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Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket. 形成细胞内袋的残基上的电荷反转对连接蛋白通道和半通道的调节作用不同。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-23 DOI: 10.1186/s40659-024-00501-5
Felipe Villanelo, Peter J Minogue, Jaime Maripillán, Mauricio Reyna-Jeldes, Joaquin Jensen-Flores, Isaac E García, Eric C Beyer, Tomás Pérez-Acle, Viviana M Berthoud, Agustín D Martínez

Background: Members of the β-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts.

Methods: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules.

Results: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels.

Conclusions: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

背景:连接蛋白β亚家族成员含有一个由四个跨膜螺旋的氨基酸残基包围的胞内袋。此前还没有人研究过 α-、γ-、δ- 和 ε 亚家族成员是否存在这个口袋。我们研究了作为α-亚家族代表的连接蛋白50(Cx50),因为它的结构已经确定,而且Cx50的突变是先天性白内障最常见的遗传原因之一:为了研究 Cx50 细胞内口袋的存在和功能,我们使用了分子动力学模拟、定点突变、缝隙连接示踪剂细胞间转移以及通过电生理学和带电分子渗透检测的半通道活性:利用分子动力学,我们确定了 Cx50 半通道细胞内口袋的存在,并确定了参与其形成的氨基酸。我们利用定点突变改变了支持胞内袋的盐桥相互作用,这种作用发生在附件蛋白家族中高度保守的两个残基 R33 和 E162 之间。在这两个位置替换相反的电荷会减少缝隙连接斑块的形成和细胞间的通讯,并适度降低半通道电流。在这些位置上同时进行电荷反转会产生形成斑块的无功能缝隙连接通道和高活性半通道:这些结果表明,细胞内袋的相互作用会影响缝隙连接通道和半通道的功能。这些相互作用的破坏可能是导致与这些位置的突变相关的疾病的原因。
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引用次数: 0
IDH1 mutation produces R-2-hydroxyglutarate (R-2HG) and induces mir-182-5p expression to regulate cell cycle and tumor formation in glioma. IDH1 突变会产生 R-2-hydroxyglutarate (R-2HG),并诱导 mir-182-5p 的表达,从而调节胶质瘤的细胞周期和肿瘤形成。
IF 4.3 2区 生物学 Q1 BIOLOGY Pub Date : 2024-05-17 DOI: 10.1186/s40659-024-00512-2
Haiting Zhao, Li Meng, Peng Du, Xinbin Liao, Xin Mo, Mengqi Gong, Jiaxin Chen, Yiwei Liao

Background: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), are present in most gliomas. IDH1 mutation is an important prognostic marker in glioma. However, its regulatory mechanism in glioma remains incompletely understood.

Results: miR-182-5p expression was increased within IDH1-mutant glioma specimens according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples. (R)-2-hydroxyglutarate ((R)-2HG) treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p inhibition partially eliminated the oncogenic effects of R-2HG upon glioma cells. By direct binding to Cyclin Dependent Kinase Inhibitor 2 C (CDKN2C) 3'UTR, miR-182-5p inhibited CDKN2C expression. Regarding cellular functions, CDKN2C knockdown promoted R-2HG-treated glioma cell viability, suppressed apoptosis, and relieved cell cycle arrest. Furthermore, CDKN2C knockdown partially attenuated the effects of miR-182-5p inhibition on cell phenotypes. Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition in cell cycle check point and apoptosis markers. The engineered CS-NPs (antagomir-182-5p) effectively encapsulated and delivered antagomir-182-5p, enhancing anti-tumor efficacy in vivo, indicating the therapeutic potential of CS-NPs(antagomir-182-5p) in targeting the miR-182-5p/CDKN2C axis against R-2HG-driven oncogenesis in mice models.

Conclusions: These insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/CDKN2C axis, offering a promising therapeutic avenue against R-2HG's oncogenic influence to glioma.

背景:大多数胶质瘤都存在异柠檬酸脱氢酶 1 和 2(IDH1 和 IDH2)突变。IDH1 突变是胶质瘤预后的重要标志。结果发现:根据 TCGA、CGGA 和在线数据集 GSE119740,以及收集的临床样本,IDH1 突变的胶质瘤标本中 miR-182-5p 表达增加。(R)-2-羟基戊二酸((R)-2HG)处理上调了 miR-182-5p 的表达,增强了胶质瘤细胞的增殖,抑制了细胞凋亡;抑制 miR-182-5p 可部分消除 R-2HG 对胶质瘤细胞的致癌作用。通过与细胞周期蛋白依赖性激酶抑制剂 2 C(CDKN2C)3'UTR 直接结合,miR-182-5p 抑制了 CDKN2C 的表达。在细胞功能方面,CDKN2C的敲除促进了R-2HG处理的胶质瘤细胞的活力,抑制了细胞凋亡,并缓解了细胞周期的停滞。此外,CDKN2C的敲除部分减弱了miR-182-5p抑制对细胞表型的影响。此外,CDKN2C敲除对细胞周期检查点和细胞凋亡标志物的影响与miR-182-5p抑制相反;CDKN2C敲除还部分削弱了miR-182-5p抑制对细胞周期检查点和细胞凋亡标志物的影响。工程化的CS-NPs(antagomir-182-5p)能有效封装和递送antagomir-182-5p,提高体内抗肿瘤疗效,表明CS-NPs(antagomir-182-5p)在靶向miR-182-5p/CDKN2C轴对抗R-2HG驱动的小鼠肿瘤发生方面具有治疗潜力:这些发现凸显了CS-NPs(antagomir-182-5p)靶向miR-182-5p/CDKN2C轴的潜力,为对抗R-2HG对胶质瘤的致癌影响提供了一条前景广阔的治疗途径。
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引用次数: 0
Therapeutic potential of oleic acid supplementation in myotonic dystrophy muscle cell models. 在肌营养不良症肌肉细胞模型中补充油酸的治疗潜力。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-17 DOI: 10.1186/s40659-024-00496-z
Nerea Moreno, Maria Sabater-Arcis, Teresa Sevilla, Manuel Perez Alonso, Jessica Ohana, Ariadna Bargiela, Ruben Artero

