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Straightforward MALDI-TOF MS based screening approach for selection of recombinant protein-expressing E. coli 直接基于MALDI-TOF MS筛选表达重组蛋白大肠杆菌的方法

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.02.004

I.N. Kravtsov , A.I. Solovyev , E.A. Potemkina , A.V. Kartashova , M.A. Dmitrieva , K.V. Danilova , I.L. Tutykhina , N.B. Polyakov , V.D. Desinov , D.A. Egorova , A.L. Gintsburg

Recombinant protein production is a milestone of modern biotechnology, drug development and scientific research. When obtaining recombinant protein producers, differences in expression levels among clones necessitate screening. Traditional widely used methods include protein electrophoresis and western blot hybridization. This protocol provides high-throughput advantages by eliminating time-consuming steps inherent to traditional methods, such as cell lysis, protein extraction, purification, antibody-based detection, and gel-based analysis. MALDI-TOF MS represents a simple, rapid and cost-effective method for bacterial species identification through protein fingerprint signature in clinical diagnostics, but not practically integrated into biotechnological workflow. This study proposes a fast and easy method for screening E. coli clones producing recombinant proteins with MALDI-TOF MS. The proposed method demonstrated efficiency in screening of E. coli producing several recombinant proteins with different properties: sfGFP; bacterial DNA binding proteins IHFα, IHFβ, HU; bacteriophage protein GP46 and camelid VHH antibody fragments.

重组蛋白的生产是现代生物技术、药物开发和科学研究的一个里程碑。在获得重组蛋白生产者时，需要筛选克隆之间表达水平的差异。传统的广泛使用的方法包括蛋白质电泳和western blot杂交。该方案通过消除传统方法固有的耗时步骤提供高通量优势，例如细胞裂解，蛋白质提取，纯化，基于抗体的检测和基于凝胶的分析。MALDI-TOF质谱在临床诊断中是一种简单、快速、具有成本效益的蛋白质指纹图谱细菌种类鉴定方法，但尚未真正融入生物技术工作流程。本研究提出了一种快速简便的利用MALDI-TOF ms筛选产生重组蛋白的大肠杆菌克隆的方法，该方法在筛选产生不同性质重组蛋白的大肠杆菌中表现出高效率：sfGFP；细菌DNA结合蛋白IHFα、IHFβ、HU；噬菌体蛋白GP46和骆驼类VHH抗体片段。

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Biotechnology Notes

Pub Date : 2025-01-01

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Tackling carbapenem-resistant Acinetobacter baumannii (CRAB) and their virulence factors using biosynthesized silver nanoparticles combined with imipenem 生物合成纳米银与亚胺培南联合处理耐碳青霉烯鲍曼不动杆菌及其毒力因子

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.07.002

Mohamed Shawky, Mohamed H. Kalaba, Gamal M. El-Sherbiny

Carbapenem-resistant Acinetobacter baumannii is an extremely hazardous microorganism due to its high level of resistance to a wide array of antibiotics, making it a significant public health concern. Herein, this study aimed to biofabricate silver nanoparticles using a cell-free filter derived from Streptomyces sp., with a focus on characterizing their physicochemical properties, and use them to combat CRAB and their virulence factors. The biofabricated Ag-NPs were predominantly spherical with an average size 50 nm, confirmed through TEM analyses, while DLS measurements showed an average hydrodynamic diameter of approximately 36.78 nm. UV–Vis spectroscopy displayed a characteristic surface plasmon resonance peak in the range of 420 nm, indicative of nanoparticle formation. XRD confirmed the crystalline structure, presenting peaks corresponding to face-centered cubic silver. FTIR spectroscopy revealed active participation of metabolite compounds derived from the Streptomyces cell-free filter in both reduction and stabilization processes. Eight clinical bacterial isolates were identified as CRAB using the Vitek-2 system, and biofilm formation with 100 % was assessed through Congo red and microplate assays. The MIC for Ag-NPs and imipenem (IMP) were found to be between 4 and 5 μg/mL and 13 and 15 μg/mL, respectively. Additionally, the fractional inhibitory concentration index (FICI) for the synergistic combinations of Ag-NPs and IMP ranged from 0.5 to 0.375, indicating a notable decrease in the MIC values for both IMP and Ag-NPs from 14 and 5 μg/mL to 1.75 and 1.25 μg/mL, respectively. The qRT-PCR demonstrated a significant reduction in the expression levels of the Bap and ompA genes by up to 4.0-fold (p ≤ 0.001). The time-killing assay confirmed that the bacterial strain was effectively eliminated through the synergistic action of Ag-NPs and IMP. Moreover, the cytotoxicity assessment of Ag-NPs and their combination with IMP revealed low toxicity of the combination of Ag-NPs and IMP, with an IC₅₀ of 26.13 ± 0.24 and 45.33 ± 0.21 μg/mL, respectively (p < 0.0019), indicating good biosafety, while the hemolysis rates were recorded at 0.4 and 0.7 at 12 and 24 h, respectively. We concluded that the combination of Ag-NPs with IMP could serve as a promising alternative strategy for treating CRAB.

