Methods最新文献_第3页

In vitro wound simulation: A high-throughput device for scratch assays 体外伤口模拟：一种用于划痕分析的高通量设备。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-18 DOI: 10.1016/j.ymeth.2025.12.007

Jacob E. Labovitz , Patrick Kulaga , Eric M. DuBois, Kunyu Li, Timothy M. O’Shea

Traumatic injury to the healthy central nervous system (CNS) causes mechanical tissue damage that results in localized cell death and blood–brain-barrier (BBB) disruption. CNS tissue damage stimulates a multicellular wound response to limit the extent of damage but fails to reestablish the normal function of injured tissue. There is strong interest in developing new strategies to augment regeneration after CNS injury. To enable therapy development, reliable assays to screen and identify molecular approaches to augment glial-based wound responses over fibrotic scarring are needed. Scratch assays, which involve mechanically removing cells from an in vitro culture, allow for the simulation of wounds with high throughput and tight control over applied treatments to mechanistically study cell migration and proliferation functions that are critical to effective repair. Current methods require researchers to individually scratch each well with a pipette tip, resulting in low throughput as well as inconsistent scratch widths, straightness, and efficacy within and between wells. Here, we describe the design of a quickly assembled (<30 min), inexpensive (<$110) scratch assay rig that readily creates uniform scratches that are straight (average tortuosity < 1.1), have tunable widths (730–1100 µm), and fully remove damaged cells from the simulated wound region. Designed for a 24-well plate, the rig allows for high-throughput screening of varied experimental conditions or for testing many replicates. Application of the scratch assay device on an in vitro culture of neural progenitor cells (NPC) demonstrates the ability to detect differences in wound closure rate for three unique media conditions. These results support the implementation of this high-throughput scratch assay rig as a method to standardize and improve the efficiency of in vitro wound healing studies.

对健康中枢神经系统（CNS）的创伤性损伤引起机械组织损伤，导致局部细胞死亡和血脑屏障（BBB）破坏。中枢神经系统组织损伤可刺激多细胞损伤反应，限制损伤程度，但不能重建损伤组织的正常功能。人们对开发新的策略来增强中枢神经系统损伤后的再生有浓厚的兴趣。为了促进治疗的发展，需要可靠的检测方法来筛选和识别分子方法，以增强基于胶质细胞的纤维化瘢痕的伤口反应。划伤试验涉及机械地从体外培养物中移除细胞，允许高通量和严格控制应用治疗的伤口模拟，以机械地研究细胞迁移和增殖功能，这对有效修复至关重要。目前的方法需要研究人员用吸管头单独刮擦每口井，这导致了低通量以及井内和井间刮擦宽度、直线度和效果不一致。在这里，我们描述了一个快速组装(

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引用次数: 0

Development of a novel microsampling device to standardize the analysis of intranasal inflammatory biomarkers 一种新型微采样装置的开发，以标准化鼻内炎症生物标志物的分析。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-18 DOI: 10.1016/j.ymeth.2025.12.006

Tanya Lupancu , Jonathan Limpah , Esrin Aydin , Timothy Fan , Vanessa Wong , Joanne Rimmer , Brian S. Wang , David M. Yen , Eldin Rostom , Adam M. Damry

Nasal fluid biomarker analysis is an emerging technique for studying sinonasal pathophysiology, monitoring therapeutic efficacy, and discovering novel drug targets. Variability in biomarker results can be contributed to non-standardized collection methodology. To address this, a novel microsampler was developed, designed to enable precise site-specific sampling, consistent volume collection, and high analyte recovery. This study aims to evaluate the performance of this new microsampler device compared to commonly utilized flocked swab, and other absorbent materials. To do so, fixed volumes of a synthetic nasal fluid mimic were deposited onto the anterior region of the inferior turbinate of a 3D-printed sinus model to assess volumetric and collection site accuracy of the nasal microsampler, in comparison to a flocked swab. Additionally, protein biomarker recovery properties of the device’s absorption membrane, Leukosorb^TM, versus experimental proprietary absorbent materials, were assessed using ELISA. The microsampler, contrasting the flocked swab, demonstrated statistically significant lower coefficient of variation for collected nasal fluid volume and greater sampling site precision. The spike and recovery study indicated that the proprietary materials had statistically significant higher biomarker recovery rates than Leukosorb^TM. Overall, the novel nasal microsampler offers significantly improved volumetric control and site-specific collection against flocked swab. All experimental proprietary absorbent materials displayed significantly higher protein recovery rates, comparing to widely accepted and utilized Leukosorb^TM. Consistent use of the novel nasal microsampler device has the potential to standardize protein recovery processes and minimize variability across studies, leading to enhanced reliability and comparability of future findings.