Background: We recently reported that upregulation of Musashi 2 (MSI2) protein in the rare neuromuscular disease myotonic dystrophy type 1 contributes to the hyperactivation of the muscle catabolic processes autophagy and UPS through a reduction in miR-7 levels. Because oleic acid (OA) is a known allosteric regulator of MSI2 activity in the biogenesis of miR-7, here we sought to evaluate endogenous levels of this fatty acid and its therapeutic potential in rescuing cell differentiation phenotypes in vitro. In this work, four muscle cell lines derived from DM1 patients were treated with OA for 24 h, and autophagy and muscle differentiation parameters were analyzed.

Results: We demonstrate a reduction of OA levels in different cell models of the disease. OA supplementation rescued disease-related phenotypes such as fusion index, myotube diameter, and repressed autophagy. This involved inhibiting MSI2 regulation of direct molecular target miR-7 since OA isoschizomer, elaidic acid (EA) could not cause the same rescues. Reduction of OA levels seems to stem from impaired biogenesis since levels of the enzyme stearoyl-CoA desaturase 1 (SCD1), responsible for converting stearic acid to oleic acid, are decreased in DM1 and correlate with OA amounts.

Conclusions: For the first time in DM1, we describe a fatty acid metabolism impairment that originated, at least in part, from a decrease in SCD1. Because OA allosterically inhibits MSI2 binding to molecular targets, reduced OA levels synergize with the overexpression of MSI2 and contribute to the MSI2 > miR-7 > autophagy axis that we proposed to explain the muscle atrophy phenotype.

背景:我们最近报告说,在罕见的神经肌肉疾病肌营养不良症1型中,Musashi 2(MSI2)蛋白的上调通过降低miR-7的水平导致肌肉分解代谢过程自噬和UPS的过度激活。由于油酸(OA)是miR-7生物生成过程中MSI2活性的已知异构调节剂,我们在此试图评估这种脂肪酸的内源性水平及其在体外挽救细胞分化表型方面的治疗潜力。在这项工作中,我们用OA处理了来自DM1患者的四种肌肉细胞系24小时,并分析了自噬和肌肉分化参数:结果:我们发现在不同的疾病细胞模型中,OA的水平都有所下降。结果:我们发现,在不同的疾病细胞模型中,OA水平都有所下降,补充OA后,疾病相关表型(如融合指数、肌管直径)得到改善,自噬功能受到抑制。这涉及抑制MSI2对直接分子靶标miR-7的调控,因为OA异构体麦饭石酸(EA)不能产生同样的挽救效果。OA水平的降低似乎源于生物生成受损,因为在DM1中,负责将硬脂酸转化为油酸的硬脂酰-CoA脱饱和酶1(SCD1)的水平降低,并与OA数量相关:我们首次在 DM1 中描述了脂肪酸代谢障碍,这种障碍至少部分源于 SCD1 的减少。由于OA会异位抑制MSI2与分子靶标的结合,因此OA水平的降低会与MSI2的过度表达产生协同作用,并促成MSI2 > miR-7 > 自噬轴,我们提出了这一轴来解释肌肉萎缩表型。
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引用次数: 0
Dorsal root ganglion-derived exosomes deteriorate neuropathic pain by activating microglia via the microRNA-16-5p/HECTD1/HSP90 axis. 背根神经节衍生的外泌体通过microRNA-16-5p/HECTD1/HSP90轴激活小胶质细胞,从而恶化神经病理性疼痛。
IF 4.3 2区 生物学 Q1 BIOLOGY Pub Date : 2024-05-15 DOI: 10.1186/s40659-024-00513-1
Yinghao Xing, Pei Li, Yuanyuan Jia, Kexin Zhang, Ming Liu, Jingjing Jiang

Background: The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism.

Methods: A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments.

Results: DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP.

