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Improving CRISPR-Cas9 directed faithful transgene integration outcomes by reducing unwanted random DNA integration. 通过减少不必要的随机 DNA 整合,改善 CRISPR-Cas9 定向忠实转基因整合结果。
IF 9 2区 医学 Q1 CELL BIOLOGY Pub Date : 2024-03-26 DOI: 10.1186/s12929-024-01020-x
Rio Hermantara, Laura Richmond, Aqeel Faisal Taqi, Sabari Chilaka, Valentine Jeantet, Ileana Guerrini, Katherine West, Adam West

Background: The field of genome editing has been revolutionized by the development of an easily programmable editing tool, the CRISPR-Cas9. Despite its promise, off-target activity of Cas9 posed a great disadvantage for genome editing purposes by causing DNA double strand breaks at off-target locations and causing unwanted editing outcomes. Furthermore, for gene integration applications, which introduce transgene sequences, integration of transgenes to off-target sites could be harmful, hard to detect, and reduce faithful genome editing efficiency.

Method: Here we report the development of a multicolour fluorescence assay for studying CRISPR-Cas9-directed gene integration at an endogenous locus in human cell lines. We examine genetic integration of reporter genes in transiently transfected cells as well as puromycin-selected stable cell lines to determine the fidelity of multiple CRISPR-Cas9 strategies.

Result: We found that there is a high occurrence of unwanted DNA integration which tarnished faithful knock-in efficiency. Integration outcomes are influenced by the type of DNA DSBs, donor design, the use of enhanced specificity Cas9 variants, with S-phase regulated Cas9 activity. Moreover, restricting Cas9 expression with a self-cleaving system greatly improves knock-in outcomes by substantially reducing the percentage of cells with unwanted DNA integration.

Conclusion: Our results highlight the need for a more stringent assessment of CRISPR-Cas9-mediated knock-in outcomes, and the importance of careful strategy design to maximise efficient and faithful transgene integration.

背景:一种易于编程的编辑工具--CRISPR-Cas9--的开发给基因组编辑领域带来了革命性的变化。尽管Cas9前景广阔,但它的脱靶活性会在脱靶位置造成DNA双链断裂,导致不必要的编辑结果,从而对基因组编辑造成极大的不利影响。此外,对于引入转基因序列的基因整合应用,转基因整合到脱靶位点可能是有害的,难以检测,并降低忠实的基因组编辑效率:方法:我们在此报告了一种多色荧光检测方法的开发情况,该方法可用于研究人体细胞系中内源基因座的 CRISPR-Cas9 引导基因整合。我们研究了瞬时转染细胞和嘌呤霉素筛选的稳定细胞系中报告基因的基因整合,以确定多种CRISPR-Cas9策略的保真度:结果:我们发现,有很多不必要的DNA整合发生,这玷污了忠实的基因敲入效率。整合结果受DNA DSBs类型、供体设计、使用特异性增强的Cas9变体以及S期调控Cas9活性的影响。此外,利用自清除系统限制Cas9的表达,可大幅降低不需要的DNA整合细胞的比例,从而大大改善基因敲入结果:我们的研究结果凸显了对 CRISPR-Cas9 介导的基因敲入结果进行更严格评估的必要性,以及精心设计策略以最大限度地提高转基因整合的效率和忠实性的重要性。
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引用次数: 0
A matter of new life and cell death: programmed cell death in the mammalian ovary 新生与细胞死亡:哺乳动物卵巢中的程序性细胞死亡
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-03-20 DOI: 10.1186/s12929-024-01017-6
Mikhail S. Chesnokov, Aygun R. Mamedova, Boris Zhivotovsky, Gelina S. Kopeina
The mammalian ovary is a unique organ that displays a distinctive feature of cyclic changes throughout the entire reproductive period. The estrous/menstrual cycles are associated with drastic functional and morphological rearrangements of ovarian tissue, including follicular development and degeneration, and the formation and subsequent atrophy of the corpus luteum. The flawless execution of these reiterative processes is impossible without the involvement of programmed cell death (PCD). PCD is crucial for efficient and careful clearance of excessive, depleted, or obsolete ovarian structures for ovarian cycling. Moreover, PCD facilitates selection of high-quality oocytes and formation of the ovarian reserve during embryonic and juvenile development. Disruption of PCD regulation can heavily impact the ovarian functions and is associated with various pathologies, from a moderate decrease in fertility to severe hormonal disturbance, complete loss of reproductive function, and tumorigenesis. This comprehensive review aims to provide updated information on the role of PCD in various processes occurring in normal and pathologic ovaries. Three major events of PCD in the ovary—progenitor germ cell depletion, follicular atresia, and corpus luteum degradation—are described, alongside the detailed information on molecular regulation of these processes, highlighting the contribution of apoptosis, autophagy, necroptosis, and ferroptosis. Ultimately, the current knowledge of PCD aberrations associated with pathologies, such as polycystic ovarian syndrome, premature ovarian insufficiency, and tumors of ovarian origin, is outlined. PCD is an essential element in ovarian development, functions and pathologies. A thorough understanding of molecular mechanisms regulating PCD events is required for future advances in the diagnosis and management of various disorders of the ovary and the female reproductive system in general.
哺乳动物的卵巢是一个独特的器官,在整个生殖周期中都会发生周期性变化。发情/月经周期与卵巢组织功能和形态的急剧变化有关,包括卵泡的发育和退化,以及黄体的形成和随后的萎缩。如果没有程序性细胞死亡(PCD)的参与,这些重复过程就不可能完美无瑕地进行。程序性细胞死亡对于高效、仔细地清除过多、耗竭或过时的卵巢结构以实现卵巢循环至关重要。此外,PCD 还有助于在胚胎和幼年发育过程中选择优质卵母细胞和形成卵巢储备。PCD 调节紊乱会严重影响卵巢功能,并与各种病症相关,从生育力中度下降到严重的激素紊乱、生殖功能完全丧失以及肿瘤发生。本综述旨在提供有关 PCD 在正常和病理卵巢的各种过程中所起作用的最新信息。文章描述了卵巢中 PCD 的三个主要事件--生殖细胞祖细胞耗竭、卵泡闭锁和黄体退化,并详细介绍了这些过程的分子调控,强调了细胞凋亡、自噬、坏死和铁凋亡的作用。最后,概述了与多囊卵巢综合征、卵巢早衰和卵巢肿瘤等病症相关的 PCD 畸变的现有知识。PCD 是卵巢发育、功能和病变的基本要素。彻底了解调控 PCD 事件的分子机制,对今后卵巢和整个女性生殖系统各种疾病的诊断和治疗都有重要意义。
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引用次数: 0
Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury. 携带 let-7a-5p 的工程细胞外囊泡用于缓解急性肺损伤的炎症反应。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-03-19 DOI: 10.1186/s12929-024-01019-4
Sin-Yu Chen, Yi-Ling Chen, Po-Chen Li, Tai-Shan Cheng, Yeh-Shiu Chu, Yi-Shan Shen, Hsin-Tung Chen, Wei-Ni Tsai, Chien-Ling Huang, Martin Sieber, Yuan-Chieh Yeh, Hsiao-Sheng Liu, Chi-Ling Chiang, Chih-Hung Chang, Andrew S Lee, Yen-Han Tseng, Ly James Lee, Hsiu-Jung Liao, Hon-Kan Yip, Chi-Ying F Huang