鼻液生物标志物分析是研究鼻腔病理生理、监测治疗效果和发现新的药物靶点的新兴技术。生物标志物结果的可变性可归因于非标准化的收集方法。为了解决这个问题，开发了一种新型微型采样器，旨在实现精确的特定地点采样，一致的体积收集和高分析物回收率。本研究旨在评估这种新型微采样器装置与常用的蜂群拭子和其他吸收材料的性能。为此，将固定体积的合成鼻模拟物沉积在3d打印鼻窦模型的下鼻甲前区，与蜂群拭子相比，评估鼻微采样器的体积和收集位置准确性。此外，使用ELISA评估了该装置的吸收膜LeukosorbTM与实验专有吸收材料的蛋白质生物标志物回收性能。与蜂群拭子相比，微采样器收集的鼻液量变异系数更低，采样点精度更高。峰值和回收率研究表明，专利材料具有统计学上显著高于LeukosorbTM的生物标志物回收率。总体而言，新型鼻腔微采样器提供了显著改善的体积控制和针对蜂群拭子的特定地点收集。与广泛接受和使用的LeukosorbTM相比，所有实验专有吸收材料都显示出显著更高的蛋白质回收率。持续使用新型鼻腔微采样器装置有可能使蛋白质回收过程标准化，并最大限度地减少研究中的可变性，从而提高未来研究结果的可靠性和可比性。

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引用次数: 0

Application of a 2A-peptide system for polycistronic human IgG production in Leishmania tarentolae 2a肽体系在产人多顺反性IgG中的应用。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-15 DOI: 10.1016/j.ymeth.2025.12.005

Jing Yi Lai , Kae Yi Tan , Choo Hock Tan , Yee Siew Choong , Theam Soon Lim

Monoclonal antibodies are commonly expressed using mammalian cell lines due to their required post-translational modifications. However, this process can become costly and requires investment in specialized equipment and facilities. The use of eukaryotic cell Leishmania tarentolae offers a cost-effective alternative while still allowing N-linked glycosylation to occur. The focus of this study explores the use of L. tarentolae as a platform for expressing a monoclonal IgG antibody against the long-chain neurotoxin (LNTX) from the venom of the Monocled Cobra, Naja kaouthia. The monoclonal antibody was isolated from a human naïve phage display library and expressed as single-chain fragment variable (scFv) in Escherichia coli with good specificity against the LNTX of Naja kaouthia. The scFv was then converted to the scFv-Fc format and full-length IgG for expression in L. tarentolae. The IgG expression was achieved using a 2A peptide-based bicistronic vector system with good expression yield. The expressed IgG demonstrated homogeneity in size and minimal degradation. Therefore, L. tarentolae can be considered a possible alternative platform for the expression of human scFv-Fc and IgG antibodies.