Conclusions: miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

背景:据报道,活化的小胶质细胞是神经病理性疼痛(NP)病理学的支柱因素,但驱动疼痛诱导的小胶质细胞活化的分子还需要进一步探索。本研究探讨了背根神经节(DRG)衍生的外泌体(Exo)对小胶质细胞活化的影响及其相关机制:方法:通过脊神经结扎术(SNL)建立NP小鼠模型,并提取DRG衍生的外泌体。通过行为测试、HE染色、免疫荧光和Western blot等方法评估了DRG-Exo对SNL小鼠NP和小胶质细胞活化的影响。接着,使用芯片分析了 DRG-Exo 处理过的小胶质细胞中不同程度富集的微 RNA(miRNA)。通过 RT-qPCR、RNA pull-down、双荧光素酶报告实验和免疫荧光来验证 miR-16-5p 与 HECTD1 的结合关系。最后,通过Co-IP、Western印迹、免疫荧光检测和挽救实验研究了HECTD1对HSP90泛素化修饰对NP进展和小胶质细胞活化的影响:结果:DRG-Exo加重了小鼠SNL导致的NP,促进了DRG中小胶质细胞的活化,加重了神经炎症。DRG-Exo 通过 miR-16-5p 和 HECTD1 之间的相互作用调节 HSP90 的泛素化。结论:DRG-Exo穿梭的miR-16-5p通过与HECTD1相互作用调节了HSP90的泛素化,从而促进了NP中的小胶质细胞活化。
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引用次数: 0
MicroRNA-721 regulates gluconeogenesis via KDM2A-mediated epigenetic modulation in diet-induced insulin resistance in C57BL/6J mice. 微RNA-721通过KDM2A介导的表观遗传调控调节C57BL/6J小鼠饮食诱导的胰岛素抵抗中的葡萄糖生成。
IF 4.3 2区 生物学 Q1 BIOLOGY Pub Date : 2024-05-14 DOI: 10.1186/s40659-024-00495-0
Shaheen Wasil Kabeer, Shivam Sharma, Shalemraju Sriramdasu, Kulbhushan Tikoo

Background: Aberrant gluconeogenesis is considered among primary drivers of hyperglycemia under insulin resistant conditions, with multiple studies pointing towards epigenetic dysregulation. Here we examine the role of miR-721 and effect of epigenetic modulator laccaic acid on the regulation of gluconeogenesis under high fat diet induced insulin resistance.

Results: Reanalysis of miRNA profiling data of high-fat diet-induced insulin-resistant mice model, GEO dataset (GSE94799) revealed a significant upregulation of miR-721, which was further validated in invivo insulin resistance in mice and invitro insulin resistance in Hepa 1-6 cells. Interestingly, miR-721 mimic increased glucose production in Hepa 1-6 cells via activation of FOXO1 regulated gluconeogenic program. Concomitantly, inhibition of miR-721 reduced glucose production in palmitate induced insulin resistant Hepa 1-6 cells by blunting the FOXO1 induced gluconeogenesis. Intriguingly, at epigenetic level, enrichment of the transcriptional activation mark H3K36me2 got decreased around the FOXO1 promoter. Additionally, identifying targets of miR-721 using miRDB.org showed H3K36me2 demethylase KDM2A as a potential target. Notably, miR-721 inhibitor enhanced KDM2A expression which correlated with H3K36me2 enrichment around FOXO1 promoter and the downstream activation of the gluconeogenic pathway. Furthermore, inhibition of miR-721 in high-fat diet-induced insulin-resistant mice resulted in restoration of KDM2A levels, concomitantly reducing FOXO1, PCK1, and G6PC expression, attenuating gluconeogenesis, hyperglycemia, and improving glucose tolerance. Interestingly, the epigenetic modulator laccaic acid also reduced the hepatic miR-721 expression and improved KDM2A expression, supporting our earlier report that laccaic acid attenuates insulin resistance by reducing gluconeogenesis.

Conclusion: Our study unveils the role of miR-721 in regulating gluconeogenesis through KDM2A and FOXO1 under insulin resistance, pointing towards significant clinical and therapeutic implications for metabolic disorders. Moreover, the promising impact of laccaic acid highlights its potential as a valuable intervention in managing insulin resistance-associated metabolic diseases.

