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Development of a screening platform to discover natural products active against SARS-CoV-2 infection using lung organoid models. 开发筛选平台,利用肺器官模型发现对 SARS-CoV-2 感染有活性的天然产品。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-03-01 DOI: 10.1186/s40824-023-00357-y
Joo-Eun Lee, Se Yun Jeong, Zijun Li, Hyun-Yi Kim, Hyun-Woo Kim, Min Jeong Yoo, Hee Joo Jang, Do-Kyun Kim, Namki Cho, Hee Min Yoo, Ki Hyun Kim

Background: Natural products can serve as one of the alternatives, exhibiting high potential for the treatment and prevention of COVID-19, caused by SARS-CoV-2. Herein, we report a screening platform to test the antiviral efficacy of a natural product library against SARS-CoV-2 and verify their activity using lung organoids.

Methods: Since SARS-CoV-2 is classified as a risk group 3 pathogen, the drug screening assay must be performed in a biosafety level 3 (BSL-3) laboratory. To circumvent this limitation, pseudotyped viruses (PVs) have been developed as replacements for the live SARS-CoV-2. We developed PVs containing spikes from Delta and Omicron variants of SARS-CoV-2 and improved the infection in an angiotensin-converting enzyme 2 (ACE2)-dependent manner. Human induced pluripotent stem cells (hiPSCs) derived lung organoids were generated to test the SARS-CoV-2 therapeutic efficacy of natural products.

Results: Flavonoids from our natural product library had strong antiviral activity against the Delta- or Omicron-spike-containing PVs without affecting cell viability. We aimed to develop strategies to discover the dual function of either inhibiting infection at the beginning of the infection cycle or reducing spike stability following SARS-CoV-2 infection. When lung cells are already infected with the virus, the active flavonoids induced the degradation of the spike protein and exerted anti-inflammatory effects. Further experiments confirmed that the active flavonoids had strong antiviral activity in lung organoid models.

Conclusion: This screening platform will open new paths by providing a promising standard system for discovering novel drug leads against SARS-CoV-2 and help develop promising candidates for clinical investigation as potential therapeutics for COVID-19.

背景:天然产物是治疗和预防由SARS-CoV-2引起的COVID-19的替代品之一,具有很高的潜力。在此,我们报告了一个筛选平台,以测试天然产品库对 SARS-CoV-2 的抗病毒功效,并使用肺器官组织验证其活性:方法:由于SARS-CoV-2被列为3类危险病原体,因此药物筛选试验必须在生物安全等级为3级(BSL-3)的实验室中进行。为了规避这一限制,人们开发了伪型病毒(PV)来替代活的 SARS-CoV-2。我们开发了含有 SARS-CoV-2 Delta 和 Omicron 变体尖峰的假病毒,并以血管紧张素转换酶 2 (ACE2) 依赖性方式改善了感染。为了测试天然产物对SARS-CoV-2的疗效,我们生成了人诱导多能干细胞(hiPSCs)衍生的肺器官组织:结果:我们的天然产品库中的黄酮类化合物对含有Delta或Omicron-spike的PV具有很强的抗病毒活性,且不影响细胞活力。我们的目标是制定策略,发现在感染周期开始时抑制感染或在感染 SARS-CoV-2 后降低尖峰稳定性的双重功能。当肺部细胞已经感染病毒时,活性黄酮类化合物会诱导尖峰蛋白降解,并发挥抗炎作用。进一步的实验证实,活性黄酮类化合物在肺器官模型中具有很强的抗病毒活性:这一筛选平台将为发现抗SARS-CoV-2的新药线索提供一个有前途的标准系统,从而开辟新的道路,并有助于开发有希望的候选药物,作为COVID-19的潜在治疗药物进行临床研究。
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引用次数: 0
Rediscovery of poly(ethylene glycol)s as a cryoprotectant for mesenchymal stem cells. 重新发现作为间充质干细胞低温保护剂的聚乙二醇。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-20 DOI: 10.1186/s40824-023-00356-z
Madhumita Patel, Jin Kyung Park, Byeongmoon Jeong

Background: A medium containing dimethyl sulfoxide (DMSO) (10% v/v) is most widely used for cell cryopreservation at -196 °C. However, residual DMSO consistently raises concerns because of its toxicity; thus, its complete removal process is required.

Method: As biocompatible polymers approved by the Food and Drug Administration for various biomedical applications for humans, poly(ethylene glycol)s (PEGs) with various molecular weights (400, 600, 1 K, 1.5 K, 5 K, 10 K, and 20 K Da) were studied as a cryoprotectant of mesenchymal stem cells (MSCs). Considering the cell permeability difference of PEGs depending on their molecular weight, the cells were preincubated for 0 h (no incubation), 2 h, and 4 h at 37 °C in the presence of PEGs at 10 wt.% before cryopreservation at -196 °C for 7 days. Then, cell recovery was assayed.

Results: We found that low molecular weight PEGs (400 and 600 Da) exhibit excellent cryoprotecting properties by 2 h preincubation, whereas intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) exhibit their cryoprotecting properties without preincubation. High molecular weight PEGs (10 K and 20 K Da) were ineffective as cryoprotectants for MSCs. Studies on ice recrystallization inhibition (IRI), ice nucleation inhibition (INI), membrane stabilization, and intracellular transport of PEGs suggest that low molecular weight PEGs (400 and 600 Da) exhibit excellent intracellular transport properties, and thus the internalized PEGs during preincubation contribute to the cryoprotection. Intermediate molecular weight PEGs (1 K, 1.5 K, and 5 K Da) worked by extracellular PEGs through IRI, INI, as well as partly internalized PEGs. High molecular weight PEGs (10 K and 20 K Da) killed the cells during preincubation and were ineffective as cryoprotectants.

