Pub Date : 2024-05-12DOI: 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。
{"title":"Combined transcriptomics and proteomics unveil the impact of vitamin C in modulating specific protein abundance in the mouse liver.","authors":"Lucie Aumailley, Antoine Bodein, Pauline Adjibade, Mickaël Leclercq, Sylvie Bourassa, Arnaud Droit, Rachid Mazroui, Michel Lebel","doi":"10.1186/s40659-024-00509-x","DOIUrl":"10.1186/s40659-024-00509-x","url":null,"abstract":"<p><strong>Background: </strong>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<sup>-/-</sup> 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<sup>-/-</sup> 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.</p><p><strong>Results: </strong>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<sup>-/-</sup> mice treated with sub-optimal and optimal ascorbate levels.</p><p><strong>Conclusions: </strong>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.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"57 1","pages":"26"},"PeriodicalIF":6.7,"publicationDate":"2024-05-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11088995/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140911169","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}
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.
{"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":"57 1","pages":"25"},"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}
Pub Date : 2024-05-06DOI: 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.
{"title":"High-fat diet, microbiome-gut-brain axis signaling, and anxiety-like behavior in male rats.","authors":"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","doi":"10.1186/s40659-024-00505-1","DOIUrl":"10.1186/s40659-024-00505-1","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"57 1","pages":"23"},"PeriodicalIF":6.7,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11071217/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140856535","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}
Pub Date : 2024-05-06DOI: 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.
{"title":"Rapid development and mass production of SARS-CoV-2 neutralizing chicken egg yolk antibodies with protective efficacy in hamsters.","authors":"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","doi":"10.1186/s40659-024-00508-y","DOIUrl":"10.1186/s40659-024-00508-y","url":null,"abstract":"<p><p>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.</p>","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"57 1","pages":"24"},"PeriodicalIF":6.7,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11071260/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140853130","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}
Pub Date : 2024-05-04DOI: 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.
{"title":"Establishment of primary prostate epithelial and tumorigenic cell lines using a non-viral immortalization approach","authors":"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","doi":"10.1186/s40659-024-00507-z","DOIUrl":"https://doi.org/10.1186/s40659-024-00507-z","url":null,"abstract":"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.","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"119 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841311","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-04DOI: 10.1186/s40659-024-00500-6
Andrea Oyarzún-Cisterna, Cristián Gidi, Fernanda Raiqueo, Roberto Amigo, Camila Rivas, Marcela Torrejón, José L. Gutiérrez
Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.
{"title":"General regulatory factors exert differential effects on nucleosome sliding activity of the ISW1a complex","authors":"Andrea Oyarzún-Cisterna, Cristián Gidi, Fernanda Raiqueo, Roberto Amigo, Camila Rivas, Marcela Torrejón, José L. Gutiérrez","doi":"10.1186/s40659-024-00500-6","DOIUrl":"https://doi.org/10.1186/s40659-024-00500-6","url":null,"abstract":"Chromatin dynamics is deeply involved in processes that require access to DNA, such as transcriptional regulation. Among the factors involved in chromatin dynamics at gene regulatory regions are general regulatory factors (GRFs). These factors contribute to establishment and maintenance of nucleosome-depleted regions (NDRs). These regions are populated by nucleosomes through histone deposition and nucleosome sliding, the latter catalyzed by a number of ATP-dependent chromatin remodeling complexes, including ISW1a. It has been observed that GRFs can act as barriers against nucleosome sliding towards NDRs. However, the relative ability of the different GRFs to hinder sliding activity is currently unknown. Considering this, we performed a comparative analysis for the main GRFs, with focus in their ability to modulate nucleosome sliding mediated by ISW1a. Among the GRFs tested in nucleosome remodeling assays, Rap1 was the only factor displaying the ability to hinder the activity of ISW1a. This effect requires location of the Rap1 cognate sequence on linker that becomes entry DNA in the nucleosome remodeling process. In addition, Rap1 was able to hinder nucleosome assembly in octamer transfer assays. Concurrently, Rap1 displayed the highest affinity for and longest dwell time from its target sequence, compared to the other GRFs tested. Consistently, through bioinformatics analyses of publicly available genome-wide data, we found that nucleosome occupancy and histone deposition in vivo are inversely correlated with the affinity of Rap1 for its target sequences in the genome. Our findings point to DNA binding affinity, residence time and location at particular translational positions relative to the nucleosome core as the key features of GRFs underlying their roles played in nucleosome sliding and assembly.","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"41 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841093","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-05-02DOI: 10.1186/s40659-024-00502-4
Omar I. Badr, Mohamed M. Kamal, Shohda A. El-Maraghy, Heba R. Ghaiad
Diabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro. We isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague–Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of β-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals. Our results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to β-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups. Our findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs.
