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Role of viscoelasticity in the appearance of low-Reynolds turbulence: considerations for modelling 粘弹性在出现低雷诺湍流中的作用:建模考虑因素
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-08 DOI: 10.1186/s13036-024-00415-6
Ivana Pajic-Lijakovic, Milan Milivojevic, Peter V. E. McClintock
Inertial effects caused by perturbations of dynamical equilibrium during the flow of soft matter constitute a hallmark of turbulence. Such perturbations are attributable to an imbalance between energy storage and energy dissipation. During the flow of Newtonian fluids, kinetic energy can be both stored and dissipated, while the flow of viscoelastic soft matter systems, such as polymer fluids, induces the accumulation of both kinetic and elastic energies. The accumulation of elastic energy causes local stiffening of stretched polymer chains, which can destabilise the flow. Migrating multicellular systems are hugely complex and are capable of self-regulating their viscoelasticity and mechanical stress generation, as well as controlling their energy storage and energy dissipation. Since the flow perturbation of viscoelastic systems is caused by the inhomogeneous accumulation of elastic energy, rather than of kinetic energy, turbulence can occur at low Reynolds numbers. This theoretical review is focused on clarifying the role of viscoelasticity in the appearance of low-Reynolds turbulence. Three types of system are considered and compared: (1) high-Reynolds turbulent flow of Newtonian fluids, (2) low and moderate-Reynolds flow of polymer solutions, and (3) migration of epithelial collectives, discussed in terms of two model systems. The models considered involve the fusion of two epithelial aggregates, and the free expansion of epithelial monolayers on a substrate matrix.
软物质流动过程中的动态平衡扰动所产生的惯性效应是湍流的一大特征。这种扰动可归因于能量储存和能量耗散之间的不平衡。在牛顿流体的流动过程中,动能既可以储存也可以耗散,而粘弹性软物质系统(如聚合物流体)的流动会导致动能和弹性能的积累。弹性能的积累会导致拉伸的聚合物链局部变硬,从而破坏流动的稳定性。迁移的多细胞系统非常复杂,能够自我调节其粘弹性和机械应力的产生,并控制其能量储存和能量耗散。由于粘弹性系统的流动扰动是由弹性能而不是动能的不均匀积累引起的,因此在低雷诺数时就会出现湍流。本理论综述的重点是阐明粘弹性在低雷诺数湍流出现中的作用。本文考虑并比较了三种类型的系统:(1) 牛顿流体的高雷诺湍流,(2) 聚合物溶液的低雷诺和中雷诺流动,以及 (3) 上皮细胞的迁移。所考虑的模型涉及两个上皮聚集体的融合,以及上皮单层在基质基体上的自由扩展。
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引用次数: 0
Eggshell waste bioprocessing for sustainable acid phosphatase production and minimizing environmental hazards 蛋壳废弃物生物处理,实现酸性磷酸酶的可持续生产并最大限度地减少环境危害
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-08 DOI: 10.1186/s13036-024-00421-8
Soad Abubakr Abdelgalil, Mohamed Mohamed Yousri Kaddah, Gaber Attia Abo-Zaid
The Environmental Protection Agency has listed eggshell waste as the 15th most significant food industry pollution hazard. Using eggshell waste as a renewable energy source has been a hot topic recently. Therefore, finding a sustainable solution for the recycling and valorization of eggshell waste by investigating its potential to produce acid phosphatase (ACP) and organic acids by the newly-discovered B. sonorensis was the target of the current investigation. Drawing on both molecular and morphological characterizations, the most potent ACP-producing B. sonorensis strain ACP2, was identified as a local bacterial strain obtained from the effluent of the paper and pulp industries. The use of consecutive statistical experimental approaches of Plackett–Burman Design (PBD) and Orthogonal Central Composite Design (OCCD), followed by pH-uncontrolled cultivation conditions in a 7 L bench-top bioreactor, revealed an innovative medium formulation that substantially improved ACP production, reaching 216 U L−1 with an ACP yield coefficient Yp/x of 18.2 and a specific growth rate (µ) of 0.1 h−1. The metals Ag+, Sn+, and Cr+ were the most efficiently released from eggshells during the solubilization process by B. sonorensis. The uncontrolled pH culture condition is the most suitable and favoured setting for improving ACP and organic acids production. Quantitative and qualitative analyses of the produced organic acids were carried out using liquid chromatography-tandem mass spectrometry (LC–MS/MS). Lactic acid, citric acid, and hydroxybenzoic acid isomer were the most common organic acids produced throughout the cultivation process. The findings of TGA, DSC, SEM, EDS, FTIR, and XRD analysis emphasize the significant influence of organic acids and ACP activity on the solubilization of eggshell particles. This study emphasized robust microbial engineering approaches for the large-scale production of a newly discovered acid phosphatase, accompanied by organic acids production from B. sonorensis. The biovalorization of the eggshell waste and the production of cost-effective ACP and organic acids were integrated into the current study, and this was done through the implementation of a unique and innovative medium formulation design for eggshell waste management, as well as scaling up ACP production on a bench-top scale.