Conclusions: PEGs can be used as cryoprotectants. However, the detailed procedures, including preincubation, should consider the effect of the molecular weight of PEGs. The recovered cells well proliferated and underwent osteo/chondro/adipogenic differentiation similar to the MSCs recovered from the traditional DMSO 10% system.

背景:含二甲基亚砜(DMSO)(10% v/v)的培养基被广泛用于-196 °C的细胞冷冻保存。然而,残留的二甲基亚砜因其毒性而一直引起人们的关注;因此,需要彻底清除二甲基亚砜:方法:作为经美国食品和药物管理局批准用于人类各种生物医学应用的生物相容性聚合物,不同分子量(400、600、1 K、1.5 K、5 K、10 K 和 20 K Da)的聚乙二醇(PEGs)被研究用作间充质干细胞(MSCs)的冷冻保护剂。考虑到不同分子量的 PEG 对细胞的渗透性不同,在-196 °C冷冻保存 7 天之前,将细胞在 10 wt.% 的 PEG 存在下于 37 °C预孵育 0 小时(不孵育)、2 小时和 4 小时。然后检测细胞的恢复情况:结果:我们发现低分子量 PEG(400 和 600 Da)在预孵育 2 小时后表现出卓越的冷冻保护特性,而中分子量 PEG(1 K、1.5 K 和 5 K Da)无需预孵育即可表现出冷冻保护特性。高分子量聚乙二醇(10 K 和 20 K Da)不能作为间充质干细胞的低温保护剂。对 PEG 的冰再结晶抑制(IRI)、冰成核抑制(INI)、膜稳定性和细胞内转运的研究表明,低分子量 PEG(400 和 600 Da)具有良好的细胞内转运特性,因此预孵育期间内化的 PEG 有助于低温保护。中等分子量的 PEG(1 K、1.5 K 和 5 K Da)通过 IRI、INI 以及部分内化的 PEG 发挥细胞外 PEG 的作用。高分子量 PEG(10 K 和 20 K Da)会在预孵育过程中杀死细胞,不能作为低温保护剂:结论:PEG 可用作低温保护剂。结论:PEGs 可用作低温保护剂,但具体操作(包括预孵育)应考虑 PEGs 分子量的影响。回收的细胞增殖良好,并进行了成骨/软骨/成脂分化,与传统的 DMSO 10% 系统回收的间充质干细胞相似。
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引用次数: 0
Identification of cell-biologic mechanisms of coronary artery spasm and its ex vivo diagnosis using peripheral blood-derived iPSCs. 利用外周血源性iPSCs鉴定冠状动脉痉挛的细胞生物学机制及其体外诊断。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-18 DOI: 10.1186/s40824-023-00345-2
Han-Mo Yang, Joo-Eun Lee, Ju-Young Kim, Jihye You, Joonoh Kim, Hak Seung Lee, Hee Min Yoo, Min Gyu Kong, Jung-Kyu Han, Hyun-Jai Cho, Kyung Woo Park, Hyun-Jae Kang, Bon-Kwon Koo, Young-Bae Park, Hyo-Soo Kim

Background: Although vasospastic angina (VSA) is known to be caused by coronary artery spasm, no study has fully elucidated the exact underlying mechanism. Moreover, in order to confirm VSA, patients should undergo invasive coronary angiography with spasm provocation test. Herein, we investigated the pathophysiology of VSA using peripheral blood-derived induced pluripotent stem cells (iPSCs) and developed an ex vivo diagnostic method for VSA.

Methods and results: With 10 mL of peripheral blood from patients with VSA, we generated iPSCs and differentiated these iPSCs into target cells. As compared with vascular smooth muscle cells (VSMCs) differentiated from iPSCs of normal subjects with negative provocation test, VSA patient-specific iPSCs-derived VSMCs showed very strong contraction in response to stimulants. Moreover, VSA patient-specific VSMCs exhibited a significant increase in stimulation-induced intracellular calcium efflux (Changes in the relative fluorescence unit [ΔF/F]; Control group vs. VSA group, 2.89 ± 0.34 vs. 10.32 ± 0.51, p < 0.01), and exclusively induced a secondary or tertiary peak of calcium efflux, suggesting that those findings could be diagnostic cut-off values for VSA. The observed hyperreactivity of VSA patient-specific VSMCs were caused by the upregulation of sarco/endoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) due to its enhanced small ubiquitin-related modifier (SUMO)ylation. This increased activity of SERCA2a was reversed by treatment with ginkgolic acid, an inhibitor of SUMOylated E1 molecules (pi/µg protein; VSA group vs. VSA + ginkgolic acid, 52.36 ± 0.71 vs. 31.93 ± 1.13, p < 0.01).

Conclusions: Our findings showed that abnormal calcium handling in sarco/endoplasmic reticulum could be induced by the enhanced SERCA2a activity in patients with VSA, leading to spasm. Such novel mechanisms of coronary artery spasm could be useful for drug development and diagnosis of VSA.

