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Journal articles on the topic 'Shona novels'
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1
Mavesera, Miidzo, and Davie E. Mutasa. "Empowerment through indigenous literature: The case of Shona novels." South African Journal of African Languages 29, no. 1 (January 2009): 74–86. http://dx.doi.org/10.1080/02572117.2009.10587318.
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Mutasa, D. E., and W. L. Chigidi. "Black writers’ Shona novels of the liberation war in Zimbabwe: an art that tells the truth of its day." Literator 31, no. 2 (July 13, 2010): 61–82. http://dx.doi.org/10.4102/lit.v31i2.47.
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Over the years Shona fiction that portrays Zimbabwe’s liberation war has been a subject of severe criticism because of its tendency to falsify and distort history. This article attempts to provide answers to the question of why authors of Shona war fiction tended to romanticise the war of liberation. In pursuance of this objective this article looks at circumstances and conditions that prevailed at the time that most of the Shona stories about Zimbabwe’s liberation war were written. These stories were published during the first decade of Zimbabwe’s independence and it is possible to look at this time and come up with a set of interdependent cultural, economic, political and ideological conditions that helped to shape writers’ perspectives on the war. The article argues that the conditions of artistic freedom that interfaced with internalised fear, the euphoria and celebration, the dominant ideology of the time, as well as the situation of competition were responsible for shaping the consciousness of the war fiction writers. In this article views expressed in interviews by some of the writers of Shona war fiction are taken into consideration. All interviews with authors referred to in the article were carried out by the researcher.
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Flora Veit-Wild. "Hearing Voices: The Linguistic and Narrative Design of Three Eminent Shona Novels." Research in African Literatures 48, no. 1 (2017): 1. http://dx.doi.org/10.2979/reseafrilite.48.1.02.
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Hassanin, Shaimaa Mohamed Mohamed. "The Influence of Rhodesia Literature Bureau on the Shona literature in Selected Zimbabwean Novels." مجلة القراءة والمعرفة 20, no. 1 (March 1, 2020): 1–21. http://dx.doi.org/10.21608/mrk.2020.100656.
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Makaudze, Godwin. "The Journey Back: Ambivalent (Re)Presentations of Pre-Colonial Women in Post-Independent Shona Novels." Journal of Literary Studies 35, no. 3 (July 3, 2019): 19–31. http://dx.doi.org/10.1080/02564718.2019.1657279.
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N., Eunitah Viriri, and Viriri Maradze. "The teaching Of Unhu/Ubuntu through Shona novels in Zimbabwean secondary schools: A case for Masvingo urban district." Journal of African Studies and Development 10, no. 8 (October 31, 2018): 101–14. http://dx.doi.org/10.5897/jasd2018.0508.
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Beach, D. N. "An Innocent Woman, Unjustly Accused? Charwe, Medium of the Nehanda Mhondoro Spirit, and the 1896–97 Central Shona Rising in Zimbabwe." History in Africa 25 (1998): 27–54. http://dx.doi.org/10.2307/3172179.
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The rising of the Ndebele and southwestern and central Shona people against colonial rule in the 1890s has become one of the classic cases of such resistance. Yet, since the independence of Zimbabwe in 1980, very little fresh research has been carried out on the subject. This paper re-examines the role of Shona religious authorities in the rising, especially that of the medium of the Nehanda spirit of the Mazowe valley in the central Shona area. In just over a century, the figure of “Mbuya Nehanda” has become the best-known popular symbol of resistance to colonial rule in modern Zimbabwe. She has been commemorated since 1980 in statues, street names, a hospital, posters, songs, novels, and poems, and is soon to be the subject of a full-length feature film. This paper examines the historical basis behind the legend.This legend runs as follows: the historical “Nehanda” was supposed to have been the daughter of the founding ancestor of the Mutapa dynasty, who lived in the fifteenth century. Her ritual incest with her brother Matope gave supernatural sanction to the power of the Mutapa state. After her death, she became a mhondoro spirit, and this spirit possessed a number of mediums (masvikiro, singular svikiro). During periods of possession by the spirit, the svikiro was regarded as speaking with the voice and personality of the original Nehanda and not with her own. In the last part of the nineteenth century one medium, Charwe, was responsible for the organization of resistance to the government of the British South Africa Company and the settlers in the Mazowe valley, and in particular for the killing of H.H. Pollard, Kunyaira, the extremely oppressive Native Commissioner of the area. This resistance began in June 1896, and from then until her capture in late 1897 the Nehanda medium was a major factor in the war. Tried and sentenced to death in March 1898, she refused to convert to Christianity and struggled right up to the moment when she was hanged.
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Mutasa, D. E., and I. Mutawi. "A philosophical interpretation of the significance of oral forms in I. Mabasa’s novel Mapenzi (1999)." Literator 29, no. 3 (July 25, 2008): 157–80. http://dx.doi.org/10.4102/lit.v29i3.130.
