SLAS Discovery最新文献_第8页

Optimized scaffold-free human 3D adipose tissue organoid culture for obesity and disease modeling 优化的无支架人体三维脂肪组织类器官培养用于肥胖和疾病建模。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-01-25 DOI: 10.1016/j.slasd.2025.100218

Rafael Dariolli , Raphael Nir , Tova Mushlam , Glauco R. Souza , Stephen R. Farmer , Miguel L Batista Jr.

Obesity and type 2 diabetes (T2D) are strongly linked to abnormal adipocyte metabolism and adipose tissue (AT) dysfunction. However, existing adipose tissue models have limitations, particularly in the stable culture of fat cells that maintain physiologically relevant phenotypes, hindering a deeper understanding of adipocyte biology and the molecular mechanisms behind differentiation. Current model systems fail to fully replicate In vivo metabolism, posing challenges in adipose tissue research. Three-dimensional (3D) AT organoids, although promising, present significant handling challenges during long-term culture. As adipocytes maturate and accumulate fat, they develop organotypic characteristics, increasing the buoyancy effect, which causes the organoids to oscillate, complicating culture manipulation and rendering multiple handling steps difficult.

Due to these challenges, most adipose spheroid and organoid models are scaffold-based, despite many cell types' ability to secrete extracellular matrix (ECM) components and self-assemble into aggregates. Scaffold-free 3D organoids have been less explored. To address the shortage of affordable and reliable AT models, we utilized magnetic bioprinting technology to develop a human-derived 3D model of adipose tissue. This system incorporates a magnetic holder that restrains organoids, preventing them from floating and minimizing the risk of loss during manipulation.

This study outlines a protocol for generating In vitro AT-derived organoid using 3D magnetic bioprinting, with a focus on manufacturing, culturing, and assessing the morpho-functional characteristics of late-stage AT organoids. Magnetic bioprinting allows for the replication of tissue structure and function In vitro without the risk of organoid loss, making it an ideal method for high-throughput AT organoid culture. Additionally, the combination of 3D scaffold-free manufacturing with In vitro disease modeling offers a valuable tool for discovering treatments for metabolic diseases such as obesity and T2D.

肥胖和2型糖尿病（T2D）与脂肪细胞代谢异常和脂肪组织（AT）功能障碍密切相关。然而，现有的脂肪组织模型存在局限性，特别是在维持生理相关表型的脂肪细胞的稳定培养方面，阻碍了对脂肪细胞生物学和分化背后的分子机制的更深入理解。目前的模型系统不能完全复制体内代谢，这给脂肪研究带来了挑战。三维（3D） AT类器官虽然很有前途，但在长期培养过程中存在重大的处理挑战。随着脂肪细胞的成熟和脂肪的积累，它们形成了器官型特征，增加了浮力效应，导致类器官振荡，使培养操作复杂化，使多个处理步骤变得困难。由于这些挑战，尽管许多细胞类型能够分泌细胞外基质（ECM）成分并自组装成聚集体，但大多数脂肪球体模型都是基于支架的。对无支架的3D类器官的探索较少。为了解决价格合理且可靠的AT模型短缺的问题，我们利用磁性生物打印技术开发了人体来源的脂肪组织3D模型。该系统包含一个磁性支架，可以抑制类器官，防止它们漂浮，并最大限度地降低操作过程中丢失的风险。本研究概述了一种使用3D磁性生物打印技术生成体外AT衍生类器官的方案，重点是制造、培养和评估晚期AT类器官的形态功能特征。磁性生物打印允许在体外复制组织结构和功能，而没有类器官损失的风险，使其成为高通量AT类器官培养的理想方法。此外，3D无支架制造与体外疾病建模的结合为发现代谢性疾病（如肥胖和T2D）的治疗方法提供了有价值的工具。

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引用次数: 0

Identification of unique binding mode anti-NTF3 antibodies from a novel long VH CDR3 phage display library 新型长VH CDR3噬菌体展示文库中独特结合模式抗ntf3抗体的鉴定

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-01-18 DOI: 10.1016/j.slasd.2025.100216