环境保护局已将蛋壳废弃物列为食品工业第 15 大污染危害。将蛋壳废弃物用作可再生能源已成为近期的热门话题。因此,通过研究新发现的B. sonorensis产生酸性磷酸酶(ACP)和有机酸的潜力,为蛋壳废弃物的回收利用和增值找到一种可持续的解决方案,是本次研究的目标。根据分子和形态特征,从造纸和纸浆工业废水中获得的本地细菌菌株 ACP2 被确定为最能产生 ACP 的 B. sonorensis 菌株。通过连续使用普拉克特-伯曼设计(PBD)和正交中心复合设计(OCCD)统计实验方法,并在 7 升台式生物反应器中采用不受 pH 值控制的培养条件,发现了一种创新的培养基配方,该配方可大幅提高 ACP 产量,达到 216 U L-1,ACP 产量系数 Yp/x 为 18.2,特定生长速率 (µ) 为 0.1 h-1。在超声波杆菌的增溶过程中,金属 Ag+、Sn+ 和 Cr+ 从蛋壳中释放的效率最高。不受控制的 pH 值培养条件是提高 ACP 和有机酸产量的最合适和最有利的环境。使用液相色谱-串联质谱法(LC-MS/MS)对所产生的有机酸进行了定量和定性分析。乳酸、柠檬酸和羟基苯甲酸异构体是整个培养过程中最常见的有机酸。TGA、DSC、SEM、EDS、FTIR 和 XRD 分析结果表明,有机酸和 ACP 活性对蛋壳颗粒的增溶具有重要影响。本研究强调了利用微生物工程学方法大规模生产新发现的酸性磷酸酶,同时利用 B. sonorensis 生产有机酸。蛋壳废弃物的生物硼化和具有成本效益的 ACP 和有机酸的生产被纳入了当前的研究中,这是通过为蛋壳废弃物管理实施独特和创新的培养基配方设计以及在台式规模上扩大 ACP 的生产来实现的。
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引用次数: 0
Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation 通过 IEDDA 介导的位点特异性白蛋白共轭增强抗体片段的治疗潜力
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-04 DOI: 10.1186/s13036-024-00418-3
Eun Byeol Go, Jae Hun Lee, Jeong Haeng Cho, Na Hyun Kwon, Jong-il Choi, Inchan Kwon
The use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties. In this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv. The antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.
利用单链可变片段(scFv)治疗癌症和免疫系统疾病等人类疾病已引起广泛关注。然而,scFv 的一个重要缺点是血清半衰期极短,这限制了其治疗潜力。因此,临床应用迫切需要延长 scFv 的血清半衰期。人血清白蛋白(HSA)是一种很有前景的血清半衰期延长剂,它是人体血清中含量最高的蛋白质,已知其血清半衰期特别长。然而,将大分子半衰期延长剂与 scFv 等小分子蛋白质共轭,往往会导致其关键特性的显著丧失。在本研究中,我们将 HSA 与 scFv 的一个允许位点共轭,以改善药代动力学特征。为了确保 HSA 连接后对 scFv 抗原结合能力的破坏最小,我们采用了一种位点特异性连接方法,使用了一种能促进硫醇-马来酰亚胺反应和反电子需求 Diels-Alder 反应(IEDDA)的杂官能团交联剂。我们选择了曲妥珠单抗衍生的 4D5scFv 作为模型蛋白,曲妥珠单抗是一种用于治疗人表皮生长因子 2(HER2)阳性乳腺癌的治疗性抗体。我们在通过计算方法预测的候选连接位点上引入了含有高活性四嗪基团(frTet)的苯丙氨酸类似物。使用连接剂 TCO-PEG4-MAL,将单个 HSA 分子与 4D5scFv (4D5scFv-HSA)定点连接。4D5scFv-HSA 共轭物的 HER2 结合亲和力与未修饰的 4D5scFv 相当。此外,在小鼠药代动力学图谱中,4D5scFv-HSA 的血清半衰期约为 12 小时,是 4D5scFv 的 85 倍。4D5scFv-HSA 的抗原结合结果和药代动力学特征表明,位点特异性白蛋白结合的 scFv 在延长血清半衰期的同时保持了其结合亲和力。总之,我们开发出了一种制备位点特异性白蛋白共轭 4D5scFv 的有效策略,可用于多种临床应用并提高疗效。
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引用次数: 0
3D-printed nanohydroxyapatite/methylacrylylated silk fibroin scaffold for repairing rat skull defects. 用于修复大鼠颅骨缺损的三维打印纳米羟基磷灰石/甲基丙烯酰化丝纤维支架。
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-03-21 DOI: 10.1186/s13036-024-00416-5
Wu Huiwen, Liang Shuai, Xie Jia, Deng Shihao, Wei Kun, Yang Runhuai, Qian Haisheng, Li Jun
<p><p>The repair of bone defects remains a major challenge in the clinic, and treatment requires bone grafts or bone replacement materials. Existing biomaterials have many limitations and cannot meet the various needs of clinical applications. To treat bone defects, we constructed a nanohydroxyapatite (nHA)/methylacrylylated silk fibroin (MASF) composite biological scaffold using photocurable 3D printing technology. In this study, scanning electron microscopy (SEM) was used to detect the changes in the morphological structure of the composite scaffold with different contents of nanohydroxyapatite, and FTIR was used to detect the functional groups and chemical bonds in the composite scaffold to determine the specific components of the scaffold. In in vitro experiments, bone marrow mesenchymal stem cells from SD rats were cocultured with scaffolds soaking solution, and the cytotoxicity, cell proliferation, Western blot analysis, Quantitative real-time PCR analysis, bone alkaline phosphatase activity and alizarin red staining of scaffolds were detected to determine the biocompatibility of scaffolds and the effect of promoting proliferation and osteogenesis