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The article critically analyses the use of Shona oral art forms in I. Mabasa’s novel “Mapenzi” (“Mad people”/“Foolish people”). It departs from the realisation that the writer identifies with Shona people’s oral experiences in the form of songs, “bembera” (satiric poetry) and folktales among others. These oral art forms provide the means by which the writer overcomes both selfcensorship and real or imagined state censorship. The article advances the argument that Mabasa uses the Shona people’s oral art forms in a manner that is ideologically and pedagogically empowering. This is consistent with the value thrust of Shona people’s epistemological assumptions. The article comes to the conclusion that Mabasa’s vision in the novel “Mapenzi” maintains the line between tradition and continuity.
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Huang, Ming-Shyan, Ya-Ling Hsu, I.-Jeng Yeh, Kuan-Ting Liu, and Meng-Chi Yen. "The Expression Profile of mRNA and tRNA Genes in Splenocytes and Neutrophils after In Vivo Delivery of Antitumor Short Hairpin RNA of Indoleamine 2,3- Dioxygenase." International Journal of Molecular Sciences 21, no. 18 (September 13, 2020): 6703. http://dx.doi.org/10.3390/ijms21186703.
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RNA-based therapeutics are considered as novel treatments for human diseases. Our previous study demonstrated that treatment with short-hairpin RNA against Ido1 (IDO shRNA) suppresses tumor growth, detects Th1-bias immune responses, and elevates expression of tryptophan transfer RNA (tRNATrp) in total splenocytes. In addition, depletion of Ly6g+ neutrophils attenuates the effect of IDO shRNA. The aim of this study was to investigate the regulatory network and the expression profile of tRNAs and other non-coding RNAs in IDO shRNA-treated spleens. The total splenocytes and magnetic bead-enriched splenic neutrophils were collected from the lung tumor bearing mice, which were treated with IDO shRNA or scramble IDO shRNA, and the collected cells were subsequently subjected to RNA sequencing. The gene ontology analysis revealed the different enrichment pathways in total splenocytes and splenic neutrophils. Furthermore, the expression of tRNA genes was identified and validated. Six isoacceptors of tRNA, with different expression patterns between total splenocytes and splenic neutrophils, were observed. In summary, our findings not only revealed novel biological processes in IDO shRNA-treated total splenocytes and splenic neutrophils, but the identified tRNAs and other non-coding RNAs may contribute to developing a novel biomarker gene set for evaluating the clinical efficiency of RNA-based cancer immunotherapies.
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Unwalla, Hoshang J., Hai-Tang Li, Ingrid Bahner, Ming-Jie Li, Donald Kohn, and John J. Rossi. "Novel Pol II Fusion Promoter Directs Human Immunodeficiency Virus Type 1-Inducible Coexpression of a Short Hairpin RNA and Protein." Journal of Virology 80, no. 4 (February 15, 2006): 1863–73. http://dx.doi.org/10.1128/jvi.80.4.1863-1873.2006.
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ABSTRACT We demonstrate a novel approach for coexpression of a short hairpin RNA (shRNA) with an open reading frame which exploits transcriptional read-through of a minimal polyadenylation signal from a Pol II promoter. We first observed efficient inducible expression of enhanced green fluorescent protein along with an anti-rev shRNA. We took advantage of this observation to test coexpression of the transdominant negative mutant (humanized) of human immunodeficiency type 1 (HIV-1) Rev (huRevM10) along with an anti-rev shRNA via an HIV-1-inducible fusion promoter. The coexpression of the shRNA and transdominant protein resulted in potent, long-term inhibition of HIV-1 gene expression and suppression of shRNA-resistant mutants. This dual expression system has broad-based potential for other shRNA applications, such as cases where simultaneous knockdown of mutant and wild-type transcripts must be accompanied by replacement of the wild-type protein.
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Pushparaj, P. N., J. J. Aarthi, J. Manikandan, and S. D. Kumar. "siRNA, miRNA, and shRNA: in vivo Applications." Journal of Dental Research 87, no. 11 (November 2008): 992–1003. http://dx.doi.org/10.1177/154405910808701109.
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RNA interference (RNAi), an accurate and potent gene-silencing method, was first experimentally documented in 1998 in Caenorhabditis elegans by Fire et al., who subsequently were awarded the 2006 Nobel Prize in Physiology/Medicine. Subsequent RNAi studies have demonstrated the clinical potential of synthetic small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) in dental diseases, eye diseases, cancer, metabolic diseases, neurodegenerative disorders, and other illnesses. siRNAs are generally from 21 to 25 base-pairs (bp) in length and have sequence-homology-driven gene-knockdown capability. RNAi offers researchers an effortless tool for investigating biological systems by selectively silencing genes. Key technical aspects—such as optimization of selectivity, stability, in vivo delivery, efficacy, and safety—need to be investigated before RNAi can become a successful therapeutic strategy. Nevertheless, this area shows a huge potential for the pharmaceutical industry around the globe. Interestingly, recent studies have shown that the small RNA molecules, either indigenously produced as microRNAs (miRNAs) or exogenously administered synthetic dsRNAs, could effectively activate a particular gene in a sequence-specific manner instead of silencing it. This novel, but still uncharacterized, phenomenon has been termed ‘RNA activation’ (RNAa). In this review, we analyze these research findings and discussed the in vivo applications of siRNAs, miRNAs, and shRNAs.