Stacey E. Chin , Pablo Gallego , Anna Aagaard , Sara Carmen , Nathalie Barrett , Marcin Wolny , Sophie Cloarec , Judy Paterson , Rohan Sivapalan , James Hunt , Thomas V. Murray , Tracy Delaney , Tove Sjögren , Frances Neal

Neurotrophic factor 3 (NTF3) is a cysteine knot protein and a member of the nerve growth factor (NGF) family of cytokines. NTF3 engages the Trk family of receptor tyrosine kinases, playing a pivotal role in the development and function of both the central and peripheral nervous systems. Its involvement in neuronal survival, differentiation, and growth links NTF3 to a spectrum of neurodegenerative diseases. Consequently, targeting NTF3 with antibodies holds promise as a first in class therapeutic opportunity for a wide range of conditions.

Specific and neutralizing antibodies against NTF3 were successfully isolated using phage display. Initial phage display selections revealed a preference of hits for a longer than average complementarity-determining region 3 (CDR3) in the heavy chain variable domain (VH). To investigate this further we developed a long loop length VH CDR3 antibody library that demonstrated increased hit rates versus a standard antibody library and allowed the isolation of IgG that demonstrated inhibition of functional activity, coupled with a favourable kinetic profile.

Structural analysis of the Fab/NTF3 interaction, via X-ray crystallography, unveiled an unconventional interaction wherein regions beyond the longer CDR loops of the Fab induced ordering in a flexible loop on NTF3, which remained disordered in its free antigenic state. This comprehensive approach not only sheds light on the therapeutic potential of NTF3-specific antibodies but also provides critical structural details that enhance our understanding of the complex NTF3-Fab interaction thus offering valuable insights for future antibody design and therapeutic development.

神经营养因子3 （NTF3）是一种半胱氨酸结蛋白，是神经生长因子（NGF）细胞因子家族的成员。NTF3参与Trk受体酪氨酸激酶家族，在中枢和周围神经系统的发育和功能中发挥关键作用。它参与神经元存活、分化和生长，将NTF3与一系列神经退行性疾病联系起来。因此，用抗体靶向NTF3有望成为广泛疾病的一流治疗机会。利用噬菌体展示技术成功分离出NTF3特异性抗体和中和抗体。初始噬菌体展示选择显示，在重可变结构域（VH）中，比平均互补决定区域3 （CDR3）更长的命中偏好。为了进一步研究这一点，我们开发了一个长环长度的CDR3抗体库，与标准抗体库相比，该抗体库显示出更高的命中率，并允许分离出抑制功能活性的IgG，同时具有良好的动力学特征。通过x射线晶体学对Fab/NTF3相互作用进行结构分析，揭示了一种非常规的相互作用，其中Fab的较长CDR环以外的区域诱导NTF3上的柔性环有序，而NTF3在其自由抗原状态下保持无序。这种全面的方法不仅揭示了ntf3特异性抗体的治疗潜力，而且提供了关键的结构细节，增强了我们对复杂的NTF3-Fab相互作用的理解，从而为未来的抗体设计和治疗开发提供了有价值的见解。

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引用次数: 0

NF-kB oscillation profiles decode response to anti-EGFR monoclonal antibodies NF-κB振荡谱解码对抗egfr单克隆抗体的应答。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-01-31 DOI: 10.1016/j.slasd.2025.100219

Donatella Romaniello , Lorenzo Dall'Olio , Martina Mazzeschi , Anna Francia , Federica Pagano , Valerio Gelfo , Gabriele D'Uva , Enrico Giampieri , Mattia Lauriola

A direct connection between an inflammatory environment and cancer has been extensively proven over the years. We previously reported that the presence of interleukin 1 (IL-1) is responsible for the lack of response to monoclonal antibody targeting epidermal growth factor receptor (EGFR) in colorectal cancer (CRC).

Considering the driver role of NF-kB in controlling the expression of IL-1, herein, we investigate the dynamics of the oscillatory profile of the NF-kB response to monoclonal antibody, on the background of an inflammatory environment. NF-kB is a typical transcription factor that displays intrinsic oscillatory behavior, whose biological relevance in term for example of decoding response to monoclonal antibodies, remains unclear.

