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Design of a wasp-inspired biopsy needle capable of self-propulsion and friction-based tissue transport. 黄蜂启发的活检针的设计,能够自我推进和基于摩擦的组织运输。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1497221
Jette Bloemberg, Suzanne van Wees, Vera G Kortman, Aimée Sakes

Percutaneous pancreatic core biopsy is conclusive but challenging due to large-diameter needles, while smaller-diameter needles used in aspiration methods suffer from buckling and clogging. Inspired by the ovipositor of parasitic wasps, which resists buckling through self-propulsion and prevents clogging via friction-based transport, research has led to the integration of these functionalities into multi-segment needle designs or tissue transport system designs. This study aimed to combine these wasp-inspired functionalities into a single biopsy needle by changing the interconnection of the needle segments. The resulting biopsy needle features six parallel needle segments interconnected by a ring passing through slots along the length of the needle segments, enabling a wasp-inspired reciprocating motion. Actuation employs a cam and follower mechanism for controlled translation of the segments. The needle prototype, constructed from nitinol rods and stainless steel rings, measures 3 mm in outer diameter and 1 mm in inner diameter. Testing in gelatin phantoms demonstrated efficient gelatin core transport (up to 69.9% ± 9.1% transport efficiency) and self-propulsion (0.842 ± 0.042 slip ratio). Future iterations should aim to reduce the outer diameter while maintaining tissue yield. The design offers a promising new avenue for wasp-inspired medical tools, potentially enhancing early pancreatic cancer detection, thus reducing healthcare costs and patient complications.

经皮胰腺核心活检是决定性的,但具有挑战性,因为大直径的针,而小直径的针在穿刺方法中遭受屈曲和堵塞。受寄生蜂产卵器的启发,研究人员将这些功能整合到多节段针设计或组织运输系统设计中。寄生蜂产卵器通过自我推进来抵抗屈曲,并防止摩擦运输造成堵塞。本研究旨在通过改变针段的互连,将这些受黄蜂启发的功能结合到单个活检针中。由此产生的活检针具有六个平行的针段,通过沿针段长度穿过槽的环相互连接,从而实现黄蜂启发的往复运动。驱动器采用凸轮和从动件机构来控制各部分的平移。针原型由镍钛诺棒和不锈钢环构成,外径3毫米,内径1毫米。在明胶模型中进行的测试显示了高效的明胶核心运输(高达69.9%±9.1%的运输效率)和自我推进(0.842±0.042的滑移比)。未来的迭代应旨在减少外径,同时保持组织产量。该设计为黄蜂启发的医疗工具提供了一条有前途的新途径,有可能提高胰腺癌的早期检测,从而降低医疗成本和患者并发症。
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引用次数: 0
Research on upper limb rehabilitation assessment model based on belief rule base. 基于信念规则库的上肢康复评估模型研究。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1469598
Dawei Jiang, Zixu Zhao, Lijun Wang, Chao Zhang, Meixuan He, Tiejun Ji

Rehabilitation assessments hold an irreplaceable role in the field of rehabilitative therapy. However, due to the subjectivity of traditional physicians and the variability of patient conditions, this leads to a lack of detailed grading and inaccurate assessment results. To address this issue, we developed an upper limb rehabilitation evaluation model. This model integrates muscle strength assessment methods and the Belief Rule Base (BRB), along with qualitative knowledge such as clinical rehabilitation theories and expert experiences. It also utilizes training data from actual patients, collected by an upper limb rehabilitation robot. We then optimized the BRB model's evaluation accuracy using the Fmincon algorithm and compared its result with commonly used methods such as the Back Propagation (BP) neural network and Support Vector Machine (SVM). This comparison validated the effectiveness and advancement of our BRB approach. This work has laid both a theoretical and practical groundwork for developing a clinical decision support system based on the BRB for upper limb rehabilitation evaluations.