Background: Acute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton's jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.

Methods: A cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI.

Results: The CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function.

Conclusion: This study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

背景:急性肺损伤(ALI)是一种危及生命的呼吸系统疾病,其特征是严重的炎症和肺组织损伤,经常导致快速呼吸衰竭和长期并发症。microRNA let-7a-5p 通过调节免疫细胞的活化和细胞因子的产生,参与肺损伤、炎症和纤维化的进展。本研究旨在利用创新的细胞电穿孔平台生成携带let-7a-5p(EV-let-7a-5p)的细胞外囊泡(EVs),EVs来源于转染的沃顿氏果冻-间充质干细胞(WJ-MSCs),作为治疗ALI的潜在基因疗法:方法:采用细胞纳米化(CNP)方法诱导转染了相关质粒DNA的WJ-间充质干细胞产生并释放EV-let-7a-5p。使用切向流过滤(TFF)系统分离条件培养基中的EV-let-7a-5p。EV的表征遵循了国际细胞外囊泡协会(International Society for Extracellular Vesicles)制定的最低共识指南。我们进行了一系列全面的治疗评估,包括使用转化生长因子β(TGF-β)诱导细胞模型的抗纤维化作用、对巨噬细胞极化的调节作用,以及 EV-let-7a-5p 在高氧诱导 ALI 大鼠模型中的影响:结果:CNP平台明显增加了转染WJ-间充质干细胞的EV分泌,工程EV中包裹的let-7a-5p明显高于未处理的WJ-间充质干细胞。这些EV-let-7a-5p不影响细胞增殖,并通过下调LL29细胞中SMAD2/3磷酸化,有效缓解了TGF-β诱导的纤维化表型。此外,EV-let-7a-5p 还能调节炎症微环境中 M2 样巨噬细胞的活化,并显著诱导白细胞介素(IL)-10 的分泌,这证明了它们对炎症的调节作用。在高氧诱导的 ALI 大鼠模型中,给予未经处理的 WJ-间充质干细胞的 EVs 可轻微改善肺功能,并增加血浆中 let-7a-5p 的表达。相比之下,EV-let-7a-5p 能显著减少巨噬细胞浸润和胶原沉积,同时增加 IL-10 的表达,从而大幅改善肺功能:本研究揭示了使用 CNP 平台刺激和转染 WJ-间充质干细胞可产生大量富含 let-7a-5p 的 EVs,这凸显了 EVs 在对抗炎症反应、纤维化活化和高氧诱导的肺损伤方面的治疗潜力。这些结果为开发创新的治疗方法提供了潜在的途径,以更有效地干预 ALI。
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引用次数: 0
The rise of big data: deep sequencing-driven computational methods are transforming the landscape of synthetic antibody design. 大数据的崛起:深度测序驱动的计算方法正在改变合成抗体设计的格局。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-03-16 DOI: 10.1186/s12929-024-01018-5
Eugenio Gallo