Using live cell luciferase techniques, we recorded NF-kB activity over time in response to cetuximab (CTX) alone or in combination with IL-1 cytokines. Our results revealed an additive effect of these two agents on NF-kB activation, which was specific to CTX responsive cells. In contrast, CTX resistant cells did not display a significant change in the NF-kB profile under the IL-1 plus CTX combination. These results suggest an immediate interactive crosstalk between IL-1 and EGFR in the activation of NF-kB signaling pathway, which may lay the basis for the development of drug tolerant persister cells (DTP), leading to CTX resistance.

多年来，炎症环境和癌症之间的直接联系已被广泛证实。我们之前报道过白介素1 （IL-1）的存在是导致结直肠癌（CRC）对靶向EGFR的单克隆抗体缺乏反应的原因。考虑到NF-κB在控制IL-1表达中的驱动作用，本文在炎症环境的背景下，研究了NF-κB对单克隆抗体反应的振荡谱动力学。NF-kB是一种典型的转录因子，表现出固有的振荡行为，其生物学相关性，例如对单克隆抗体的解码反应，尚不清楚。使用活细胞荧光素酶技术，我们记录了西妥昔单抗（CTX）单独或与IL-1细胞因子联合作用时NF-κB随时间的活性。我们的研究结果显示，这两种药物对NF-κB活化具有叠加效应，这是CTX应答细胞所特有的。相比之下，在IL-1 + CTX联合作用下，CTX耐药细胞的NF-κB谱没有明显变化。这些结果表明，在NF-κB信号通路的激活过程中，IL-1和EGFR之间存在直接的相互作用串扰，这可能为耐药持久细胞（DTP）的发展奠定基础，从而导致CTX耐药。

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引用次数: 0

Green chemistry: Modern therapies using nanocarriers for treating rare brain cancer metastasis from colon cancer 绿色化学：利用纳米载体治疗结肠癌转移性脑癌的现代疗法。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-01-16 DOI: 10.1016/j.slasd.2025.100213

Doaa S․R․ Khafaga , Ghazala Muteeb , Darin․W․ Aswa , Mohammad Aatif , Mohd Farhan , Salma Allam

Brain metastasis (BM) from colon cancer is associated with a poor prognosis and restricted treatment alternatives, largely due to issues related to blood-brain barrier (BBB) permeability and the negative effects of standard chemotherapy. Nanotechnology improves treatment efficacy by enabling targeted and controlled drug delivery. This review article evaluates the potential of nanotechnology-based therapies for treating colon cancer BM, emphasizing their capacity to cross the BBB, diminish metastatic growth, and enhance overall survival rates. A review of multiple studies evaluated nanoparticles (NPs) as carriers for chemotherapy, focusing on parameters including particle size, surface charge, and drug-loading capacity. The study also reviewed studies that examined BBB penetration, in vitro tumor accumulation, and in vivo tumor growth inhibition. In vitro findings indicated that NPs accumulate more efficiently in BM tissue than in healthy brain tissue and show significant BBB penetration. In vivo, nanotherapy markedly inhibited tumor growth and prolonged survival relative to conventional chemotherapy or control treatments while also exhibiting reduced side effects. Recent studies demonstrated that plant extracts can effectively and safely synthesize nanomaterials, positioning them as a viable and environmentally friendly precursor for nanomaterial production. Nanotechnology-based therapies demonstrate significant potential in the treatment of colon cancer BM by minimizing systemic toxicity, enhancing therapeutic efficacy, and facilitating more targeted drug delivery. Further research is required to confirm these findings and implement them in clinical practice.