单克隆抗体通常在哺乳动物细胞系中表达，因为它们需要翻译后修饰。然而，这个过程可能会变得昂贵，需要投资专门的设备和设施。使用真核细胞利什曼绦虫提供了一种具有成本效益的替代方案，同时仍然允许n -链糖基化发生。本研究的重点是探索利用L. tarentolae作为表达单克隆IgG抗体的平台，抗来自单眼眼镜蛇Naja kaouthia毒液的长链神经毒素（LNTX）。该单克隆抗体从人naïve噬菌体展示文库中分离得到，在大肠杆菌中以单链片段变量（single-chain fragment variable, scFv）的形式表达，具有较好的特异性。然后将scFv转化为scFv- fc格式和全长IgG，在猪乳杆菌中表达。IgG的表达采用基于2A肽的双链载体系统，表达量高。表达的IgG大小均匀，降解程度最低。因此，可以将链乳杆菌视为表达人scFv-Fc和IgG抗体的可能替代平台。

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引用次数: 0

Sensitivity-improving CRISPR-Cas strategies for non-nucleic acid targets detection 提高非核酸靶点检测灵敏度的CRISPR-Cas策略

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-15 DOI: 10.1016/j.ymeth.2025.12.004

Huijuan Qi , Yujin Yang , Xiangting Hou , Yuanyuan Chen , Shaohua Gong

CRISPR-Cas systems have revolutionized non-nucleic acid targets detection across diverse applications. Nevertheless, the relatively low enzymatic turnover rate of activated Cas nucleases during substrate cleavage remains a critical bottleneck, limiting the sensitivity of such detection methods. To address this challenge, numerous innovative strategies have been proposed to enhance the sensitivity of CRISPR-Cas systems, enabling high-sensitive non-nucleic acid targets detection. This review systematically summarizes the sensitivity-enhancing methodologies for non-nucleic acid targets detection using CRISPR-Cas technologies. We first delineate the working mechanisms of various CRISPR-Cas systems and the signal transduction pathways specific to non-nucleic acid targets. Subsequently, we detail diverse sensitivity-improving approaches, including nucleic acid amplification-facilitated strategies, multimolecular labeling techniques, dual-enzyme cascade methods, and multiplex amplification methodologies. Additionally, the current challenges and future perspectives in this field are discussed, aiming to inspire researchers to develop more ingenious solutions and facilitate real-world applications of CRISPR-Cas system for non-nucleic acid targets detection.

CRISPR-Cas系统在各种应用中彻底改变了非核酸靶标检测。然而，在底物裂解过程中，活化Cas核酸酶相对较低的酶周转率仍然是一个关键瓶颈，限制了这种检测方法的灵敏度。为了应对这一挑战，已经提出了许多创新策略来提高CRISPR-Cas系统的灵敏度，从而实现高灵敏度的非核酸靶点检测。本文系统总结了利用CRISPR-Cas技术提高非核酸靶点检测灵敏度的方法。我们首先描述了各种CRISPR-Cas系统的工作机制以及针对非核酸靶点的信号转导途径。随后，我们详细介绍了各种提高灵敏度的方法，包括核酸扩增促进策略、多分子标记技术、双酶级联方法和多重扩增方法。此外，还讨论了该领域目前面临的挑战和未来的前景，旨在启发研究人员开发更巧妙的解决方案，促进CRISPR-Cas系统在非核酸靶点检测中的实际应用。

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引用次数: 0

Denaturation, rapid dilution refolding, and single-step purification of the core histones using desalting size-exclusion chromatography 变性，快速稀释，再折叠，和一步纯化核心组蛋白使用脱盐尺寸排除色谱。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-11 DOI: 10.1016/j.ymeth.2025.12.003

Olufola O. Ige , Thordur Hendrickson-Rebizant , Wenxia Luo , Marvellous Oyeyode , Stefan Jacobson , Fryda Malinalli Ortiz Bada , Michael J. Rowley , Rui Wen Liu , James R Davie , Adam Frankel , Ted M. Lakowski