Synthetic antibodies (Abs) represent a category of artificial proteins capable of closely emulating the functions of natural Abs. Their in vitro production eliminates the need for an immunological response, streamlining the process of Ab discovery, engineering, and development. These artificially engineered Abs offer novel approaches to antigen recognition, paratope site manipulation, and biochemical/biophysical enhancements. As a result, synthetic Abs are fundamentally reshaping conventional methods of Ab production. This mirrors the revolution observed in molecular biology and genomics as a result of deep sequencing, which allows for the swift and cost-effective sequencing of DNA and RNA molecules at scale. Within this framework, deep sequencing has enabled the exploration of whole genomes and transcriptomes, including particular gene segments of interest. Notably, the fusion of synthetic Ab discovery with advanced deep sequencing technologies is redefining the current approaches to Ab design and development. Such combination offers opportunity to exhaustively explore Ab repertoires, fast-tracking the Ab discovery process, and enhancing synthetic Ab engineering. Moreover, advanced computational algorithms have the capacity to effectively mine big data, helping to identify Ab sequence patterns/features hidden within deep sequencing Ab datasets. In this context, these methods can be utilized to predict novel sequence features thereby enabling the successful generation of de novo Ab molecules. Hence, the merging of synthetic Ab design, deep sequencing technologies, and advanced computational models heralds a new chapter in Ab discovery, broadening our comprehension of immunology and streamlining the advancement of biological therapeutics.

合成抗体(Abs)是一类能够近似天然抗体功能的人造蛋白质。它们在体外生产,无需免疫反应,简化了抗体的发现、工程和开发过程。这些人工合成的 Abs 为抗原识别、副配位位点操作和生化/生物物理增强提供了新的方法。因此,合成 Abs 正在从根本上重塑传统的 Ab 生产方法。这反映了分子生物学和基因组学因深度测序而发生的革命,深度测序可对 DNA 和 RNA 分子进行快速、低成本的大规模测序。在这一框架内,深度测序能够探索整个基因组和转录组,包括感兴趣的特定基因片段。值得注意的是,合成 Ab 发现与先进深度测序技术的融合正在重新定义当前的 Ab 设计和开发方法。这种结合提供了详尽探索抗体库的机会,可快速跟踪抗体发现过程,并加强合成抗体工程。此外,先进的计算算法还能有效挖掘大数据,帮助识别隐藏在深度测序 Ab 数据集中的 Ab 序列模式/特征。在这种情况下,可以利用这些方法预测新的序列特征,从而成功生成新的 Ab 分子。因此,合成 Ab 设计、深度测序技术和先进计算模型的融合预示着 Ab 发现将翻开新的篇章,拓宽我们对免疫学的理解,促进生物疗法的发展。
{"title":"The rise of big data: deep sequencing-driven computational methods are transforming the landscape of synthetic antibody design.","authors":"Eugenio Gallo","doi":"10.1186/s12929-024-01018-5","DOIUrl":"10.1186/s12929-024-01018-5","url":null,"abstract":"<p><p>Synthetic antibodies (Abs) represent a category of artificial proteins capable of closely emulating the functions of natural Abs. Their in vitro production eliminates the need for an immunological response, streamlining the process of Ab discovery, engineering, and development. These artificially engineered Abs offer novel approaches to antigen recognition, paratope site manipulation, and biochemical/biophysical enhancements. As a result, synthetic Abs are fundamentally reshaping conventional methods of Ab production. This mirrors the revolution observed in molecular biology and genomics as a result of deep sequencing, which allows for the swift and cost-effective sequencing of DNA and RNA molecules at scale. Within this framework, deep sequencing has enabled the exploration of whole genomes and transcriptomes, including particular gene segments of interest. Notably, the fusion of synthetic Ab discovery with advanced deep sequencing technologies is redefining the current approaches to Ab design and development. Such combination offers opportunity to exhaustively explore Ab repertoires, fast-tracking the Ab discovery process, and enhancing synthetic Ab engineering. Moreover, advanced computational algorithms have the capacity to effectively mine big data, helping to identify Ab sequence patterns/features hidden within deep sequencing Ab datasets. In this context, these methods can be utilized to predict novel sequence features thereby enabling the successful generation of de novo Ab molecules. Hence, the merging of synthetic Ab design, deep sequencing technologies, and advanced computational models heralds a new chapter in Ab discovery, broadening our comprehension of immunology and streamlining the advancement of biological therapeutics.</p>","PeriodicalId":15365,"journal":{"name":"Journal of Biomedical Science","volume":null,"pages":null},"PeriodicalIF":11.0,"publicationDate":"2024-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10943851/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140140321","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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: 0
Tick-borne encephalitis virus transmitted singly and in duo with Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum bacteria by ticks as pathogens modifying lipid metabolism in human blood. 蜱传脑炎病毒作为病原体,通过蜱单独传播或与博氏包虫病和噬细胞阿纳疟原虫共同传播,改变人体血液中的脂质代谢。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-03-04 DOI: 10.1186/s12929-024-01016-7
Marta Dobrzyńska, Anna Moniuszko-Malinowska, Piotr Radziwon, Sławomir Pancewicz, Agnieszka Gęgotek, Elżbieta Skrzydlewska