of bone marrow mesenchymal stem cells in vitro. In the in vivo experiment, the skull defect was constructed by adult SD rats, and the scaffold was implanted into the skull defect site. After 4 weeks and 8 weeks of culture, the specific osteogenic effect of the scaffold in the skull defect site was detected by animal micro-CT, hematoxylin and eosin (HE) staining and Masson's staining. Through the analysis of the morphological structure of the scaffold, we found that the frame supported good retention of the lamellar structure of silk fibroin, when mixed with nHA, the surface of the stent was rougher, the cell contact area increased, and cell adhesion and lamellar microstructure for cell migration and proliferation of the microenvironment provided a better space. FTIR results showed that the scaffold completely retained the β -folded structure of silk fibroin, and the scaffold composite was present without obvious impurities. The staining results of live/dead cells showed that the constructed scaffolds had no significant cytotoxicity, and thw CCK-8 assay also showed that the constructed scaffolds had good biocompatibility. The results of osteogenic induction showed that the scaffold had good osteogenic induction ability. Moreover, the results also showed that the scaffold with a MASF: nHA ratio of 1: 0.5 (SFH) showed better osteogenic ability. The micro-CT and bone histometric results were consistent with the in vitro results after stent implantation, and there was more bone formation at the bone defect site in the SFH group.This research used photocurable 3D printing technology to successfully build an osteogenesis bracket. The results show that the constructed nHA/MASF biological composite material, has good biocompatibility and good osteogenesis function. At the same time, in the microenvironment
骨缺损的修复仍然是临床上的一大挑战,治疗需要骨移植或骨替代材料。现有的生物材料有很多局限性,无法满足临床应用的各种需求。为了治疗骨缺损,我们采用光固化三维打印技术构建了纳米羟基磷灰石(nHA)/甲基丙烯酰化丝纤维素(MASF)复合生物支架。该研究利用扫描电子显微镜(SEM)检测不同纳米羟基磷灰石含量的复合支架形态结构的变化,并利用傅立叶变换红外光谱(FTIR)检测复合支架中的官能团和化学键,以确定支架的具体成分。在体外实验中,将 SD 大鼠的骨髓间充质干细胞与支架浸泡液共培养,检测支架的细胞毒性、细胞增殖、Western 印迹分析、定量实时 PCR 分析、骨碱性磷酸酶活性和茜素红染色,以确定支架的生物相容性和促进骨髓间充质干细胞增殖和成骨的效果。在体内实验中,用成年 SD 大鼠构建颅骨缺损,将支架植入颅骨缺损部位。分别培养4周和8周后,通过动物显微CT、苏木精和伊红(HE)染色以及马森氏染色检测支架在颅骨缺损部位的特异性成骨效应。通过对支架形态结构的分析,我们发现框架支撑的丝纤维素层状结构保留良好,当与 nHA 混合后,支架表面更粗糙,细胞接触面积增大,细胞粘附和层状微结构为细胞迁移和增殖提供了更好的微环境空间。傅立叶变换红外光谱(FTIR)结果表明,支架完全保留了蚕丝纤维素的β折叠结构,支架复合材料无明显杂质。活/死细胞染色结果表明,所构建的支架没有明显的细胞毒性,CCK-8 试验也表明所构建的支架具有良好的生物相容性。成骨诱导结果表明,该支架具有良好的成骨诱导能力。此外,结果还显示,MASF:nHA 比为 1:0.5(SFH)的支架具有更好的成骨能力。支架植入后的显微 CT 和骨组织测定结果与体外结果一致,SFH 组在骨缺损部位有更多的骨形成。结果表明,构建的 nHA/MASF 生物复合材料具有良好的生物相容性和成骨功能。同时,在微环境中,该材料还能促进骨缺损修复,可作为骨缺损填充材料用于骨再生应用。
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引用次数: 0
Construction and applications of the EOMA spheroid model of Kaposiform hemangioendothelioma. 卡波斯状血管内皮瘤 EOMA 球形模型的构建与应用。
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-03-14 DOI: 10.1186/s13036-024-00417-4
Yanan Li, Xinglong Zhu, Li Li, Chunjuan Bao, Qin Liu, Ning Zhang, Ziyan He, Yi Ji, Ji Bao

Background: Kaposiform hemangioendothelioma (KHE) is a rare intermediate vascular tumor with unclear pathogenesis. Recently, three dimensional (3D) cell spheroids and organoids have played an indispensable role in the study of many diseases, such as infantile hemangioma and non-involuting congenital hemangiomas. However, few research on KHE are based on the 3D model. This study aims to evaluate the 3D superiority, the similarity with KHE and the ability of drug evaluation of EOMA spheroids as an in vitro 3D KHE model.