背景:在胰岛素抵抗条件下,畸形的糖元生成被认为是高血糖的主要驱动因素之一,而多项研究都指向表观遗传失调。在此,我们研究了 miR-721 的作用以及表观遗传调节剂漆树酸对高脂饮食诱导的胰岛素抵抗条件下糖元生成调节的影响:结果:重新分析高脂饮食诱导的胰岛素抵抗小鼠模型的 miRNA 图谱数据、GEO 数据集(GSE94799)发现,miR-721 有显著上调,这在小鼠体内胰岛素抵抗和 Hepa 1-6 细胞体内胰岛素抵抗中得到了进一步验证。有趣的是,miR-721 模拟物通过激活 FOXO1 调控的葡萄糖生成程序增加了 Hepa 1-6 细胞的葡萄糖生成。同时,通过抑制 FOXO1 诱导的葡萄糖生成,抑制 miR-721 可减少棕榈酸酯诱导的胰岛素抵抗 Hepa 1-6 细胞的葡萄糖生成。耐人寻味的是,在表观遗传学水平上,FOXO1 启动子周围的转录激活标记 H3K36me2 的富集减少了。此外,利用 miRDB.org 确定 miR-721 的靶标显示,H3K36me2 去甲基化酶 KDM2A 是一个潜在靶标。值得注意的是,miR-721抑制剂增强了KDM2A的表达,这与FOXO1启动子周围的H3K36me2富集以及葡萄糖生成途径的下游激活相关。此外,在高脂饮食诱导的胰岛素抵抗小鼠中抑制 miR-721 可恢复 KDM2A 的水平,同时降低 FOXO1、PCK1 和 G6PC 的表达,减轻糖原生成、高血糖和改善糖耐量。有趣的是,表观遗传调节剂莱卡酸也降低了肝脏miR-721的表达,改善了KDM2A的表达,这支持了我们之前的报告,即莱卡酸通过减少葡萄糖生成来减轻胰岛素抵抗:我们的研究揭示了 miR-721 在胰岛素抵抗下通过 KDM2A 和 FOXO1 调节葡萄糖生成的作用,这对代谢性疾病具有重要的临床和治疗意义。此外,莱卡酸的积极影响凸显了其作为一种有价值的干预措施来控制胰岛素抵抗相关代谢疾病的潜力。
{"title":"MicroRNA-721 regulates gluconeogenesis via KDM2A-mediated epigenetic modulation in diet-induced insulin resistance in C57BL/6J mice.","authors":"Shaheen Wasil Kabeer, Shivam Sharma, Shalemraju Sriramdasu, Kulbhushan Tikoo","doi":"10.1186/s40659-024-00495-0","DOIUrl":"10.1186/s40659-024-00495-0","url":null,"abstract":"<p><strong>Background: </strong>Aberrant gluconeogenesis is considered among primary drivers of hyperglycemia under insulin resistant conditions, with multiple studies pointing towards epigenetic dysregulation. Here we examine the role of miR-721 and effect of epigenetic modulator laccaic acid on the regulation of gluconeogenesis under high fat diet induced insulin resistance.</p><p><strong>Results: </strong>Reanalysis of miRNA profiling data of high-fat diet-induced insulin-resistant mice model, GEO dataset (GSE94799) revealed a significant upregulation of miR-721, which was further validated in invivo insulin resistance in mice and invitro insulin resistance in Hepa 1-6 cells. Interestingly, miR-721 mimic increased glucose production in Hepa 1-6 cells via activation of FOXO1 regulated gluconeogenic program. Concomitantly, inhibition of miR-721 reduced glucose production in palmitate induced insulin resistant Hepa 1-6 cells by blunting the FOXO1 induced gluconeogenesis. Intriguingly, at epigenetic level, enrichment of the transcriptional activation mark H3K36me2 got decreased around the FOXO1 promoter. Additionally, identifying targets of miR-721 using miRDB.org showed H3K36me2 demethylase KDM2A as a potential target. Notably, miR-721 inhibitor enhanced KDM2A expression which correlated with H3K36me2 enrichment around FOXO1 promoter and the downstream activation of the gluconeogenic pathway. Furthermore, inhibition of miR-721 in high-fat diet-induced insulin-resistant mice resulted in restoration of KDM2A levels, concomitantly reducing FOXO1, PCK1, and G6PC expression, attenuating gluconeogenesis, hyperglycemia, and improving glucose tolerance. Interestingly, the epigenetic modulator laccaic acid also reduced the hepatic miR-721 expression and improved KDM2A expression, supporting our earlier report that laccaic acid attenuates insulin resistance by reducing gluconeogenesis.</p><p><strong>Conclusion: </strong>Our study unveils the role of miR-721 in regulating gluconeogenesis through KDM2A and FOXO1 under insulin resistance, pointing towards significant clinical and therapeutic implications for metabolic disorders. Moreover, the promising impact of laccaic acid highlights its potential as a valuable intervention in managing insulin resistance-associated metabolic diseases.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":null,"pages":null},"PeriodicalIF":4.3,"publicationDate":"2024-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11092102/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140921314","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver. 转录组学和蛋白质组学的结合揭示了维生素 C 对调节小鼠肝脏中特定蛋白质丰度的影响。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-12 DOI: 10.1186/s40659-024-00509-x
Lucie Aumailley, Antoine Bodein, Pauline Adjibade, Mickaël Leclercq, Sylvie Bourassa, Arnaud Droit, Rachid Mazroui, Michel Lebel

Background: Vitamin C (ascorbate) is a water-soluble antioxidant and an important cofactor for various biosynthetic and regulatory enzymes. Mice can synthesize vitamin C thanks to the key enzyme gulonolactone oxidase (Gulo) unlike humans. In the current investigation, we used Gulo-/- mice, which cannot synthesize their own ascorbate to determine the impact of this vitamin on both the transcriptomics and proteomics profiles in the whole liver. The study included Gulo-/- mouse groups treated with either sub-optimal or optimal ascorbate concentrations in drinking water. Liver tissues of females and males were collected at the age of four months and divided for transcriptomics and proteomics analysis. Immunoblotting, quantitative RT-PCR, and polysome profiling experiments were also conducted to complement our combined omics studies.