耐碳青霉烯鲍曼不动杆菌是一种极其危险的微生物，因为它对多种抗生素具有高度耐药性，使其成为一个重大的公共卫生问题。本研究旨在利用链霉菌（Streptomyces sp.）衍生的无细胞过滤器生物制备纳米银，重点研究其物理化学性质，并利用其对抗螃蟹及其毒力因子。通过TEM分析证实，生物制备的Ag-NPs主要为球形，平均尺寸为50 nm，而DLS测量显示其平均水动力直径约为36.78 nm。紫外可见光谱显示，表面等离子体共振峰在420 nm范围内，表明纳米颗粒形成。XRD证实其晶体结构为面心立方银。FTIR光谱显示，链霉菌无细胞过滤器衍生的代谢物化合物积极参与还原和稳定过程。使用Vitek-2系统鉴定8株临床分离细菌为CRAB，通过刚果红和微孔板检测100%的生物膜形成情况。Ag-NPs和亚胺培南（IMP）的MIC分别为4 ~ 5 μg/mL和13 ~ 15 μg/mL。此外，Ag-NPs和IMP协同组合的分数抑制浓度指数（FICI）在0.5 ~ 0.375之间，表明IMP和Ag-NPs的MIC值分别从14和5 μg/mL显著降低到1.75和1.25 μg/mL。qRT-PCR结果显示，Bap和ompA基因的表达水平显著降低了4.0倍（p≤0.001）。时间杀伤实验证实，Ag-NPs与IMP的协同作用可有效杀灭该菌株，细胞毒性评价显示Ag-NPs与IMP联用毒性较低，IC50分别为26.13±0.24和45.33±0.21 μg/mL (p <；0.0019)，生物安全性较好，12 h和24 h溶血率分别为0.4和0.7。我们的结论是Ag-NPs与IMP的联合治疗可能是一种有希望的治疗螃蟹的替代策略。

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Modeling and simulation of soft bio-mimetic fingers with a novel soft thumb design for bionic hand applications using ANN 基于人工神经网络的柔性仿生手指建模与仿真研究

Biotechnology Notes

Pub Date : 2025-01-01 DOI: 10.1016/j.biotno.2025.05.002

Eman R.A. Soliman , Ayman Nada , Hiroyuki Ishii , Ahmed M.R. Fathelbeb

The paper presents a novel design for a soft bio-mimetic finger and soft thumb structure for bionic hand applications. It introduces an anthropomorphic pneumatic flexible finger system using a PneuNets framework to enhance flexibility and maneuverability. The research investigates the influence of geometric variations (wall thickness, chamber number, and spacing) on finger deformation, demonstrating that reduced wall thickness and augmented chambers substantially improve flexibility. A key innovation is the soft thumb design that accurately replicates the complex movements of the Carpometacarpal (CMC) joint, enabling natural opposition and dexterity. Eight models were developed for four fingers and two models for the thumb. Simulation results indicate that models with thinner walls (2 mm) achieve bending angles exceeding 80° at 120 KPa, whereas 3 mm models remain below 50°. Moreover, increasing the number of chambers enhances deformation, with each added chamber contributing approximately 41 % more flexibility. For the thumb models, we successfully mapped the motion ranges and accurately mimicked the base joint, enabling natural opposition and dexterity. Furthermore, the paper also integrates Artificial Neural Networks (ANNs) to model forward kinematics, improving the estimation of bending angles and end-tip positions, which enhances the overall adaptability and control of the system.

本文提出了一种适用于仿生手的柔性仿生手指和柔性拇指结构设计。采用PneuNets框架设计了一种拟人气动柔性手指系统，提高了系统的灵活性和可操作性。该研究调查了几何变化（壁厚、室数和间距）对手指变形的影响，表明减少壁厚和增加室可大大提高灵活性。一个关键的创新是柔软的拇指设计，准确地复制了复杂的腕掌骨（CMC）关节的运动，使自然对立和灵巧。为四个手指开发了八个模型，为拇指开发了两个模型。仿真结果表明，在120 KPa下，壁较薄（2 mm）的模型的弯曲角超过80°，而3 mm的模型的弯曲角低于50°。此外，增加腔室的数量可以增强变形，每增加一个腔室，可使弹性提高约41%。对于拇指模型，我们成功地绘制了运动范围，并准确地模拟了基础关节，实现了自然的对立和灵巧性。此外，本文还将人工神经网络（ann）集成到机器人的正运动学建模中，改进了机器人弯曲角度和端尖位置的估计，增强了系统的整体适应性和控制能力。

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