Results: After two days, relatively uniform morphology and high viability of EOMA spheroids were generated by the rotating cell culture system (RCCS). Through transcriptome analysis, compared with 2D EOMA cells, focal adhesion-related genes such as Itgb4, Flt1, VEGFC, TNXB, LAMA3, VWF, and VEGFD were upregulated in EOMA spheroids. Meanwhile, the EOMA spheroids injected into the subcutaneous showed more obvious KMP than 2D EOMA cells. Furthermore, EOMA spheroids possessed the similar characteristics to the KHE tissues and subcutaneous tumors, such as diagnostic markers (CD31 and LYVE-1), cell proliferation (Ki67), hypoxia (HIF-1α) and cell adhesion (E-cadherin and N-cadherin). Based on the EOMA spheroid model, we discovered that sirolimus, the first-line drug for treating KHE, could inhibit EOMA cell proliferation and downregulate the VEGFC expression. Through the extra addition of VEGFC, the effect of sirolimus on EOMA spheroid could be weakened.

Conclusion: With a high degree of similarity of the KHE, 3D EOMA spheroids generated by the RCCS can be used as a in vitro model for basic researches of KHE, generating subcutaneous tumors and drug screening.

背景:汲状血管内皮瘤(KHE)是一种罕见的中间血管肿瘤,发病机制尚不清楚。近来,三维(3D)细胞球和器官组织在婴儿血管瘤和非膨胀性先天性血管瘤等多种疾病的研究中发挥了不可或缺的作用。然而,有关 KHE 的研究很少基于三维模型。本研究旨在评估EOMA球体作为体外三维KHE模型的三维优越性、与KHE的相似性以及药物评估能力:结果:两天后,旋转细胞培养系统(RCCS)生成了形态相对均匀、存活率较高的EOMA球体。通过转录组分析,与二维EOMA细胞相比,EOMA球形细胞中的Itgb4、Flt1、VEGFC、TNXB、LAMA3、VWF和VEGFD等局灶粘附相关基因上调。同时,与二维EOMA细胞相比,注射到皮下的EOMA球体表现出更明显的KMP。此外,EOMA球形细胞具有与KHE组织和皮下肿瘤相似的特征,如诊断标志物(CD31和LYVE-1)、细胞增殖(Ki67)、缺氧(HIF-1α)和细胞粘附(E-cadherin和N-cadherin)。基于EOMA球体模型,我们发现治疗KHE的一线药物西罗莫司可抑制EOMA细胞增殖并下调VEGFC的表达。通过额外添加 VEGFC,西罗莫司对 EOMA 球形细胞的作用可以减弱:结论:RCCS生成的三维EOMA球体与KHE高度相似,可作为KHE基础研究、皮下肿瘤生成和药物筛选的体外模型。
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引用次数: 0
Structural and practical identifiability analysis in bioengineering: a beginner's guide. 生物工程中的结构和实用可识别性分析:初学者指南。
IF 5.7 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-03-04 DOI: 10.1186/s13036-024-00410-x
Linda Wanika, Joseph R Egan, Nivedhitha Swaminathan, Carlos A Duran-Villalobos, Juergen Branke, Stephen Goldrick, Mike Chappell

Advancements in digital technology have brought modelling to the forefront in many disciplines from healthcare to architecture. Mathematical models, often represented using parametrised sets of ordinary differential equations, can be used to characterise different processes. To infer possible estimates for the unknown parameters, these models are usually calibrated using associated experimental data. Structural and practical identifiability analyses are a key component that should be assessed prior to parameter estimation. This is because identifiability analyses can provide insights as to whether or not a parameter can take on single, multiple, or even infinitely or countably many values which will ultimately have an impact on the reliability of the parameter estimates. Also, identifiability analyses can help to determine whether the data collected are sufficient or of good enough quality to truly estimate the parameters or if more data or even reparameterization of the model is necessary to proceed with the parameter estimation process. Thus, such analyses also provide an important role in terms of model design (structural identifiability analysis) and the collection of experimental data (practical identifiability analysis). Despite the popularity of using data to estimate the values of unknown parameters, structural and practical identifiability analyses of these models are often overlooked. Possible reasons for non-consideration of application of such analyses may be lack of awareness, accessibility, and usability issues, especially for more complicated models and methods of analysis. The aim of this study is to introduce and perform both structural and practical identifiability analyses in an accessible and informative manner via application to well established and commonly accepted bioengineering models. This will help to improve awareness of the importance of this stage of the modelling process and provide bioengineering researchers with an understanding of how to utilise the insights gained from such analyses in future model development.

