Analytical biochemistry最新文献_第4页

A high-throughout PCR test strip method for the rapid identification of four placentas 一种快速鉴定四种胎盘的高通量PCR试纸条方法。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-31 DOI: 10.1016/j.ab.2025.116006

Guangxin Yuan , Meng Xia , Ya Li , Honglei Tian , Jianyu Zhu , Yanshuang Wang

Deer placenta and Placenta Hominis are valued traditional Chinese medicines often adulterated with cheaper pig or cow placenta. Current identification methods frequently lack the throughput, speed, or simplicity for effective field monitoring. This study aimed to develop a high-throughput, rapid method to simultaneously identify deer, human, pig, and cow placenta. The approach involved screening a rapid genomic DNA extraction protocol and designing specific primers for the mitochondrial cytochrome b gene of each species, with primers labeled with FAM and Biotin. Following the optimization of PCR amplification conditions, results were visually detected using immunocolloidal gold test strips. The method was rigorously evaluated for its specificity, reproducibility, sensitivity, and stability. The results showed that the extracted DNA was of satisfactory concentration, purity, and integrity. Under optimized conditions (59 °C annealing temperature, 20 cycles), authentic samples for all four placenta types produced two distinct red bands on the test strips, while adulterated and blank controls showed only one control band. Agarose gel electrophoresis confirmed specific amplification for each target with no cross-reactivity. The method demonstrated strong specificity, excellent reproducibility and stability, and a high sensitivity of 0.01 ng μL⁻¹, surpassing conventional electrophoresis. In conclusion, this PCR-based test strip method enables the visual, simultaneous authentication of the four placenta types in a single test, making it highly suitable for on-site rapid monitoring and quality control.

鹿胎素和人胎素是很有价值的中药，经常掺入便宜的猪或牛胎素。目前的识别方法往往缺乏吞吐量，速度，或简单有效的现场监测。本研究旨在建立高通量、快速同时鉴定鹿、人、猪和牛胎盘的方法。该方法包括筛选快速基因组DNA提取方案，并为每个物种的线粒体细胞色素b基因设计特异性引物，引物标记为FAM和生物素。优化PCR扩增条件后，用免疫胶体金试纸目测结果。对该方法的特异性、重复性、敏感性和稳定性进行了严格的评价。结果表明，提取的DNA具有满意的浓度、纯度和完整性。在优化条件下（59°C退火温度，20个循环），所有四种胎盘类型的真实样本在试纸上产生两条明显的红色条带，而掺杂和空白对照仅显示一条控制带。琼脂糖凝胶电泳证实了每个目标的特异性扩增，无交叉反应性。该方法特异性强，重现性和稳定性好，灵敏度为0.01 ng·μL-1，优于常规电泳。综上所述，基于pcr的试纸方法可在一次检测中同时对四种胎盘类型进行可视化鉴定，非常适合现场快速监测和质量控制。

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引用次数: 0

Advancing a sensitive method for measuring mitochondrial ATP production in small muscle biopsy samples 提出一种在小肌肉活检样本中测量线粒体ATP生成的灵敏方法。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-30 DOI: 10.1016/j.ab.2025.116004

Rolf Wibom , David Alsina , Karin Naess , Martin Engvall , Christoph Freyer , Anna Wedell , Anna Wredenberg

We present an optimised luminometric method for measuring muscle mitochondrial ATP production rate (MAPR), adapted to a 96-well microplate format. The enhanced assay enables quantification of ATP production from 12 or more substrate combinations within 15 min, using only 10 μL of isolated mitochondria. The method demonstrates high accuracy and precision, with a validated measurement range of 0.3–70 nmol/min/L. To support clinical interpretation, a reference dataset was established from 92 individuals aged seven months to 79 years. All these individuals were referred for muscle biopsy but were subsequently deemed unlikely to have a mitochondrial disorder following comprehensive clinical evaluation. An overview of the current version of our assays for oxidative phosphorylation (OXPHOS) enzymes is also provided.

As proof of concept, we present three patients carrying pathogenic variants in mitochondrial DNA (ATP6 and MT-TL1) and the nuclear PDHA1 gene. All exhibited decreased MAPR with one or more substrates, along with additional clinical, biochemical, and morphological features consistent with mitochondrial disease.