结肠癌脑转移（BM）与预后不良和治疗方案受限相关，主要是由于血脑屏障（BBB）通透性和标准化疗的负面影响。纳米技术通过实现靶向和受控的药物递送来提高治疗效果。这篇综述文章评估了纳米技术治疗结肠癌转移瘤的潜力，强调了它们穿越血脑屏障、减少转移性生长和提高总体生存率的能力。对纳米颗粒（NPs）作为化疗载体的多项研究进行了回顾，重点关注颗粒大小、表面电荷和载药能力等参数。该研究还回顾了血脑屏障渗透、体外肿瘤积累和体内肿瘤生长抑制的研究。体外研究结果表明，NPs在脑梗死组织中比在健康脑组织中更有效地积累，并表现出明显的血脑屏障渗透。在体内，与常规化疗或对照治疗相比，纳米疗法显著抑制肿瘤生长，延长生存期，同时也显示出更少的副作用。最近的研究表明，植物提取物可以有效和安全地合成纳米材料，使其成为一种可行的、环保的纳米材料前体。基于纳米技术的治疗方法通过最小化全身毒性、提高治疗效果和促进更有针对性的药物传递，在结肠癌BM的治疗中显示出巨大的潜力。需要进一步的研究来证实这些发现并将其应用于临床实践。

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引用次数: 0

Rapid luminescence-based screening method for SARS- CoV-2 inhibitors discovery 基于荧光快速筛选SARS- CoV-2抑制剂的方法。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-01-15 DOI: 10.1016/j.slasd.2025.100211

Abdeldjalil Madani, Nadine Alvarez, Steven Park, Madhuvika Murugan, David S. Perlin

The COVID-19 pandemic has emphasized the necessity for rapid and adaptable drug screening platforms against live pathogenic viruses that require high levels of biosafety containment. Conventional antiviral testing is time-consuming and labor-intensive. Here, we outline the design and validation of a semi-automated drug-screening platform for SARS-CoV-2 that utilizes multiple liquid handlers, a stable A549 cell line expressing ACE2 and TMPRSS2 receptors, and a recombinant SARS-CoV-2 strain harboring the nano-luciferase gene. This platform allows for accelerated low-, mid-, and high-throughput screenings by bypassing the virus inactivation and the staining steps compared to assays utilizing fluorescent reporter viruses or immunofluorescence. First, we demonstrated that the luminescence signal obtained at 24 h post-infection is robust and can be used as a surrogate for fluorescent reporter viruses and immunofluorescence assays that require 48 h incubation post infection. We confirmed the susceptibility of the reporter virus to a panel of reference drugs and validated the luminescence signal in 96- and 384-well plates in accordance with NIH criteria for high-throughput screening. The validation assays showed reproducible results, robust Z factor of ≥0.5, and a coefficient of variation of <20% achieved in both 96 and 384-well plate formats. Lastly, we assessed the assay's performance by screening 240 compounds from the MMV Global Health Library, using the 384-well plate format and remdesivir as a control compound. The single point screening resulted in the identification of 48 hits that inhibited more than 50% of the viral growth. We selected the 15 most active compounds to evaluate their inhibitory concentration and their cytotoxicity, which resulted in the confirmation of the 3 most potent and least toxic compounds that were never reported as antivirals. These results confirm that our platform can be reliably employed for rapid drug screening against SARS-CoV-2 and can be easily adapted to other nano-luciferase reporter viruses.

COVID-19大流行强调了针对活致病性病毒的快速和适应性强的药物筛选平台的必要性，这些平台需要高水平的生物安全控制。传统的抗病毒检测既耗时又费力。在这里，我们概述了一个半自动化的SARS-CoV-2药物筛选平台的设计和验证，该平台利用多个液体处理器、表达ACE2和TMPRSS2受体的稳定A549细胞系和含有纳米荧光素酶基因的重组SARS-CoV-2菌株。与使用荧光报告病毒或免疫荧光的检测相比，该平台可以通过绕过病毒灭活和染色步骤来加速低、中、高通量筛选。首先，我们证明了在感染后24小时获得的发光信号是稳健的，可以用作荧光报告病毒和免疫荧光检测的替代品，这些检测需要在感染后48小时孵育。我们确认了报告病毒对一组参考药物的敏感性，并根据NIH高通量筛选标准在96孔和384孔板上验证了发光信号。验证试验结果重复性好，稳健性Z因子≥0.5，变异系数为

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引用次数: 0

Comparative evaluation of cell-based assay technologies for scoring drug-induced condensation of SARS-CoV-2 nucleocapsid protein 基于细胞的药物诱导的SARS-CoV-2核衣壳蛋白凝集检测技术的比较评价。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2025-02-01 DOI: 10.1016/j.slasd.2025.100220