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Yugawa, Takashi, Keisuke Handa, Mako Narisawa-Saito, Shin-ichi Ohno, Masatoshi Fujita, and Tohru Kiyono. "Regulation of Notch1 Gene Expression by p53 in Epithelial Cells." Molecular and Cellular Biology 27, no. 10 (March 12, 2007): 3732–42. http://dx.doi.org/10.1128/mcb.02119-06.
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ABSTRACT The E6 protein of cervical cancer-associated human papillomaviruses (HPVs) is known to suppress keratinocyte differentiation through unidentified mechanisms. Notch1 is a determinant of keratinocyte differentiation and functions as a tumor suppressor in mammalian epidermis. Here, we report that the Notch1 gene is a novel target of p53 and can be down-regulated by E6 through p53 degradation in normal human epithelial cells. Thus, inactivation of p53 by E6 or short-hairpin RNA (shRNA) resulted in reduced Notch1 expression at the transcription level, and a p53-responsive element could be identified in the Notch1 promoter. The expression of E6, p53 shRNA, or Notch1 shRNA suppressed both spontaneous keratinocyte differentiation in culture and its induction upon DNA damage. Furthermore, the induction of Notch1 and differentiation makers as well as thickening of the epidermal layer upon UV irradiation was observed in wild-type but not in p53-deficient mouse skin. Together, our findings not only demonstrate a novel link between p53 and Notch1 in keratinocyte differentiation upon genotoxic stress but also suggest a novel tumor suppressor mechanism of p53 in the development of squamous cell carcinomas, including HPV-induced tumors.
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Montazeri, Hesam, Mairene Coto-Llerena, Gaia Bianco, Ehsan Zangene, Stephanie Taha-Mehlitz, Viola Paradiso, Sumana Srivatsa, et al. "Systematic identification of novel cancer genes through analysis of deep shRNA perturbation screens." Nucleic Acids Research 49, no. 15 (July 27, 2021): 8488–504. http://dx.doi.org/10.1093/nar/gkab627.
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Abstract Systematic perturbation screens provide comprehensive resources for the elucidation of cancer driver genes. The perturbation of many genes in relatively few cell lines in such functional screens necessitates the development of specialized computational tools with sufficient statistical power. Here we developed APSiC (Analysis of Perturbation Screens for identifying novel Cancer genes) to identify genetic drivers and effectors in perturbation screens even with few samples. Applying APSiC to the shRNA screen Project DRIVE, APSiC identified well-known and novel putative mutational and amplified cancer genes across all cancer types and in specific cancer types. Additionally, APSiC discovered tumor-promoting and tumor-suppressive effectors, respectively, for individual cancer types, including genes involved in cell cycle control, Wnt/β-catenin and hippo signalling pathways. We functionally demonstrated that LRRC4B, a putative novel tumor-suppressive effector, suppresses proliferation by delaying cell cycle and modulates apoptosis in breast cancer. We demonstrate APSiC is a robust statistical framework for discovery of novel cancer genes through analysis of large-scale perturbation screens. The analysis of DRIVE using APSiC is provided as a web portal and represents a valuable resource for the discovery of novel cancer genes.
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Wermke, Martin, Aylin Camgoz, Maciej Paszkowski-Rogacz, Sebastian Thieme, Malte von Bonin, Andreas Dahl, Mirko Theis, et al. "A Genome Wide shRNA Screen In Primary Human AML Cells Identifies ROCK1 As a Novel Therapeutic Target." Blood 122, no. 21 (November 15, 2013): 170. http://dx.doi.org/10.1182/blood.v122.21.170.170.