Furthermore, we illustrate the age-dependent development of MAPR in muscle across the human lifespan, demonstrating a 60–80 % higher maximal capacity for oxidative ATP production in adults compared with young children. In contrast, MAPR supported by fatty acid-derived substrates remains unchanged over the same period.

In conclusion, the improved MAPR assay offers a robust and efficient tool for assessing mitochondrial function in both clinical diagnostics and research. Its high-throughput format and reliable performance make it particularly well-suited for the investigation of suspected mitochondrial disorders.

我们提出了一种优化的光度法来测量肌肉线粒体ATP产生率（MAPR），适用于96孔微孔板格式。增强型分析可以在15分钟内定量12种或更多底物组合的ATP产量，仅使用10 μL的分离线粒体。该方法具有较高的准确度和精密度，测量范围为0.3 ~ 70 nmol/min/L。为了支持临床解释，我们建立了92个年龄在7个月至79岁之间的个体的参考数据集。所有这些人都进行了肌肉活检，但在综合临床评估后，随后被认为不太可能患有线粒体疾病。还提供了我们氧化磷酸化（OXPHOS）酶测定的当前版本的概述。为了证明这一概念，我们提出了三名携带线粒体DNA （ATP6和MT-TL1）和核PDHA1基因致病变异的患者。所有患者都表现出一种或多种底物的MAPR下降，以及与线粒体疾病一致的其他临床、生化和形态学特征。此外，我们说明了在整个人类生命周期中，肌肉中MAPR的年龄依赖性发展，证明了与幼儿相比，成人氧化ATP生产的最大能力高60-80%。相比之下，由脂肪酸衍生底物支持的MAPR在同一时期保持不变。总之，改进的MAPR检测为临床诊断和研究中评估线粒体功能提供了一个强大而有效的工具。其高通量格式和可靠的性能使其特别适合于可疑线粒体疾病的调查。

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引用次数: 0

A novel sandwich hybridization assay method and probe sets for cyanobacterial harmful algal bloom detection 一种新的蓝细菌有害藻华检测夹心杂交检测方法及探针装置

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-29 DOI: 10.1016/j.ab.2025.116002

Katelyn M. Brown , Dianne I. Greenfield , George S. Bullerjahn

A novel, rapid (2.5 h) sandwich hybridization assay (SHA) method for the detection of harmful cyanobacterial genera has been developed based on oligonucleotide hybridization to genus-specific sequences within the 16S ribosomal RNA. Unlike previously-developed 96-well plate SHA formats, this modification does not involve a robotic processor to maneuver reagents across the plate. Rather, it requires manual pipetting each reagent sequentially into the same well and washing the 96-well plate between each addition, thereby increasing sample throughput. Another key difference is the colorimetric reporter molecule. Yet, it enables specific detection of Planktothrix, Raphidiopsis and the Anabaena-Dolichospermum-Aphanizomenon (ADA) clade of the Nostocales. As these are the most common planktonic bloom-forming cyanobacteria, the method can be used to assess potential threats posed by these taxa, specifically cyanotoxins that are associated with particular bloom-forming taxa. The assays detected target groups and standard curves were created from cultured cyanobacteria biomass. Here, the SHA method, calibration, and the field performance of the assay on cyanobacterial bloom biomass is described. Linear standard curves were established for each assay, with a Planktothrix and Raphidiopsis quantification range of 10–500 mm³ L⁻¹ 100 μL homogenate⁻¹ and a range of 50–500 mm³ L⁻¹ 100 μL homogenate⁻¹ for the ADA clade assay. In environmental samples from three Ohio waterbodies, the assays detected the target genus in a mixed cyanobacterial community. This new SHA protocol will be highly beneficial to both management and research communities because it considerably broadens the capability to rapidly detect multiple cyanobacterial genera that cause persistent toxic blooms.