康复评估在康复治疗领域具有不可替代的作用。然而,由于传统医生的主观性和患者病情的可变性,这导致缺乏详细的分级和不准确的评估结果。为了解决这个问题,我们开发了一个上肢康复评估模型。该模型融合了肌力评估方法和信念规则库(Belief Rule Base, BRB),以及临床康复理论和专家经验等定性知识。它还利用上肢康复机器人收集的实际患者的训练数据。然后,我们使用Fmincon算法优化了BRB模型的评估精度,并将其结果与常用方法如BP神经网络和支持向量机(SVM)进行了比较。这个比较验证了我们的BRB方法的有效性和先进性。本研究为开发基于BRB的上肢康复评估临床决策支持系统奠定了理论和实践基础。
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引用次数: 0
Comparative analysis of solvent-based and solvent-free (melting) methods for fabricating 3D-printed polycaprolactone-hydroxyapatite composite bone scaffolds: physicochemical/mechanical analyses and in vitro cytocompatibility. 制备3d打印聚己内酯-羟基磷灰石复合骨支架的溶剂基和无溶剂(熔融)方法的比较分析:理化/力学分析和体外细胞相容性。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1473777
Brigita De Vega, Abir Dutta, Aisha Mumtaz, Bob C Schroeder, Craig Gerrand, Ashleigh S Boyd, Deepak M Kalaskar

Purpose: The study conducts a comparative analysis between two prominent methods for fabricating composites for bone scaffolds-the (solid) solvent method and the solvent-free (melting) method. While previous research has explored these methods individually, this study provides a direct comparison of their outcomes in terms of physicochemical properties, cytocompatibility, and mechanical strength. We also analyse their workflow and scalability potentials.

Design/methodology/approach: Polycaprolactone (PCL) and hydroxyapatite (HA) composites were prepared using solvent (chloroform) and melting (180°C) methods, then 3D-printed using an extrusion-based 3D printer to fabricate scaffolds (8 × 8 × 4 mm). Rheology, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), accelerated degradation, mechanical/compression test, wettability/contact angle, live/dead assay, and DNA quantification (Picogreen) assays were evaluated.

Findings: The study finds that scaffolds made via the solid solvent method have higher mechanical strength and degradation rate as compared to those from the melting method, while both methods ensure adequate cytocompatibility and homogenous hydroxyapatite distribution, supporting their use in bone tissue engineering.

Originality: This research investigates the utility of chloroform as a solvent for PCL composite in a direct comparison with the melting method. It also highlights the differences in workflows between the two methods and their scalability implications, emphasizing the importance of considering workflow efficiency and the potential for automation in scaffold fabrication processes for bone tissue engineering applications.

目的:对骨支架复合材料的两种主要制备方法——(固体)溶剂法和无溶剂(熔融)法进行比较分析。虽然以前的研究已经分别探索了这些方法,但本研究在物理化学性质、细胞相容性和机械强度方面提供了直接比较。我们还分析了它们的工作流程和可扩展性潜力。设计/方法/方法:采用溶剂(氯仿)和熔融(180℃)的方法制备聚己内酯(PCL)和羟基磷灰石(HA)复合材料,然后使用基于挤压的3D打印机3D打印制造支架(8 × 8 × 4 mm)。对流变学、扫描电镜(SEM)、能量色散x射线光谱(EDX)、傅里叶变换红外光谱(FTIR)、x射线衍射(XRD)、热重分析(TGA)、差示扫描量热法(DSC)、加速降解、力学/压缩试验、润湿性/接触角、活/死试验和DNA定量(Picogreen)试验进行了评估。研究发现:固体溶剂法制备的支架比熔融法制备的支架具有更高的机械强度和降解率,且两种方法均具有良好的细胞相容性和羟基磷灰石均匀分布,支持其在骨组织工程中的应用。独创性:本研究考察了氯仿作为PCL复合材料溶剂的实用性,并与熔融法进行了直接比较。它还强调了两种方法之间工作流程的差异及其可扩展性的影响,强调了考虑工作流程效率的重要性以及骨组织工程应用中支架制造过程自动化的潜力。
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引用次数: 0
Validation of the efficacy of the porous medium model in hemodynamic analysis of iliac vein compression syndrome. 多孔介质模型在髂静脉压迫综合征血流动力学分析中的有效性验证。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1481336
Lingling Wei, Ke Hu, Jiaqiu Wang, Shuang Zhang, Xiaoxiao Yang, Yuanli Chen, Chenshu Li, Xinwu Lu, Kaichuang Ye, Peng Qiu, Yanqing Zhan

