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The IBEX knowledge-base a community resource enabling adoption and development of immunofluorescence imaging methods. IBEX知识库是一种社区资源,能够采用和开发免疫荧光成像方法。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-23 DOI: 10.7554/eLife.105737
Ziv Yaniv, Ifeanyichukwu U Anidi, Leanne Arakkal, Armando J Arroyo-Mejías, Rebecca T Beuschel, Katy Börner, Colin J Chu, Beatrice H Clark, Menna R Clatworthy, Jake Colautti, Fabian Coscia, Joshua Croteau, Saven Denha, Rose Dever, Walderez O Dutra, Sonja Fritzsche, Spencer Fullam, Michael Y Gerner, Anita Gola, Kenneth J Gollob, Jonathan M Hernandez, Jyh Liang Hor, Hiroshi Ichise, Zhixin Jing, Danny Jonigk, Evelyn Kandov, Wolfgang Kastenmüller, Joshua F E Koenig, Aanandita Kothurkar, Rosa K Kortekaas, Alexandra Y Kreins, Ian T Lamborn, Yuri Lin, Katia Luciano Pereira Morais, Aleksandra Lunich, Jean C S Luz, Ryan B MacDonald, Chen Makranz, Vivien I Maltez, John E McDonough, Ryan V Moriarty, Juan M Ocampo-Godinez, Vitoria Murakami Olyntho, Annette Oxenius, Kartika Padhan, Kirsten Remmert, Nathan Richoz, Edward C Schrom, Wanjing Shang, Lihong Shi, Rochelle M Shih, Emily Speranza, Salome Stierli, Sarah A Teichmann, Tibor Z Verse, Megan Vierhout, Brianna T Wachter, Adam K Wade-Vallance, Margaret Williams, Nathan Zangger, Ronald N Germain, Andrea J Radtke

The iterative bleaching extends multiplexity (IBEX) Knowledge-Base is a central portal for researchers adopting IBEX and related 2D and 3D immunofluorescence imaging methods. The design of the Knowledge-Base is modeled after efforts in the open-source software community and includes three facets: a development platform (GitHub), static website, and service for data archiving. The Knowledge-Base facilitates the practice of open science throughout the research life cycle by providing validation data for recommended and non-recommended reagents, such as primary and secondary antibodies. In addition to reporting negative data, the Knowledge-Base empowers method adoption and evolution by providing a venue for sharing protocols, videos, datasets, software, and publications. A dedicated discussion forum fosters a sense of community among researchers while addressing questions not covered in published manuscripts. Together, scientists from around the world are advancing scientific discovery at a faster pace, reducing wasted time and effort, and instilling greater confidence in the resulting data.

迭代漂白扩展多重性(IBEX)知识库是研究人员采用IBEX及相关的二维和三维免疫荧光成像方法的中心门户。知识库的设计借鉴了开源软件社区的努力,包括三个方面:开发平台(GitHub)、静态网站和数据归档服务。该知识库通过提供推荐和非推荐试剂(如一抗和二抗)的验证数据,促进了开放科学在整个研究生命周期中的实践。除了报告负面数据外,知识库还通过提供共享协议、视频、数据集、软件和出版物的场所,支持方法的采用和发展。一个专门的讨论论坛促进了研究人员之间的社区意识,同时解决了未在发表的手稿中涵盖的问题。来自世界各地的科学家共同努力,以更快的速度推进科学发现,减少浪费的时间和精力,并增强对结果数据的信心。
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引用次数: 0
Doubling dolutegravir dosage reduces the viral reservoir in ART-treated people with HIV. 加倍多替格拉韦剂量可减少接受抗逆转录病毒治疗的艾滋病毒感染者体内的病毒库。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-23 DOI: 10.7554/eLife.106931
Céline Fombellida-Lopez, Aurelija Valaitienė, Lee Winchester, Nathalie Maes, Patricia Dellot, Céline Vanwinge, Aurélie Ladang, Etienne Cavalier, Fabrice Susin, Dolores Vaira, Marie-Pierre Hayette, Catherine Reenaers, Michel Moutschen, Courtney V Fletcher, Alexander O Pasternak, Gilles Darcis