Nucleosomes, composed of DNA and histone octamers, regulate gene expression through histone modifications such as lysine acetylation and methylation. These modifications are added by writers, removed by erasers, and interpreted by readers to control gene expression, chromatin structure, and essential cellular processes such as differentiation and development. Accurate Post Translational modifications analysis requires high-purity histones due to the sensitivity of epigenetic assays. Recombinant histones are expressed in E. coli as inclusion bodies, requiring denaturation, refolding, and purification. These traditional purification methods involve complicated and lengthy protocols taking days and potentially exposing the histones to oxidation and proteolytic degradation. We developed a rapid method for refolding histones from inclusion bodies in a one-step purification using a desalting column achieving > 90 % purity. This method is compared to our previous High Performance Liquid Chromatography (HPLC)-based protocol. Our single-step desalting purification reduces purification time from multiple days to one day, lowers operational cost, and eliminates the need for reverse-phase HPLC, making high-purity histone production accessible to laboratories without specialized chromatography infrastructure.

核小体由DNA和组蛋白八聚体组成，通过组蛋白修饰如赖氨酸乙酰化和甲基化来调节基因表达。这些修饰由编辑器添加，由橡皮擦删除，并由读取器解释，以控制基因表达，染色质结构和基本细胞过程，如分化和发育。由于表观遗传检测的敏感性，准确的PTM分析需要高纯度的组蛋白。重组组蛋白以包涵体形式在大肠杆菌中表达，需要变性、重折叠和纯化。这些传统的纯化方法涉及复杂和冗长的程序，需要数天时间，并且可能使组蛋白暴露于氧化和蛋白水解降解中。我们开发了一种快速从包涵体中折叠组蛋白的方法，使用脱盐柱一步纯化，纯度达到 > 90 %。该方法与我们之前基于hplc的协议进行了比较。我们的单步脱盐纯化将纯化时间从几天缩短到一天，降低了操作成本，并且消除了反相高效液相色谱的需要，使高纯度组蛋白生产在没有专门色谱基础设施的实验室中变得容易。

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引用次数: 0

Investigation on structure–property relationships of MgO-SrO containing silicate-based bioactive glasses: An experimental and molecular dynamics simulation study 含MgO-SrO硅酸盐基生物活性玻璃的结构-性能关系研究：实验和分子动力学模拟研究。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-07 DOI: 10.1016/j.ymeth.2025.12.001

Amirhossein Moghanian , Ramin Farmani , Niloufar Kolivand , Arman Tayebi , Sirus Safaee

Molecular dynamics (MD) simulations and experimental analysis were performed on MgO–SrO containing bioactive glasses (MSBGs) with the composition of 60SiO₂–(31–x)CaO–4P₂O₅–5MgO–xSrO (mol%) (x = 0, 1, 3, 5, 8, 10, 15, 20; M5S0–M5S20) to evaluate the structural properties, ion clustering, and dissolution behavior as a function of SrO content. Simulation employed the Buckingham potential for short-range interactions and Coulombic potentials for long-range forces. MSBGs had Si–O and P–O bond lengths of 1.609 Å and 1.491 Å, with O–Si–O and O–P–O bond angles centered at ∼109.3° and 109.4°, respectively, confirming tetrahedral SiO₄/PO₄ coordination. Across all compositions, Si–O–Si bonds dominated the majority of the distribution (88–89 %), with Si–O–P at 11–12 % and P–O–P negligible (∼0.3 %). Densities decreased from 2.913 g·cm⁻³ (M5S20) to 2.631 g·cm⁻³ (M5S0), reflecting network loosening with SrO substitution. Qⁿ distribution remained stable, with Q³/Q⁴ fractions of 38–43 % and 26–30 % for Si-based tetrahedra. R-factor analysis revealed optimal homogeneity for M5S5 (

R_{S i / P}^{S r}

= 0.838252), balancing reduced cation clustering and moderate network stability. ICP-AES showed M5S5 with a sustained release of Si⁴⁺, Mg²⁺, and Sr²⁺ over 24 h. Meanwhile, antibacterial study resulted in statistically significant increase in efficiency for M5S5 compared to M5S0 (***p < 0.001). The combined computational and experimental findings identify M5S5 as the most promising candidate for biomedical applications requiring structural benefits, controlled ion release, and antibacterial efficiency.