{"title":"The effect of diabetes mellitus on differentiation of mesenchymal stem cells into insulin-producing cells","authors":"Omar I. Badr, Mohamed M. Kamal, Shohda A. El-Maraghy, Heba R. Ghaiad","doi":"10.1186/s40659-024-00502-4","DOIUrl":"https://doi.org/10.1186/s40659-024-00502-4","url":null,"abstract":"Diabetes mellitus (DM) is a global epidemic with increasing incidences. DM is a metabolic disease associated with chronic hyperglycemia. Aside from conventional treatments, there is no clinically approved cure for DM up till now. Differentiating mesenchymal stem cells (MSCs) into insulin-producing cells (IPCs) is a promising approach for curing DM. Our study was conducted to investigate the effect of DM on MSCs differentiation into IPCs in vivo and in vitro. We isolated adipose-derived mesenchymal stem cells (Ad-MSCs) from the epididymal fat of normal and STZ-induced diabetic Sprague–Dawley male rats. Afterwards, the in vitro differentiation of normal-Ad-MSCs (N-Ad-MSCs) and diabetic-Ad-MSCs (DM-Ad-MSCs) into IPCs was compared morphologically then through determining the gene expression of β-cell markers including neurogenin-3 (Ngn-3), homeobox protein (Nkx6.1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA), and insulin-1 (Ins-1) and eventually, through performing glucose-stimulated insulin secretion test (GSIS). Finally, the therapeutic potential of N-Ad-MSCs and DM-Ad-MSCs transplantation was compared in vivo in STZ-induced diabetic animals. Our results showed no significant difference in the characteristics of N-Ad-MSCs and DM-Ad-MSCs. However, we demonstrated a significant difference in their abilities to differentiate into IPCs in vitro morphologically in addition to β-cell markers expression, and functional assessment via GSIS test. Furthermore, the abilities of both Ad-MSCs to control hyperglycemia in diabetic rats in vivo was assessed through measuring fasting blood glucose (FBGs), body weight (BW), histopathological examination of both pancreas and liver and immunoexpression of insulin in pancreata of study groups. Our findings reveal the effectiveness of N-Ad-MSCs in differentiating into IPCs in vitro and controlling the hyperglycemia of STZ-induced diabetic rats in vivo compared to DM-Ad-MSCs. ","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"1 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841327","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-30DOI: 10.1186/s40659-024-00503-3
Mariela Puebla, Manuel F. Muñoz, Mauricio A. Lillo, Jorge E. Contreras, Xavier F. Figueroa
Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.
星形胶质细胞的 Ca2+ 信号在调节神经元功能方面发挥着核心作用。突触活动增加时释放的谷氨酸会激活代谢型谷氨酸受体(mGluR),从而触发星形胶质细胞中协调的 Ca2+ 信号。重要的是,星形胶质细胞表达依赖 Ca2+ 的一氧化氮(NO)合成酶 eNOS 和 nNOS,它们可能通过激活连接素 43(Cx43)半桥通道、pannexin-1(Panx-1)通道或 Ca2+ 平衡调节器 1(CALHM1)通道触发 Ca2+ 流入或 ATP 释放,从而促进 Ca2+ 信号。因此,我们旨在评估 NO 在大鼠大脑皮层星形胶质细胞原代培养物中参与由 mGluR 刺激引发的星形胶质细胞 Ca2+ 信号转导的情况。用谷氨酸或 t-ACPD 刺激星形胶质细胞,评估[Ca2+]i 和 ATP 释放的 NO 依赖性变化。此外,还分析了 Cx43 半通道、Panx-1 通道和 CALHM1 通道的活性。免疫荧光分析证实了 Cx43、Panx-1 和 CALHM1 在星形胶质细胞中的表达,谷氨酸和 t-ACPD 通过直接 S-亚硝基化诱导了 NO 介导的 CALHM1 通道活化。药理阻断或 siRNA 介导的 CALHM1 表达抑制显示,这些通道的开放为 ATP 释放以及随后嘌呤能受体依赖性激活 Cx43 半ich通道和 Panx-1 通道提供了途径,从而进一步促进了星形胶质细胞的 Ca2+ 信号转导。我们的研究结果表明,在体外星形胶质细胞中通过 NO 介导的 S-亚硝基化激活 CALHM1 通道对于谷氨酸引发的星形胶质细胞 Ca2+ 信号的产生至关重要。
{"title":"Control of astrocytic Ca2+ signaling by nitric oxide-dependent S-nitrosylation of Ca2+ homeostasis modulator 1 channels","authors":"Mariela Puebla, Manuel F. Muñoz, Mauricio A. Lillo, Jorge E. Contreras, Xavier F. Figueroa","doi":"10.1186/s40659-024-00503-3","DOIUrl":"https://doi.org/10.1186/s40659-024-00503-3","url":null,"abstract":"Astrocytes Ca2+ signaling play a central role in the modulation of neuronal function. Activation of metabotropic glutamate receptors (mGluR) by glutamate released during an increase in synaptic activity triggers coordinated Ca2+ signals in astrocytes. Importantly, astrocytes express the Ca2+-dependent nitric oxide (NO)-synthetizing enzymes eNOS and nNOS, which might contribute to the Ca2+ signals by triggering Ca2+ influx or ATP release through the activation of connexin 43 (Cx43) hemichannels, pannexin-1 (Panx-1) channels or Ca2+ homeostasis modulator 1 (CALHM1) channels. Hence, we aim to evaluate the participation of NO in the astrocytic Ca2+ signaling initiated by stimulation of mGluR in primary cultures of astrocytes from rat brain cortex. Astrocytes were stimulated with glutamate or t-ACPD and NO-dependent changes in [Ca2+]i and ATP release were evaluated. In addition, the activity of