Rui Tong Quek , Cyna R. Shirazinejad , Christina L. Young , Kierra S. Hardy , Samuel Lim , Phillip J. Elms , David T. McSwiggen , Timothy J. Mitchison , Pamela A. Silver

Protein-nucleic acid phase separation has been implicated in many diseases such as viral infections, neurodegeneration, and cancer. There is great interest in identifying condensate modulators (CMODs), which are small molecules that alter the dynamics and functions of phase-separated condensates, as a potential therapeutic modality. Most CMODs were identified in cellular high-content screens (HCS) where micron-scale condensates were characterized by fluorescence microscopy. These approaches lack information on protein dynamics, are limited by microscope resolution, and are insensitive to subtle condensation phenotypes missed by overfit analysis pipelines. Here, we evaluate two alternative cell-based assays: high-throughput single molecule tracking (htSMT) and proximity-based condensate biosensors using NanoBIT (split luciferase) and NanoBRET (bioluminescence resonance energy transfer) technologies. We applied these methods to evaluate condensation of the SARS-CoV-2 nucleocapsid (N) protein under GSK3 inhibitor treatment, which we had previously identified in our HCS campaign to induce condensation with well-defined structure-activity relationships (SAR). Using htSMT, we observed robust changes in N protein diffusion as early as 3 h post GSK3 inhibition. Proximity-based N biosensors also reliably reported on condensation, enabling the rapid assaying of large compound libraries with a readout independent of imaging. Both htSMT and proximity-based biosensors performed well in a screening format and provided information on CMOD activity that was complementary to HCS. We expect that this expanded toolkit for interrogating phase-separated proteins will accelerate the identification of CMODs for important therapeutic targets.

蛋白质-核酸相分离涉及许多疾病，如病毒感染、神经变性和癌症。凝析物调节剂（CMODs）是一种可以改变相分离凝析物的动力学和功能的小分子，是一种潜在的治疗方式。大多数CMODs是在细胞高含量筛选（HCS）中鉴定的，其中微米尺度的凝聚物是用荧光显微镜表征的。这些方法缺乏蛋白质动力学的信息，受显微镜分辨率的限制，并且对过拟合分析管道遗漏的细微冷凝表型不敏感。在这里，我们评估了两种可选的基于细胞的检测方法：高通量单分子跟踪（htSMT）和基于邻近的冷凝生物传感器，使用NanoBIT（分裂荧光素酶）和NanoBRET（生物发光共振能量转移）技术。我们应用这些方法来评估GSK3抑制剂处理下SARS-CoV-2核衣壳(N)蛋白的缩聚，我们之前在HCS活动中发现了这种方法，可以诱导具有明确结构-活性关系（SAR）的缩聚。使用htSMT，我们观察到早在GSK3抑制后3小时N蛋白扩散的强劲变化。基于接近度的N生物传感器也可靠地报告了缩合，使大型化合物文库的快速分析具有独立于成像的读数。htSMT和基于邻近的生物传感器在筛选格式中表现良好，并提供了CMOD活性的信息，这是对HCS的补充。我们期望这个扩展的工具可以用于询问相分离蛋白，从而加速识别用于重要治疗靶点的CMODs。

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引用次数: 0

Development of a DNA-encoded library screening method “DEL Zipper” to empower the study of RNA-targeted chemical matter 开发一种dna编码文库筛选方法“DEL Zipper”，使rna靶向化学物质的研究成为可能。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-03-01 Epub Date: 2024-12-21 DOI: 10.1016/j.slasd.2024.100204

Zhongyao Ma , Bin Zou , Jiannan Zhao , Rui Zhang , Qiaoqiao Zhu , Xiaofeng Wang , Linan Xu , Xiang Gao , Xinyue Hu , Wei Feng , Wen Luo , Min Wang , Yunyun He , Zhifeng Yu , Weiren Cui , Qi Zhang , Letian Kuai , Wenji Su

To date, RNA-targeted chemical matter is under explored due to a lack of robust screening assays. In this study, we present a novel RNA-targeted small molecule screening approach using a specialized DNA-encoded library (DEL). Our findings reveal that the specialized DEL library, called “DEL Zipper”, can significantly reduce single-stranded DNA-RNA region interaction signals during various kinds of RNA selection. By performing the selection against both G-quadruplex, we have identified novel hits that interact with RNA targets and the results are validated through binding. This study demonstrates that the “DEL Zipper” method is a robust screening assay that has potential for discovering small molecule ligands for diverse RNA targets.