采用分子动力学（MD）模拟和实验分析了60SiO2-(31-x)CaO-4P2O5-5MgO-xSrO (mol%) （x = 0,1,3,5,8,10,15,20;M5S0-M5S20）的MgO-SrO生物活性玻璃（MSBGs）的结构性质、离子聚类和溶解行为与SrO含量的关系。模拟使用白金汉势来模拟短程相互作用，使用库仑势来模拟远距离力。MSBGs的Si-O和P-O键长分别为1.609 Å和1.491 Å， O-Si-O和O-P-O键角中心分别为~ 109.3°和109.4°，证实了SiO4/PO4的四面体配位。在所有成分中，Si-O-Si键占据了大部分分布（88-89 %），Si-O-P占11-12 %，P-O-P可以忽略不计（~ 0.3 %）。密度从2.913 g·cm-3 （M5S20）下降到2.631 g·cm-3 (M5S0)，反映了SrO取代导致的网络松动。Qn分布稳定，硅基四面体Q3/Q4分数分别为38-43 %和26-30 %。r因子分析显示，M5S5的均匀性最佳（RSi/PSr = 0.838252），平衡了阳离子聚类减少和适度的网络稳定性。ICP-AES显示M5S5的Si4+、Mg2+和Sr2+的持续释放时间超过24 h。同时，抗菌研究结果显示，M5S5比M5S0的抗菌效率有统计学意义（***p）

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引用次数: 0

MDM2/p53-based live-cell quantitative FRET imaging for apoptosis drug discovery 基于MDM2/p53的活细胞定量FRET成像用于细胞凋亡药物发现。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-04 DOI: 10.1016/j.ymeth.2025.12.002

Zhiyu Xiao, Lingmin Xie, Ziru Wu, Xinghong Cai, Zhengfei Zhuang, Tongsheng Chen

Targeting the interaction between P53 and MDM2 to re-activate P53 to induce apoptosis is an important strategy for cancer treatment. In this study, based on the unique advantages of in situ visualization, dynamic imaging, and quantitative analysis of living cell FRET imaging, a method for screening apoptotic drugs targeting p53-MDM2 interaction was developed. A stable model of Nutlin-3-induced apoptosis was established in MCF-7 cells, which was verified by reducing mitochondrial membrane potential and increasing the proportion of nuclear chromatin condensation (from 9.16 % to 50.55 %). Biochemical methods such as WB analysis found that after activating P53, BAX expression was up-regulated through a Puma-independent pathway, which promoted BAX oligomerization. Live-cell quantitative FRET imaging found that the maximum donor center FRET efficiency (E_Dmax) of CFP-p53 and YFP-MDM2 decreased from 0.50 to 0.22 after Nutlin-3 treatment, and the co-localization coefficient decreased significantly from 83 % to 22 %, confirmed that Nutlin-3 directly disrupted the interaction between P53/MDM2, promoting P53 nuclear translocation and apoptosis. This indicated that Nutlin-3 was a direct inhibitor of the P53/MDM2 interaction. Apoptosis drug screening was performed in MCF-7 cells, and we found that the E_Dmax was 0.29 and 0.31 for the cells treated with DOX and RSV, respectively, and 0.48 for the IKE-treated cells and 0.43 for the SOR-treated cells, indicating that DOX and RSV, but not IKE and SOR, were potential P53/MDM2-dependent apoptotic drugs. In addition, Nutlin-3 treatment decreased the E_Dma_x value in p53 wild-type U2OS cells from 0.43 to 0.20. In summary, our method can identify p53-MDM2 interaction inhibitors in living cells, providing a quantitative in vivo supplement for traditional target-based drug discovery.