背景:虽然血管痉挛性心绞痛(VSA)已知是由冠状动脉痉挛引起的,但没有研究完全阐明其确切的潜在机制。此外,为了确认VSA,患者应进行有创冠状动脉造影和痉挛激发试验。在此,我们使用外周血源性诱导多能干细胞(iPSCs)研究VSA的病理生理,并开发了VSA的体外诊断方法。方法和结果:用10 mL VSA患者外周血制备iPSCs,并将其分化为靶细胞。与阴性激发试验的正常受试者iPSCs分化的血管平滑肌细胞(VSMCs)相比,VSA患者特异性iPSCs衍生的血管平滑肌细胞在兴奋剂作用下表现出非常强的收缩反应。此外,VSA患者特异性VSMCs表现出刺激诱导的细胞内钙外排显著增加(相对荧光单位的变化[ΔF/F];对照组与VSA组相比,p 2+- atp酶2a (SERCA2a)的差异为2.89±0.34比10.32±0.51,这是由于其小泛素相关修饰子(SUMO)的磷酸化增强。这种增加的SERCA2a活性被银杏酸处理逆转,银杏酸是SUMOylated E1分子的抑制剂(pi/µg蛋白;VSA组vs. VSA +银杏酸组,52.36±0.71 vs. 31.93±1.13,p结论:VSA患者SERCA2a活性增强可诱导sarco/内质网钙处理异常,导致痉挛。这种新的冠状动脉痉挛机制可能对VSA的药物开发和诊断有用。
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引用次数: 0
A novel DNA double-strand breaks biosensor based on fluorescence resonance energy transfer. 基于荧光共振能量转移的新型DNA双链断裂生物传感器。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-17 DOI: 10.1186/s40824-023-00354-1
Jung-Soo Suh, Tae-Jin Kim

Revealing the spatiotemporal behavior of DNA double-strand breaks (DSBs) is crucial for understanding the processes of DNA damage and repair. Traditionally, γH2AX and DNA damage response (DDR) factors have been used to detect DSBs using classical biochemical assays, such as antibody-based immunostaining. However, a reliable method to visualize and assess DSB activity real-time in living cells is yet to be established. Herein, we developed a novel DNA double-strand breaks biosensor (DSBS) based on fluorescence resonance energy transfer (FRET) by employing the H2AX and BRCT1 domains. By applying FRET imaging with DSBS, we show that DSBS specifically reacts to drug- or ionizing radiation (IR)-induced γH2AX activity, allowing for the quantification of DSB events at high spatiotemporal resolutions. Taken together, we provide a new experimental tool to evaluate the spatiotemporal dynamics of DNA double-strand breaks. Ultimately, our biosensor can be useful for elucidating the molecular mechanisms underlying DNA damage and repair processes.

揭示DNA双链断裂(DSBs)的时空行为对于理解DNA损伤和修复过程至关重要。传统上,使用γ - h2ax和DNA损伤反应(DDR)因子使用传统的生化分析,如基于抗体的免疫染色来检测dsb。然而,一种可靠的方法来可视化和实时评估DSB活性的活细胞尚未建立。在此,我们利用H2AX和BRCT1结构域开发了一种基于荧光共振能量转移(FRET)的DNA双链断裂生物传感器(DSBS)。通过应用DSBS的FRET成像,我们发现DSBS对药物或电离辐射(IR)诱导的γ - h2ax活性有特异性反应,从而可以在高时空分辨率下量化DSB事件。总之,我们提供了一个新的实验工具来评估DNA双链断裂的时空动力学。最终,我们的生物传感器可以用于阐明DNA损伤和修复过程的分子机制。
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引用次数: 0
Effect of chitooligosaccharide on the inhibition of SARS-CoV-2 main protease. 壳寡糖对SARS-CoV-2主要蛋白酶的抑制作用。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-17 DOI: 10.1186/s40824-023-00351-4
Qian Wang, Yuanyuan Song, Mungu Kim, Sei Kwang Hahn, Ge Jiang

Background: The main protease (Mpro) is a crucial target for severe acute respiratory syndrome coronavirus (SARS-CoV-2). Chitooligosaccharide (CS) has broad-spectrum antiviral activity and can effectively inhibit the activity of SARS-CoV. Here, based on the high homology between SARS-CoV-2 and SARS-CoV, this study explores the effect and mechanism of CS with various molecular weights on the activity of SARS-CoV-2 Mpro.

Methods: We used fluorescence resonance energy transfer (FRET), UV-Vis, synchronous fluorescence spectroscopy, circular dichroism (CD) spectroscopy and computational simulation to investigate the molecular interaction and the interaction mechanism between CS and SARS-CoV-2 Mpro.

Results: Four kinds of CS with different molecular weights significantly inhibited the activity of Mpro by combining the hydrogen bonding and the salt bridge interaction to form a stable complex. Glu166 appeared to be the key amino acid. Among them, chitosan showed the highest inhibition effect on Mpro enzyme activity and the greatest impact on the spatial structure of protein. Chitosan would be one of the most potential anti-viral compounds.

Conclusion: This study provides the theoretical basis to develop targeted Mpro inhibitors for the screening and application of anti-novel coronavirus drugs.