迄今为止，由于缺乏可靠的筛选分析，rna靶向化学物质尚处于探索阶段。在这项研究中，我们提出了一种新的rna靶向小分子筛选方法，使用专门的dna编码文库（DEL）。我们的研究结果表明，在各种RNA选择过程中，称为“DEL Zipper”的特殊DEL文库可以显著减少单链DNA-RNA区域相互作用信号。通过对g -四重体进行选择，我们发现了与RNA靶点相互作用的新靶点，并通过结合验证了结果。这项研究表明，“DEL Zipper”方法是一种强大的筛选试验，具有发现不同RNA靶标的小分子配体的潜力。

引用次数: 0

Optimization and calibration of 384-well kinetic Ca2+ mobilization assays for the human transient receptor potential cation channels TRPM8, TRPV1, and TRPA1 人瞬时受体电位阳离子通道TRPM8、TRPV1和TRPA1 384孔动态Ca2+动员实验的优化和校准

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-01-01 Epub Date: 2024-12-31 DOI: 10.1016/j.slasd.2024.100207

David A. Close , V. Blair Journigan , Paul A. Johnston

Development, optimization, and calibration of human transient receptor potential (TRP) channel Ca²⁺ mobilization assays for TRPM8, TRPV1, and TRPA1 are described. Heterologous expression of hTRPM8 in HEK293T cells was required for anti-TRPM8 antibody staining and TRPM8 agonist induced Ca²⁺ mobilization signals which were both used to optimize transfection efficiency. FLIPR Calcium 6 dye concentration, loading time, and TRPM8 transfected cell seeding density were optimized and a DMSO tolerance of ≤0.2 % was set. The resting baseline relative fluorescent unit (RFUs) signals of the TRPM8 Ca²⁺ mobilization assay exhibited substantial well-to-well variability, even though such differences were small relative to maximal agonist induced responses. Maximum RFU, cumulative RFU sum, or area under the curve values were extracted from Ca²⁺ mobilization kinetic data to plot curves and calculate EC₅₀ and IC₅₀ values. Fold over baseline (FOB) ratio data processing eliminated well-to-well differences in resting baseline signals, reduced error bars, improved curve fits and reduced 95 % confidence interval EC₅₀ and IC₅₀ ranges. FOB ratio data processing decreased variability and improved the precision of repeat measurements in single experimental sessions thereby reducing the minimum threshold difference in EC₅₀ or IC₅₀ values required to distinguish compound potencies. EC₅₀ and IC₅₀ values of TRPM8 agonists and antagonists determined in single experiments were strongly aligned to those from multiple independent experiments. Benchmark TRPM8, TRPV1, and TRPA1 EC₅₀ and IC₅₀ values were within the ranges previously reported for agonist and antagonist standards. The improved precision and accuracy of the TRP Ca2+ mobilization assays afforded by FOB ratio data processing enhances their utility for investigating structure activity relationships.