数字技术的进步使建模技术在从医疗到建筑的众多学科中占据了前沿地位。数学模型通常使用参数化的常微分方程组来表示,可用于描述不同的过程。为了推断未知参数的可能估计值,这些模型通常使用相关的实验数据进行校准。结构和实际可识别性分析是参数估计前应评估的关键部分。这是因为,可识别性分析可以帮助人们深入了解参数是否可以取单个、多个、甚至无限或可数的值,而这些值最终将对参数估计的可靠性产生影响。此外,可识别性分析还有助于确定所收集的数据是否足够或质量是否足够好,从而真正估算出参数,或者是否需要更多的数据,甚至对模型进行重新参数化,以继续参数估计过程。因此,此类分析在模型设计(结构可识别性分析)和实验数据收集(实际可识别性分析)方面也发挥着重要作用。尽管利用数据估计未知参数值的做法很流行,但这些模型的结构和实际可识别性分析却常常被忽视。不考虑应用此类分析的可能原因可能是缺乏认识、可及性和可用性问题,特别是对于较为复杂的模型和分析方法。本研究的目的是通过应用于成熟和普遍接受的生物工程模型,以易于理解和信息丰富的方式介绍和执行结构和实际可识别性分析。这将有助于提高对建模过程中这一阶段重要性的认识,并使生物工程研究人员了解如何在未来的模型开发中利用从此类分析中获得的见解。
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引用次数: 0
An innovative lab-scale production for a novel therapeutic DNA vaccine candidate against rheumatoid arthritis 以创新的实验室规模生产治疗类风湿性关节炎的新型 DNA 候选疫苗
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-02-27 DOI: 10.1186/s13036-024-00411-w
Juan Long, Xiao Zhao, Fei Liang, Yang Zeng, Nan Liu, Yuying Sun, Yongzhi Xi
Recent therapeutic-plasmid DNA vaccine strategies for rheumatoid arthritis (RA) have significantly improved. Our pcDNA-CCOL2A1 vaccine is the most prominent and the first antigen-specific tolerising DNA vaccine with potent therapeutic and prophylactic effects compared with methotrexate (MTX), the current “gold standard” treatment for collagen-induced arthritis (CIA). This study developed a highly efficient, cost-effective, and easy-to-operate system for the lab-scale production of endotoxin-free supercoiled plasmids with high quality and high yield. Based on optimised fermentation culture, we obtained a high yield of pcDNA-CCOL2A1 vaccine by PEG/MgCl2 precipitation and TRION-114. We then established a method for quality control of the pcDNA-CCOL2A1 vaccine. Collagen-induced arthritis (CIA) model rats were subjected to intramuscular injection of the pcDNA-CCOL2A1 vaccine (300 μg/kg) to test its biological activity. An average yield of 11.81 ± 1.03 mg purified supercoiled plasmid was obtained from 1 L of fermentation broth at 670.6 ± 57.42 mg/L, which was significantly higher than that obtained using anion exchange column chromatography and a commercial purification kit. Our supercoiled plasmid had high purity, biological activity, and yield, conforming to the international guidelines for DNA vaccines. The proposed innovative downstream process for the pcDNA-CCOL2A1 vaccine can not only provide a large-scale high-quality supercoiled plasmid DNA for preclinical research but also facilitate further pilot-scale and even industrial-scale production of pcDNA-CCOL2A1 vaccine.