建立了一种新的快速（2.5 h）夹心杂交法（SHA）检测有害蓝藻属的方法，该方法基于16S核糖体RNA中属特异性序列的寡核苷酸杂交。与以前开发的96孔板SHA格式不同，这种修改不涉及机器人处理器来操纵试剂穿过板。相反，它需要手动将每种试剂依次移液到同一孔中，并在每次添加之间清洗96孔板，从而提高样品吞吐量。另一个关键的区别是比色报告分子。然而，它可以特异性检测浮游thrix， Raphidiopsis和Nostocales的anabaena - dolichosperum - aphanizomena （ADA）分支。由于这些是最常见的浮游藻华形成蓝藻，该方法可用于评估这些分类群构成的潜在威胁，特别是与特定藻华形成分类群相关的蓝藻毒素。实验检测的目标群体和标准曲线是由培养的蓝藻生物量创建的。在这里，SHA方法，校准和现场性能的分析蓝藻华生物量描述。每项检测均建立线性标准曲线，其中浮游thrix和Raphidiopsis的定量范围为10-500 mm3 L−1 100 μL匀浆−1，ADA分支检测的定量范围为50-500 mm3 L−1 100 μL匀浆−1。在俄亥俄州三个水体的环境样本中，检测到混合蓝藻群落的目标属。这种新的SHA协议将对管理和研究界都非常有益，因为它大大扩大了快速检测导致持续有毒繁殖的多种蓝藻属的能力。

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引用次数: 0

Direct detection of a target nucleic acid on a surface by spraying DNA-nano-tweezers-based biosensing molecules 通过喷射基于dna纳米镊子的生物传感分子直接检测表面上的目标核酸。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-28 DOI: 10.1016/j.ab.2025.116003

Hisakage Funabashi, Hiroya Hatano, Hirotaka Nakamura, Reiji Shigematsu, Akio Kuroda

We propose a new concept to detect a target molecule on a solid surface directly by spraying biosensing molecules. First, a filter paper as a model of a solid surface was spotted with a target nucleic acid. Then, the biosensing molecule, a DNA-nano-tweezers-structure that exhibits a peroxidase activity recognizing the target, was sprayed with hemin as a cofactor, followed by a second spray containing luminol as a substrate for a light-glowing signal. Finally, an image was recorded using a smartphone. The images revealed that only the places where the corresponding targets were spotted glowed a blue light.

我们提出了一种通过喷射生物传感分子直接检测固体表面目标分子的新概念。首先，将滤纸作为固体表面的模型，并在上面标记目标核酸。然后，生物传感分子，一种dna纳米镊子结构，表现出识别目标的过氧化物酶活性，被喷上血红蛋白作为辅助因子，随后第二次喷雾含有发光氨作为发光信号的底物。最后，用智能手机拍了一张照片。图像显示，只有相应目标被发现的地方才会发出蓝光。

引用次数: 0

3DCOOR-Kace: A 3-d spatial coordinates representation method for lysine acetylation site identification 一种用于赖氨酸乙酰化位点识别的三维空间坐标表示方法。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-16 DOI: 10.1016/j.ab.2025.115994

Lichao Zhang , Xue Wang , Liang Kong

Lysine acetylation (Kace) is an important post-translational modification. Although structure information has been proven to be a key for improving model effectiveness, it is difficult to obtain in bulk due to experiments limitations. In this study, we propose a spatial coordinates representation using a 2-order tensor according to defined property sequence to address the existing limitations and depicting amino acid position in 3-d space. Based on the proposed coordinates, we construct optimal complex networks to extract network-derived features. Compared to existing network construction methods, protein contact networks (PCN), the features achieve superior performance, demonstrating the proposed spatial coordinates could effectively capture biological global information. Meanwhile, we proposed a computational model, named 3DCOOR-Kace, by fusing sequence and structure information based on DenseNet and Squeeze-and-Excitation layer. The 3DCOOR-Kace achieved satisfactory MCC with 0.7358. Compared with MusiteDeep and TransPTM by the independent testing set, the MCC is 0.4261 higher than MusiteDeep and 0.1660 higher than TransPTM, which demonstrates 3DCOOR-Kace are effective for integrating structure and sequence information for improving Kace site identification. Instead of doing biological experiments, the 3-d spatial coordinates representation could give sites positions directly, which could address the experiments limitation and be convenient for computational methods and biological function research.

赖氨酸乙酰化是一种重要的翻译后修饰。虽然结构信息已被证明是提高模型有效性的关键，但由于实验的限制，很难大量获得。在本研究中，我们根据定义的性质序列，提出了一种使用二阶张量的空间坐标表示，以解决现有的局限性，并描绘了氨基酸在三维空间中的位置。基于所提出的坐标，我们构建了最优的复杂网络来提取网络衍生的特征。与现有的蛋白质接触网络（protein contact networks， PCN）网络构建方法相比，该特征具有更优越的性能，表明所提出的空间坐标能够有效捕获生物全局信息。同时，我们提出了基于DenseNet和Squeeze-and-Excitation层融合序列和结构信息的计算模型3DCOOR-Kace。3DCOOR-Kace的MCC达到了令人满意的0.7358。与独立测试集的MusiteDeep和TransPTM相比，MCC比MusiteDeep高0.4261，比TransPTM高0.1660，说明3DCOOR-Kace能够有效整合结构和序列信息，提高Kace位点的识别。三维空间坐标表示代替了生物实验，直接给出了位点的位置，解决了实验的局限性，方便了计算方法和生物功能研究。