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Abstract In spite of recent advances, the prognosis especially of elderly AML patients remains unsatisfactory with survival rates of less than 10 % at 10 years. Genome-wide RNA-interference screens systematically interrogating the specific vulnerabilities of leukemic cells could be a valuable tool to identify novel therapeutic targets in this patient population. So far, such screens have only been done in immortalized cell lines and / or at sub-genome scale, which limits their transferability to individual patients. Therefore, we set out to establish an unbiased genome-wide pooled shRNA screen in primary human AML cells to prove the feasibility and test the possible clinical implications of such an approach. Lentiviral transduction of primary leukemic blasts from a 67-year old patient with AML FAB M1 with a pooled shRNA library (Mission TRC shRNA library SP1, Sigma) according to a specifically optimized protocol resulted in a transduction rate of 25 %, thus rendering multiple integrants unlikely. An aliquot of the cells was separated for DNA extraction directly after removal of viral supernatant (day 0) and after 9 days of suspension culture (day 9). ShRNA barcodes integrated into the genome of the host cells were read out using PCR-coupled next-generation sequencing (HiSeq 2000, Illumina). Of 7709 shRNA contained in the library, 6626 were recovered with at least 10 reads in the day 0 sample. After 9 days of culture, 25 shRNA targeting a total of 12 genes were identified as potentially lethal to the patient's AML-cells (Table 1). All of these shRNA were subjected to single-shRNA transduction experiments using leukemic cells from the same donor. In fact, 18 of 25 shRNA were validated with respect to viability. Knockdown specificity was documented for all validated shRNA by qPCR. For further analyses we focused only on those 7 genes in which more than 50% of the shRNA identified in the pooled screen could be validated (Table 1). These genes were assessed for druggability using publicly available databases. For exploration of the potential therapeutic implications of our screen we chose ROCK1 as a potential target, because Fasudil, a specific ROCK1 inhibitor, has already been licensed for the treatment of pulmonary hypertension in humans.Table.Table1No. shRNAs >100 reads day 0No. scoring shRNA in pooled screenNo. valdiated shRNA in single shRNA experimentsOverall gene validation statusBNIPL322ValidatedC7orf16322ValidatedCCRL1321Not validatedDGAT2321Not validatedDUSP14320Not validatedMAP3K6422ValidatedROCK1532ValidatedRPS13322ValidatedSF3A1321Not validatedSNX27422ValidatedSTK3422ValidatedWDHD1220Not validated Knockdown of ROCK1 in primary leukemic blasts led to rapid cell-cycle arrest and cell-death. Treatment with Fasudil proved to be equally effective in killing leukemic cells. Compared to primary leukemic cells from the original as well as from other AML patients, Fasudil seemed to be less toxic to hematopoietic cells derived from healthy volunteer donors. RNA-sequencing revealed that in comparison to the healthy controls none of the studied AML patients demonstrated a significant overexpression of ROCK1. Moreover there was no indication for a functional ROCK1 mutation in the analyzed AML samples. Feeder based long-term culture initiating cell (LTC-IC) assays further suggested that Fasudil had a significant negative effect on the self-renewal capacity of primary human leukemic stem/progenitor cells (Figure 1). Studies in xenograft-models to assess the stem cell toxicity of ROCK1 inhibition in more detail are currently ongoing.Figure.Figure. Taken together our results show that pooled shRNA screens in primary patient-derived leukemic cells are feasible and able to pinpoint novel therapeutic targets, which might be missed in mutation- or overexpression-based approaches. Further optimization of transduction and screening protocols might enable such screens to assist physicians in the selection of optimal therapeutic strategies especially in poor risk AML. Disclosures: No relevant conflicts of interest to declare.
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Keerthivasan, Ganesan, Jing Yang, Piu Wong, John Doench, David E. Root, and Peng Ji. "Targeted ShRNA Screening Identified Critical Role of Pleckstrin-2 in Erythropoiesis." Blood 120, no. 21 (November 16, 2012): 3199. http://dx.doi.org/10.1182/blood.v120.21.3199.3199.
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Abstract Abstract 3199 Mammalian erythropoiesis is globally regulated by erythropoietin (Epo). Epo binds to its receptor on the cell surface of erythroid precursor; induces a series of downstream pathways that promote cell differentiation and inhibit apoptosis. Recent genome wide transcriptional profile study demonstrated that over 500 genes are up-regulated during erythropoiesis. Many of these genes encode erythroid specific proteins that play well-known functions in red cells. However, the functions of the most other genes in the erythroid cells are still unknown. To identify novel genes in erythropoiesis, we infected mouse fetal liver erythroblasts with lentiviruses containing mammalian shRNA knockdown library that selectively includes the most highly upregulated 100 genes with unknown functions in erythroid cells. The infected cells were cultured in two different conditions for the characterization of early and late stage erythropoiesis using a high throughput flow cytometry based analysis. With these methods, we identified 33 novel genes that regulate cell differentiation or apoptosis in early stage erythropoisis; 20 genes play important roles in late stage erythropoiesis including enucleation. Significantly, there is an overlap of 16 genes that function in both early and late stage erythropoiesis. We focused on pleckstrin-2, which is specifically and abundantly expressed in erythroid cells, to further characterize its detailed functions in red cell development. We found that knockdown of pleckstrin-2 leads to dramatic apoptosis in early stage erythropoiesis. Knockdown of pleckstrin-2 in late stage erythropoiesis blocks enucleation with no apparent effects on cell differentiation, proliferation or apoptosis. We further discovered that pleckstrin-2 deficiency in early and late erythroblasts disrupts normal actin cytoskeleton as evidenced by super-resolution immunofluorescence microscope. To elucidate the detailed mechanisms of the functions of pleckstrin-2 in different stages of erythropoiesis, we performed proteomic studies and identified candidate proteins that interact with pleckstrin-2 that may contribute to the phenotypes of apoptosis and enucleation defects. In summary, our study identified pleckstrin-2 as a critical regulator of mammalian erythropoiesis and proved the significance of large-scale shRNA screening in the discovery of novel genes in development. Disclosures: No relevant conflicts of interest to declare.