背景:主要蛋白酶(Mpro)是严重急性呼吸综合征冠状病毒(SARS-CoV-2)的关键靶点。壳寡糖(Chitooligosaccharide, CS)具有广谱抗病毒活性,能有效抑制SARS-CoV的活性。本研究基于SARS-CoV-2与SARS-CoV的高度同源性,探讨不同分子量的CS对SARS-CoV-2 Mpro活性的影响及其机制。方法:采用荧光共振能量转移(FRET)、紫外可见、同步荧光光谱、圆二色(CD)光谱和计算模拟等方法研究CS与SARS-CoV-2 Mpro的分子相互作用及其机制。结果:4种不同分子量的CS通过结合氢键和盐桥相互作用形成稳定的配合物,显著抑制了Mpro的活性。Glu166似乎是关键氨基酸。其中,壳聚糖对Mpro酶活性的抑制作用最大,对蛋白质空间结构的影响最大。壳聚糖可能是最有潜力的抗病毒化合物之一。结论:本研究为开发靶向Mpro抑制剂,为新型冠状病毒药物的筛选和应用提供了理论依据。
{"title":"Effect of chitooligosaccharide on the inhibition of SARS-CoV-2 main protease.","authors":"Qian Wang,&nbsp;Yuanyuan Song,&nbsp;Mungu Kim,&nbsp;Sei Kwang Hahn,&nbsp;Ge Jiang","doi":"10.1186/s40824-023-00351-4","DOIUrl":"https://doi.org/10.1186/s40824-023-00351-4","url":null,"abstract":"<p><strong>Background: </strong>The main protease (Mpro) is a crucial target for severe acute respiratory syndrome coronavirus (SARS-CoV-2). Chitooligosaccharide (CS) has broad-spectrum antiviral activity and can effectively inhibit the activity of SARS-CoV. Here, based on the high homology between SARS-CoV-2 and SARS-CoV, this study explores the effect and mechanism of CS with various molecular weights on the activity of SARS-CoV-2 Mpro.</p><p><strong>Methods: </strong>We used fluorescence resonance energy transfer (FRET), UV-Vis, synchronous fluorescence spectroscopy, circular dichroism (CD) spectroscopy and computational simulation to investigate the molecular interaction and the interaction mechanism between CS and SARS-CoV-2 Mpro.</p><p><strong>Results: </strong>Four kinds of CS with different molecular weights significantly inhibited the activity of Mpro by combining the hydrogen bonding and the salt bridge interaction to form a stable complex. Glu166 appeared to be the key amino acid. Among them, chitosan showed the highest inhibition effect on Mpro enzyme activity and the greatest impact on the spatial structure of protein. Chitosan would be one of the most potential anti-viral compounds.</p><p><strong>Conclusion: </strong>This study provides the theoretical basis to develop targeted Mpro inhibitors for the screening and application of anti-novel coronavirus drugs.</p>","PeriodicalId":9079,"journal":{"name":"Biomaterials Research","volume":"27 1","pages":"13"},"PeriodicalIF":11.3,"publicationDate":"2023-02-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9935244/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9293509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Basic amino acid-mediated cationic amphiphilic surfaces for antimicrobial pH monitoring sensor with wound healing effects. 碱性氨基酸介导的阳离子两亲性表面用于具有伤口愈合作用的抗菌pH监测传感器。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-17 DOI: 10.1186/s40824-023-00355-0
Dong Uk Lee, Se-Chang Kim, Dong Yun Choi, Won-Kyo Jung, Myung Jun Moon

Background: The wound healing process is a complex cascade of physiological events, which are vulnerable to both our body status and external factors and whose impairment could lead to chronic wounds or wound healing impediments. Conventional wound healing materials are widely used in clinical management, however, they do not usually prevent wounds from being infected by bacteria or viruses. Therefore, simultaneous wound status monitoring and prevention of microbial infection are required to promote healing in clinical wound management.

Methods: Basic amino acid-modified surfaces were fabricated in a water-based process via a peptide coupling reaction. Specimens were analyzed and characterized by X-ray photoelectron spectroscopy, Kelvin probe force microscopy, atomic force microscopy, contact angle, and molecular electrostatic potential via Gaussian 09. Antimicrobial and biofilm inhibition tests were conducted on Escherichia coli and Staphylococcus epidermidis. Biocompatibility was determined through cytotoxicity tests on human epithelial keratinocytes and human dermal fibroblasts. Wound healing efficacy was confirmed by mouse wound healing and cell staining tests. Workability of the pH sensor on basic amino acid-modified surfaces was evaluated on normal human skin and Staphylococcus epidermidis suspension, and in vivo conditions.

Results: Basic amino acids (lysine and arginine) have pH-dependent zwitterionic functional groups. The basic amino acid-modified surfaces had antifouling and antimicrobial properties similar to those of cationic antimicrobial peptides because zwitterionic functional groups have intrinsic cationic amphiphilic characteristics. Compared with untreated polyimide and modified anionic acid (leucine), basic amino acid-modified polyimide surfaces displayed excellent bactericidal, antifouling (reduction ~ 99.6%) and biofilm inhibition performance. The basic amino acid-modified polyimide surfaces also exhibited wound healing efficacy and excellent biocompatibility, confirmed by cytotoxicity and ICR mouse wound healing tests. The basic amino acid-modified surface-based pH monitoring sensor was workable (sensitivity 20 mV pH-1) under various pH and bacterial contamination conditions.

Conclusion: Here, we developed a biocompatible and pH-monitorable wound healing dressing with antimicrobial activity via basic amino acid-mediated surface modification, creating cationic amphiphilic surfaces. Basic amino acid-modified polyimide is promising for monitoring wounds, protecting them from microbial infection, and promoting their healing. Our findings are expected to contribute to wound management and could be expanded to various wearable healthcare devices for clinical, biomedical, and healthcare applications.