Background: Ticks are vectors of various pathogens, including tick-borne encephalitis virus causing TBE and bacteria such as Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum causing e.g. viral-bacterial co-infections (TBE + LB/HGA), which pose diagnostic and therapeutic problems. Since these infections are usually accompanied by inflammation and oxidative stress causing metabolic modifications, including phospholipids, the aim of the study was to assess the level of polyunsaturated fatty acids and their metabolism (ROS- and enzyme-dependent) products in the blood plasma of patients with TBE and TBE + LB/HGA before and after pharmacotherapy.

Methods: The total antioxidant status was determined using 2,20-azino-bis-3-ethylbenzothiazolin-6-sulfonic acid. The phospholipid and free fatty acids were analysed by gas chromatography. Lipid peroxidation was estimated by measuring small molecular weight reactive aldehyde, malondialdehyde and neuroprostanes. The reactive aldehyde was determined using gas chromatography coupled with mass spectrometry. The activity of enzymes was examined spectrophotometrically. An analysis of endocannabinoids and eicosanoids was performed using a Shimadzu UPLC system coupled with an electrospray ionization source to a Shimadzu 8060 Triple Quadrupole system. Receptor expression was measured using an enzyme-linked immunosorbent assay (ELISA).

Results: The reduced antioxidant status as a result of infection was accompanied by a decrease in the level of phospholipid arachidonic acid (AA) and docosahexaenoic acid (DHA) in TBE, an increase in DHA in co-infection and in free DHA in TBE with an increase in the level of lipid peroxidation products. The enhanced activity of enzymes metabolizing phospholipids and free PUFAs increased the level of endocannabinoids and eicosanoids, while decreased 15-PGJ2 and PGE2 was accompanied by activation of granulocyte receptors before pharmacotherapy and only tending to normalize after treatment.

Conclusion: Since classical pharmacotherapy does not prevent disorders of phospholipid metabolism, the need to support treatment with antioxidants may be suggested.

背景:蜱虫是各种病原体的载体,包括引起蜱传脑炎的蜱传脑炎病毒(TBE)和引起病毒-细菌混合感染(TBE + LB/HGA)的细菌(如晚期鲍曼不动杆菌(Borrelia burgdorferi sensu lato)和噬胞嗜血杆菌(Anaplasma phagocytophilum)),这给诊断和治疗带来了难题。由于这些感染通常伴有炎症和氧化应激,导致包括磷脂在内的代谢改变,因此本研究旨在评估药物治疗前后 TBE 和 TBE + LB/HGA 患者血浆中多不饱和脂肪酸及其代谢(ROS 和酶依赖性)产物的水平:用2,20-偶氮-双-3-乙基苯并噻唑啉-6-磺酸测定总抗氧化状态。采用气相色谱法分析磷脂和游离脂肪酸。通过测量小分子量活性醛、丙二醛和神经前列素来估计脂质过氧化情况。活性醛采用气相色谱-质谱法测定。用分光光度法检测酶的活性。使用岛津 8060 三重四极杆系统与电喷雾离子源耦合的岛津超高效液相色谱(UPLC)系统对内源性大麻素和二十烷酸进行了分析。使用酶联免疫吸附试验(ELISA)测量受体表达:结果:感染导致的抗氧化状态降低伴随着TBE中磷脂花生四烯酸(AA)和二十二碳六烯酸(DHA)水平的降低,合并感染时DHA水平的升高和TBE中游离DHA水平的升高以及脂质过氧化产物水平的升高。磷脂和游离 PUFAs 代谢酶的活性增强提高了内源性大麻素和二十酸的水平,而 15-PGJ2 和 PGE2 的降低则伴随着药物治疗前粒细胞受体的激活,治疗后才趋于正常:结论:由于传统的药物治疗无法预防磷脂代谢紊乱,因此建议使用抗氧化剂辅助治疗。
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引用次数: 0
Integration of transcription regulation and functional genomic data reveals lncRNA SNHG6's role in hematopoietic differentiation and leukemia. 转录调控和功能基因组数据的整合揭示了 lncRNA SNHG6 在造血分化和白血病中的作用。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-02-28 DOI: 10.1186/s12929-024-01015-8
Joshua M Hazan, Raziel Amador, Tahleel Ali-Nasser, Tamar Lahav, Stav Roni Shotan, Miryam Steinberg, Ziv Cohen, Dvir Aran, David Meiri, Yehuda G Assaraf, Roderic Guigó, Assaf C Bester