Results: Principal component analyses revealed distinctive differences in the mRNA and protein profiles as a function of sex between all the mouse cohorts. Despite such sexual dimorphism, Spearman analyses of transcriptomics data from females and males revealed correlations of hepatic ascorbate levels with transcripts encoding a wide array of biological processes involved in glucose and lipid metabolisms as well as in the acute-phase immune response. Moreover, integration of the proteomics data showed that ascorbate modulates the abundance of various enzymes involved in lipid, xenobiotic, organic acid, acetyl-CoA, and steroid metabolism mainly at the transcriptional level, especially in females. However, several proteins of the mitochondrial complex III significantly correlated with ascorbate concentrations in both males and females unlike their corresponding transcripts. Finally, poly(ribo)some profiling did not reveal significant enrichment difference for these mitochondrial complex III mRNAs between Gulo-/- mice treated with sub-optimal and optimal ascorbate levels.

Conclusions: Thus, the abundance of several subunits of the mitochondrial complex III are regulated by ascorbate at the post-transcriptional levels. Our extensive omics analyses provide a novel resource of altered gene expression patterns at the transcriptional and post-transcriptional levels under ascorbate deficiency.

背景:维生素 C(抗坏血酸)是一种水溶性抗氧化剂,也是各种生物合成和调节酶的重要辅助因子。与人类不同的是,小鼠可以通过关键酶古洛内酯氧化酶(Gulo)合成维生素 C。在目前的研究中,我们使用了不能自己合成抗坏血酸的 Gulo/- 小鼠,以确定这种维生素对全肝脏转录组学和蛋白质组学特征的影响。研究包括在饮用水中添加次优或最优抗坏血酸浓度的 Gulo-/- 小鼠组。在小鼠四个月大时收集雌性和雄性小鼠的肝脏组织,并进行转录组学和蛋白质组学分析。我们还进行了免疫印迹、定量 RT-PCR 和多聚体分析实验,以补充我们的综合全息研究:结果:主成分分析表明,所有小鼠组群的 mRNA 和蛋白质图谱在性别功能上存在明显差异。尽管存在这种性别二态性,但对雌性和雄性的转录组学数据进行斯皮尔曼分析后发现,肝脏抗坏血酸水平与编码葡萄糖和脂质代谢以及急性期免疫反应所涉及的一系列生物过程的转录本存在相关性。此外,蛋白质组学数据整合显示,抗坏血酸主要在转录水平上调节参与脂质、异生物、有机酸、乙酰-CoA 和类固醇代谢的各种酶的丰度,尤其是在雌性动物中。然而,线粒体复合体 III 的几种蛋白质与抗坏血酸浓度在雄性和雌性中都有显著的相关性,这与其相应的转录物不同。最后,聚核糖谱分析结果显示,在接受次优和最优抗坏血酸水平治疗的 Gulo-/- 小鼠中,这些线粒体复合体 III mRNA 的富集差异并不明显:因此,线粒体复合体 III 的几个亚基的丰度在转录后水平上受抗坏血酸的调控。我们广泛的全局分析为抗坏血酸缺乏时转录和转录后水平基因表达模式的改变提供了新的资源。
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引用次数: 0
Novel role of LLGL2 silencing in autophagy: reversing epithelial-mesenchymal transition in prostate cancer. LLGL2沉默在自噬中的新作用:逆转前列腺癌的上皮-间质转化。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-08 DOI: 10.1186/s40659-024-00499-w
Geum-Lan Hong, Kyung-Hyun Kim, Yae-Ji Kim, Hui-Ju Lee, Sung-Pil Cho, Seung-Yun Han, Seung Woo Yang, Jong-Soo Lee, Shin-Kwang Kang, Jae-Sung Lim, Ju-Young Jung

Purpose: Prostate cancer (PCa) is a major urological disease that is associated with significant morbidity and mortality in men. LLGL2 is the mammalian homolog of Lgl. It acts as a tumor suppressor in breast and hepatic cancer. However, the role of LLGL2 and the underlying mechanisms in PCa have not yet been elucidated. Here, we investigate the role of LLGL2 in the regulation of epithelial-mesenchymal transition (EMT) in PCa through autophagy in vitro and in vivo.

Methods: PC3 cells were transfected with siLLGL2 or plasmid LLGL2 and autophagy was examined. Invasion, migration, and wound healing were assessed in PC3 cells under autophagy regulation. Tumor growth was evaluated using a shLLGL2 xenograft mouse model.

Results: In patients with PCa, LLGL2 levels were higher with defective autophagy and increased EMT. Our results showed that the knockdown of LLGL2 induced autophagy flux by upregulating Vps34 and ATG14L. LLGL2 knockdown inhibits EMT by upregulating E-cadherin and downregulating fibronectin and α-SMA. The pharmacological activation of autophagy by rapamycin suppressed EMT, and these effects were reversed by 3-methyladenine treatment. Interestingly, in a shLLGL2 xenograft mouse model, tumor size and EMT were decreased, which were improved by autophagy induction and worsened by autophagy inhibition.