本文描述了TRPM8、TRPV1和TRPA1的人类瞬时受体电位（TRP）通道Ca2+动员测定的开发、优化和校准。HEK293T细胞需要异源表达hTRPM8，以获得抗TRPM8抗体染色和TRPM8激动剂诱导的Ca2+动员信号，两者都用于优化转染效率。优化FLIPR钙6染料浓度、加载时间、TRPM8转染细胞播种密度，设定DMSO耐受性≤0.2%。TRPM8 Ca2+动员试验的静息基线相对荧光单位（RFUs）信号显示出大量的井间变异性，尽管这种差异相对于最大激动剂诱导的反应来说很小。从Ca2+动员动力学数据中提取最大RFU、累计RFU总和或曲线下面积，绘制曲线并计算EC50和IC50值。折叠基线（FOB）比率数据处理消除了井与井之间静息基线信号的差异，减少了误差条，改善了曲线拟合，降低了95%置信区间EC50和IC50范围。FOB比率数据处理降低了可变性，提高了单次实验中重复测量的精度，从而降低了区分化合物效价所需的EC50或IC50值的最小阈值差异。单次实验测定的TRPM8激动剂和拮抗剂的EC50和IC50值与多个独立实验的结果高度一致。基准TRPM8、TRPV1和TRPA1的EC50和IC50值均在先前报道的激动剂和拮抗剂标准范围内。FOB比率数据处理提高了TRP Ca2+动员测定的精度和准确性，提高了它们在研究结构活性关系方面的实用性。

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引用次数: 0

Miniaturization and characterization of patient derived hepatocellular carcinoma tumor organoid cultures for cancer drug discovery applications 用于癌症药物发现应用的病人来源的肝细胞癌肿瘤类器官培养的小型化和特性。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-01-01 Epub Date: 2024-12-09 DOI: 10.1016/j.slasd.2024.100201

David A. Close , Paul A. Johnston

Patient derived tumor organoid (PDTO) models retain the structural, morphological, genetic, and clonal heterogeneity of the original tumors. The ability to efficiently generate, expand, and biobank PDTOs has the potential to make the clinical diversity of cancer accessible for personalized medicine assay guided therapeutic drug selection and drug discovery. We describe the miniaturization and growth in 96- and 384-well formats of a single non-tumor liver and two Hepatocellular carcinoma (HCC) organoids derived from cryopreserved PDTO cells and the application of high content imaging (HCI) to characterize the models and enhance drug sensitivity testing. Non-invasive sequentially acquired transmitted light images showed that seeding cryopreserved cells from non-tumoral and HCC PDTOs into 96- or 384-well plates in reduced growth factor Matrigel (rgf-MG) that were fed with growth medium every 3 days supported organoid growth up to 15 days. The number and sizes of organoids increased with longer times in culture. HCC PDTO's had more heterogeneous morphologies than non-tumor organoids with respect to size, shape, and optical density. Organoids cultured in rgf-MG could be stained in situ with HCI reagents without mechanical, chemical or enzymatic disruption of the hydrogel matrices and quantitative data extracted by image analysis. Hoechst and live/dead reagents provided organoid numbers and viability comparisons. HCC PDTO's stained with phalloidin or immuno-stained with α-tubulin antibodies revealed F-actin and microtubule cytoskeleton organization. HCC PDTO's stained with antibodies to signaling pathway proteins and their phosphorylation status allowed comparisons of relative expression levels and inference of pathway activation. Images of HCC PDTO's exposed to ellipticine showed that drugs penetrate Matrigel hydrogels and accumulate in organoid cells. 9-day 384-well HCC organoid cultures exhibited robust and reproducible growth signals suitable for cancer drug testing. Complimenting cell viability readouts with multiple HCI parameters including morphological features and dead cell staining improved the analysis of drug impact and enhanced the value that could be extracted from these more physiologically relevant three-dimensional HCC organoid cultures.