Whether antiretroviral therapy (ART) is always completely suppressive, or HIV might continue to replicate at low levels despite ART in some people with HIV (PWH), is still debated. Here, we intensified the ART regimen by doubling dolutegravir (DTG) dosage and investigated the impact of this strategy on HIV blood and tissue reservoirs, immune activation, and inflammation. Twenty HIV-infected adults, who had received a triple ART consisting of 50 mg DTG/600 mg abacavir/300 mg lamivudine pre-intensification and had been suppressed on ART for at least 2 years, were enrolled in a phase 2 randomized clinical trial (https://clinicaltrials.gov/ identifier: NCT05351684). Half of them received an additional 50 mg of DTG for a period of 84 days. As expected, plasma and tissue DTG concentrations significantly increased during the study period in the intensified group but not in the control group. Accordingly, significant decreases in total HIV DNA, intact HIV DNA, and cell-associated unspliced (US) HIV RNA in peripheral blood mononuclear cells, as well as in the US RNA/total DNA ratio, were observed in the intensified group but not in the control group. Intensification also modestly reduced markers of immune activation and exhaustion but had no measurable impact on systemic or tissue inflammation. Together with this, intensification resulted in a temporary decrease in the CD4/CD8 ratio that returned to baseline by day 84. Our results strongly suggest that the pre-intensification ART regimen was not completely suppressive. If confirmed in larger clinical trials, these results could have an impact on the clinical management of PWH and HIV curative strategies.

抗逆转录病毒治疗(ART)是否总是完全抑制,或者艾滋病毒可能继续在低水平复制,尽管抗逆转录病毒治疗在一些艾滋病毒感染者(PWH)中,仍然存在争议。在这里,我们通过加倍多替格拉韦(DTG)剂量来加强抗逆转录病毒治疗方案,并研究这一策略对HIV血液和组织储库、免疫激活和炎症的影响。20名接受了由50 mg DTG/600 mg阿巴卡韦/300 mg拉米夫定预强化组成的三联抗逆转录病毒治疗的艾滋病毒感染成人被纳入了一项2期随机临床试验(https://clinicaltrials.gov/标识码:NCT05351684)。其中一半的人在84天的时间里额外服用了50毫克的DTG。正如预期的那样,在研究期间,强化组的血浆和组织DTG浓度显著增加,而对照组则没有。因此,在强化组中观察到外周血单个核细胞中总HIV DNA、完整HIV DNA和细胞相关的未剪接(US) HIV RNA以及US RNA/总DNA比率显著降低,而在对照组中则没有。强化也适度降低了免疫激活和衰竭的标志物,但对全身或组织炎症没有可测量的影响。与此同时,强化导致CD4/CD8比值暂时下降,到第84天恢复到基线水平。我们的结果强烈提示强化前ART治疗方案并不是完全抑制。如果在更大规模的临床试验中得到证实,这些结果可能对PWH的临床管理和HIV治疗策略产生影响。
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引用次数: 0
Computational modelling identifies key determinants of subregion-specific dopamine dynamics in the striatum. 计算模型确定了纹状体中亚区域特异性多巴胺动力学的关键决定因素。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-23 DOI: 10.7554/eLife.105214
Aske Ejdrup, Jakob Kisbye Dreyer, Matthew D Lycas, Søren H Jørgensen, Trevor W Robbins, Jeffrey Dalley, Freja Herborg, Ulrik Gether

Striatal dopamine (DA) release regulates reward-related learning and motivation and is believed to consist of a short-lived phasic and continuous tonic component. Here, we build a large-scale three-dimensional model of extracellular DA dynamics in dorsal (DS) and ventral striatum (VS). The model predicts rapid dynamics in DS with little to no basal DA and slower dynamics in the VS enabling build-up of tonic DA levels. These regional differences do not reflect release-related phenomena but rather differential dopamine transporter (DAT) activity. Interestingly, our simulations posit DAT nanoclustering as a possible regulator of this activity. Receptor binding simulations show that D1 receptor occupancy follows extracellular DA concentration with milliseconds delay, while D2 receptors do not respond to brief pauses in firing but rather integrate DA signal over seconds. Summarised, our model distills recent experimental observations into a computational framework that challenges prevailing paradigms of striatal DA signalling.