Iliac Vein Compression Syndrome (IVCS) is a common risk factor for deep vein thrombosis in the lower extremities. The objective of this study was to investigate whether employing a porous medium model to simulate the compressed region of an iliac vein could improve the reliability and accuracy of Computational Fluid Dynamics (CFD) analysis outcomes of IVCS. Pre-operative Computed Tomography (CT) scan images of patients with IVCS were utilized to reconstruct models illustrating both the compression and collateral circulation of the iliac vein. A porous medium model was employed to simulate the compressed region of the iliac vein. The agreements of times to peak between discrete phase particles in CFD analysis and contrast agent particles in Digital Subtraction Angiography (DSA) were compared. Furthermore, comparisons were made between the CFD analysis results that incorporated the porous media and those that did not. The results revealed that in the CFD analysis incorporating the porous media model, more than 80% of discrete phase particles reached the inferior vena cava via collateral circulation. Additionally, the concentration variation curve of discrete phase particles demonstrated a high concordance rate of 92.4% compared to that obtained in DSA. In comparison to CFD analysis conducted without the porous medium model, the incorporation of the porous medium model resulted in a substantial decrease in blood flow velocity by 87.5% within the compressed region, a significant increase in pressure gradient of 141 Pa between the inferior vena cava and left iliac vein, and a wider distribution of wall shear stress exceeding 2.0 Pa in collateral vessels rather than in the compressed region. The study suggests that the introduction of a porous medium model improves the hemodynamic analysis of patients with IVCS, resulting in a closer alignment with clinical observations. This provides a novel theoretical framework for the assessment and treatment of patients with IVCS.

髂静脉压迫综合征(IVCS)是下肢深静脉血栓形成的常见危险因素。本研究的目的是探讨采用多孔介质模型模拟髂静脉受压区域是否可以提高IVCS计算流体动力学(CFD)分析结果的可靠性和准确性。利用IVCS患者术前CT扫描图像重建髂静脉压迫和侧支循环的模型。采用多孔介质模型模拟髂静脉受压区。比较了CFD分析中离散相颗粒与数字减影血管造影(DSA)中造影剂颗粒的峰值时间一致性。此外,还比较了考虑多孔介质和未考虑多孔介质的CFD分析结果。结果表明,在采用多孔介质模型的CFD分析中,超过80%的离散相颗粒通过侧支循环到达下腔静脉。与DSA相比,离散相颗粒浓度变化曲线的一致性高达92.4%。与未加入多孔介质模型的CFD分析相比,加入多孔介质模型后,压缩区域内的血流速度大幅下降87.5%,下腔静脉与左髂静脉之间的压力梯度显著增加141 Pa,侧枝血管壁面剪应力分布更广,超过2.0 Pa。该研究表明,多孔介质模型的引入改善了IVCS患者的血流动力学分析,使其与临床观察结果更加吻合。这为IVCS患者的评估和治疗提供了一个新的理论框架。
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引用次数: 0
Editorial: Micro/nano devices and technologies for neural science and medical applications. 编辑:神经科学和医学应用的微/纳米设备和技术。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1545853
Juntao Liu, Zhugen Yang, Yang Wang, Li Wang, Ziyue Li
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引用次数: 0
Effects of polylactic acid scaffolds with various orientations and diameters on osteogenesis and angiogenesis. 不同取向和直径聚乳酸支架对骨生成和血管生成的影响。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1495810
Yun Rong Xu, Dai Yuan Tang, Zhen Ping Xiao, Zai Tian Huang, Heng Rui Zhang, Zi Wen Tang, Fei He