Background: Long non-coding RNAs (lncRNAs) are pivotal players in cellular processes, and their unique cell-type specific expression patterns render them attractive biomarkers and therapeutic targets. Yet, the functional roles of most lncRNAs remain enigmatic. To address the need to identify new druggable lncRNAs, we developed a comprehensive approach integrating transcription factor binding data with other genetic features to generate a machine learning model, which we have called INFLAMeR (Identifying Novel Functional LncRNAs with Advanced Machine Learning Resources).

Methods: INFLAMeR was trained on high-throughput CRISPR interference (CRISPRi) screens across seven cell lines, and the algorithm was based on 71 genetic features. To validate the predictions, we selected candidate lncRNAs in the human K562 leukemia cell line and determined the impact of their knockdown (KD) on cell proliferation and chemotherapeutic drug response. We further performed transcriptomic analysis for candidate genes. Based on these findings, we assessed the lncRNA small nucleolar RNA host gene 6 (SNHG6) for its role in myeloid differentiation. Finally, we established a mouse K562 leukemia xenograft model to determine whether SNHG6 KD attenuates tumor growth in vivo.

Results: The INFLAMeR model successfully reconstituted CRISPRi screening data and predicted functional lncRNAs that were previously overlooked. Intensive cell-based and transcriptomic validation of nearly fifty genes in K562 revealed cell type-specific functionality for 85% of the predicted lncRNAs. In this respect, our cell-based and transcriptomic analyses predicted a role for SNHG6 in hematopoiesis and leukemia. Consistent with its predicted role in hematopoietic differentiation, SNHG6 transcription is regulated by hematopoiesis-associated transcription factors. SNHG6 KD reduced the proliferation of leukemia cells and sensitized them to differentiation. Treatment of K562 leukemic cells with hemin and PMA, respectively, demonstrated that SNHG6 inhibits red blood cell differentiation but strongly promotes megakaryocyte differentiation. Using a xenograft mouse model, we demonstrate that SNHG6 KD attenuated tumor growth in vivo.

Conclusions: Our approach not only improved the identification and characterization of functional lncRNAs through genomic approaches in a cell type-specific manner, but also identified new lncRNAs with roles in hematopoiesis and leukemia. Such approaches can be readily applied to identify novel targets for precision medicine.

背景:长非编码 RNA(lncRNA)是细胞过程中的关键角色,其独特的细胞类型特异性表达模式使其成为具有吸引力的生物标记物和治疗靶标。然而,大多数 lncRNA 的功能作用仍然是个谜。为了满足识别新的可药用lncRNAs的需求,我们开发了一种综合方法,将转录因子结合数据与其他遗传特征整合在一起,生成一个机器学习模型,我们称之为INFLAMeR(利用先进的机器学习资源识别新型功能性lncRNAs):INFLAMeR是在七个细胞系的高通量CRISPR干扰(CRISPRi)筛选中训练出来的,该算法基于71个遗传特征。为了验证预测结果,我们在人类 K562 白血病细胞系中选择了候选 lncRNA,并确定了它们的基因敲除(KD)对细胞增殖和化疗药物反应的影响。我们进一步对候选基因进行了转录组分析。基于这些发现,我们评估了 lncRNA 小核 RNA 宿主基因 6(SNHG6)在髓样细胞分化中的作用。最后,我们建立了小鼠K562白血病异种移植模型,以确定SNHG6 KD是否能抑制体内肿瘤的生长:INFLAMeR模型成功地重建了CRISPRi筛选数据,并预测了之前被忽视的功能性lncRNA。对 K562 中的近 50 个基因进行了深入的细胞学和转录组学验证,发现 85% 预测的 lncRNA 具有细胞类型特异性功能。在这方面,我们基于细胞和转录组的分析预测了 SNHG6 在造血和白血病中的作用。与预测的造血分化作用相一致,SNHG6 的转录受造血相关转录因子的调控。SNHG6 KD可减少白血病细胞的增殖,并使其对分化敏感。用hemin和PMA分别处理K562白血病细胞,结果表明SNHG6抑制红细胞分化,但强烈促进巨核细胞分化。我们利用异种移植小鼠模型证明,SNHG6 KD抑制了体内肿瘤的生长:我们的方法不仅通过基因组学方法以细胞类型特异性的方式改进了功能性lncRNA的鉴定和表征,而且还发现了在造血和白血病中发挥作用的新lncRNA。这种方法可用于识别精准医疗的新靶点。
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引用次数: 0
Reduced interleukin-18 secretion by human monocytic cells in response to infections with hyper-virulent Streptococcus pyogenes. 人类单核细胞在感染高致病性化脓性链球菌后分泌的白细胞介素-18减少。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-02-27 DOI: 10.1186/s12929-024-01014-9
Lea A Tölken, Antje D Paulikat, Lana H Jachmann, Alexander Reder, Manuela Gesell Salazar, Laura M Palma Medina, Stephan Michalik, Uwe Völker, Mattias Svensson, Anna Norrby-Teglund, Katharina J Hoff, Michael Lammers, Nikolai Siemens