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Liu, Qiao, Michelle Garcia, Shaoyuan Wang, and Chun-Wei Chen. "Therapeutic Target Discovery Using High-Throughput Genetic Screens in Acute Myeloid Leukemia." Cells 9, no. 8 (August 12, 2020): 1888. http://dx.doi.org/10.3390/cells9081888.
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The development of high-throughput gene manipulating tools such as short hairpin RNA (shRNA) and CRISPR/Cas9 libraries has enabled robust characterization of novel functional genes contributing to the pathological states of the diseases. In acute myeloid leukemia (AML), these genetic screen approaches have been used to identify effector genes with previously unknown roles in AML. These AML-related genes centralize alongside the cellular pathways mediating epigenetics, signaling transduction, transcriptional regulation, and energy metabolism. The shRNA/CRISPR genetic screens also realized an array of candidate genes amenable to pharmaceutical targeting. This review aims to summarize genes, mechanisms, and potential therapeutic strategies found via high-throughput genetic screens in AML. We also discuss the potential of these findings to instruct novel AML therapies for combating drug resistance in this genetically heterogeneous disease.
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Bot, Ilze, Jian Guo, Miranda Van Eck, Peter J. Van Santbrink, Pieter H. E. Groot, Reeni B. Hildebrand, Jurgen Seppen, Theo J. C. Van Berkel, and Erik A. L. Biessen. "Lentiviral shRNA silencing of murine bone marrow cell CCR2 leads to persistent knockdown of CCR2 function in vivo." Blood 106, no. 4 (August 15, 2005): 1147–53. http://dx.doi.org/10.1182/blood-2004-12-4839.
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AbstractA major barrier in hematopoietic gene function studies is posed by the laborious and time-consuming generation of knockout mice with an appropriate genetic background. Here we present a novel lentivirus-based strategy for the in situ generation of hematopoietic knockdowns. A short hairpin RNA (shRNA) was designed targeting murine CC-chemokine receptor 2 (CCR2), which was able to specifically blunt CCR2 expression at the mRNA, protein, and functional levels in vitro. Reconstitution of irradiated recipient mice with autologous bone marrow that had been ex vivo transduced with shRNA lentivirus led to persistent down-regulation of CCR2 expression, which translated into a 70% reduction in CCR2-dependent recruitment of macrophages to an inflamed peritoneal cavity without noticeable side effects on related chemokine receptors or general inflammation status. These findings clearly demonstrate the potential of shRNA lentivirus–infected bone marrow transplantation as a rapid and effective method to generate hematopoietic knockdowns for leukocyte gene function studies.
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Ariefa, Nina Alia, and Andhika Pratiwi. "Normative Women and Patriarchal Hegemony in Ariyoshi Sawako’s Hanaoka Seishu no Tsuma (1966)." IZUMI 10, no. 1 (May 11, 2021): 143–55. http://dx.doi.org/10.14710/izumi.10.1.143-155.
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This research examines the depiction of normative women in the Edo period (1603-1868) in the novel entitled Hanaoka Seishu no Tsuma (1966) by Ariyoshi Sawako, a Japanese female writer in the post World War II Showa era. Reflecting on the novel’s normative female characters, it analyzes the silenced voices of women. It will contribute to the discussion on how the normative female figures criticizing the patriarchal hegemony that has not been revealed in the literary canon of the Edo period. This research shows how normative women characters are presented in the text as a feminine strategy to criticize this hegemony. The researchers use feminist criticism theory from Butler’s gender performativity (1990). The study concludes that although normative women characters are commonly represented as men dominating women, those can also be used to criticize the patriarchal hegemony.
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Schuster, A., V. Neirinckx, E. Klein, P. V. Nazarov, A. Oudin, A. Muller, F. Azuaje, C. Herold-Mende, B. Klink, and S. P. Niclou. "P11.26 Genome-wide shRNA screen identifies candidate genes driving glioblastoma invasion." Neuro-Oncology 21, Supplement_3 (August 2019): iii48. http://dx.doi.org/10.1093/neuonc/noz126.172.