纹状体多巴胺(DA)的释放调节与奖励相关的学习和动机,被认为是由一个短暂的阶段性和连续的强直成分组成。在此,我们建立了背侧纹状体(DS)和腹侧纹状体(VS)细胞外DA动力学的大尺度三维模型。该模型预测了DS的快速动态,几乎没有基础DA,而VS的缓慢动态使强直DA水平得以建立。这些区域差异并不反映释放相关现象,而是反映多巴胺转运体(DAT)活性的差异。有趣的是,我们的模拟假设DAT纳米聚类可能是这种活动的调节器。受体结合模拟表明,D1受体的占用伴随着细胞外DA浓度的毫秒延迟,而D2受体对放电的短暂暂停没有反应,而是在几秒钟内整合DA信号。总之,我们的模型将最近的实验观察提炼成一个计算框架,挑战纹状体数据传导的主流范式。
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引用次数: 0
Robust assessment of asymmetric division in colon cancer cells. 结肠癌细胞不对称分裂的可靠评估。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-23 DOI: 10.7554/eLife.104528
Domenico Caudo, Chiara Giannattasio, Simone Scalise, Valeria de Turris, Fabio Giavazzi, Giancarlo Ruocco, Giorgio Gosti, Giovanna Peruzzi, Mattia Miotto

Asymmetric partition of fate determinants during cell division is a hallmark of cell differentiation. Recent work suggested that such a mechanism is hijacked by cancer cells to increase both their phenotypic heterogeneity and plasticity and, in turn, their fitness. To quantify fluctuations in the partitioning of cellular elements, imaging-based approaches are used, whose accuracy is limited by the difficulty of detecting cell divisions. Our work addresses this gap, proposing a general method based on high-throughput flow cytometry measurements coupled with a theoretical framework. We applied our method to a panel of both normal and cancerous human colon cells, showing that different kinds of colon adenocarcinoma cells display very distinct extents of fluctuations in their cytoplasm partition, explained by an asymmetric division of their size. To test the accuracy of our population-level protocol, we directly measure the inherited fractions of cellular elements from extensive time lapses of live-cell laser scanning microscopy, finding excellent agreement across the cell types. Ultimately, our flow cytometry-based method promises to be accurate and easily applicable to a wide range of biological systems where the quantification of partition fluctuations would help account for the observed phenotypic heterogeneity and plasticity.

细胞分裂过程中命运决定因子的不对称分裂是细胞分化的标志。最近的研究表明,这种机制被癌细胞劫持,增加了它们的表型异质性和可塑性,进而增加了它们的适应性。为了量化细胞元素分裂的波动,使用了基于成像的方法,其准确性受到检测细胞分裂的困难的限制。我们的工作解决了这一差距,提出了一种基于高通量流式细胞术测量和理论框架的通用方法。我们将我们的方法应用于一组正常和癌变的人类结肠细胞,结果表明,不同种类的结肠腺癌细胞在其细胞质分裂中表现出非常不同程度的波动,这可以用它们大小的不对称分裂来解释。为了测试我们的群体水平协议的准确性,我们直接从活细胞激光扫描显微镜的大量时间间隔中测量细胞元素的遗传部分,在细胞类型中发现了极好的一致性。最终,我们基于流式细胞术的方法有望准确且易于适用于广泛的生物系统,其中分区波动的量化将有助于解释观察到的表型异质性和可塑性。
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引用次数: 0
HIV-specific CD8+ T-cell proliferative response 24 weeks after early antiretroviral therapy initiation is associated with the subsequent reduction in the viral reservoir. 早期抗逆转录病毒治疗开始后24周的hiv特异性CD8+ t细胞增殖反应与随后病毒库的减少有关。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-23 DOI: 10.7554/eLife.106402
Pien Margien van Paassen, Alexander O Pasternak, Dita C Bolluyt, Karel A van Dort, Ad C van Nuenen, Irma Maurer, Brigitte Boeser-Nunnink, Ninée V E J Buchholtz, Tokameh Mahmoudi, Cynthia Lungu, Reinout van Crevel, Casper Rokx, Jori Symons, Monique Nijhuis, Annelou L I P van der Veen, Liffert Vogt, Michelle J Klouwens, Jan M Prins, Neeltje A Kootstra, Godelieve J de Bree