Background: Streptococcus pyogenes (group A streptococcus, GAS) causes a variety of diseases ranging from mild superficial infections of the throat and skin to severe invasive infections, such as necrotizing soft tissue infections (NSTIs). Tissue passage of GAS often results in mutations within the genes encoding for control of virulence (Cov)R/S two component system leading to a hyper-virulent phenotype. Dendritic cells (DCs) are innate immune sentinels specialized in antigen uptake and subsequent T cell priming. This study aimed to analyze cytokine release by DCs and other cells of monocytic origin in response to wild-type and natural covR/S mutant infections.

Methods: Human primary monocyte-derived (mo)DCs were used. DC maturation and release of pro-inflammatory cytokines in response to infections with wild-type and covR/S mutants were assessed via flow cytometry. Global proteome changes were assessed via mass spectrometry. As a proof-of-principle, cytokine release by human primary monocytes and macrophages was determined.

Results: In vitro infections of moDCs and other monocytic cells with natural GAS covR/S mutants resulted in reduced secretion of IL-8 and IL-18 as compared to wild-type infections. In contrast, moDC maturation remained unaffected. Inhibition of caspase-8 restored secretion of both molecules. Knock-out of streptolysin O in GAS strain with unaffected CovR/S even further elevated the IL-18 secretion by moDCs. Of 67 fully sequenced NSTI GAS isolates, 28 harbored mutations resulting in dysfunctional CovR/S. However, analyses of plasma IL-8 and IL-18 levels did not correlate with presence or absence of such mutations.

Conclusions: Our data demonstrate that strains, which harbor covR/S mutations, interfere with IL-18 and IL-8 responses in monocytic cells by utilizing the caspase-8 axis. Future experiments aim to identify the underlying mechanism and consequences for NSTI patients.

背景:化脓性链球菌(A 组链球菌,GAS)可引起多种疾病,从轻微的咽喉和皮肤浅表感染到严重的侵袭性感染,如坏死性软组织感染(NSTI)。组织通过 GAS 往往会导致编码控制毒力(Cov)R/S 双组分系统的基因发生突变,从而形成高毒力表型。树突状细胞(DC)是先天性免疫哨兵,专门从事抗原摄取和随后的 T 细胞引物。本研究旨在分析 DC 和其他单核细胞来源的细胞因子在野生型和天然 covR/S 突变型感染中的释放情况。方法:使用人类原代单核细胞衍生(mo)DC,通过流式细胞仪评估DC对野生型和covR/S突变体感染的成熟度和促炎细胞因子的释放。通过质谱法评估了全局蛋白质组的变化。作为原理验证,还测定了人类原代单核细胞和巨噬细胞的细胞因子释放量:结果:与野生型感染相比,moDCs 和其他单核细胞体外感染天然 GAS covR/S 突变体会导致 IL-8 和 IL-18 的分泌减少。相比之下,moDC 的成熟仍然不受影响。抑制 caspase-8 可恢复这两种分子的分泌。在CovR/S未受影响的GAS菌株中敲除链溶菌素O甚至进一步提高了moDCs的IL-18分泌。在 67 个完全测序的 NSTI GAS 分离物中,28 个存在导致 CovR/S 功能障碍的突变。然而,血浆中IL-8和IL-18水平的分析与是否存在此类突变无关:我们的数据表明,携带covR/S突变的菌株会利用caspase-8轴干扰单核细胞中的IL-18和IL-8反应。未来的实验旨在确定潜在的机制和对 NSTI 患者的影响。
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引用次数: 0
Metabolism-regulating non-coding RNAs in breast cancer: roles, mechanisms and clinical applications 乳腺癌中的代谢调节非编码 RNA:作用、机制和临床应用
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-02-26 DOI: 10.1186/s12929-024-01013-w
Shiliang Xu, Lingxia Wang, Yuexin Zhao, Tong Mo, Bo Wang, Jun Lin, Huan Yang
Breast cancer is one of the most common malignancies that pose a serious threat to women's health. Reprogramming of energy metabolism is a major feature of the malignant transformation of breast cancer. Compared to normal cells, tumor cells reprogram metabolic processes more efficiently, converting nutrient supplies into glucose, amino acid and lipid required for malignant proliferation and progression. Non-coding RNAs(ncRNAs) are a class of functional RNA molecules that are not translated into proteins but regulate the expression of target genes. NcRNAs have been demonstrated to be involved in various aspects of energy metabolism, including glycolysis, glutaminolysis, and fatty acid synthesis. This review focuses on the metabolic regulatory mechanisms and clinical applications of metabolism-regulating ncRNAs involved in breast cancer. We summarize the vital roles played by metabolism-regulating ncRNAs for endocrine therapy, targeted therapy, chemotherapy, immunotherapy, and radiotherapy resistance in breast cancer, as well as their potential as therapeutic targets and biomarkers. Difficulties and perspectives of current targeted metabolism and non-coding RNA therapeutic strategies are discussed.
乳腺癌是最常见的恶性肿瘤之一,严重威胁着妇女的健康。能量代谢重编程是乳腺癌恶性转化的一个主要特征。与正常细胞相比,肿瘤细胞能更有效地重塑代谢过程,将营养物质转化为葡萄糖、氨基酸和脂质,以满足恶性肿瘤增殖和发展的需要。非编码 RNA(ncRNA)是一类功能性 RNA 分子,它们不会被翻译成蛋白质,但能调节靶基因的表达。NcRNA 已被证实参与能量代谢的各个方面,包括糖酵解、谷氨酰胺酵解和脂肪酸合成。这篇综述主要探讨了乳腺癌相关代谢调节 ncRNA 的代谢调节机制和临床应用。我们总结了调节代谢的 ncRNA 在乳腺癌的内分泌治疗、靶向治疗、化疗、免疫治疗和放疗耐药中发挥的重要作用,以及它们作为治疗靶点和生物标志物的潜力。还讨论了目前靶向代谢和非编码 RNA 治疗策略的难点和前景。
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引用次数: 0
Genetic and pharmacologic p32-inhibition rescue CHCHD2-linked Parkinson's disease phenotypes in vivo and in cell models. 基因和药物 p32 抑制可在体内和细胞模型中挽救与 CHCHD2 相关的帕金森病表型。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-02-23 DOI: 10.1186/s12929-024-01010-z
Murni Tio, Rujing Wen, Cai Ning Choo, Jian Bin Tan, Aaron Chua, Bin Xiao, Jeyapriya Rajameenakshi Sundaram, Christine Hui Shan Chan, Eng-King Tan