Researchers in the field of regenerative medicine have consistently focused on the biomimetic design of engineered bone materials on the basis of the microstructure of natural bone tissue. Additionally, the effects of the micromorphological characteristics of these materials on angiogenesis have garnered increasing attention. In vitro, the orientation and diameter of scaffold materials can exert different effects on osteogenesis and vascularisation. However, more comprehensive investigations, including in vivo studies, are required to confirm the results observed in vitro. Accordingly, in the present study, fibre scaffolds with various orientations and diameters were prepared by electrospinning with polylactic acid. The effects of the micromorphological characteristics of these scaffolds with different orientations and diameters on osteogenesis and vascularisation were systematically studied via in vivo experiments. The scaffolds with aligned micromorphological features positively affected osteogenesis and vascularisation, which indicated that such characteristics could be considered crucial factors when designing materials for bone repair.

再生医学领域的研究人员一直致力于在天然骨组织微观结构的基础上进行工程骨材料的仿生设计。此外,这些材料的微形态特征对血管生成的影响已经引起了越来越多的关注。在体外,支架材料的取向和直径对成骨和血管形成有不同的影响。然而,需要更全面的研究,包括体内研究,来证实在体外观察到的结果。因此,本研究采用聚乳酸静电纺丝法制备了不同取向和直径的纤维支架。通过体内实验系统研究了不同取向和直径支架的微形态特征对成骨和血管化的影响。具有排列的微形态特征的支架对成骨和血管化有积极的影响,这表明这些特征可以被认为是设计骨修复材料时的关键因素。
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引用次数: 0
The influence of different crosslinking agents onto the physical properties, integration behavior and immune response of collagen-based barrier membranes. 不同交联剂对胶原基屏障膜物理性能、整合行为及免疫反应的影响。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1506433
Yanru Ren, Said Alkildani, Kim Burckhardt, Alexander Köwitsch, Milena Radenkovic, Sanja Stojanovic, Stevo Najman, Ole Jung, Luo Liu, Mike Barbeck

This study investigates the mechanical properties as well as in vitro and in vivo cyto- and biocompatibility of collagen membranes cross-linked with glutaraldehyde (GA), proanthocyanidins (PC), hexamethylendiisocyanate (HMDI) and 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide (EC/NHS). A non-crosslinked membrane was used as reference control (RF). The initial in vitro cytotoxic analyses revealed that the PC, EC, and HMDI crosslinked membranes were cytocompatible, while the GA crosslinked membrane was cytotoxic and thus selected as positive control in the further in vivo study. Cross-linking enhances the tensile strength and collagenase resistance, effectively prolonging the membrane's standing time in vivo. Using (immune-) histochemistry and histomorphometrical analyses, the cellular inflammatory responses, tissue integration and vascularization patterns at 10-, 30-, and 90-day post-implantation in a subcutaneous implantation model in rats were analyzed. The PC membrane elicited the mildest inflammatory cell levels, akin to the RF membrane, while other groups induced an M1-dominated macrophage response and numerous multinucleated giant cells throughout the study period. EC membranes maintained structural stability up to 30 days post-implantation, similar to the GA group, whereas others collapsed prematurely. Concurrent with membrane collapse, transmembrane vascularization occurred across all groups. Histopathological and histomorphometry results reveal the intricate interplay of inflammatory cell populations in vascularization. These findings offer valuable insights into the pivotal role of cross-linkers in modulating mechanical properties and tissue responses of collagen membranes.

