1动力学，并确定了柠檬酸盐的IC50，与先前的报道一致。这种简单、快速、可靠的检测方法，只需要一个紫外可见分光光度计，将加速ACOD1调节剂的筛选，加快治疗开发。

引用次数: 0

Oxford Nanopore third generation sequencing for analysis of FMR1 5′UTR CGG repeat expansions 牛津纳米孔第三代测序分析fmr15 ' utr CGG重复扩增。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-11 DOI: 10.1016/j.ab.2025.115931

Xu Yang , Bowei Han , Jie Huang , Min Zhang , Shi Weng , Guojun Ouyang , Wanqing Han , Wenyu Wang , Li Zhang , Juanjuan Chen , Juan Du , Yingsong Wu , Xuexi Yang

Objective

Fragile X syndrome is mainly caused by the expansion of GC-rich cytosine-guanine-guanine (CGG) repeat in FMR1 5′UTR region, as well as rare gene point mutations or deletions in its open reading frame. Currently, third-generation long-read sequencing is a potential technology for simultaneously detecting CGG repeat expansions, point mutations, and deletions. However, a major challenge remains in obtaining the target long-fragment CGG repeat region with ultra-high GC content for sequencing.

Methods

We developed a novel approach combining long-fragment ultra-high GC polymerase chain reaction (PCR) amplification with Oxford Nanopore sequencing to detect the full spectrum of FMR1 5′UTR CGG repeat mutations. The method was validated using 10 standard cell line samples (males: n_normal = 1, n_intermediate = 1, n_pre-mutation = 1, and n_{full mutation} = 2; females: n_normal = 1, n_intermediate = 1, n_pre-mutation = 2, and n_{full mutation} = 1) and 53 retrospective clinical blood samples (males: n_normal = 7, n_pre-mutation = 3, n_{full mutation} = 15, and n_mosaic _mutaion = 2; females: n_normal = 9, n_pre-mutation = 13, and n_{full mutation} = 4).

Results

Our method demonstrated that the 100 % concordance with the triplet repeat-primed PCR and Southern blot analysis in genotyping 10 cell line samples and 53 clinical samples. Additionally, CGG repeat numbers showed strong correlation with reference mehods (male cell lines, n = 5, R² = 0.9996; female cell lines, n = 5, R² = 0.9972; clinical male samples, n = 26, R² = 1.0000; clinical female samples, n = 25, R² = 0.9854).

Conclusion

This study presents a simple and cost-effective strategy for preparing FMR1 5′UTR CGG repeat regions with long-fragment ultra-high GC content for third-generation sequencing. The approach could serve as a model for detecting other challenging disorders caused by short tandem repeat expansions, such as myotonic dystrophy and Huntington’ s disease.

目的：脆性X综合征主要由fmr15 ' utr区富含gc - cytosin -guanine-guanine （CGG）重复扩增以及其开放阅读框中罕见的基因点突变或缺失引起。目前，第三代长读测序是同时检测CGG重复扩增、点突变和缺失的潜在技术。然而，获得具有超高GC含量的目标长片段CGG重复区域进行测序仍然是一个主要的挑战。方法：我们开发了一种结合长片段超高GC聚合酶链反应（PCR）扩增和牛津纳米孔测序的新方法来检测fmr15 ' utr CGG重复突变的全谱。使用10个标准细胞系样本（雄性：非正常=1，中间=1，预突变=1，完全突变=2）验证方法；女性：异常=1，非中间=1,npre-mutation=2， nfull突变=1)，53份回顾性临床血样(男性：异常=7,npre-mutation=3， nfull突变=15，nmosaic突变=2；女性：nnormal=9, npremutation =13, nfull mutation=4)。结果：对10份细胞系样本和53份临床样本进行基因分型，结果与三联体重复引物PCR和Southern blot分析的一致性为100%。此外，CGG重复数与参考方法有很强的相关性(雄性细胞系，n=5, R2=0.9996；雌性细胞系，n=5, R2=0.9972；临床男性样本，n=26, R2=1.0000；临床女性样本，n=25, R2=0.9854)。结论：本研究为第三代测序提供了一种简单、经济的fmr15 ' utr CGG重复区长片段超高GC含量制备策略。这种方法可以作为一种模型，用于检测由短串联重复扩增引起的其他具有挑战性的疾病，如肌强直性营养不良和亨廷顿病。