背景:创面愈合过程是一个复杂的级联生理事件,易受机体状态和外界因素的影响,其损伤可导致慢性创面或创面愈合障碍。传统的伤口愈合材料在临床治疗中被广泛使用,然而,它们通常不能防止伤口被细菌或病毒感染。因此,在临床创面管理中,需要同时监测创面状态和预防微生物感染,以促进创面愈合。方法:通过多肽偶联反应,在水基法制备碱性氨基酸修饰表面。采用x射线光电子能谱、开尔文探针力显微镜、原子力显微镜、接触角和分子静电势等方法对样品进行了分析和表征。对大肠杆菌和表皮葡萄球菌进行了抑菌试验和生物膜抑制试验。通过人上皮角质形成细胞和人真皮成纤维细胞的细胞毒性试验确定生物相容性。小鼠创面愈合及细胞染色试验证实创面愈合效果。在正常人体皮肤和表皮葡萄球菌悬浮液以及体内条件下,评估了pH传感器在碱性氨基酸修饰表面的可加工性。结果:碱性氨基酸(赖氨酸和精氨酸)具有ph依赖的两性离子官能团。由于两性离子官能团具有固有的阳离子两亲性,碱性氨基酸修饰的表面具有与阳离子抗菌肽相似的防污和抗菌性能。与未处理的聚酰亚胺和改性阴离子酸(亮氨酸)相比,碱性氨基酸改性聚酰亚胺表面表现出优异的杀菌、防污(还原~ 99.6%)和生物膜抑制性能。细胞毒性和ICR小鼠伤口愈合实验证实,碱性氨基酸修饰的聚酰亚胺表面也表现出良好的伤口愈合效果和生物相容性。碱性氨基酸修饰的表面pH监测传感器在各种pH和细菌污染条件下均可工作,灵敏度为20 mV pH-1。结论:通过碱性氨基酸介导的表面修饰,我们开发了一种具有生物相容性和ph可监测的抗菌活性伤口愈合敷料,形成了阳离子两亲性表面。碱性氨基酸修饰聚酰亚胺有望监测伤口,保护他们免受微生物感染,并促进其愈合。我们的研究结果有望为伤口管理做出贡献,并可扩展到临床、生物医学和医疗保健应用的各种可穿戴医疗设备。
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引用次数: 0
Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases. 磁性转铁蛋白纳米颗粒(MTNs)检测是一种分离神经疾病外泌体生物标志物的新方法。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-16 DOI: 10.1186/s40824-023-00353-2
Yoon Ok Jang, Hee-Sung Ahn, Thuy Nguyen Thi Dao, JeongYeon Hong, Wangyong Shin, Young-Min Lim, Sun Ju Chung, Jae-Hong Lee, Huifang Liu, Bonhan Koo, Myoung Gyu Kim, Kyunggon Kim, Eun-Jae Lee, Yong Shin

Background: Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples.

Methods: The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers.

Results: Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases.

Conclusion: The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.

背景:释放到血液中的脑源性外泌体被认为是研究病理生理状态的液体活检,反映了神经系统疾病中大脑病理进展的异常异质途径。脑源性血外泌体为神经系统疾病的诊断提供了良好的前景,为此类疾病的早期和敏感诊断提供了令人兴奋的可能性。然而,不能保证传统的外泌体分离方法能够特异性地分离血液外泌体,并通过高通量蛋白质组学对神经系统疾病患者临床标本的脑源性血外泌体蛋白进行表征。我们报道了磁性转铁蛋白纳米颗粒(MTNs)测定,将转铁蛋白和磁性纳米颗粒结合,从临床样品中分离脑源性血液外泌体。方法:mtn检测的原理是通过mtn上的转铁蛋白和外泌体上的转铁蛋白受体进行配体-受体相互作用,通过带正电的mtn和带负电的外泌体进行静电相互作用,分离脑源性血外泌体。结果:通过比较统计分析,我们发现21个候选蛋白质生物标志物在三组神经退行性疾病中存在显著差异。结论:MTNs检测是一种方便的方法,可以从体液中特异性分离细胞外囊泡,用于神经系统疾病的微创诊断。
{"title":"Magnetic transferrin nanoparticles (MTNs) assay as a novel isolation approach for exosomal biomarkers in neurological diseases.","authors":"Yoon Ok Jang,&nbsp;Hee-Sung Ahn,&nbsp;Thuy Nguyen Thi Dao,&nbsp;JeongYeon Hong,&nbsp;Wangyong Shin,&nbsp;Young-Min Lim,&nbsp;Sun Ju Chung,&nbsp;Jae-Hong Lee,&nbsp;Huifang Liu,&nbsp;Bonhan Koo,&nbsp;Myoung Gyu Kim,&nbsp;Kyunggon Kim,&nbsp;Eun-Jae Lee,&nbsp;Yong Shin","doi":"10.1186/s40824-023-00353-2","DOIUrl":"https://doi.org/10.1186/s40824-023-00353-2","url":null,"abstract":"<p><strong>Background: </strong>Brain-derived exosomes released into the blood are considered a liquid biopsy to investigate the pathophysiological state, reflecting the aberrant heterogeneous pathways of pathological progression of the brain in neurological diseases. Brain-derived blood exosomes provide promising prospects for the diagnosis of neurological diseases, with exciting possibilities for the early and sensitive diagnosis of such diseases. However, the capability of traditional exosome isolation assays to specifically isolate blood exosomes and to characterize the brain-derived blood exosomal proteins by high-throughput proteomics for clinical specimens from patients with neurological diseases cannot be assured. We report a magnetic transferrin nanoparticles (MTNs) assay, which combined transferrin and magnetic nanoparticles to isolate brain-derived blood exosomes from clinical samples.</p><p><strong>Methods: </strong>The principle of the MTNs assay is a ligand-receptor interaction through transferrin on MTNs and transferrin receptor on exosomes, and electrostatic interaction via positively charged MTNs and negatively charged exosomes to isolate brain-derived blood exosomes. In addition, the MTNs assay is simple and rapid (< 35 min) and does not require any large instrument. We confirmed that the MTNs assay accurately and efficiently isolated exosomes from serum samples of humans with neurodegenerative diseases, such as dementia, Parkinson's disease (PD), and multiple sclerosis (MS). Moreover, we isolated exosomes from serum samples of 30 patients with three distinct neurodegenerative diseases and performed unbiased proteomic analysis to explore the pilot value of brain-derived blood protein profiles as biomarkers.</p><p><strong>Results: </strong>Using comparative statistical analysis, we found 21 candidate protein biomarkers that were significantly different among three groups of neurodegenerative diseases.</p><p><strong>Conclusion: </strong>The MTNs assay is a convenient approach for the specific and affordable isolation of extracellular vesicles from body fluids for minimally-invasive diagnosis of neurological diseases.</p>","PeriodicalId":9079,"journal":{"name":"Biomaterials Research","volume":"27 1","pages":"12"},"PeriodicalIF":11.3,"publicationDate":"2023-02-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9936675/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10800117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 5
Gelatin methacryloyl (GelMA) loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells(ADSCs) conditioned medium promotes wound healing and vascular regeneration in aged skin. 明胶甲基丙烯酰(GelMA)装载浓缩缺氧预处理脂肪源性间充质干细胞(ADSCs)条件培养基,促进老化皮肤的伤口愈合和血管再生。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-13 DOI: 10.1186/s40824-023-00352-3
Shiyi Li, Jiachen Sun, Jinxiu Yang, Yi Yang, Hongfan Ding, Boya Yu, Kui Ma, Minliang Chen