Background: Mutations in CHCHD2 have been linked to Parkinson's disease, however, their exact pathophysiologic roles are unclear. The p32 protein has been suggested to interact with CHCHD2, however, the physiological functions of such interaction in the context of PD have not been clarified.

Methods: Interaction between CHCHD2 and p32 was confirmed by co-immunoprecipitation experiments. We studied the effect of p32-knockdown in the transgenic Drosophila and Hela cells expressing the wild type and the pathogenic variants of hCHCHD2. We further investigated the rescue ability of a custom generated p32-inhibitor in these models as well as in the human fibroblast derived neural precursor cells and the dopaminergic neurons harboring hCHCHD2-Arg145Gln.

Results: Our results showed that wildtype and mutant hCHCHD2 could bind to p32 in vitro, supported by in vivo interaction between human CHCHD2 and Drosophila p32. Knockdown of p32 reduced mutant hCHCHD2 levels in Drosophila and in vitro. In Drosophila hCHCHD2 models, inhibition of p32 through genetic knockdown and pharmacological treatment using a customized p32-inhibitor restored dopaminergic neuron numbers and improved mitochondrial morphology. These were correlated with improved locomotor function, reduced oxidative stress and decreased mortality. Consistently, Hela cells expressing mutant hCHCHD2 showed improved mitochondrial morphology and function after treatment with the p32-inhibitor. As compared to the isogenic control cells, large percentage of the mutant neural precursor cells and dopaminergic neurons harboring hCHCHD2-Arg145Gln contained fragmented mitochondria which was accompanied by lower ATP production and cell viability. The NPCs harboring hCHCHD2-Arg145Gln also had a marked increase in α-synuclein expression. The p32-inhibitor was able to ameliorate the mitochondrial fragmentation, restored ATP levels, increased cell viability and reduced α-synuclein level in these cells.

Conclusions: Our study identified p32 as a modulator of CHCHD2, possibly exerting its effects by reducing the toxic mutant hCHCHD2 expression and/or mitigating the downstream effects. Inhibition of the p32 pathway can be a potential therapeutic intervention for CHCHD2-linked PD and diseases involving mitochondrial dysfunction.