Antiretroviral therapy (ART) initiated in the acute phase of HIV infection (AHI) results in a smaller viral reservoir. However, the impact of early HIV-specific T-cell responses on long-term reservoir dynamics is less well characterized. Therefore, we measured the size of the viral reservoir and functionality of HIV-specific CD8+ T-cell responses after the acute phase at 24 and 156 weeks after ART initiation in people with HIV who started treatment during AHI. A significant reduction in total and defective HIV DNA and a trend toward a reduction in intact HIV DNA were observed between 24 and 156 weeks. Functional CD8+ T-cell responses against HIV peptides Env, Gag, Nef, and Pol were maintained over 3 years after treatment initiation. The proliferative capacity of HIV-specific CD8+ T-cells at 24 weeks of ART was predictive of the degree of reduction in total and defective HIV DNA between 24 and 156 weeks, suggesting HIV-specific CD8+ T-cells may at least partially drive the decline of the viral reservoir. Therefore, enforcing HIV-specific immune responses as early as possible after diagnosis of AHI should be a central focus of HIV cure strategies.

在HIV感染(AHI)的急性期开始抗逆转录病毒治疗(ART)导致更小的病毒库。然而,早期hiv特异性t细胞反应对长期蓄水池动力学的影响尚不清楚。因此,我们测量了病毒库的大小和艾滋病毒特异性CD8+ t细胞反应的功能,在急性期,在开始抗逆转录病毒治疗后24周和156周,在AHI期间开始治疗的艾滋病毒感染者中。在24周至156周期间,观察到总HIV DNA和缺陷HIV DNA的显著减少以及完整HIV DNA减少的趋势。CD8+ t细胞对HIV多肽Env、Gag、Nef和Pol的功能反应在治疗开始后维持3年以上。抗逆转录病毒治疗24周时,HIV特异性CD8+ t细胞的增殖能力可预测24周至156周期间总HIV DNA和缺陷HIV DNA的减少程度,这表明HIV特异性CD8+ t细胞可能至少部分驱动病毒库的下降。因此,在诊断出AHI后尽早实施HIV特异性免疫反应应该是HIV治愈策略的中心重点。
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引用次数: 0
Feedback of peripheral saccade targets to early foveal cortex. 外周扫视目标向早期中央凹皮层反馈。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-22 DOI: 10.7554/eLife.107053
Luca Kämmer, Lisa M Kroell, Tomas Knapen, Martin Rolfs, Martin N Hebart