Background: Aging skin is characterized by a disturbed structure and lack of blood supply, which makes it difficult to heal once injured. ADSCs secrete large amounts of cytokines, which promote wound healing and vascular regeneration through paracrine secretion, and the number of cytokines can be elevated by hypoxic pretreating. However, the components of ADSCs are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel synthesized from gelatin and has recently emerged as a potentially attractive material for tissue engineering applications. GelMA loaded with concentrated hypoxic pretreated ADSCs conditioned medium could provide a new method of treating wounds in aged skin.

Methods: Primary ADSCs were isolated from human adipose tissue and characterized by flow cytometry and differentiation test. ADSCs in passages 4-6 were pretreated in the hypoxic and normoxic environments to collect conditioned medium, the conditioned medium was then concentrated to prepare concentrated ADSCs conditioned medium(cADSC-CM)(the one collected from ADSCs under hypoxia was called hypo-CM ,and the one from normoxia was called nor-CM). The concentration of cytokines was detected. After treated with cADSC-CM, the abilities of proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs) were assayed, and Akt/mTOR and MAPK signal pathway was detected using western blotting. GelMA+hypo-CM hydrogel was prepared, and a comprehensive evaluation of morphology, protein release efficiency, degradation rate, mechanical properties, and rheology properties were performed. Full-thickness skin wounds were created on the backs of 20-month-old mice. After surgery, GelMA, GelMA+F12, GelMA+hypo-CM, and GelMA+nor-CM were applied to the wound surface respectively. H&E, Masson, and immunohistochemistry staining were performed, and a laser Doppler perfusion imager was used to evaluate the blood perfusion. The student's t-test was used for analysis between two groups and a one-way analysis of variance (ANOVA) was used for analysis among multi groups.

Results: Our results revealed that 1) wounds in aged skin healed more slowly than that in young skin and exhibited poorer perfusion; 2) hypoxic pretreated ADSCs secreted more cytokines including VEGF by activating HIF1α; 3) hypo-CM promoted proliferation and migration of HUVECs through VEGF/Akt/mTOR and MAPK signal pathway; 4) GelMA-hypoCM accelerated wound healing and angiogenesis in aged skin in vivo.

Conclusion: GelMA loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells conditioned medium could accelerate wound healing in aged skin by promoting angiogenesis.