最近,治疗类风湿性关节炎(RA)的质粒 DNA 疫苗策略有了显著改善。我们的 pcDNA-CCOL2A1 疫苗是最突出的,也是第一个具有抗原特异性耐受性的 DNA 疫苗,与甲氨蝶呤(MTX)(目前治疗胶原诱导性关节炎(CIA)的 "金标准")相比,具有强大的治疗和预防效果。这项研究开发了一种高效、经济、易于操作的系统,用于在实验室规模生产高质量、高产量的无内毒素超螺旋质粒。在优化发酵培养的基础上,我们通过 PEG/MgCl2 沉淀和 TRION-114 获得了高产率的 pcDNA-CCOL2A1 疫苗。随后,我们建立了 pcDNA-CCOL2A1 疫苗的质量控制方法。对胶原诱导关节炎(CIA)模型大鼠肌肉注射 pcDNA-CCOL2A1 疫苗(300 μg/kg),以测试其生物活性。从 1 升发酵液中平均获得 11.81 ± 1.03 毫克纯化的超螺旋质粒,含量为 670.6 ± 57.42 毫克/升,明显高于使用阴离子交换柱色谱法和商业纯化试剂盒获得的含量。我们的超螺旋质粒具有高纯度、高生物活性和高产量,符合 DNA 疫苗的国际准则。所提出的 pcDNA-CCOL2A1 疫苗下游创新工艺不仅能为临床前研究提供大规模高质量的超螺旋质粒 DNA,还能促进 pcDNA-CCOL2A1 疫苗的进一步中试甚至工业化生产。
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引用次数: 0
Chitosan as a promising materials for the construction of nanocarriers for diabetic retinopathy: an updated review. 壳聚糖是一种用于构建糖尿病视网膜病变纳米载体的前景广阔的材料:最新综述。
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-02-22 DOI: 10.1186/s13036-024-00414-7
Yan Lv, Chenglei Zhai, Gang Sun, Yangfang He

Diabetic retinopathy (DR) is a condition that causes swelling of the blood vessels of the retina and leaks blood and fluids. It is the most severe form of diabetic eye disease. It causes vision loss in its advanced stage. Diabetic retinopathy is responsible for causing 26% of blindness. Very insufficient therapies are accessible for the treatment of DR. As compared to the conventional therapies, there should be enhanced research on the controlled release, shorter duration, and cost-effective therapy of diabetic retinopathy. The expansion of advanced nanocarriers-based drug delivery systems has been now employed to exploit as well as regulate the transport of many therapeutic agents to target sites via the increase in penetration or the extension of the duration of contact employing production by enclosing as well as distributing tiny molecules in nanostructured formulation. Various polymers have been utilized for the manufacturing of these nanostructured formulations. Chitosan possesses incredible biological and chemical properties, that have led to its extensive use in pharmaceutical and biomedical applications. Chitosan has been used in many studies because of its enhanced mucoadhesiveness and non-toxicity. Multiple studies have used chitosan as the best candidate for manufacturing nanocarriers and treating diabetic retinopathy. Numerous nanocarriers have been formulated by using chitosan such as nanostructured lipid carriers, solid lipid nanoparticles, liposomes, and dendrimers for treating diabetic retinopathy. This current review elaborates on the recent advancements of chitosan as a promising approach for the manufacturing of nanocarriers that can be used for treating diabetic retinopathy.

糖尿病视网膜病变(DR)是一种导致视网膜血管肿胀并渗出血液和液体的疾病。它是糖尿病眼病中最严重的一种。晚期会导致视力丧失。糖尿病视网膜病变导致了 26% 的失明。目前可用于治疗糖尿病视网膜病变的疗法非常有限。与传统疗法相比,应加强对糖尿病视网膜病变的控释、短效和成本效益疗法的研究。目前,先进的纳米载体给药系统已被广泛应用,通过在纳米结构配方中包裹和分布微小分子,增加渗透性或延长接触时间,从而利用和调节许多治疗药物向目标部位的运输。各种聚合物已被用于制造这些纳米结构制剂。壳聚糖具有令人难以置信的生物和化学特性,因此被广泛应用于制药和生物医学领域。由于壳聚糖具有更强的粘附性和无毒性,已被许多研究采用。多项研究将壳聚糖作为制造纳米载体和治疗糖尿病视网膜病变的最佳候选材料。利用壳聚糖配制的纳米载体不计其数,如用于治疗糖尿病视网膜病变的纳米结构脂质载体、固体脂质纳米颗粒、脂质体和树枝状分子等。本综述阐述了壳聚糖作为一种有望用于治疗糖尿病视网膜病变的纳米载体制造方法的最新进展。
{"title":"Chitosan as a promising materials for the construction of nanocarriers for diabetic retinopathy: an updated review.","authors":"Yan Lv, Chenglei Zhai, Gang Sun, Yangfang He","doi":"10.1186/s13036-024-00414-7","DOIUrl":"10.1186/s13036-024-00414-7","url":null,"abstract":"<p><p>Diabetic retinopathy (DR) is a condition that causes swelling of the blood vessels of the retina and leaks blood and fluids. It is the most severe form of diabetic eye disease. It causes vision loss in its advanced stage. Diabetic retinopathy is responsible for causing 26% of blindness. Very insufficient therapies are accessible for the treatment of DR. As compared to the conventional therapies, there should be enhanced research on the controlled release, shorter duration, and cost-effective therapy of diabetic retinopathy. The expansion of advanced nanocarriers-based drug delivery systems has been now employed to exploit as well