Conclusion: Defective expression of LLGL2 leads to attenuation of EMT due to the upregulation of autophagy flux in PCa. Our results suggest that LLGL2 is a novel target for alleviating PCa via the regulation of autophagy.

目的:前列腺癌(PCa)是一种主要的泌尿系统疾病,与男性严重的发病率和死亡率有关。LLGL2 是哺乳动物 Lgl 的同源物。它是乳腺癌和肝癌的肿瘤抑制因子。然而,LLGL2在PCa中的作用及其内在机制尚未阐明。在此,我们研究了LLGL2在PCa中通过体外和体内自噬调节上皮-间质转化(EMT)的作用:方法:用 siLLGL2 或质粒 LLGL2 转染 PC3 细胞并检测自噬。在自噬调控下,对 PC3 细胞的侵袭、迁移和伤口愈合进行了评估。使用 shLLGL2 异种移植小鼠模型评估肿瘤生长情况:结果:在PCa患者中,LLGL2水平较高,自噬缺陷和EMT增加。我们的研究结果表明,敲除LLGL2可通过上调Vps34和ATG14L诱导自噬通量。LLGL2敲除可通过上调E-cadherin、下调纤连蛋白和α-SMA来抑制EMT。雷帕霉素对自噬的药理激活抑制了EMT,而3-甲基腺嘌呤处理则逆转了这些效应。有趣的是,在shLLGL2异种移植小鼠模型中,肿瘤大小和EMT均有所减小,自噬诱导可改善肿瘤大小和EMT,而自噬抑制则会恶化肿瘤大小和EMT:结论:LLGL2表达缺陷会导致自噬通量上调,从而减轻PCa的EMT。我们的研究结果表明,LLGL2是通过调节自噬缓解PCa的一个新靶点。
{"title":"Novel role of LLGL2 silencing in autophagy: reversing epithelial-mesenchymal transition in prostate cancer.","authors":"Geum-Lan Hong, Kyung-Hyun Kim, Yae-Ji Kim, Hui-Ju Lee, Sung-Pil Cho, Seung-Yun Han, Seung Woo Yang, Jong-Soo Lee, Shin-Kwang Kang, Jae-Sung Lim, Ju-Young Jung","doi":"10.1186/s40659-024-00499-w","DOIUrl":"10.1186/s40659-024-00499-w","url":null,"abstract":"<p><strong>Purpose: </strong>Prostate cancer (PCa) is a major urological disease that is associated with significant morbidity and mortality in men. LLGL2 is the mammalian homolog of Lgl. It acts as a tumor suppressor in breast and hepatic cancer. However, the role of LLGL2 and the underlying mechanisms in PCa have not yet been elucidated. Here, we investigate the role of LLGL2 in the regulation of epithelial-mesenchymal transition (EMT) in PCa through autophagy in vitro and in vivo.</p><p><strong>Methods: </strong>PC3 cells were transfected with siLLGL2 or plasmid LLGL2 and autophagy was examined. Invasion, migration, and wound healing were assessed in PC3 cells under autophagy regulation. Tumor growth was evaluated using a shLLGL2 xenograft mouse model.</p><p><strong>Results: </strong>In patients with PCa, LLGL2 levels were higher with defective autophagy and increased EMT. Our results showed that the knockdown of LLGL2 induced autophagy flux by upregulating Vps34 and ATG14L. LLGL2 knockdown inhibits EMT by upregulating E-cadherin and downregulating fibronectin and α-SMA. The pharmacological activation of autophagy by rapamycin suppressed EMT, and these effects were reversed by 3-methyladenine treatment. Interestingly, in a shLLGL2 xenograft mouse model, tumor size and EMT were decreased, which were improved by autophagy induction and worsened by autophagy inhibition.</p><p><strong>Conclusion: </strong>Defective expression of LLGL2 leads to attenuation of EMT due to the upregulation of autophagy flux in PCa. Our results suggest that LLGL2 is a novel target for alleviating PCa via the regulation of autophagy.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":null,"pages":null},"PeriodicalIF":6.7,"publicationDate":"2024-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11077766/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140891243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"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
High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats. 雄性大鼠的高脂饮食、微生物组-肠-脑轴信号传导和焦虑样行为。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-06 DOI: 10.1186/s40659-024-00505-1
Sylvana I S Rendeiro de Noronha, Lauro Angelo Gonçalves de Moraes, James E Hassell, Christopher E Stamper, Mathew R Arnold, Jared D Heinze, Christine L Foxx, Margaret M Lieb, Kristin E Cler, Bree L Karns, Sophia Jaekel, Kelsey M Loupy, Fernanda C S Silva, Deoclécio Alves Chianca-Jr, Christopher A Lowry, Rodrigo Cunha de Menezes