背景:CHCHD2突变与帕金森病有关,但其确切的病理生理作用尚不清楚。有人认为 p32 蛋白与 CHCHD2 相互作用,但这种作用在帕金森病中的生理功能尚未明确:方法:通过共免疫沉淀实验证实了CHCHD2和p32之间的相互作用。我们研究了在表达hCHCHD2野生型和致病变体的转基因果蝇和Hela细胞中敲除p32的效果。我们还进一步研究了在这些模型以及人成纤维细胞衍生的神经前体细胞和携带 hCHCHD2-Arg145Gln 的多巴胺能神经元中定制生成的 p32 抑制剂的拯救能力:结果:我们的研究结果表明,野生型和突变型hCHCHD2可在体外与p32结合,人CHCHD2与果蝇p32的体内相互作用也证实了这一点。p32被敲除后,果蝇和体外的突变型hCHCHD2水平降低。在hCHCHD2果蝇模型中,通过基因敲除和使用定制的p32抑制剂进行药物治疗来抑制p32,可以恢复多巴胺能神经元的数量并改善线粒体形态。这与运动功能改善、氧化应激减少和死亡率降低相关。同样,表达突变型 hCHCHD2 的 Hela 细胞在使用 p32 抑制剂处理后,线粒体形态和功能也得到了改善。与同源对照细胞相比,携带 hCHCHD2-Arg145Gln 的突变神经前体细胞和多巴胺能神经元中有很大比例的线粒体破碎,同时 ATP 产量和细胞存活率也较低。携带 hCHCHD2-Arg145Gln 的神经元还明显增加了 α-突触核蛋白的表达。p32抑制剂能够改善这些细胞的线粒体破碎、恢复ATP水平、提高细胞活力并降低α-突触核蛋白水平:我们的研究发现 p32 是 CHCHD2 的调节剂,可能通过减少毒性突变体 hCHCHD2 的表达和/或减轻下游效应来发挥其作用。抑制p32通路可能是治疗与CHCHD2相关的帕金森病和涉及线粒体功能障碍的疾病的一种潜在干预措施。
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引用次数: 0
The role of pregnancy associated plasma protein-A in triple negative breast cancer: a promising target for achieving clinical benefits. 妊娠相关血浆蛋白-A在三阴性乳腺癌中的作用:有望实现临床获益的靶点。
IF 11 2区 医学 Q1 Medicine Pub Date : 2024-02-23 DOI: 10.1186/s12929-024-01012-x
Arpita Poddar, Farah Ahmady, Sushma R Rao, Revati Sharma, George Kannourakis, Prashanth Prithviraj, Aparna Jayachandran

Pregnancy associated plasma protein-A (PAPP-A) plays an integral role in breast cancer (BC), especially triple negative breast cancer (TNBC). This subtype accounts for the most aggressive BC, possesses high tumor heterogeneity, is least responsive to standard treatments and has the poorest clinical outcomes. There is a critical need to address the lack of effective targeted therapeutic options available. PAPP-A is a protein that is highly elevated during pregnancy. Frequently, higher PAPP-A expression is detected in tumors than in healthy tissues. The increase in expression coincides with increased rates of aggressive cancers. In BC, PAPP-A has been demonstrated to play a role in tumor initiation, progression, metastasis including epithelial-mesenchymal transition (EMT), as well as acting as a biomarker for predicting patient outcomes. In this review, we present the role of PAPP-A, with specific focus on TNBC. The structure and function of PAPP-A, belonging to the pappalysin subfamily, and its proteolytic activity are assessed. We highlight the link of BC and PAPP-A with respect to the IGFBP/IGF axis, EMT, the window of susceptibility and the impact of pregnancy. Importantly, the relevance of PAPP-A as a TNBC clinical marker is reviewed and its influence on immune-related pathways are explored. The relationship and mechanisms involving PAPP-A reveal the potential for more treatment options that can lead to successful immunotherapeutic targets and the ability to assist with better predicting clinical outcomes in TNBC.

妊娠相关血浆蛋白-A(PAPP-A)在乳腺癌(BC),尤其是三阴性乳腺癌(TNBC)中发挥着不可或缺的作用。该亚型是侵袭性最强的乳腺癌,具有高度的肿瘤异质性,对标准治疗反应最小,临床疗效最差。目前亟需解决缺乏有效靶向治疗方案的问题。PAPP-A 是一种在怀孕期间高度升高的蛋白质。在肿瘤中检测到的 PAPP-A 表达通常高于健康组织。这种表达的增加与侵袭性癌症发病率的增加相吻合。在 BC 中,PAPP-A 已被证实在肿瘤的发生、发展、转移(包括上皮-间质转化(EMT))中发挥作用,并可作为预测患者预后的生物标记物。在本综述中,我们将介绍 PAPP-A 的作用,并特别关注 TNBC。我们评估了属于 pappalysin 亚家族的 PAPP-A 的结构和功能及其蛋白水解活性。我们强调了BC与PAPP-A在IGFBP/IGF轴、EMT、易感性窗口期和妊娠影响方面的联系。重要的是,我们回顾了 PAPP-A 作为 TNBC 临床标志物的相关性,并探讨了它对免疫相关途径的影响。涉及 PAPP-A 的关系和机制揭示了更多治疗选择的潜力,这些治疗选择可能导致成功的免疫治疗目标,并能帮助更好地预测 TNBC 的临床结果。
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引用次数: 0
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