Cx43 hemichannels, Panx-1 channels and CALHM1 channels was also analyzed. The expression of Cx43, Panx-1 and CALHM1 in astrocytes was confirmed by immunofluorescence analysis and both glutamate and t-ACPD induced NO-mediated activation of CALHM1 channels via direct S-nitrosylation, which was further confirmed by assessing CALHM1-mediated current using the two-electrode voltage clamp technique in Xenopus oocytes. Pharmacological blockade or siRNA-mediated inhibition of CALHM1 expression revealed that the opening of these channels provides a pathway for ATP release and the subsequent purinergic receptor-dependent activation of Cx43 hemichannels and Panx-1 channels, which further contributes to the astrocytic Ca2+ signaling. Our findings demonstrate that activation of CALHM1 channels through NO-mediated S-nitrosylation in astrocytes in vitro is critical for the generation of glutamate-initiated astrocytic Ca2+ signaling.","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"45 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841308","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2024-04-26DOI: 10.1186/s40659-024-00498-x
Gloria Arriagada, Johan Quezada, Nicolas Merino-Veliz, Fernando Avilés, Diana Tapia-Cammas, Jorge Gomez, Daniela Curotto, Juan A. Valdes, Pablo A. Oyarzún, Cristian Gallardo-Escárate, Michael J. Metzger, Marco Alvarez
Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.
{"title":"Identification and expression analysis of two steamer-like retrotransposons in the Chilean blue mussel (Mytilus chilensis)","authors":"Gloria Arriagada, Johan Quezada, Nicolas Merino-Veliz, Fernando Avilés, Diana Tapia-Cammas, Jorge Gomez, Daniela Curotto, Juan A. Valdes, Pablo A. Oyarzún, Cristian Gallardo-Escárate, Michael J. Metzger, Marco Alvarez","doi":"10.1186/s40659-024-00498-x","DOIUrl":"https://doi.org/10.1186/s40659-024-00498-x","url":null,"abstract":"Disseminated neoplasia (DN) is a proliferative cell disorder of the circulatory system of bivalve mollusks. The disease is transmitted between individuals and can also be induced by external chemical agents such as bromodeoxyuridine. In Mya arenaria, we have cloned and characterized an LTR-retrotransposon named Steamer. Steamer mRNA levels and gene copy number correlates with DN and can be used as a marker of the disease. So far, the only mollusk where a retrotransposon expression relates to DN is Mya arenaria. On the other hand, it has been reported that the Chilean blue mussel Mytilus chilensis can also suffers DN. Our aim was to identify retrotransposons in Mytilus chilensis and to study their expression levels in the context of disseminated neoplasia. Here we show that 7.1% of individuals collected in August 2018, from two farming areas, presents morphological characteristics described in DN. Using Steamer sequence to interrogate the transcriptome of M. chilensis we found two putative retrotransposons, named Steamer-like elements (MchSLEs). MchSLEs are present in the genome of M. chilensis and MchSLE1 is indeed an LTR-retrotransposon. Neither expression, nor copy number of the reported MchSLEs correlate with DN status but both are expressed at different levels among individual animals. We also report that in cultured M. chilensis haemocytes MchSLEs1 expression can be induced by bromodeoxyuridine. We conclude that SLEs present in Mytilus chilensis are differentially expressed among individuals and do not correlate with disseminated neoplasia. Treatment of haemocytes with a stressor like bromodeoxyuridine induces expression of MchSLE1 suggesting that in Mytilus chilensis environmental stressors can induce activation of LTR-retrotransposon.","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"39 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140801070","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as “junk DNA”. Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.
{"title":"Noncoding RNAs in skeletal development and disorders","authors":"Qing Yao, Tailin He, Jian-You Liao, Rongdong Liao, Xiaohao Wu, Lijun Lin, Guozhi Xiao","doi":"10.1186/s40659-024-00497-y","DOIUrl":"https://doi.org/10.1186/s40659-024-00497-y","url":null,"abstract":"Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as “junk DNA”. Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.","PeriodicalId":9084,"journal":{"name":"Biological Research","volume":"135 1","pages":""},"PeriodicalIF":6.7,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140634361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}