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引用次数: 0

Electrochemical strategies for decoding serotonin using advanced carbonaceous and 5th generation materials: A comprehensive review 利用先进的碳质和第五代材料解码血清素的电化学策略：综合综述。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-15 DOI: 10.1016/j.ab.2025.115990

Rimpa Mondal , Sk Faruque Ahmed , Nillohit Mukherjee

Carbonaceous materials are placed in the category of advanced and future materials for their very unique physicochemical attributes like tuneable electronic, electrical and optical properties associated with good chemical reactivity, biocompatibility and high surface to volume ratio that make them good candidates for various device applications including biosensors. Recently, such carbonaceous materials and their composites are being widely used as the electrode materials for enzyme-free electrochemical detection of various bioanalytes. Among the bioanalytes, the neurotransmitters are known to play crucial roles in maintaining proper balance in the human nervous systems. However, there are many challenges like selectivity, sensitivity and rapidity; that are normally faced during the detection of such neurotransmitters using the conventional enzymatic way. Electrochemical biosensors made up of advanced carbonaceous materials could be a single-key answer to all such challenges. Serotonin, a catecholamine group neurotransmitter, also known as the “happy hormone”, is known to control human emotions. So, real-time monitoring of its level in human serum carries immense importance, however; it comes with various challenges. This review article covers the developments of advanced carbonaceous, as well as, the 5th generation materials like Mxenes and related composites in the enzyme-free electrochemical sensing of serotonin since the inception of such materials in electrochemical biosensing. A detailed literature review exhibited that, impressive results, like very low limit of detection (LoD) of 8.0 nM could be achieved using multiwalled carbon nanotube (MWCNT) based electrodes, whereas, nitrogen incorporated graphene quantum dot (N-GQD) modified electrodes can help to reach a notably high sensitivity of 24.0 μAμM⁻¹.cm⁻². Again, carbon spheres (CS) have been established as a good candidate to bring a balance between LoD (4.0 nM) and sensitivity (5.5 μAμM⁻¹.cm⁻²) in combination with metal oxide electrode.

碳质材料因其独特的物理化学特性而被列入先进和未来材料的范畴，如可调谐的电子、电气和光学特性，以及良好的化学反应性、生物相容性和高表面体积比，使其成为包括生物传感器在内的各种设备应用的良好候选者。近年来，这类碳质材料及其复合材料被广泛用作各种生物分析物无酶电化学检测的电极材料。在生物分析物中，神经递质在维持人类神经系统的适当平衡中起着至关重要的作用。然而，有许多挑战，如选择性，灵敏度和速度；在使用传统的酶法检测这些神经递质时，通常会遇到这些问题。由先进的碳质材料组成的电化学生物传感器可能是解决所有这些挑战的一个关键答案。血清素是一种儿茶酚胺类神经递质，也被称为“快乐激素”，它控制着人类的情绪。因此，实时监测其在人类血清中的水平具有极其重要的意义；它伴随着各种各样的挑战。本文综述了先进的碳质材料及第五代材料Mxenes及其复合材料在5 -羟色胺无酶电化学传感领域的研究进展。详细的文献综述表明，使用多壁碳纳米管（MWCNT）电极可以实现8.0 nM的极低检测限（LoD），而氮掺杂石墨烯量子点（N-GQD）修饰电极可以达到24.0 μAμ m- 1 cm-2的高灵敏度。同样，碳球（CS）已被确定为在LoD （4.0 nM）和灵敏度（5.5 μAμM-1）之间取得平衡的良好候选者。Cm-2)与金属氧化物电极结合。

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引用次数: 0

Exploring biosensors: Distinctive features and emerging applications 探索生物传感器：独特的特点和新兴的应用。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-14 DOI: 10.1016/j.ab.2025.115993