Human vision is characterized by frequent eye movements and constant shifts in visual input, yet our perception of the world remains remarkably stable. Here, we directly demonstrate image-specific foveal feedback to primary visual cortex in the context of saccadic eye movements. To this end, we used a gaze-contingent fMRI paradigm, in which peripheral saccade targets disappeared before they could be fixated. Despite no direct foveal stimulation, we were able to decode peripheral saccade targets from foveal retinotopic areas, demonstrating that image-specific feedback during saccade preparation may underlie this effect. Decoding was sensitive to shape but not semantic category of natural images, indicating feedback of only low-to-mid-level information. Cross-decoding to a control condition with foveal stimulus presentation indicates a shared representational format between foveal feedback and direct stimulation. Moreover, eccentricity-dependent analyses showed a U-shaped decoding curve, confirming that these results are not explained by spillover of peripheral activity or large receptive fields. Finally, fluctuations in foveal decodability covaried with activity in the intraparietal sulcus, thus providing a candidate region for driving foveal feedback. These findings suggest that foveal cortex predicts the features of incoming stimuli through feedback from higher cortical areas, which offers a candidate mechanism underlying stable perception.

人类视觉的特点是频繁的眼球运动和视觉输入的不断变化,但我们对世界的感知仍然非常稳定。在这里,我们直接证明了在跳眼运动的背景下,初级视觉皮层的图像特异性中央凹反馈。为此,我们使用了注视-偶然fMRI范式,其中外周扫视目标在被注视之前就消失了。尽管没有直接的中央凹刺激,但我们能够从中央凹视网膜定位区域解码外周扫视目标,证明在扫视准备过程中的图像特异性反馈可能是这种效果的基础。解码对自然图像的形状敏感,但对语义类别不敏感,表明仅对中低层次信息进行反馈。交叉解码到具有中央凹刺激呈现的控制条件表明中央凹反馈与直接刺激之间具有共同的表征格式。此外,与偏心率相关的分析显示了u型解码曲线,证实了这些结果不能用外周活动溢出或大的接受野来解释。最后,中央凹可解码性的波动与顶叶内沟的活动共同变化,从而提供了驱动中央凹反馈的候选区域。这些发现表明,中央凹皮层通过来自高层皮质区的反馈来预测传入刺激的特征,这为稳定感知提供了一种候选机制。
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引用次数: 0
A titin missense variant drives atrial electrical remodeling and is associated with atrial fibrillation. 一种titin错义变体驱动心房电重构并与心房颤动相关。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-22 DOI: 10.7554/eLife.104719
Mahmud Arif Pavel, Hanna Chen, Michael Hill, Arvind Sridhar, Miles Barney, Jaime DeSantiago, Abhinaya Baskaran, Asia Owais, Shashank Sandu, Faisal A Darbar, Aylin Ornelas Loredo, Bahaa Al-Azzam, Brandon Chalazan, Jalees Rehman, Dawood Darbar

Rare and common genetic variants contribute to the risk of atrial fibrillation (AF). Although ion channels were among the first AF candidate genes identified, rare loss-of-function variants in structural genes, such as TTN, have also been implicated in AF pathogenesis, partly through the development of atrial myopathy; however, the underlying mechanisms are poorly understood. While TTN truncating variants (TTNtvs) have been causally linked to arrhythmia and cardiomyopathy syndromes, the role of missense variants (mvs) remains unclear. We show that rare TTNmvs are associated with worse clinical outcomes in a single-center ethnic minority clinical cohort and uncover a pathogenic mechanism by which the T32756I variant drives AF. Modeling the TTN-T32756I variant using human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-aCMs) revealed that the mutant cells display aberrant contractility, increased activity of a cardiac potassium channel (KCNQ1, Kv7.1), and dysregulated calcium homeostasis without compromising the sarcomeric integrity of the atrial cardiomyocytes. We also show that a titin-binding protein, the Four-and-a-Half Lim domains 2 (FHL2), has increased binding with KCNQ1 and its modulatory subunit KCNE1 in the TTN-T32756I-iPSC-aCMs, enhancing the slow delayed rectifier potassium current (Iks). Suppression of FHL2 in mutant iPSC-aCMs normalized the Iks, supporting FHL2 as an Iks modulator. Our findings demonstrate that a single amino acid substitution in titin not only impairs its function but also remodels ion channels, contributing to AF. These findings underscore the importance of high-throughput screening to assess the pathogenicity of TTNmvs and establish a mechanistic connection between titin, potassium ion channels, and sarcomeric proteins, which may represent a novel therapeutic target.