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Abstract BACKGROUND A major hallmark of glioblastoma (GBM) is its highly invasive capacity, contributing to its aggressive behaviour. Since invasive cells cannot be easily removed by surgery or irradiation, they are left behind and eventually result in lethal recurrence. Therefore, a better understanding of the invasion process and of the key molecular players underlying the invasive capacities of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM sphere cultures. The phenotype of the most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cell lines was compared with overexpression in non-invasive cells. RNA sequencing of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS Through a whole genome shRNA screen, a zinc-finger containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong decrease in invasion capacity in two highly invasive GBM cell lines. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of either one or both zinc-finger motifs led to decreased invasion indicating that the two zinc-finger motifs are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through a transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent a novel promising target to overcome invasive capacities in GBM.
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Coblentz, Philip D., Bumsoo Ahn, Linda F. Hayward, Jeung-Ki Yoo, Demetra D. Christou, and Leonardo F. Ferreira. "Small-hairpin RNA and pharmacological targeting of neutral sphingomyelinase prevent diaphragm weakness in rats with heart failure and reduced ejection fraction." American Journal of Physiology-Lung Cellular and Molecular Physiology 316, no. 4 (April 1, 2019): L679—L690. http://dx.doi.org/10.1152/ajplung.00516.2018.
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Heart failure with reduced ejection fraction (HFREF) increases neutral sphingomyelinase (NSMase) activity and mitochondrial reactive oxygen species (ROS) emission and causes diaphragm weakness. We tested whether a systemic pharmacological NSMase inhibitor or short-hairpin RNA (shRNA) targeting NSMase isoform 3 (NSMase3) would prevent diaphragm abnormalities induced by HFREF caused by myocardial infarction. In the pharmacological intervention, we used intraperitoneal injection of GW4869 or vehicle. In the genetic intervention, we injected adeno-associated virus serotype 9 (AAV9) containing shRNA targeting NSMase3 or a scrambled sequence directly into the diaphragm. We also studied acid sphingomyelinase-knockout mice. GW4869 prevented the increase in diaphragm ceramide content, weakness, and tachypnea caused by HFREF. For example, maximal specific forces (in N/cm2) were vehicle [sham 31 ± 2 and HFREF 26 ± 2 ( P < 0.05)] and GW4869 (sham 31 ± 2 and HFREF 31 ± 1). Respiratory rates were (in breaths/min) vehicle [sham 61 ± 3 and HFREF 84 ± 11 ( P < 0.05)] and GW4869 (sham 66 ± 2 and HFREF 72 ± 2). AAV9-NSMase3 shRNA prevented heightening of diaphragm mitochondrial ROS and weakness [in N/cm2, AAV9-scrambled shRNA: sham 31 ± 2 and HFREF 27 ± 2 ( P < 0.05); AAV9-NSMase3 shRNA: sham 30 ± 1 and HFREF 30 ± 1] but displayed tachypnea. Both wild-type and ASMase-knockout mice with HFREF displayed diaphragm weakness. Our study suggests that activation of NSMase3 causes diaphragm weakness in HFREF, presumably through accumulation of ceramide and elevation in mitochondrial ROS. Our data also reveal a novel inhibitory effect of GW4869 on tachypnea in HFREF likely mediated by changes in neural control of breathing.
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Mockenhaupt, Stefan, Stefanie Grosse, Daniel Rupp, Ralf Bartenschlager, and Dirk Grimm. "Alleviation of off-target effects from vector-encoded shRNAs via codelivered RNA decoys." Proceedings of the National Academy of Sciences 112, no. 30 (July 13, 2015): E4007—E4016. http://dx.doi.org/10.1073/pnas.1510476112.
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Exogenous RNAi triggers such as shRNAs ideally exert their activities exclusively via the antisense strand that binds and silences designated target mRNAs. However, in principle, the sense strand also possesses silencing capacity that may contribute to adverse RNAi side effects including off-target gene regulation. Here, we address this concern with a novel strategy that reduces sense strand activity of vector-encoded shRNAs via codelivery of inhibitory tough decoy (TuD) RNAs. Using various shRNAs for proof of concept, we validate that coexpression of TuDs can sequester and inactivate shRNA sense strands in human cells selectively without affecting desired antisense activities from the same shRNAs. Moreover, we show how coexpressed TuDs can alleviate shRNA-mediated perturbation of global gene expression by specifically de-repressing off-target transcripts carrying seed matches to the shRNA sense strand. Our combination of shRNA and TuD in a single bicistronic gene transfer vector derived from Adeno-associated virus (AAV) enables a wide range of applications, including gene therapies. To this end, we engineered our constructs in a modular fashion and identified simple hairpin design rules permitting adaptation to preexisting or new shRNAs. Finally, we demonstrate the power of our vectors for combinatorial RNAi strategies by showing robust suppression of hepatitis C virus (HCV) with an AAV expressing a bifunctional TuD against an anti-HCV shRNA sense strand and an HCV-related cellular miRNA. The data and tools reported here represent an important step toward the next generation of RNAi triggers with increased specificity and thus ultimately safety in humans.