Obesity, associated with the intake of a high-fat diet (HFD), and anxiety are common among those living in modern urban societies. Recent studies suggest a role of microbiome-gut-brain axis signaling, including a role for brain serotonergic systems in the relationship between HFD and anxiety. Evidence suggests the gut microbiome and the serotonergic brain system together may play an important role in this response. Here we conducted a nine-week HFD protocol in male rats, followed by an analysis of the gut microbiome diversity and community composition, brainstem serotonergic gene expression (tph2, htr1a, and slc6a4), and anxiety-related defensive behavioral responses. We show that HFD intake decreased alpha diversity and altered the community composition of the gut microbiome in association with obesity, increased brainstem tph2, htr1a and slc6a4 mRNA expression, including in the caudal part of the dorsomedial dorsal raphe nucleus (cDRD), a subregion previously associated with stress- and anxiety-related behavioral responses, and, finally, increased anxiety-related defensive behavioral responses. The HFD increased the Firmicutes/Bacteroidetes ratio relative to control diet, as well as higher relative abundances of Blautia, and decreases in Prevotella. We found that tph2, htr1a and slc6a4 mRNA expression were increased in subregions of the dorsal raphe nucleus in the HFD, relative to control diet. Specific bacterial taxa were associated with increased serotonergic gene expression in the cDRD. Thus, we propose that HFD-induced obesity is associated with altered microbiome-gut-serotonergic brain axis signaling, leading to increased anxiety-related defensive behavioral responses in rats.

肥胖(与摄入高脂肪饮食(HFD)有关)和焦虑是生活在现代城市社会中的人的共同特征。最近的研究表明,微生物组-肠道-大脑轴信号传导,包括大脑血清素能系统在高脂饮食与焦虑之间的关系中发挥作用。有证据表明,肠道微生物组和大脑血清素能系统可能在这一反应中共同发挥重要作用。在此,我们对雄性大鼠进行了为期九周的高氟日粮方案,随后分析了肠道微生物组的多样性和群落组成、脑干血清素能基因表达(ph2、htr1a 和 slc6a4)以及与焦虑相关的防御性行为反应。我们的研究表明,摄入高氟日粮会降低肠道微生物组的α多样性并改变其群落组成,从而导致肥胖;增加脑干tph2、htr1a和slc6a4 mRNA的表达,包括在背内侧背侧剑突核(ctRD)尾部的表达,该亚区以前与压力和焦虑相关的行为反应有关;最后,增加焦虑相关的防御性行为反应。与对照组饮食相比,HFD 增加了固着菌/类杆菌的比例,并提高了 Blautia 的相对丰度,降低了 Prevotella 的丰度。我们发现,与对照组饮食相比,HFD 食物中背侧剑突核亚区域的 tph2、htr1a 和 slc6a4 mRNA 表达增加。特定细菌类群与 cDRD 中血清素能基因表达的增加有关。因此,我们认为高密度脂蛋白胆固醇诱导的肥胖与微生物组-肠道-血清素能脑轴信号的改变有关,从而导致大鼠焦虑相关防御性行为反应的增加。
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引用次数: 0
Rapid development and mass production of SARS-CoV-2 neutralizing chicken egg yolk antibodies with protective efficacy in hamsters. 快速开发和大规模生产对仓鼠具有保护效力的 SARS-CoV-2 中和性鸡卵黄抗体。
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-06 DOI: 10.1186/s40659-024-00508-y
Binan Zhao, Haoran Peng, Yanjing Zhang, Jie Zhang, Desheng Kong, Sai Cao, Yan Li, Dan Yang, Chuanwen Sun, Xinyi Pu, Ping Zhao, Yan Xu, Kai Zhao, Liangzhi Xie

Despite the record speed of developing vaccines and therapeutics against the SARS-CoV-2 virus, it is not a given that such success can be secured in future pandemics. In addition, COVID-19 vaccination and application of therapeutics remain low in developing countries. Rapid and low cost mass production of antiviral IgY antibodies could be an attractive alternative or complementary option for vaccine and therapeutic development. In this article, we rapidly produced SARS-CoV-2 antigens, immunized hens and purified IgY antibodies in 2 months after the SARS-CoV-2 gene sequence became public. We further demonstrated that the IgY antibodies competitively block RBD binding to ACE2, neutralize authentic SARS-CoV-2 virus and effectively protect hamsters from SARS-CoV-2 challenge by preventing weight loss and lung pathology, representing the first comprehensive study with IgY antibodies. The process of mass production can be easily implemented in most developing countries and hence could become a new vital option in our toolbox for combating viral pandemics. This study could stimulate further studies, optimization and potential applications of IgY antibodies as therapeutics and prophylactics for human and animals.