本研究研究了与戊二醛(GA)、原花青素(PC)、六亚甲基二异氰酸酯(HMDI)和1-乙基-3-(3-二甲氨基丙基)碳二亚胺/ n -羟基琥珀酰亚胺(EC/NHS)交联的胶原膜的力学性能以及体外和体内细胞和生物相容性。非交联膜作为对照(RF)。最初的体外细胞毒性分析显示,PC、EC和HMDI交联膜具有细胞相容性,而GA交联膜具有细胞毒性,因此在进一步的体内研究中被选为阳性对照。交联增强了膜的抗拉强度和抗胶原酶能力,有效延长了膜在体内的停留时间。采用(免疫)组织化学和组织形态计量学分析,分析了大鼠皮下植入模型植入后10、30和90天的细胞炎症反应、组织整合和血管化模式。与RF膜类似,PC膜诱导的炎症细胞水平最轻,而其他组在整个研究期间诱导了m1主导的巨噬细胞反应和大量多核巨细胞。EC膜在植入后30天保持结构稳定,与GA组相似,而其他组则过早崩溃。在膜塌陷的同时,所有组都发生了跨膜血管形成。组织病理学和组织形态学结果揭示了炎症细胞群在血管形成过程中的复杂相互作用。这些发现为交联剂在调节胶原膜的力学性能和组织反应中的关键作用提供了有价值的见解。
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引用次数: 0
Editorial: The application of bioactive materials in bone repair. 编辑:生物活性材料在骨修复中的应用。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1539142
Yori Endo, Elena Giunta, Jessica Mroueh, William McCarthy, Nina Graf
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引用次数: 0
Development of an interpretable model for foot soft tissue stiffness based on gait plantar pressure analysis. 基于步态足底压力分析的足部软组织刚度可解释模型的建立。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1482382
Xiaotian Bai, Xiao Hou, Dazhi Lv, Jialin Wei, Yiling Song, Zhengyan Tang, Hongfeng Huo, Jingmin Liu

Purpose: Plantar soft tissue properties affect foot biomechanics during movement. This study aims to explore the relationship between plantar pressure features and soft tissue stiffness through interpretable neural network model. The findings could inform orthotic insole design.

Methods: A sample of 30 healthy young male subjects with normal feet were recruited (age 23.56 ± 3.28 years, height 1.76 ± 0.04 m, weight 72.21 ± 5.69 kg). Plantar pressure data were collected during 5 trials at the subjects' preferred walking speed (1.15 ± 0.04 m/s). Foot soft tissue stiffness was recorded using a MyotonPRO biological soft tissue stiffness meter before each walking trial. A backpropagation neural network, optimized by integrating particle swarm optimization and genetic algorithm, was constructed to predict foot soft tissue stiffness using plantar pressure data collected during walking. Mean impact value analysis was conducted in parallel to investigate the relative importance of different plantar pressure features.

Results: The predicted values for the training set are slightly higher than the actual values (MBE = 0.77N/m, RMSE = 11.89 N/m), with a maximum relative error of 7.82% and an average relative error of 1.98%, and the predicted values for the test set are slightly lower than the actual values (MBE = -4.43N/m, RMSE = 14.73 N/m), with a maximum relative error of 7.35% and an average relative error of 2.55%. Regions with highest contribution rates to foot soft tissue stiffness prediction were the third metatarsal (13.58%), fourth metatarsal (14.71%), midfoot (12.43%) and medial heel (12.58%) regions, which accounted for 53.3% of total contribution.

Conclusion: The pressure features in the medial heel, midfoot area, and lateral mid-metatarsal regions during walking can better reflect plantar soft tissue stiffness. Future studies should ensure measurement stability of this region and refine insole designs to mitigate plantar soft tissue fatigue in the specified areas.