Shweta Mishra , Ashlesha J. Chauhan , Jayaveersinh Mahida

Biosensors have become essential analytical tools that integrate biological recognition elements with physical transducers to detect specific analytes with remarkable sensitivity. This review delves into the applications of biosensors in healthcare, environmental monitoring, and other fields. Notable advancements include nanotechnology-enhanced biosensors, wearable devices, and point-of-care systems, all of which are characterized by miniaturization and multiplexing capabilities. In healthcare, biosensors facilitate disease diagnosis and continuous monitoring, whereas in environmental applications, they aid in pollutant detection. In analytical technology, biosensors merge biological recognition with physical transduction, offering unparalleled sensitivity in biological and environmental systems. This review examines the characteristics and applications of biosensors in various fields. They support disease diagnosis and health surveillance in healthcare and enable real-time pollutant monitoring for environmental protection purposes. Recent advances have incorporated nanotechnology to enhance performance through miniaturization and label-free detection. Wearable biosensors have revolutionized point-of-care diagnostics by providing real-time health data outside clinical settings. The integration of artificial intelligence and microfluidics has led to the development of more adaptive biosensing platforms. Despite challenges such as biological component stability and nonspecific binding, innovations in materials science offer potential solutions. The convergence of expertise positions biosensors as vital instruments for improving health outcomes and ensuring environmental sustainability, thereby transforming diagnostics and ecological preservation.

生物传感器已成为必不可少的分析工具，它将生物识别元件与物理传感器结合起来，以显着的灵敏度检测特定的分析物。本文综述了生物传感器在医疗保健、环境监测等领域的应用。值得注意的进步包括纳米技术增强的生物传感器、可穿戴设备和护理点系统，所有这些都以小型化和多路复用能力为特征。在医疗保健中，生物传感器有助于疾病诊断和持续监测，而在环境应用中，它们有助于污染物检测。在分析技术中，生物传感器将生物识别与物理转导相结合，在生物和环境系统中提供无与伦比的灵敏度。本文综述了生物传感器的特点及其在各个领域的应用。它们支持医疗保健中的疾病诊断和健康监测，并为环境保护目的实现实时污染物监测。最近的进展已纳入纳米技术，以提高性能，通过小型化和无标签检测。可穿戴生物传感器通过在临床环境之外提供实时健康数据，彻底改变了护理点诊断。人工智能和微流控技术的结合促进了自适应生物传感平台的发展。尽管面临生物成分稳定性和非特异性结合等挑战，材料科学的创新提供了潜在的解决方案。专业知识的汇集使生物传感器成为改善健康结果和确保环境可持续性的重要工具，从而改变诊断和生态保护。

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引用次数: 0

Revisiting phenylketonuria: Do high brain glycine levels caused by chronic hyperphenylalanemia contribute to brain dysfunction by modulating D-serine levels and NMDA receptor activity? 重新审视苯丙酮尿症：慢性高苯贫血引起的高脑甘氨酸水平是否通过调节d -丝氨酸水平和NMDA受体活性而导致脑功能障碍？

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-14 DOI: 10.1016/j.ab.2025.115992

Gerald A. Dienel

Phenylketonuria (PKU) is an inborn error of metabolism owing to deficits in phenylalanine hydroxylase (PAH) activity. PKU children acquire irreversible brain damage if newborns are not identified and treated with a phenylalanine-restricted diet. In spite of decades of research, the mechanisms underlying PKU brain dysfunction are not adequately understood. Competition of phenylalanine with large neutral amino acids (LNAAs) for carrier-mediated uptake into brain, causing lower brain LNAA levels and reduced neurotransmitter synthesis from tyrosine and tryptophan, is a long-favored mechanism for brain dysfunction. Here, glycine is hypothesized to contribute to phenylalanine-evoked brain disorders. All PKU animal models exhibit elevated brain glycine levels similar to mouse models of nonketotic hyperglycinemia. Glycine is synthesized from l-serine; it is a co-agonist of N-methyl-d-aspartate receptors (NMDARs) and an inhibitory neurotransmitter. l-Serine is synthesized from glucose in astrocytes, exported to neurons, and converted by serine racemase to d-serine, an NMDAR co-agonist. Increased glycine level enhances its inhibition of serine racemase and reduces levels of d-serine. l-Serine-glycine-d-serine interactions can be linked to PAH deficits because elevated brain phenylalanine concentration causes its metabolism by minor pathways to generate phenyllactate. If phenyllactate and l-serine synthesis are coupled via transaminase and redox reactions, the stoichiometry is 1:1. These findings support the following hypothesis: (i) phenylalanine disrupts glycine and d-serine homeostasis during brain maturation, irreversibly altering neuronal development and circuit formation, and (ii) high glycine and low d-serine levels in PKU adults contribute to cognitive and behavioral dysfunction. Suggested new directions for future studies of PKU focus on glycine neurotoxicity.