尽管针对 SARS-CoV-2 病毒的疫苗和疗法的开发速度创下了纪录,但并不意味着未来的大流行病也能取得这样的成功。此外,在发展中国家,COVID-19 疫苗的接种率和疗法的应用率仍然很低。快速、低成本地大规模生产抗病毒 IgY 抗体可能是疫苗和疗法开发的一个有吸引力的替代或补充方案。在这篇文章中,我们在 SARS-CoV-2 基因序列公开后的 2 个月内快速制备了 SARS-CoV-2 抗原、免疫母鸡并纯化了 IgY 抗体。我们进一步证明了 IgY 抗体能竞争性地阻断 RBD 与 ACE2 的结合,中和真实的 SARS-CoV-2 病毒,并能有效地保护仓鼠免受 SARS-CoV-2 的挑战,防止体重减轻和肺部病变,这是首次用 IgY 抗体进行的全面研究。大规模生产的过程在大多数发展中国家都很容易实现,因此可以成为我们抗击病毒大流行工具箱中的一个新的重要选择。这项研究可以促进进一步研究、优化和潜在应用 IgY 抗体作为人类和动物的治疗和预防药物。
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引用次数: 0
Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach 利用非病毒永生化方法建立原代前列腺上皮细胞系和肿瘤细胞系
IF 6.7 2区 生物学 Q1 Agricultural and Biological Sciences Pub Date : 2024-05-04 DOI: 10.1186/s40659-024-00507-z
Simon Lange, Anna Kuntze, Neele Wüstmann, Theresa Reckers, Verena Humberg, Wilhelm G. Dirks, Sebastian Huss, Julia Vieler, Andres Jan Schrader, Martin Bögemann, Katrin Schlack, Christof Bernemann
Research on prostate cancer is mostly performed using cell lines derived from metastatic disease, not reflecting stages of tumor initiation or early progression. Establishment of cancer cell lines derived from the primary tumor site has not been described so far. By definition, cancer cells are able to be cultured indefinitely, whereas normal epithelial cells undergo senescence in vitro. Epithelial cells can be immortalized, accomplished by using viral integration of immortalization factors. Viral approaches, however, might be impaired by regulatory and safety issues as well as random integration into regulatory genetic elements, modifying precise gene expression. We intend to use surgical specimen of prostate cancer patients to (i) prove for establishment of cancer cell lines, and (ii) perform non-viral, Sleeping Beauty (SB) transposase-based immortalization of prostate epithelial cells. Radical prostatectomy samples of prostate cancer patients (n = 4) were dissociated and cultured in vitro. Cells were cultivated either without or after non-viral, Sleeping-Beauty transposase-based stable transfection with immortalization factors SV40LT and hTERT. Established cell lines were analyzed in vitro and in vivo for characteristics of prostate (cancer) cells. Initial cell cultures without genetic manipulation underwent senescence within ≤ 15 passages, demonstrating inability to successfully derive primary prostate cancer cell lines. By using SB transposase-based integration of immortalization factors, we were able to establish primary prostate cell lines. Three out of four cell lines displayed epithelial characteristics, however without expression of prostate (cancer) characteristics, e.g., androgen receptor. In vivo, one cell line exhibited tumorigenic potential, yet characteristics of prostate adenocarcinoma were absent. Whereas no primary prostate cancer cell line could be established, we provide for the first-time immortalization of primary prostate cells using the SB transposase system, thereby preventing regulatory and molecular issues based on viral immortalization approaches. Although, none of the newly derived cell lines demonstrated prostate cancer characteristics, tumor formation was observed in one cell line. Given the non-prostate adenocarcinoma properties of the tumor, cells have presumably undergone oncogenic transformation rather than prostate cancer differentiation. Still, these cell lines might be used as a tool for research on prostate cancer initiation and early cancer progression.
对前列腺癌的研究大多使用来自转移性疾病的细胞系,不能反映肿瘤的起始阶段或早期进展。迄今为止,还没有人描述过从原发肿瘤部位提取的癌细胞系的建立情况。根据定义,癌细胞可以无限期培养,而正常上皮细胞在体外会衰老。上皮细胞可以通过病毒整合永生化因子实现永生化。然而,病毒方法可能会受到监管和安全问题的影响,也可能会随机整合到调控遗传元件中,从而改变精确的基因表达。我们打算利用前列腺癌患者的手术标本:(i) 证明癌症细胞系的建立;(ii) 对前列腺上皮细胞进行基于睡美人(SB)转座酶的非病毒永生化。对前列腺癌患者的前列腺根治术样本(n = 4)进行离体和体外培养。培养细胞时不使用病毒,也不使用基于睡美人转座酶的稳定转染永生化因子 SV40LT 和 hTERT。对建立的细胞系进行了体外和体内分析,以确定前列腺(癌)细胞的特征。未进行基因操作的初始细胞培养物在传代次数不超过 15 次时就会衰老,这表明无法成功培育出原代前列腺癌细胞系。通过使用基于 SB 转座酶的永生化因子整合,我们建立了原代前列腺细胞系。四个细胞系中有三个显示出上皮特征,但没有前列腺(癌症)特征(如雄激素受体)的表达。在体内,一种细胞系具有致瘤潜能,但没有前列腺腺癌的特征。虽然无法建立原代前列腺癌细胞系,但我们首次利用 SB 转座酶系统实现了原代前列腺细胞的永生化,从而避免了基于病毒永生化方法的调控和分子问题。虽然新获得的细胞系都没有表现出前列腺癌的特征,但在一个细胞系中观察到了肿瘤的形成。鉴于肿瘤的非前列腺腺癌特性,细胞可能发生了致癌转化,而非前列腺癌分化。不过,这些细胞系仍可作为研究前列腺癌发生和早期癌症进展的工具。
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