目的:足底软组织特性影响运动过程中的足部生物力学。本研究旨在通过可解释神经网络模型探讨足底压力特征与软组织刚度之间的关系。研究结果可以为矫形鞋垫的设计提供参考。方法:选取30名健康青年男性,年龄23.56±3.28岁,身高1.76±0.04 m,体重72.21±5.69 kg。在受试者首选步行速度(1.15±0.04 m/s)下进行5次试验,收集足底压力数据。在每次步行试验前,使用MyotonPRO生物软组织刚度计记录足部软组织刚度。利用步行过程中采集的足底压力数据,构建了融合粒子群算法和遗传算法优化的反向传播神经网络,预测足部软组织刚度。同时进行平均影响值分析,探讨不同足底压力特征的相对重要性。结果:训练集预测值略高于实际值(MBE = 0.77N/m, RMSE = 11.89 N/m),最大相对误差为7.82%,平均相对误差为1.98%;测试集预测值略低于实际值(MBE = -4.43N/m, RMSE = 14.73 N/m),最大相对误差为7.35%,平均相对误差为2.55%。对足部软组织刚度预测贡献率最高的区域为第三跖(13.58%)、第四跖(14.71%)、足中部(12.43%)和足跟内侧(12.58%),占总贡献率的53.3%。结论:行走时足跟内侧、足中区、外侧跖中区压力特征能较好地反映足底软组织僵硬度。未来的研究应确保该区域的测量稳定性,并改进鞋垫设计以减轻特定区域的足底软组织疲劳。
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引用次数: 0
Optogenetic control of mitochondrial aggregation and function. 线粒体聚集和功能的光遗传学控制。
IF 4.3 3区 工程技术 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Pub Date : 2025-01-06 eCollection Date: 2024-01-01 DOI: 10.3389/fbioe.2024.1500343
Luhao Zhang, Xuechun Liu, Min Zhu, Yuanfa Yao, Zhichao Liu, Xianming Zhang, Xin Deng, Yi Wang, Liting Duan, Xiaogang Guo, Junfen Fu, Yingke Xu

The balance of mitochondrial fission and fusion plays an important role in maintaining the stability of cellular homeostasis. Abnormal mitochondrial fission and fragmentation have been shown to be associated with oxidative stress, which causes a variety of human diseases from neurodegeneration disease to cancer. Therefore, the induction of mitochondrial aggregation and fusion may provide an alternative approach to alleviate these conditions. Here, an optogenetic-based mitochondrial aggregation system (Opto-MitoA) developed, which is based on the CRY2clust/CIBN light-sensitive module. Upon blue light illumination, CRY2clust relocates from the cytosol to mitochondria where it induces mitochondrial aggregation by CRY2clust homo-oligomerization and CRY2clust-CIBN hetero-dimerization. Our functional experiments demonstrate that Opto-MitoA-induced mitochondrial aggregation potently alleviates niclosamide-caused cell dysfunction in ATP production. This study establishes a novel optogenetic-based strategy to regulate mitochondrial dynamics in cells, which may provide a potential therapy for treating mitochondrial-related diseases.

线粒体分裂和融合的平衡对维持细胞稳态的稳定起着重要作用。线粒体异常分裂和断裂已被证明与氧化应激有关,氧化应激可导致从神经退行性疾病到癌症等多种人类疾病。因此,诱导线粒体聚集和融合可能为缓解这些疾病提供了另一种方法。本文基于CRY2clust/CIBN光敏模块,开发了基于光遗传学的线粒体聚集系统(Opto-MitoA)。在蓝光照射下,CRY2clust从细胞质溶胶迁移到线粒体,并通过CRY2clust同质寡聚化和CRY2clust- cibn异二聚化诱导线粒体聚集。我们的功能实验表明,opto - mitoa诱导的线粒体聚集有效地缓解了氯硝胺引起的细胞ATP产生功能障碍。本研究建立了一种新的基于光遗传学的策略来调节细胞中的线粒体动力学,这可能为线粒体相关疾病的治疗提供潜在的治疗方法。
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引用次数: 0
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