苯丙酮尿症（PKU）是由于苯丙氨酸羟化酶（PAH）活性不足而导致的先天性代谢错误。如果新生儿未被识别并以苯丙氨酸限制饮食治疗，PKU儿童将获得不可逆的脑损伤。尽管经过数十年的研究，PKU脑功能障碍的机制仍未得到充分的了解。苯丙氨酸与大中性氨基酸（LNAAs）在载体介导的脑摄取中竞争，导致脑LNAA水平降低，酪氨酸和色氨酸合成神经递质减少，这是脑功能障碍的一个长期被认为的机制。在这里，甘氨酸被假设有助于苯丙氨酸诱发的脑部疾病。所有PKU动物模型均表现出脑甘氨酸水平升高，与非酮症型高甘氨酸血症小鼠模型相似。甘氨酸是由l -丝氨酸合成的；它是n -甲基- d -天冬氨酸受体（NMDARs）的协同激动剂和一种抑制性神经递质。l -丝氨酸由星形胶质细胞中的葡萄糖合成，输出到神经元，并通过丝氨酸消旋酶转化为d -丝氨酸，一种NMDAR共激动剂。甘氨酸水平的升高增强了其对丝氨酸消旋酶的抑制作用，降低了d -丝氨酸水平。l -丝氨酸-甘氨酸- d -丝氨酸相互作用可能与多环芳烃缺陷有关，因为脑苯丙氨酸浓度升高导致其通过次要途径代谢产生苯乳酸。如果苯乳酸和l -丝氨酸的合成通过转氨酶和氧化还原反应偶联，则化学计量比为1:1。这些发现支持以下假设：(i)苯丙氨酸破坏脑成熟过程中甘氨酸和d -丝氨酸的稳态，不可逆转地改变神经元发育和电路形成；（ii） PKU成人中高甘氨酸和低d -丝氨酸水平导致认知和行为功能障碍。提出甘氨酸神经毒性是今后研究的新方向。

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引用次数: 0

A sensitive ELISA made with recombinant antibodies for cynomolgus IL-8 in serum ELISA检测食蟹蟹血清中IL-8的重组抗体

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-13 DOI: 10.1016/j.ab.2025.115991

Leting Yang , Xia Han , Xiao Zhu , Yuhao Wang , Junjiu Xu , Yan Chen , Qiuping Qin , Likun Gong

Accurate measurement of interleukin-8 (IL-8) in non-human primates is crucial for research into inflammation and tumors; however, sensitive enzyme-linked immunosorbent assays (ELISAs) for non-clinical studies remain limited. We aimed to develop and validate a sensitive method for IL-8 quantification in cynomolgus monkey serum. Using two recombinant IL-8-specific antibodies and a surrogate matrix, a sandwich-type ELISA was developed, involving sequential incubations with samples, biotinylated antibody, and horseradish peroxidase-labeled Streptavidin (SA-HRP). The assay was validated against regulatory guidelines, achieving a lower limit of quantification of 4.69 pg/mL as well as good accuracy and precision. The assay was neither affected by serum matrix nor hemolysis, nor did it cross-react with other cytokines. Furthermore, the assay showed dilution linearity, no hook effect, and good recovery and parallelism. Finally, IL-8 in monkey serum exhibited sufficient stability for analysis. After validation, the assay was applied to 19 healthy cynomolgus monkey serum samples, and the results correlated well with those from a commercial assay. In conclusion, the developed ELISA is a sensitive, accurate, and reliable method for IL-8 quantification in cynomolgus monkey serum, suitable for non-clinical studies in cynomolgus monkeys.