靶向P53与MDM2的相互作用，重新激活P53诱导细胞凋亡是癌症治疗的重要策略。本研究基于活细胞FRET成像在原位可视化、动态成像、定量分析等方面的独特优势，开发了一种靶向p53-MDM2相互作用的凋亡药物筛选方法。在MCF-7细胞中建立了稳定的nutlin -3诱导凋亡模型，通过降低线粒体膜电位和增加核染色质凝聚比例（从9.16%增加到50.55%）来验证。WB分析等生化方法发现，激活P53后，BAX的表达通过不依赖puma的途径上调，促进BAX寡聚化。活细胞定量FRET成像发现，经Nutlin-3处理后，CFP-p53和YFP-MDM2的最大供体中心FRET效率（EDmax）从0.50下降到0.22，共定位系数从83%下降到22%，证实了Nutlin-3直接破坏了P53/MDM2之间的相互作用，促进了P53核转运和凋亡。这表明Nutlin-3是P53/MDM2相互作用的直接抑制剂。在MCF-7细胞中进行凋亡药物筛选，我们发现DOX和RSV处理细胞的EDmax分别为0.29和0.31，IKE处理细胞的EDmax为0.48，SOR处理细胞的EDmax为0.43，这表明DOX和RSV是潜在的P53 / mdm2依赖性凋亡药物，而IKE和SOR则不是。此外，Nutlin-3处理使p53野生型U2OS细胞的EDmax值从0.43降低到0.20。总之，我们的方法可以在活细胞中鉴定p53-MDM2相互作用抑制剂，为传统的基于靶标的药物发现提供了定量的体内补充。

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引用次数: 0

Development and characterization of a membrane-permeant GRASP65-mimetic peptide that inhibits Golgi unlinking and cell cycle progression 抑制高尔基解联和细胞周期进展的膜渗透grasp65模拟肽的开发和表征。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-12-01 DOI: 10.1016/j.ymeth.2025.11.008

Romina Ines Cervigni , Raffaella Bonavita , Maria Luisa Barretta , Daniela Spano , Inmaculada Ayala , Fabiola Mascanzoni , Roberta Iannitti , Petra Henklein , Alessandra Monti , Maurizio Renna , Nunzianna Doti , Antonino Colanzi

The Golgi complex is central to cellular homeostasis and serves as a key processing and sorting hub for protein trafficking. In many cell types, the Golgi complex is organized as interconnected stacks of cisternae, forming a structure known as the Golgi ribbon. This ribbon undergoes dynamic remodelling during physiological processes, such as cell division, and under pathological conditions, including cancer and neurodegeneration. A critical step in the unlinking of the Golgi ribbon involves the phosphorylation of the stacking protein GRASP65, which leads to the separation of the ribbon into individual stacks, a process necessary for the G2/M transition of the cell cycle. However, existing tools for selectively manipulating the GRASP65 role in ribbon organization are limited by non-specific effects or technical challenges.

Here, we present the development and characterization of a membrane-permeable peptide, R₈-GRASP65-S277, derived from GRASP65 and containing the phosphorylation site Ser277, which is essential for Golgi unlinking. This peptide effectively inhibited Golgi unlinking and mitotic entry in several cell lines, including cancer models. In contrast, a control peptide with a non-phosphorylatable alanine substitution (R₈-GRASP65-S277A) showed no such effect, confirming the specificity of the tool. Furthermore, the R₈-GRASP65-S277 peptide reversed Golgi unlinking induced by the chemotherapeutic agent doxorubicin, demonstrating its utility in studying stress-induced Golgi disassembly.

These findings establish the R8-GRASP65-S277 peptide as a specific, potent, and scalable tool for probing the molecular mechanisms of Golgi unlinking, its regulation of cell cycle progression, and its potential contributions to pathological states.