患者源性肿瘤类器官（PDTO）模型保留了原始肿瘤的结构、形态、遗传和克隆异质性。有效地生成、扩展和生物库pdto的能力有可能使癌症的临床多样性可用于个性化医学检测指导的治疗药物选择和药物发现。我们描述了96孔和384孔格式的单个非肿瘤肝脏和两个肝细胞癌（HCC）类器官的小型化和生长，这些器官来自冷冻保存的PDTO细胞，并应用高含量成像（HCI）来表征模型并增强药物敏感性测试。非侵入性序列获得的透射光图像显示，将非肿瘤和HCC pto中冷冻保存的细胞接种到96孔或384孔的rgf-MG培养皿中，每3天添加一次生长培养基，可支持类器官生长长达15天。类器官的数量和大小随培养时间的延长而增加。HCC的PDTO在大小、形状和光密度方面比非肿瘤类器官具有更多的异质形态。在rgf-MG中培养的类器官可以用HCI试剂原位染色，而不会对水凝胶基质造成机械、化学或酶的破坏，也不会通过图像分析提取定量数据。赫斯特试剂和活/死试剂提供类器官数量和活力比较。phalloidin染色或α-微管蛋白抗体免疫染色显示F-actin和微管细胞骨架组织。肝癌PDTO的信号通路蛋白抗体染色及其磷酸化状态可以比较相对表达水平和途径激活的推断。肝细胞癌PDTO暴露于椭圆线的图像显示药物穿透基质水凝胶并在类器官细胞中积累。9天384孔肝细胞癌类器官培养显示出适合癌症药物测试的稳健且可重复的生长信号。用多种HCI参数（包括形态学特征和死细胞染色）补充细胞活力数据，改善了药物影响的分析，并增强了从这些更具有生理学相关性的三维HCC类器官培养中提取的价值。

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引用次数: 0

Drug repurposing screen for the rare disease ataxia-telangiectasia 罕见疾病共济失调-毛细血管扩张的药物再利用筛选。

IF 2.7 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

SLAS Discovery

Pub Date : 2025-01-01 Epub Date: 2024-12-04 DOI: 10.1016/j.slasd.2024.100200

Namrata Jayanth , Gurvan Mahé , Matthew Campbell , Mike Lipkin , Shushant Jain , Rhea van de Bospoort , Jennifer Thornton , Brad Margus , David F. Fischer

Ataxia Telangiectasia (A-T) is a rare, autosomal recessive genetic disorder characterized by a variety of symptoms, including progressive neurodegeneration, telangiectasia, immunodeficiency, and an increased susceptibility to cancer. It is caused by bi-allelic mutations impacting a gene encoding a serine/threonine kinase ATM (Ataxia Telangiectasia Mutated), which plays a crucial role in DNA repair and maintenance of genomic stability. The disorder primarily affects the nervous system, leading to a range of neurological issues, including cerebellar ataxia. The cause of neurodegeneration due to mutations in ATM is still an area of investigation, and currently there is no known treatment to slow down or stop the progression of the neurological problems.

In this collaboration of the A-T Children's Project (ATCP) with Charles River Discovery, we successfully developed a high-throughput assay using induced pluripotent stem cells (iPSC) from A-T donors to measure DNA damage response (DDR). By measuring the changes in levels of activated phosphorylated CHK2 (p-CHK2), which is a downstream signaling event of ATM, we were able to identify compounds that restore this response in the DDR pathway in A-T derived patient cells. Over 6,000 compounds from small molecule drug repurposing libraries were subsequently screened in the assay developed, leading to identification of several promising in vitro hits.

Using the assay developed and the identified hits opens avenues to investigate potential therapeutics for A-T.

共济失调毛细血管扩张症（a - t）是一种罕见的常染色体隐性遗传病，其特征是多种症状，包括进行性神经变性、毛细血管扩张、免疫缺陷和对癌症的易感性增加。它是由双等位基因突变影响编码丝氨酸/苏氨酸激酶ATM（共济失调毛细血管扩张突变）的基因引起的，该基因在DNA修复和基因组稳定性的维持中起着至关重要的作用。这种疾病主要影响神经系统，导致一系列神经问题，包括小脑性共济失调。由ATM突变引起的神经退行性变的原因仍是一个研究领域，目前还没有已知的治疗方法来减缓或阻止神经问题的进展。在a -t儿童项目（ATCP）与Charles River Discovery的合作中，我们成功开发了一种使用来自a -t供体的诱导多能干细胞（iPSC）来测量DNA损伤反应（DDR）的高通量测定方法。通过测量活化磷酸化CHK2 （p-CHK2）水平的变化，这是ATM的下游信号事件，我们能够识别在a - t衍生的患者细胞中恢复DDR途径中这种反应的化合物。随后，从小分子药物再利用文库中筛选了6000多种化合物，并确定了几种有前景的体外命中。使用开发的检测方法和确定的命中点为研究A-T的潜在治疗方法开辟了道路。

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引用次数: 0