罕见和常见的基因变异有助于心房颤动(AF)的风险。虽然离子通道是最早发现的房颤候选基因之一,但结构基因中罕见的功能缺失变异,如TTN,也与房颤发病有关,部分是通过心房肌病的发展;然而,人们对其潜在机制知之甚少。虽然TTN截断变异(ttntv)与心律失常和心肌病综合征有因果关系,但错义变异(mvs)的作用仍不清楚。我们发现,在单中心少数民族临床队列中,罕见的TTNmvs与较差的临床结果相关,并揭示了T32756I变体驱动AF的致病机制。利用人诱导多能干细胞来源的心房心肌细胞(iPSC-aCMs)对TTN-T32756I变体进行建模发现,突变细胞表现出异常的收缩性,增加了心脏钾通道的活性(KCNQ1, Kv7.1),在不损害心房心肌细胞肌体完整性的情况下,钙稳态失调。我们还发现,在TTN-T32756I-iPSC-aCMs中,一种titin结合蛋白,4 -半Lim结构域2 (FHL2),增加了与KCNQ1及其调节亚基KCNE1的结合,增强了慢延迟整流钾电流(Iks)。突变iPSC-aCMs中FHL2的抑制使Iks正常化,支持FHL2作为Iks调节剂。我们的研究结果表明,titin中单个氨基酸的取代不仅会损害其功能,还会重塑离子通道,从而导致AF。这些发现强调了高通量筛选对评估TTNmvs的致病性的重要性,并建立了titin、钾离子通道和肌肉蛋白之间的机制联系,这可能是一种新的治疗靶点。
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引用次数: 0
The cellular logic of limb regeneration. 肢体再生的细胞逻辑。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-22 DOI: 10.7554/eLife.110316
Matthew Cherubino, Catherine D McCusker

Hierarchical communication between cells with distinct positional memories orchestrates the regeneration of missing limb structures in axolotls.

具有不同位置记忆的细胞之间的层次交流协调了蝾螈缺失肢体结构的再生。
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引用次数: 0
Anti-resonance in developmental signaling regulates cell fate decisions. 发育信号中的反共振调节细胞命运决定。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-21 DOI: 10.7554/eLife.107794
Samuel J Rosen, Olivier Witteveen, Naomi Baxter, Ryan S Lach, Erik Hopkins, Marianne Bauer, Maxwell Z Wilson

Cells process dynamic signaling inputs to regulate fate decisions during development. While oscillations or waves in key developmental pathways, such as Wnt, have been widely observed, the principles governing how cells decode these signals remain unclear. By leveraging optogenetic control of the Wnt signaling pathway in both HEK293T cells and H9 human embryonic stem cells, we systematically map the relationship between signal frequency and downstream pathway activation. We find that cells exhibit a minimal response to Wnt at certain frequencies, a behavior we term anti-resonance. We developed both detailed biochemical and simplified hidden variable models that explain how anti-resonance emerges from the interplay between fast and slow pathway dynamics. Remarkably, we find that frequency directly influences cell fate decisions involved in human gastrulation; signals delivered at anti-resonant frequencies result in dramatically reduced mesoderm differentiation. Our work reveals a previously unknown mechanism of how cells decode dynamic signals and how anti-resonance may filter against spurious activation. These findings establish new insights into how cells decode dynamic signals with implications for tissue engineering, regenerative medicine, and cancer biology.