{"title":"Oxford Nanopore third generation sequencing for analysis of FMR1 5′UTR CGG repeat expansions","authors":"Xu Yang , Bowei Han , Jie Huang , Min Zhang , Shi Weng , Guojun Ouyang , Wanqing Han , Wenyu Wang , Li Zhang , Juanjuan Chen , Juan Du , Yingsong Wu , Xuexi Yang","doi":"10.1016/j.ab.2025.115931","DOIUrl":"10.1016/j.ab.2025.115931","url":null,"abstract":"<div><h3>Objective</h3><div>Fragile X syndrome is mainly caused by the expansion of GC-rich cytosine-guanine-guanine (CGG) repeat in <em>FMR1</em> 5′UTR region, as well as rare gene point mutations or deletions in its open reading frame. Currently, third-generation long-read sequencing is a potential technology for simultaneously detecting CGG repeat expansions, point mutations, and deletions. However, a major challenge remains in obtaining the target long-fragment CGG repeat region with ultra-high GC content for sequencing.</div></div><div><h3>Methods</h3><div>We developed a novel approach combining long-fragment ultra-high GC polymerase chain reaction (PCR) amplification with Oxford Nanopore sequencing to detect the full spectrum of <em>FMR1</em> 5′UTR CGG repeat mutations. The method was validated using 10 standard cell line samples (males: n<sub>normal</sub> = 1, n<sub>intermediate</sub> = 1, n<sub>pre-mutation</sub> = 1, and n<sub>full mutation</sub> = 2; females: n<sub>normal</sub> = 1, n<sub>intermediate</sub> = 1, n<sub>pre-mutation</sub> = 2, and n<sub>full mutation</sub> = 1) and 53 retrospective clinical blood samples (males: n<sub>normal</sub> = 7, n<sub>pre-mutation</sub> = 3, n<sub>full mutation</sub> = 15, and n<sub>mosaic</sub> <sub>mutaion</sub> = 2; females: n<sub>normal</sub> = 9, n<sub>pre-mutation</sub> = 13, and n<sub>full mutation</sub> = 4).</div></div><div><h3>Results</h3><div>Our method demonstrated that the 100 % concordance with the triplet repeat-primed PCR and Southern blot analysis in genotyping 10 cell line samples and 53 clinical samples. Additionally, CGG repeat numbers showed strong correlation with reference mehods (male cell lines, n = 5, R<sup>2</sup> = 0.9996; female cell lines, n = 5, R<sup>2</sup> = 0.9972; clinical male samples, n = 26, R<sup>2</sup> = 1.0000; clinical female samples, n = 25, R<sup>2</sup> = 0.9854).</div></div><div><h3>Conclusion</h3><div>This study presents a simple and cost-effective strategy for preparing <em>FMR1</em> 5′UTR CGG repeat regions with long-fragment ultra-high GC content for third-generation sequencing. The approach could serve as a model for detecting other challenging disorders caused by short tandem repeat expansions, such as myotonic dystrophy and Huntington’ s disease.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115931"},"PeriodicalIF":2.6,"publicationDate":"2025-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144625289","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

Mass spectrometric identification of novel truncated α-synuclein species following optimized immunoprecipitation from human brain tissue 优化免疫沉淀后人脑组织中新型截断α-突触核蛋白的质谱鉴定。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-09 DOI: 10.1016/j.ab.2025.115942

Kim-Thanh Van , Fatimah Nabeebaccus , Foudil Lamari , Susana Boluda , François Fenaille , Nicolas Villain , François Becher