高尔基复合体是细胞稳态的核心，是蛋白质运输的关键加工和分类中心。在许多细胞类型中，高尔基复合体被组织成相互连接的池池堆叠，形成一个被称为高尔基带的结构。在生理过程（如细胞分裂）和病理条件（包括癌症和神经变性）中，该带经历动态重塑。高尔基带解联的一个关键步骤涉及堆叠蛋白GRASP65的磷酸化，这导致高尔基带分离成单独的堆叠，这是细胞周期G2/M转变所必需的过程。然而，现有的选择性操纵GRASP65在带状组织中的作用的工具受到非特异性效应或技术挑战的限制。在这里，我们开发和表征了一种膜渗透肽R8-GRASP65-S277，它来源于GRASP65，含有磷酸化位点Ser277，这是高尔基解联所必需的。这种肽有效地抑制高尔基解联和有丝分裂进入几种细胞系，包括癌症模型。相比之下，具有非磷酸化丙氨酸取代的对照肽（R8-GRASP65-S277A）没有这种效果，证实了该工具的特异性。此外，R8-GRASP65-S277肽逆转了化疗药物阿霉素诱导的高尔基体解联，证明了其在研究应激诱导的高尔基体解联中的实用性。这些发现证实了R8-GRASP65-S277肽是一种特异的、有效的、可扩展的工具，可用于探索高尔基体解联的分子机制、对细胞周期进程的调节以及对病理状态的潜在贡献。

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引用次数: 0

Sensitive and specific detection of ctDNA using Copper-Free click chemistry and magnetic bead Technology 利用无铜点击化学和磁珠技术对ctDNA进行灵敏和特异的检测。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-11-30 DOI: 10.1016/j.ymeth.2025.11.010

Reza Didarian , Dilek Kanarya , Sonya Sahin , Canan Özyurt , Serap Evran , Dilek Odaci , Nimet Yildirim-Tirgil

Liquid biopsy, particularly the analysis of circulating tumor DNA (ctDNA), offers immense potential for non-invasive cancer diagnosis and monitoring. It provides a less invasive alternative to traditional tissue biopsies, enabling earlier detection and real-time assessment of disease progression. However, a significant hurdle in its widespread adoption is the extremely low concentration of ctDNA in biological samples, especially during the early stages of cancer, making sensitive and specific detection challenging. This work addresses the critical problem of developing a highly sensitive and specific method for low abundance ctDNA detection.

We developed a novel, highly sensitive, and specific method for ctDNA analysis, employing copper-free click chemistry (strain-promoted azide-alkyne cycloaddition, SPAAC) for enzyme-free amplification, coupled with magnetic bead-assisted fluorometric detection. This enzyme-free approach significantly enhanced specificity and reduced background noise. We meticulously optimized parameters, including primer length and annealing temperature, finding that 30-base primers and a 50 °C annealing temperature yielded optimal amplification efficiency. Our method successfully detected ctDNA at concentrations as low as 10 pM (15 bp primer). Agarose gel electrophoresis confirmed highly specific amplification with minimal non-specific products, and the assay demonstrated excellent allelic discrimination, accurately distinguishing single-nucleotide mutations. Importantly, the method proved robust in complex human serum samples, demonstrating its practical applicability.

This innovative, cost-effective, and enzyme-free platform overcomes many limitations of current ctDNA detection technologies. By enabling highly sensitive and specific detection of low abundance ctDNA, this methodology represents a significant leap forward for non-invasive cancer diagnostics, paving the way for earlier disease detection, improved treatment monitoring, and the broader implementation of personalized medicine.

液体活检，特别是循环肿瘤DNA （ctDNA）的分析，为非侵入性癌症诊断和监测提供了巨大的潜力。它为传统的组织活检提供了一种侵入性较小的替代方法，能够更早地发现和实时评估疾病进展。然而，其广泛采用的一个重大障碍是生物样品中ctDNA的浓度极低，特别是在癌症的早期阶段，使得敏感和特异性检测具有挑战性。这项工作解决了开发低丰度ctDNA检测的高灵敏度和特异性方法的关键问题。我们开发了一种新的、高灵敏度和特异性的ctDNA分析方法，采用无铜点击化学（菌株促进叠氮化物-炔环加成，SPAAC）进行无酶扩增，结合磁珠辅助荧光检测。这种无酶的方法显著提高了特异性并降低了背景噪声。我们精心优化了引物长度和退火温度等参数，发现30碱基的引物和50 °C的退火温度产生了最佳的扩增效率。我们的方法成功地检测到浓度低至10 pM（15 bp引物）的ctDNA。琼脂糖凝胶电泳证实了高特异性扩增和最小的非特异性产物，并且该试验显示了出色的等位基因区分，准确区分单核苷酸突变。重要的是，该方法在复杂的人血清样本中证明了鲁棒性，证明了其实用性。这种创新、经济、无酶的平台克服了当前ctDNA检测技术的许多局限性。通过实现低丰度ctDNA的高灵敏度和特异性检测，该方法代表了非侵入性癌症诊断的重大飞跃，为早期疾病检测、改进治疗监测和更广泛地实施个性化医疗铺平了道路。