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Liu-Sullivan, Nancy, Bhavneet Bhinder, David Shum, Christina Ramirez, Constantin Radu, Hakim Djaballah, and Mark G. Frattini. "Preclinical Assessment of a Novel CDC7 Inhibitor: Genomewide RNAi Screening Identifies Unique Synergetic and Resistance Genes,." Blood 118, no. 21 (November 18, 2011): 3570. http://dx.doi.org/10.1182/blood.v118.21.3570.3570.
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Abstract Abstract 3570 Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems. While insufficient drug-gene engagement leads to drug failure, de novo escape mutations give rise to patients relapsing, calling the need for systemic studies on how genes influence drug responsiveness. Towards this end, we have explored a functional short hairpin RNA (shRNA) based genomic screening platform aimed at interrogating drug-gene engagement and assessing its consequences on signaling pathways. We propose this concept as a novel way to evaluate drug candidates prior to clinical trials enabling liability assessment and predicting clinical outcome. We took advantage of the arrayed shRNA library produced in lentiviral particles and characterized by several obvious advantageous features including shRNA targeting one hairpin at a time and on the fly high content whole well microscopy imaging analysis. We carried out three parallel genomewide shRNA screens in the absence or presence of the novel CDC7 kinase inhibitor (MSK-777) at its IC20 and IC50 and have identified several gene candidates that influence MSK-777 sensitivity and resistance. These include synergizers that enhance MSK-777 sensitivity and rescuers that confer MSK-777 resistance. IPA analysis mapped clusters of these hits to multiple major pathways among them were the NF-kB pathway, the ubiquitin-proteasome pathway, DNA replication, and several epigenetic regulatory genes. We will present and discuss this concept together with the emerging pathways as a means to identify both key therapeutic targets and biomarkers of sensitivity and resistance. Thus, allowing for not only a broader applicability of assessing candidate genes that modulate specific drug agents, but also for the identification of a tailored and more efficacious therapeutic regimen to treat cancer. Disclosures: No relevant conflicts of interest to declare.
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Gu, Jun, Hao Li, Zhen Bi, Kai Li, Zhiquan Li, Deping Song, Zhen Ding, et al. "Plasmids Expressing shRNAs Specific to the Nucleocapsid Gene Inhibit the Replication of Porcine Deltacoronavirus In Vivo." Animals 11, no. 5 (April 23, 2021): 1216. http://dx.doi.org/10.3390/ani11051216.
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Porcine deltacoronavirus (PDCoV) is a novel enteric coronavirus and is becoming one of the major causative agents of diarrhea in pig herds in recent years. To date, there are no commercial vaccines or antiviral pharmaceutical agents available to control PDCoV infection. Therefore, developing a reliable strategy against PDCoV is urgently needed. In this study, to observe the antiviral activity of RNA interference (RNAi), four short hairpin RNAs (shRNAs) specific to the nucleocapsid (N) gene of PDCoV were designed and tested in vitro. Of these, a double-shRNA-expression vector, designated as pSil-double-shRNA-N1, was the most effectively expressed, and the inhibition of PDCoV replication was then further evaluated in neonatal piglets. Our preliminary results reveal that plasmid-based double-shRNA-expression targeting the N gene of PDCoV can significantly protect LLC-PK1 cells and piglets from pathological lesions induced by PDCoV. Our study could benefit the investigation of the specific functions of viral genes related to PDCoV infection and offer a possible methodology of RNAi-based therapeutics for PDCoV infection.
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Li, Shirong, Jing Fu, Jordan M. Schecter, Caisheng Lu, Markus Y. Mapara, and Suzanne Lentzsch. "Inducible Silencing Of eIF4E Using a Tet-On System Results In Myeloma Growth In Vivo That Correlates With eIF4E Expression." Blood 122, no. 21 (November 15, 2013): 3164. http://dx.doi.org/10.1182/blood.v122.21.3164.3164.