α-synuclein is a protein central to neurodegenerative diseases, and its functions are affected by multiple posttranslational modifications. Mass spectrometry is powerful for the characterization of α-synuclein forms but requires prior efficient immunoprecipitation conditions. In this study, we refined the immunoprecipitation of α-synuclein from human brain tissues by evaluating key parameters that influence recovery and specificity. We assessed the performance of tosyl-activated magnetic beads versus sheep antibody beads, identifying the optimal bead type for enhanced binding efficiency. Various elution conditions were rigorously tested to maximize protein yield. We also evaluated a range of antibodies specific to α-synuclein and delineated the effects of antibody amount and bead volume on the recovery of α-synuclein. The optimized immunoprecipitation protocol was effectively combined with high-resolution mass spectrometry for characterizing brain-derived α-synuclein from Parkinson's disease patients and controls. The assay identified a total of 38 N- or C-terminal truncated α-synuclein forms, including 22 novel sites. Our findings provide analytical tools for the reliable enrichment and characterization of α-synuclein from complex biological matrices, with potential applications in biomarker discovery and the investigation of pathogenic mechanisms underlying synucleinopathies.

α-突触核蛋白是神经退行性疾病的核心蛋白，其功能受到多种翻译后修饰的影响。质谱法是表征α-突触核蛋白形式的有力手段，但需要事先有效的免疫沉淀条件。在这项研究中，我们通过评估影响回收率和特异性的关键参数，完善了从人脑组织中提取α-突触核蛋白的免疫沉淀。我们评估了toyl活化磁珠与绵羊抗体磁珠的性能，确定了提高结合效率的最佳磁珠类型。严格测试了各种洗脱条件，以最大限度地提高蛋白质产量。我们还评估了一系列α-synuclein特异性抗体，并描绘了抗体数量和头体积对α-synuclein恢复的影响。优化后的免疫沉淀方案与高分辨率质谱法有效结合，用于表征帕金森病患者和对照组脑源性α-突触核蛋白。该分析共鉴定出38种N端或c端截断的α-突触核蛋白形式，包括22个新位点。我们的研究结果为从复杂的生物基质中可靠地富集和表征α-突触核蛋白提供了分析工具，在发现生物标志物和研究突触核蛋白病的致病机制方面具有潜在的应用价值。

{"title":"Mass spectrometric identification of novel truncated α-synuclein species following optimized immunoprecipitation from human brain tissue","authors":"Kim-Thanh Van , Fatimah Nabeebaccus , Foudil Lamari , Susana Boluda , François Fenaille , Nicolas Villain , François Becher","doi":"10.1016/j.ab.2025.115942","DOIUrl":"10.1016/j.ab.2025.115942","url":null,"abstract":"<div><div>α-synuclein is a protein central to neurodegenerative diseases, and its functions are affected by multiple posttranslational modifications. Mass spectrometry is powerful for the characterization of α-synuclein forms but requires prior efficient immunoprecipitation conditions. In this study, we refined the immunoprecipitation of α-synuclein from human brain tissues by evaluating key parameters that influence recovery and specificity. We assessed the performance of tosyl-activated magnetic beads versus sheep antibody beads, identifying the optimal bead type for enhanced binding efficiency. Various elution conditions were rigorously tested to maximize protein yield. We also evaluated a range of antibodies specific to α-synuclein and delineated the effects of antibody amount and bead volume on the recovery of α-synuclein. The optimized immunoprecipitation protocol was effectively combined with high-resolution mass spectrometry for characterizing brain-derived α-synuclein from Parkinson's disease patients and controls. The assay identified a total of 38 N- or C-terminal truncated α-synuclein forms, including 22 novel sites. Our findings provide analytical tools for the reliable enrichment and characterization of α-synuclein from complex biological matrices, with potential applications in biomarker discovery and the investigation of pathogenic mechanisms underlying synucleinopathies.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115942"},"PeriodicalIF":2.6,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144615792","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

Rapid and accurate detection of urinary iodine by single crystal silver iodide sensor 单晶碘化银传感器快速准确检测尿碘