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引用次数: 0

Fabrication of advanced microneedle-based targeted intravaginal drug delivery devices: therapeutic opportunities and translational challenges 制造先进的基于微针的阴道内靶向给药装置：治疗机会和转化挑战。

IF 4.3 3区生物学 Q1 BIOCHEMICAL RESEARCH METHODS

Methods

Pub Date : 2025-11-28 DOI: 10.1016/j.ymeth.2025.11.009

Tejas S. Patil , Deepvardhan P. Chaudhari , Utkarsh U. Bhamare , Mahesh B. Palkar , Mahendra R. Mahajan , Sopan N. Nangare

Millions of women worldwide suffer from a variety of health conditions, including vaginal bacterial and yeast infections, sexually transmitted infections (STIs), urinary tract infections, pelvic inflammatory disorders, and hormonal abnormalities. Despite major advances in biomedical research, traditional intravaginal drug administration systems such as gels, creams, and suppositories frequently encounter issues such as fast drug clearance, leakage, and uneven mucosal retention, reducing therapeutic effectiveness. Microneedles, a painless and less invasive drug delivery technology, represent a viable alternative to standard formulations because they allow for accurate, controlled, and localized drug administration. Therefore, the present review focuses on possibility of microneedle-based techniques for intravaginal medication administration. In brief, it covers the need for breakthroughs in vaginal drug administration. It provides an overview of several types of microneedles, including solid, hollow, dissolving, coated, and hydrogel-forming, and their manufacturing procedures. Then, it delves into their use in intravaginal drug administration, highlighting their capacity to improve drug penetration and retention. Finally, it discusses future problems, prospective advancements, and the larger implications of microneedle technology in vaginal therapy. Microneedle-based intravaginal medication delivery is a huge step forward in targeted vaginal infection treatment. Notably, microneedles easily cross the cervicovaginal mucus barrier, increasing drug absorption at the target region while being minimally invasive. Future studies should focus on improving microneedle formulations, assessing long-term safety, and investigating their potential for wider clinical applications.

全世界数以百万计的妇女患有各种健康问题，包括阴道细菌和酵母菌感染、性传播感染、尿路感染、盆腔炎性疾病和激素异常。尽管生物医学研究取得了重大进展，但传统的阴道内给药系统，如凝胶、乳膏和栓剂，经常遇到药物快速清除、渗漏和粘膜保留不均匀等问题，降低了治疗效果。微针是一种无痛、无创给药技术，是标准配方的可行替代方案，因为它们允许精确、可控和局部给药。因此，本综述的重点是基于微针的阴道内给药技术的可能性。简而言之，它涵盖了在阴道给药方面取得突破的需要。它提供了几种类型的微针的概述，包括固体，空心，溶解，涂层和水凝胶形成，以及它们的制造程序。然后，深入研究了它们在阴道内给药中的应用，强调了它们提高药物渗透和保留的能力。最后，它讨论了未来的问题，前瞻性的进展，以及微针技术在阴道治疗中的更大的影响。基于微针的阴道内给药是阴道感染靶向治疗的巨大进步。值得注意的是，微针很容易穿过宫颈阴道粘液屏障，增加了靶区药物的吸收，同时微创。未来的研究应侧重于改进微针配方，评估长期安全性，并调查其更广泛临床应用的潜力。

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