as regulate the transport of many therapeutic agents to target sites via the increase in penetration or the extension of the duration of contact employing production by enclosing as well as distributing tiny molecules in nanostructured formulation. Various polymers have been utilized for the manufacturing of these nanostructured formulations. Chitosan possesses incredible biological and chemical properties, that have led to its extensive use in pharmaceutical and biomedical applications. Chitosan has been used in many studies because of its enhanced mucoadhesiveness and non-toxicity. Multiple studies have used chitosan as the best candidate for manufacturing nanocarriers and treating diabetic retinopathy. Numerous nanocarriers have been formulated by using chitosan such as nanostructured lipid carriers, solid lipid nanoparticles, liposomes, and dendrimers for treating diabetic retinopathy. This current review elaborates on the recent advancements of chitosan as a promising approach for the manufacturing of nanocarriers that can be used for treating diabetic retinopathy.</p>","PeriodicalId":15053,"journal":{"name":"Journal of Biological Engineering","volume":"18 1","pages":"18"},"PeriodicalIF":5.6,"publicationDate":"2024-02-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10885467/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139931174","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Preparation of bilayer tissue-engineered polyurethane/poly-L-lactic acid nerve conduits and their in vitro characterization for use in peripheral nerve regeneration 制备用于外周神经再生的双层组织工程聚氨酯/聚左旋乳酸神经导管及其体外表征
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-02-22 DOI: 10.1186/s13036-024-00412-9
Mehran Nabipour, Amir Mellati, Mozhgan Abasi, Somayeh Ebrahimi Barough, Ayoob Karimizade, Parnian Banikarimi, Elham Hasanzadeh
Due to loss of peripheral nerve structure and/or function resulting from trauma, accidents, and other causes, peripheral nerve injuries continue to be a major clinical problem. These injuries can cause partial or total loss of sensory, motor, and autonomic capabilities as well as neuropathic pain. PNI affects between 13 and 23 out of every 100,000 people annually in developed countries. Regeneration of damaged nerves and restoration of function after peripheral nerve injury remain significant therapeutic challenges. Although autologous nerve graft transplantation is a viable therapy option in several clinical conditions, donor site morbidity and a lack of donor tissue often hinder full functional recovery. Biomimetic conduits used in tissue engineering to encourage and direct peripheral nerve regeneration by providing a suitable microenvironment for nerve ingrowth are only one example of the cutting-edge methods made possible by this field. Many innate extracellular matrix (ECM) structures of different tissues can be successfully mimicked by nanofibrous scaffolds. Nanofibrous scaffolds can closely mimic the surface structure and morphology of native ECMs of many tissues. In this study, we have produced bilayer nanofibrous nerve conduit based on poly-lactic acid/polyurethane/multiwall carbon nanotube (PLA/PU/MWCNT), for application as composite scaffolds for static nerve tissue engineering. The contact angle was indicated to show the hydrophilicity properties of electrospun nanofibers. The SEM images were analyzed to determine the fiber’s diameters, scaffold morphology, and endometrial stem cell adhesion. Moreover, MTT assay and DAPI staining were used to show the viability and proliferation of endometrial stem cells. The constructed bilayer PLA/PU/MWCNT scaffolds demonstrated the capacity to support cell attachment, and the vitality of samples was assessed using SEM, MTT assay, and DAPI staining technique. According to an in vitro study, electrospun bilayer PLA/PU/MWCNT scaffolds can encourage the adhesion and proliferation of human endometrial stem cells (hEnSCs) and create the ideal environment for increasing cell survival.