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-09 DOI: 10.1016/j.ab.2025.115939

Jianmin Yang, Kai Tang, Shuyan Xiong, Liang Shi, Aidong Tang

Iodine deficiency or excess impacts thyroid health, necessitating accurate urinary iodine monitoring. However, existing detection methods face challenges including complicated sample handling, poor anti-interference ability, and the detection range does not cover the range of human urinary iodine concentration. Conventional iodide ion-selective electrode is based on polycrystalline membranes, which has high detection limit and is susceptible to chloride ion interference. It is also not suitable for accurate detection of human urinary iodine. In this study, we introduced a single-crystal AgI electrode for rapid and interference-free detection of urinary iodine. The dense (220) crystal plane of the single-crystal AgI electrode provided excellent selectivity and sensitivity, with a detection limit as low as 59 μg L⁻¹ and good linearity in the range of 100–1000 μg L⁻¹. This fully covers the optimal range of urinary iodine concentrations recommended by the World Health Organization (100–500 μg L⁻¹). Importantly, the single-crystal AgI electrode has excellent resistance to chloride interference, which is a significant improvement over conventional polycrystallaline iodide ion-selective electrodes. Our results show that the electrode has excellent precision, stability and accuracy without interference from common urine ions. This work addresses the limitations of existing methods and provides a rapid, inexpensive and reliable method for urinary iodine testing.

碘缺乏或过量影响甲状腺健康，需要准确的尿碘监测。然而，现有的检测方法面临着样品处理复杂、抗干扰能力差、检测范围不能覆盖人体尿碘浓度范围等挑战。传统的碘离子选择电极是基于多晶膜的，检出限高，易受氯离子干扰。它也不适合用于人体尿碘的准确检测。在这项研究中，我们介绍了一种用于快速无干扰检测尿碘的单晶AgI电极。单晶AgI电极的致密（220）晶面具有良好的选择性和灵敏度，检测限低至59 μg L−1，在100-1000 μg L−1范围内具有良好的线性关系。这完全涵盖了世界卫生组织推荐的尿碘浓度的最佳范围（100-500 μg L−1）。重要的是，单晶AgI电极具有优异的抗氯化物干扰能力，这是传统多晶碘离子选择电极的显著改进。结果表明，该电极具有良好的精密度、稳定性和准确度，不受常见尿液离子的干扰。这项工作解决了现有方法的局限性，提供了一种快速、廉价、可靠的尿碘检测方法。

{"title":"Rapid and accurate detection of urinary iodine by single crystal silver iodide sensor","authors":"Jianmin Yang, Kai Tang, Shuyan Xiong, Liang Shi, Aidong Tang","doi":"10.1016/j.ab.2025.115939","DOIUrl":"10.1016/j.ab.2025.115939","url":null,"abstract":"<div><div>Iodine deficiency or excess impacts thyroid health, necessitating accurate urinary iodine monitoring. However, existing detection methods face challenges including complicated sample handling, poor anti-interference ability, and the detection range does not cover the range of human urinary iodine concentration. Conventional iodide ion-selective electrode is based on polycrystalline membranes, which has high detection limit and is susceptible to chloride ion interference. It is also not suitable for accurate detection of human urinary iodine. In this study, we introduced a single-crystal AgI electrode for rapid and interference-free detection of urinary iodine. The dense (220) crystal plane of the single-crystal AgI electrode provided excellent selectivity and sensitivity, with a detection limit as low as 59 μg L<sup>−1</sup> and good linearity in the range of 100–1000 μg L<sup>−1</sup>. This fully covers the optimal range of urinary iodine concentrations recommended by the World Health Organization (100–500 μg L<sup>−1</sup>). Importantly, the single-crystal AgI electrode has excellent resistance to chloride interference, which is a significant improvement over conventional polycrystallaline iodide ion-selective electrodes. Our results show that the electrode has excellent precision, stability and accuracy without interference from common urine ions. This work addresses the limitations of existing methods and provides a rapid, inexpensive and reliable method for urinary iodine testing.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115939"},"PeriodicalIF":2.6,"publicationDate":"2025-07-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144604946","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