非人类灵长类动物白细胞介素-8 （IL-8）的精确测量对炎症和肿瘤的研究至关重要；然而，用于非临床研究的敏感酶联免疫吸附测定（elisa）仍然有限。本研究旨在建立并验证食蟹猴血清中IL-8的灵敏定量方法。使用两种重组il -8特异性抗体和替代基质，开发了三明治型ELISA，包括样品、生物素化抗体和辣根过氧化物酶标记的链亲和素（SA-HRP）的顺序培养。该方法根据监管指南进行了验证，定量下限为4.69 pg/mL，具有良好的准确性和精密度。该试验不受血清基质和溶血的影响，也不与其他细胞因子交叉反应。稀释度线性，无钩效应，回收率和平行度好。最后，猴子血清中IL-8具有足够的稳定性。验证后，将该方法应用于19个健康食蟹猴血清样品，结果与商业试验结果具有良好的相关性。综上所述，所建立的酶联免疫吸附测定法是一种灵敏、准确、可靠的食蟹猴血清中IL-8定量方法，适用于食蟹猴非临床研究。

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引用次数: 0

Glutaminase I and glutamine transaminase–ω-amidase pathways in colorectal cancer: Metabolic reprogramming and emerging therapeutic strategies 谷氨酰胺酶I和谷氨酰胺转氨酶ω-氨基酶途径在结直肠癌中的作用：代谢重编程和新的治疗策略。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-10-10 DOI: 10.1016/j.ab.2025.115989

Sarah F. Voor, Arthur J.L. Cooper, John T. Pinto

Colorectal cancer (CRC) cells exhibit a pronounced dependence on l-glutamine to support anabolic growth, redox balance, and mitochondrial metabolism, a phenomenon known as “glutamine addiction.” The canonical glutaminase I pathway is mediated by a liver type glutaminase isozyme (LGA; GLS1), a kidney type glutaminase isozyme (KGA; GLS2), and a shortened form (glutaminase C, GAC). GLS1 and GLS2 convert glutamine to glutamate and subsequently to α-ketoglutarate (α-KG) by glutamate dehydrogenase, fueling the TCA cycle. GLS1 inhibitors, such as CB-839 (Telaglenastat), are under clinical evaluation and shows promise in treatment of CRC, particularly in combination therapies. In addition to the canonical pathway, the glutamine transaminase–ω-amidase (GTωA) pathway, a noncanonical route involving transamination of glutamine to α-ketoglutaramate (KGM) by KYAT1/2 and subsequent hydrolysis by ω-amidase (NIT2), offers metabolic flexibility under hypoxic or nutrient-limited conditions. Preclinical studies suggest that GTωA may compensate for GLS1 inhibition, contributing to therapeutic resistance. This review explores the dual roles of glutamine metabolism in CRC, emphasizing the GTωA pathway as a potentially targetable metabolic route that may contribute to therapeutic resistance. While GLS1 inhibitors are under clinical evaluation, emerging evidence suggests that dual targeting of both pathways may enhance treatment efficacy by overcoming metabolic compensation. Understanding the regulatory mechanisms driving the “glutamine shift” between these pathways is critical for developing effective metabolic interventions in CRC.

结直肠癌（CRC）细胞明显依赖l -谷氨酰胺来支持合成代谢生长、氧化还原平衡和线粒体代谢，这种现象被称为“谷氨酰胺成瘾”。典型谷氨酰胺酶I途径由肝型谷氨酰胺酶同工酶（LGA; GLS1）、肾型谷氨酰胺酶同工酶（KGA; GLS2）和缩短形式（谷氨酰胺酶C， GAC）介导。GLS1和GLS2通过谷氨酸脱氢酶将谷氨酰胺转化为谷氨酸，随后转化为α-酮戊二酸（α-KG），促进TCA循环。GLS1抑制剂，如CB-839 (Telaglenastat)，正在进行临床评估，并显示出治疗结直肠癌的希望，特别是在联合治疗中。除了典型途径外，谷氨酰胺转氨酶-ω-氨基酶（gt -ω-a）途径是一种非典型途径，涉及由KYAT1/2将谷氨酰胺转氨酶转化为α-酮戊二酸盐（KGM），随后被ω-氨基酶（NIT2）水解，在缺氧或营养受限条件下提供代谢灵活性。临床前研究表明，GTωA可能补偿GLS1抑制，有助于治疗耐药。这篇综述探讨了谷氨酰胺代谢在结直肠癌中的双重作用，强调GTωA途径可能是一个潜在的靶向代谢途径，可能有助于治疗耐药。虽然GLS1抑制剂仍处于临床评估阶段，但新出现的证据表明，双重靶向这两种途径可能通过克服代谢代偿来提高治疗效果。了解这些途径之间驱动“谷氨酰胺转移”的调节机制对于开发有效的CRC代谢干预措施至关重要。

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