由于外伤、事故和其他原因导致周围神经结构和/或功能丧失,周围神经损伤仍然是一个主要的临床问题。这些损伤可导致感觉、运动和自主神经能力的部分或完全丧失以及神经性疼痛。在发达国家,每年每 10 万人中就有 13 到 23 人受到周围神经损伤的影响。周围神经损伤后,受损神经的再生和功能恢复仍是重大的治疗难题。虽然自体神经移植在多种临床情况下都是可行的治疗方案,但供体部位的发病率和供体组织的缺乏往往会阻碍功能的完全恢复。组织工程中使用的仿生导管可为神经生长提供合适的微环境,从而促进和引导周围神经再生,这只是该领域最前沿方法的一个例子。纳米纤维支架可以成功模拟不同组织的许多固有细胞外基质(ECM)结构。纳米纤维支架可以近似模拟许多组织的原生 ECM 的表面结构和形态。在这项研究中,我们制备了基于聚乳酸/聚氨酯/多壁碳纳米管(PLA/PU/MWCNT)的双层纳米纤维神经导管,作为静态神经组织工程的复合支架。接触角显示了电纺纳米纤维的亲水性能。通过分析扫描电镜图像,确定了纤维的直径、支架形态和子宫内膜干细胞粘附性。此外,还利用 MTT 试验和 DAPI 染色来显示子宫内膜干细胞的活力和增殖情况。所构建的双层聚乳酸/聚氨酯/MWCNT 支架具有支持细胞附着的能力,并利用扫描电镜、MTT 试验和 DAPI 染色技术评估了样品的活力。体外研究表明,电纺双层聚乳酸/聚氨酯/MWCNT支架可促进人子宫内膜干细胞(hEnSCs)的粘附和增殖,并为提高细胞存活率创造理想的环境。
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引用次数: 0
Proteomic analysis of decellularized mice liver and kidney extracellular matrices 脱细胞小鼠肝脏和肾脏细胞外基质的蛋白质组分析
IF 5.6 3区 生物学 Q1 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-02-22 DOI: 10.1186/s13036-024-00413-8
Anna-Maria Diedrich, Assal Daneshgar, Peter Tang, Oliver Klein, Annika Mohr, Olachi A. Onwuegbuchulam, Sabine von Rueden, Kerstin Menck, Annalen Bleckmann, Mazen A. Juratli, Felix Becker, Igor M. Sauer, Karl H. Hillebrandt, Andreas Pascher, Benjamin Struecker
The extracellular matrix (ECM) is a three-dimensional network of proteins that encases and supports cells within a tissue and promotes physiological and pathological cellular differentiation and functionality. Understanding the complex composition of the ECM is essential to decrypt physiological processes as well as pathogenesis. In this context, the method of decellularization is a useful technique to eliminate cellular components from tissues while preserving the majority of the structural and functional integrity of the ECM. In this study, we employed a bottom-up proteomic approach to elucidate the intricate network of proteins in the decellularized extracellular matrices of murine liver and kidney tissues. This approach involved the use of a novel, perfusion-based decellularization protocol to generate acellular whole organ scaffolds. Proteomic analysis of decellularized mice liver and kidney ECM scaffolds revealed tissue-specific differences in matrisome composition, while we found a predominantly stable composition of the core matrisome, consisting of collagens, glycoproteins, and proteoglycans. Liver matrisome analysis revealed unique proteins such as collagen type VI alpha-6, fibrillin-2 or biglycan. In the kidney, specific ECM-regulators such as cathepsin z were detected. The identification of distinct proteomic signatures provides insights into how different matrisome compositions might influence the biological properties of distinct tissues. This experimental workflow will help to further elucidate the proteomic landscape of decellularized extracellular matrix scaffolds of mice in order to decipher complex cell–matrix interactions and their contribution to a tissue-specific microenvironment.
细胞外基质(ECM)是由蛋白质组成的三维网络,它包裹并支持组织内的细胞,促进生理和病理细胞的分化和功能。了解 ECM 的复杂组成对于解密生理过程和发病机制至关重要。在这种情况下,脱细胞方法是一种有用的技术,它可以消除组织中的细胞成分,同时保留 ECM 的大部分结构和功能完整性。在本研究中,我们采用了一种自下而上的蛋白质组学方法,以阐明脱细胞小鼠肝脏和肾脏组织细胞外基质中错综复杂的蛋白质网络。这种方法采用了一种新颖的、基于灌流的脱细胞方案来生成无细胞的全器官支架。对脱细胞小鼠肝脏和肾脏 ECM 支架进行的蛋白质组分析表明,不同组织的基质组组成存在差异,而我们发现核心基质组的组成主要是稳定的,由胶原蛋白、糖蛋白和蛋白多糖组成。肝脏基质组分析发现了独特的蛋白质,如α-6型胶原蛋白、纤连蛋白-2或biglycan。在肾脏中,检测到了特定的 ECM 调节因子,如 cathepsin z。通过鉴定不同的蛋白质组特征,可以深入了解不同的基质组组成如何影响不同组织的生物特性。这一实验工作流程将有助于进一步阐明小鼠脱细胞细胞外基质支架的蛋白质组图谱,从而破解复杂的细胞-基质相互作用及其对组织特异性微环境的贡献。
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