Multi-protein-triggered, aptamer-tethered auto-cycling proximity recorder in formalin-fixed paraffin-embedded tissues 福尔马林固定石蜡包埋组织中多蛋白触发、适配体系住的自动循环接近记录仪

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-07-08 DOI: 10.1016/j.ab.2025.115940

Xiaofang Zheng , Daikun Zheng , Linfeng Zheng , Peng Wang , Yong Liu , Xixi You , Xiaoling Zheng , Xinyuan Cao , Jiao Luo , Wansong Chen , Guoli Li , Ruizi Peng

Unraveling tumor-associated membrane proteins' spatial proximity holds a significance for clinically relevant multiple biomarker detection. Herein, based on aptamer binding to target proteins in clinical formalin-fixed paraffin-embedded (FFPE) tissue samples, we developed aptamer-tethered auto-cycling proximity recording (APR) probes as a proof-of-concept methodology for biosensing membrane proteins' spatial proximity. This APR enables continuous and repetitive recording of spatial neighboring pairs of DNA probes in FFPE breast cancer tissue samples in situ, without altering the probes themselves. Finally, this aptamer-based auto-cycling proximity recorder is able to identify spatial proximity of three membrane proteins in FFPE breast cancer tissues. Our research establishes a foundation for proximity-driven biosensing platform and provides a perspective for exploration in aptamer-based bioimaging in clinical tissue samples.

揭示肿瘤相关膜蛋白的空间接近性对临床相关的多种生物标志物检测具有重要意义。在此，基于适体与临床福尔马林固定石蜡包埋（FFPE）组织样本中靶蛋白的结合，我们开发了适体系住的自动循环接近记录（APR）探针，作为生物传感膜蛋白空间接近的概念验证方法。该APR能够在原位连续重复记录FFPE乳腺癌组织样本中的空间相邻DNA探针对，而不改变探针本身。最后，这种基于适配体的自动循环接近记录器能够识别FFPE乳腺癌组织中三种膜蛋白的空间接近性。我们的研究为接近驱动的生物传感平台奠定了基础，并为临床组织样本中基于适配体的生物成像的探索提供了前景。

{"title":"Multi-protein-triggered, aptamer-tethered auto-cycling proximity recorder in formalin-fixed paraffin-embedded tissues","authors":"Xiaofang Zheng , Daikun Zheng , Linfeng Zheng , Peng Wang , Yong Liu , Xixi You , Xiaoling Zheng , Xinyuan Cao , Jiao Luo , Wansong Chen , Guoli Li , Ruizi Peng","doi":"10.1016/j.ab.2025.115940","DOIUrl":"10.1016/j.ab.2025.115940","url":null,"abstract":"<div><div>Unraveling tumor-associated membrane proteins' spatial proximity holds a significance for clinically relevant multiple biomarker detection. Herein, based on aptamer binding to target proteins in clinical formalin-fixed paraffin-embedded (FFPE) tissue samples, we developed aptamer-tethered auto-cycling proximity recording (APR) probes as a proof-of-concept methodology for biosensing membrane proteins' spatial proximity. This APR enables continuous and repetitive recording of spatial neighboring pairs of DNA probes in FFPE breast cancer tissue samples in situ, without altering the probes themselves. Finally, this aptamer-based auto-cycling proximity recorder is able to identify spatial proximity of three membrane proteins in FFPE breast cancer tissues. Our research establishes a foundation for proximity-driven biosensing platform and provides a perspective for exploration in aptamer-based bioimaging in clinical tissue samples.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"706 ","pages":"Article 115940"},"PeriodicalIF":2.6,"publicationDate":"2025-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144605504","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

A nucleic acid-based strategy for highly specific discrimination between mutant and wild-type sequences 基于核酸的突变型和野生型序列高度特异性区分策略。

IF 2.6 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-06-30 DOI: 10.1016/j.ab.2025.115930