细胞在发育过程中处理动态信号输入来调节命运决定。虽然在关键的发育通路(如Wnt)中振荡或波动已被广泛观察到,但控制细胞如何解码这些信号的原理仍不清楚。通过利用HEK293T细胞和H9人胚胎干细胞中Wnt信号通路的光遗传学控制,我们系统地绘制了信号频率与下游通路激活之间的关系。我们发现细胞在某些频率下对Wnt表现出最小的反应,我们称之为反共振。我们开发了详细的生化和简化的隐变量模型,解释了反共振如何从快速和慢速途径动力学之间的相互作用中出现。值得注意的是,我们发现频率直接影响人类原肠胚形成过程中的细胞命运决定;以反共振频率传递的信号导致中胚层分化显著降低。我们的工作揭示了一种以前未知的机制,即细胞如何解码动态信号以及抗共振如何过滤虚假激活。这些发现为细胞如何解码动态信号建立了新的见解,对组织工程、再生医学和癌症生物学具有重要意义。
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引用次数: 0
Ω-Loop mutations control dynamics of the active site by modulating the hydrogen-bonding network in PDC-3 β-lactamase. Ω-Loop突变通过调节PDC-3 β-内酰胺酶的氢键网络来控制活性位点的动力学。
IF 6.4 1区 生物学 Q1 BIOLOGY Pub Date : 2026-01-21 DOI: 10.7554/eLife.107688
Shuang Chen, Andrew R Mack, Andrea M Hujer, Christopher R Bethel, Robert A Bonomo, Shozeb Haider

The expression of antibiotic-inactivating enzymes, such as Pseudomonas-derived cephalosporinase-3 (PDC-3), is a major mechanism of intrinsic resistance in bacteria. Using reinforcement learning-driven molecular dynamics simulations and constant pH MD, we investigate how clinically observed mutations in the Ω-loop (at residues V211, G214, E219, and Y221) alter the structure and function of PDC-3. Our findings reveal that these substitutions modulate the dynamic flexibility of the Ω-loop and the R2-loop, reshaping the cavity of the active site. In particular, E219K and Y221A disrupt the tridentate hydrogen bond network around K67, thus lowering its pKa and promoting proton transfer to the catalytic residue S64. Markov state models reveal that E219K achieves enhanced catalysis by adopting stable, long-lived 'active' conformations, whereas Y221A facilitates activity by rapidly toggling between bond-formed and bond-broken states. In addition, substitutions influence key hydrogen bonds that control the opening and closure of the active-site pocket, consequently influencing the overall size. The pocket expands in all nine clinically identified variants, creating additional space to accommodate bulkier R1 and R2 cephalosporin side chains. Taken together, these results provide a mechanistic basis for how single residue substitutions in the Ω-loop affect catalytic activity. Insights into the structural dynamics of the catalytic site advance our understanding of emerging β-lactamase variants and can inform the rational design of novel inhibitors to combat drug-resistant P. aeruginosa.

抗生素失活酶的表达,如假单胞菌衍生的头孢菌素酶-3 (PDC-3),是细菌内在耐药的主要机制。使用强化学习驱动的分子动力学模拟和恒定pH MD,我们研究了临床观察到的Ω-loop(残基V211, G214, E219和Y221)突变如何改变PDC-3的结构和功能。我们的研究结果表明,这些取代调节了Ω-loop和r2环的动态灵活性,重塑了活性位点的空腔。特别是E219K和Y221A破坏了K67周围的三叉氢键网络,从而降低了K67的pKa,促进了质子向催化残基S64的转移。马尔可夫状态模型显示,E219K通过采用稳定、长寿命的“活性”构象实现了增强的催化作用,而Y221A通过在键形成和键断裂状态之间快速切换来促进活性。此外,取代会影响控制活性位点口袋打开和关闭的关键氢键,从而影响整体尺寸。在所有9种临床鉴定的变异中,囊袋都会扩大,从而产生额外的空间来容纳体积较大的R1和R2头孢菌素侧链。综上所述,这些结果为Ω-loop中单残基取代如何影响催化活性提供了机理基础。对催化位点结构动力学的深入了解促进了我们对新出现的β-内酰胺酶变体的理解,并可以为合理设计新型抑制剂来对抗耐药铜绿假单胞菌提供信息。
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