背景:皮肤老化的特点是结构紊乱,血液供应不足,一旦受伤就很难愈合。ADSCs分泌大量细胞因子,通过旁分泌促进创面愈合和血管再生,低氧预处理可提高细胞因子的数量。然而,ADSCs的成分很难在伤口中保留。明胶甲基丙烯酸酯(GelMA)是一种由明胶合成的光聚合水凝胶,近年来作为一种潜在的有吸引力的材料应用于组织工程。GelMA负载高浓度缺氧预处理的ADSCs条件培养基,为治疗老化皮肤创面提供了一种新的方法。方法:从人脂肪组织中分离原代ADSCs,采用流式细胞术和分化实验对其进行鉴定。将4 ~ 6代ADSCs分别在缺氧和常氧环境下进行预处理,收集条件培养基,将条件培养基浓缩制备ADSCs浓缩条件培养基(cADSC-CM)(缺氧条件下收集的ADSCs称为hypoo - cm,常氧条件下收集的ADSCs称为normo - cm)。检测细胞因子浓度。经cADSC-CM处理后,检测人脐血管内皮细胞(HUVECs)的增殖、迁移和成管能力,并采用western blotting检测Akt/mTOR和MAPK信号通路。制备GelMA+低- cm水凝胶,对其形态、蛋白质释放效率、降解率、力学性能、流变性能进行综合评价。在20个月大的小鼠背部创造了全层皮肤伤口。术后分别将GelMA、GelMA+F12、GelMA+低cm、GelMA+无cm涂抹于创面。行H&E、Masson、免疫组化染色,激光多普勒灌注成像仪评估血流灌注。两组间采用学生t检验,多组间采用单因素方差分析(ANOVA)。结果:1)老龄皮肤创面愈合速度慢于年轻皮肤创面,血流灌注较差;2)缺氧预处理的ADSCs通过激活HIF1α分泌更多的VEGF等细胞因子;3)低浓度cm通过VEGF/Akt/mTOR和MAPK信号通路促进HUVECs的增殖和迁移;4) GelMA-hypoCM在体内加速老化皮肤的伤口愈合和血管生成。结论:装载浓缩缺氧预处理脂肪间充质干细胞条件培养基的GelMA可通过促进血管生成来加速老化皮肤创面愈合。
{"title":"Gelatin methacryloyl (GelMA) loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells(ADSCs) conditioned medium promotes wound healing and vascular regeneration in aged skin.","authors":"Shiyi Li,&nbsp;Jiachen Sun,&nbsp;Jinxiu Yang,&nbsp;Yi Yang,&nbsp;Hongfan Ding,&nbsp;Boya Yu,&nbsp;Kui Ma,&nbsp;Minliang Chen","doi":"10.1186/s40824-023-00352-3","DOIUrl":"https://doi.org/10.1186/s40824-023-00352-3","url":null,"abstract":"<p><strong>Background: </strong>Aging skin is characterized by a disturbed structure and lack of blood supply, which makes it difficult to heal once injured. ADSCs secrete large amounts of cytokines, which promote wound healing and vascular regeneration through paracrine secretion, and the number of cytokines can be elevated by hypoxic pretreating. However, the components of ADSCs are difficult to retain in wounds. Gelatin methacrylate (GelMA) is a photopolymerizable hydrogel synthesized from gelatin and has recently emerged as a potentially attractive material for tissue engineering applications. GelMA loaded with concentrated hypoxic pretreated ADSCs conditioned medium could provide a new method of treating wounds in aged skin.</p><p><strong>Methods: </strong>Primary ADSCs were isolated from human adipose tissue and characterized by flow cytometry and differentiation test. ADSCs in passages 4-6 were pretreated in the hypoxic and normoxic environments to collect conditioned medium, the conditioned medium was then concentrated to prepare concentrated ADSCs conditioned medium(cADSC-CM)(the one collected from ADSCs under hypoxia was called hypo-CM ,and the one from normoxia was called nor-CM). The concentration of cytokines was detected. After treated with cADSC-CM, the abilities of proliferation, migration, and tube formation of human umbilical vascular endothelial cells (HUVECs) were assayed, and Akt/mTOR and MAPK signal pathway was detected using western blotting. GelMA+hypo-CM hydrogel was prepared, and a comprehensive evaluation of morphology, protein release efficiency, degradation rate, mechanical properties, and rheology properties were performed. Full-thickness skin wounds were created on the backs of 20-month-old mice. After surgery, GelMA, GelMA+F12, GelMA+hypo-CM, and GelMA+nor-CM were applied to the wound surface respectively. H&E, Masson, and immunohistochemistry staining were performed, and a laser Doppler perfusion imager was used to evaluate the blood perfusion. The student's t-test was used for analysis between two groups and a one-way analysis of variance (ANOVA) was used for analysis among multi groups.</p><p><strong>Results: </strong>Our results revealed that 1) wounds in aged skin healed more slowly than that in young skin and exhibited poorer perfusion; 2) hypoxic pretreated ADSCs secreted more cytokines including VEGF by activating HIF1α; 3) hypo-CM promoted proliferation and migration of HUVECs through VEGF/Akt/mTOR and MAPK signal pathway; 4) GelMA-hypoCM accelerated wound healing and angiogenesis in aged skin in vivo.</p><p><strong>Conclusion: </strong>GelMA loaded with concentrated hypoxic pretreated adipose-derived mesenchymal stem cells conditioned medium could accelerate wound healing in aged skin by promoting angiogenesis.</p>","PeriodicalId":9079,"journal":{"name":"Biomaterials Research","volume":"27 1","pages":"11"},"PeriodicalIF":11.3,"publicationDate":"2023-02-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9926638/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10734496","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 7
Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine. 脱细胞细胞外基质支架的免疫原性:组织工程和再生医学的瓶颈。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-09 DOI: 10.1186/s40824-023-00348-z
Mohammadreza Kasravi, Armin Ahmadi, Amirhesam Babajani, Radman Mazloomnejad, Mohammad Reza Hatamnejad, Siavash Shariatzadeh, Soheyl Bahrami, Hassan Niknejad

Tissue-engineered decellularized extracellular matrix (ECM) scaffolds hold great potential to address the donor shortage as well as immunologic rejection attributed to cells in conventional tissue/organ transplantation. Decellularization, as the key process in manufacturing ECM scaffolds, removes immunogen cell materials and significantly alleviates the immunogenicity and biocompatibility of derived scaffolds. However, the application of these bioscaffolds still confronts major immunologic challenges. This review discusses the interplay between damage-associated molecular patterns (DAMPs) and antigens as the main inducers of innate and adaptive immunity to aid in manufacturing biocompatible grafts with desirable immunogenicity. It also appraises the impact of various decellularization methodologies (i.e., apoptosis-assisted techniques) on provoking immune responses that participate in rejecting allogenic and xenogeneic decellularized scaffolds. In addition, the key research findings regarding the contribution of ECM alterations, cytotoxicity issues, graft sourcing, and implantation site to the immunogenicity of decellularized tissues/organs are comprehensively considered. Finally, it discusses practical solutions to overcome immunogenicity, including antigen masking by crosslinking, sterilization optimization, and antigen removal techniques such as selective antigen removal and sequential antigen solubilization.