Xueqiang Wu , Xuyang Pu , Yinmei Zhang , Heming Wu , Zhikang Yu , Wei He , Yibiao Chen

This study presents a novel Terminal Self-Competitive Nucleic Acid Probe (TSCP) for rapid, precise detection of mutant and wild-type gene sequences. The TSCP probe employs overlapping binding domains and inhibition strands, enabling highly specific competitive hybridization. Combined with hybridization chain reaction (HCR) for signal amplification, the method achieves nanomolar sensitivity without requiring proteases, temperature control, or complex instrumentation. Experimental validation in blood samples demonstrated distinct fluorescent signals for mutant and wild-type sequences, allowing accurate target differentiation. The probe is cost-effective and scalable, as it only requires nucleic acid synthesis for new mutations, ensuring rapid adaptation to emerging variants. This straightforward, versatile approach facilitates simultaneous detection of genetic variations, making it highly suitable for molecular diagnostics and genetic research. Its high specificity, rapid response, and low-cost production underscore its potential as a practical tool for advancing mutation detection in clinical and research settings. By eliminating the need for specialized equipment and enabling quick deployment, the TSCP-based method offers a widely accessible solution for identifying genetic mutations, enhancing both diagnostic accuracy and research efficiency.

本研究提出了一种新型的末端自竞争核酸探针（TSCP），用于快速、精确地检测突变型和野生型基因序列。TSCP探针采用重叠结合域和抑制链，实现高度特异性的竞争性杂交。结合杂交链反应（HCR）的信号放大，该方法达到纳摩尔灵敏度，不需要蛋白酶，温度控制，或复杂的仪器。血液样本的实验验证表明突变型和野生型序列的荧光信号不同，允许准确的目标分化。该探针具有成本效益和可扩展性，因为它只需要为新的突变合成核酸，确保快速适应新出现的变异。这种简单，通用的方法有助于同时检测遗传变异，使其非常适合分子诊断和遗传研究。它的高特异性、快速反应和低成本生产强调了它作为在临床和研究环境中推进突变检测的实用工具的潜力。通过消除对专用设备的需求和实现快速部署，基于tscp的方法为识别基因突变提供了一种广泛可用的解决方案，提高了诊断准确性和研究效率。

{"title":"A nucleic acid-based strategy for highly specific discrimination between mutant and wild-type sequences","authors":"Xueqiang Wu , Xuyang Pu , Yinmei Zhang , Heming Wu , Zhikang Yu , Wei He , Yibiao Chen","doi":"10.1016/j.ab.2025.115930","DOIUrl":"10.1016/j.ab.2025.115930","url":null,"abstract":"<div><div>This study presents a novel Terminal Self-Competitive Nucleic Acid Probe (TSCP) for rapid, precise detection of mutant and wild-type gene sequences. The TSCP probe employs overlapping binding domains and inhibition strands, enabling highly specific competitive hybridization. Combined with hybridization chain reaction (HCR) for signal amplification, the method achieves nanomolar sensitivity without requiring proteases, temperature control, or complex instrumentation. Experimental validation in blood samples demonstrated distinct fluorescent signals for mutant and wild-type sequences, allowing accurate target differentiation. The probe is cost-effective and scalable, as it only requires nucleic acid synthesis for new mutations, ensuring rapid adaptation to emerging variants. This straightforward, versatile approach facilitates simultaneous detection of genetic variations, making it highly suitable for molecular diagnostics and genetic research. Its high specificity, rapid response, and low-cost production underscore its potential as a practical tool for advancing mutation detection in clinical and research settings. By eliminating the need for specialized equipment and enabling quick deployment, the TSCP-based method offers a widely accessible solution for identifying genetic mutations, enhancing both diagnostic accuracy and research efficiency.</div></div>","PeriodicalId":7830,"journal":{"name":"Analytical biochemistry","volume":"705 ","pages":"Article 115930"},"PeriodicalIF":2.6,"publicationDate":"2025-06-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144551708","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