组织工程脱细胞细胞外基质(ECM)支架在解决传统组织/器官移植中供体短缺和细胞免疫排斥方面具有很大的潜力。脱细胞是制造ECM支架的关键过程,它去除了免疫原细胞材料,显著降低了衍生支架的免疫原性和生物相容性。然而,这些生物支架的应用仍然面临着重大的免疫学挑战。本文讨论了损伤相关分子模式(DAMPs)和抗原之间的相互作用,作为先天免疫和适应性免疫的主要诱导剂,以帮助制造具有理想免疫原性的生物相容性移植物。它还评估了各种脱细胞方法(即细胞凋亡辅助技术)对激发免疫反应的影响,这些免疫反应参与排斥同种异体和异种脱细胞支架。此外,还全面考虑了ECM改变、细胞毒性问题、移植物来源和植入部位对脱细胞组织/器官免疫原性的贡献等关键研究结果。最后,讨论了克服免疫原性的实际解决方案,包括交联抗原掩蔽,灭菌优化和抗原去除技术,如选择性抗原去除和顺序抗原溶解。
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引用次数: 26
Drug-loaded microbubble delivery system to enhance PD-L1 blockade immunotherapy with remodeling immune microenvironment. 通过重塑免疫微环境增强PD-L1阻断免疫治疗的载药微泡递送系统。
IF 11.3 1区 医学 Q1 Medicine Pub Date : 2023-02-09 DOI: 10.1186/s40824-023-00350-5
Jun Zheng, Ju Huang, Liang Zhang, Mengna Wang, Lihong Xu, Xiaoyun Dou, Xiaojing Leng, Mingxiao Fang, Yang Sun, Zhigang Wang

Background: Although programmed cell death protein 1 (PD-1)/ programmed cell death-ligand protein 1 (PD-L1) checkpoint blockade immunotherapy demonstrates great promise in cancer treatment, poor infiltration of T cells resulted from tumor immunosuppressive microenvironment (TIME) and insufficient accumulation of anti-PD-L1 (αPD-L1) in tumor sites diminish the immune response. Herein, we reported a drug-loaded microbubble delivery system to overcome these obstacles and enhance PD-L1 blockade immunotherapy.

Methods: Docetaxel (DTX) and imiquimod (R837)-loaded microbubbles (RD@MBs) were synthesized via a typical rotary evaporation method combined with mechanical oscillation. The targeted release of drugs was achieved by using the directional "bursting" capability of ultrasound-targeted microbubble destruction (UTMD) technology. The antitumor immune response by RD@MBs combining αPD-L1 were evaluated on 4T1 and CT26 tumor models.

Results: The dying tumor cells induced by DTX release tumor-associated antigens (TAAs), together with R837, promoted the activation, proliferation and recruitment of T cells. Besides, UTMD technology and DTX enhanced the accumulation of αPD-L1 in tumor sites. Moreover, RD@MBs remolded TIME, including the polarization of M2-phenotype tumor-associated macrophages (TAMs) to M1-phenotype, and reduction of myeloid-derived suppressor cells (MDSCs). The RD@MBs + αPD-L1 synergistic therapy not only effectively inhibited the growth of primary tumors, but also significantly inhibited the mimic distant tumors as well as lung metastases.

Conclusion: PD-L1 blockade immunotherapy was enhanced by RD@MBs delivery system.

背景:尽管程序性细胞死亡蛋白1 (PD-1)/程序性细胞死亡配体蛋白1 (PD-L1)检查点阻断免疫疗法在癌症治疗中显示出巨大的前景,但由于肿瘤免疫抑制微环境(TIME)导致T细胞浸润不良和抗PD-L1 (αPD-L1)在肿瘤部位的积累不足,降低了免疫应答。在此,我们报道了一种载药微泡递送系统来克服这些障碍并增强PD-L1阻断免疫治疗。方法:采用典型的旋转蒸发法结合机械振荡合成多西他赛(DTX)和咪喹莫德(R837)负载微泡(RD@MBs)。利用超声靶向微泡破坏(UTMD)技术的定向“爆破”能力实现药物的靶向释放。观察RD@MBs联合αPD-L1对4T1和CT26肿瘤模型的抗肿瘤免疫反应。结果:DTX诱导垂死的肿瘤细胞释放肿瘤相关抗原(tumor associated antigens, TAAs),与R837共同促进T细胞的活化、增殖和募集。此外,UTMD技术和DTX增强了αPD-L1在肿瘤部位的积累。此外,RD@MBs重塑了TIME,包括m2表型肿瘤相关巨噬细胞(tam)向m1表型的极化,以及髓源性抑制细胞(MDSCs)的减少。RD@MBs + αPD-L1协同治疗不仅能有效抑制原发肿瘤的生长,还能显著抑制模拟远处肿瘤及肺转移。结论:RD@MBs给药系统对PD-L1阻断免疫治疗有增强作用。
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引用次数: 7
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Biomaterials Research
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