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Abstract Introduction Overexpression and/or activation of eukaryotic initiation factor 4E (eIF4E) is critical for oncogenic protein synthesis. Mutations in genes related to mRNA translation are involved in the pathogenesis of multiple myeloma (Chapman, Lawrence et al. 2011). Recently, we found that MM cells express high levels of eIF4E protein compared to normal plasma cells and overexpression of eIF4E induces transcription factors such as c-myc critical for the growth of multiple myeloma cells (Li, Fu et al. 2011,2012). The understanding of the mechanisms that control protein synthesis is an emerging new research area in MM with significant potential for developing innovative therapies. Here we show the critical role of eIF4E driven protein synthesis by using an inducible knockdown system to silence eIF4E gene expression and confirm the critical role of eIF4E in multiple myeloma growth in vivo and in vitro. Methods and Results We stably infected U266, RPMI-8226, IM-9 and MM.1S cells with a robust inducible single-lentiviral knockdown vector pLKO-Tet-On containing either control non-targeting shRNA or eIF4E targeting shRNA sequences. Doxycycline-induced eIF4E shRNA expression resulted in significant decrease of eIF4E mRNA and protein in eIF4E-shRNA but not the control shRNA infected MM cells. To determine the effects of eIF4E knockdown on MM cell growth and viability, stably transfected cell lines were grown in the presence or absence of doxycycline. Silencing of eIF4E by doxycycline induction of eIF4E shRNA in RPMI-8226 cells significantly inhibited (>72%,P<0.01) cell growth accompanied by a decrease of c-myc, cyclin D1, C/EBP beta and IRF4 all critical for myeloma cell growth. Cell cycle analysis revealed increased cells population in G0/G1 phase (62% vs 80%) in doxycycline-induced eIF4E shRNA cells with a significant reduction (P<0.001) of clonogenic tumor growth reflected by a decrease in colony numbers (27.6 ± 4.2 vs 5.3 ± 3.4) and size. To determine the role of high expression of eIF4E in MM tumor growth in vivo, we generated subcutaneous MM xenografts in severe combined immunodeficient x beige (SCID/bg) mice using the inducible U266-Tet-CT-shRNA and U266-Tet-eIF4E-shRNA cells. In contrast to vehicle or doxycycline-treated control shRNA tumors, doxycycline treated animals bearing U266-Tet-eIF4E-shRNA xenografts showed a significant inhibition (P<0.001) of tumor growth by 80% after 21 days. The transient inhibition of tumor growth correlated with the transient doxycycline-induced eIF4E knockdown further confirming the critical role of eIF4E. Immunohistochemical staining of tumors confirmed the decreased of eIF4E expression in doxycycline-treated mice bearing U266-Tet-eIF4E-shRNA tumors compared with tumors of vehicle-treated or non-doxycyclin treated mice. Conclusion Here we show that eIF4E, a key player in the translational machinery, promotes multiple myeloma cell growth. We found that high eIF4E expression is indispensable for the growth of MM cells both in vitro and in vivo. Silencing of eIF4E decreases protein expression of a subset of transcripts encoding regulators of the cell cycle and proliferation, and resulted in tumor inhibition. Our study indicated that targeting transcriptional initiating factor eIF4E may represent a novel therapeutic strategy for MM treatment. Disclosures: Schecter: Seattle Genetics: Honoraria, Research Funding. Lentzsch:Celgene: Research Funding.
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Schuster, Anne, Virginie Neirinckx, Eliane Klein, Petr V. Nazarov, Anais Oudin, Arnaud Muller, Fransisco Azuaje, Christel Herold-Mende, Barbara Klink, and Simone Niclou. "ANGI-02. GENOME-WIDE shRNA SCREEN IDENTIFIES CANDIDATE GENES DRIVING GLIOBLASTOMA INVASION." Neuro-Oncology 21, Supplement_6 (November 2019): vi30. http://dx.doi.org/10.1093/neuonc/noz175.113.
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Abstract BACKGROUND A major hallmark of glioblastoma (GBM) is its invasive capacity, contributing to its aggressive behaviour. Invasive cells cannot be easily removed by surgery or irradiation and eventually result in lethal recurrence. A better understanding of the invasion process and the key molecular players underlying the invasive potential of GBM may lead to the identification of new therapeutic targets for GBM patients. MATERIAL AND METHODS To identify candidate genes responsible for invasion, a genome-wide shRNA screen was performed in patient-derived GBM cultures. The most promising candidate was validated in in vitro invasion assays, ex vivo brain slice cultures and in vivo orthotopic xenografts in mice. Gene knockdown in invasive GBM cells was compared with overexpression in non-invasive cells. RNAseq of knockdown cells, along with the generation of deletion constructs were applied to uncover the mechanisms regulating invasion. RESULTS A zinc-finger domain containing protein was identified as an invasion essential candidate gene. Knockdown of this gene confirmed a strong impact on invasion in highly invasive GBM cells. In contrast, gene overexpression switched non-invasive GBM cells to an invasive phenotype. Deletion of one or both zinc-finger motifs decreased invasion indicating that both are essential for regulating invasion. Mutation of the nuclear localisation signal resulted in retention of the protein in the cytoplasm and loss of the invasion phenotype demonstrating that the protein activity is required in the nucleus. Gene expression analyses revealed that invasion-related genes are significantly regulated by the candidate gene once it is localized in the nucleus. CONCLUSION We identified a zinc-finger containing protein as a novel driver of GBM invasion, presumably through transcription factor activity resulting in the induction of an invasive transcriptional program. This protein and its downstream pathway may represent novel promising targets to overcome invasive capacities in GBM.
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Futami, Muneyoshi, Toshiyuki Hatano, Yasushi Soda, Seiichiro Kobayashi, Makoto Miyagishi, and Arinobu Tojo. "ShRNA Targeting p190BCR-ABL Successfully Eliminates Ph-ALL Cells with or without ABL Kinase Domain Mutation." Blood 108, no. 11 (November 16, 2006): 1838. http://dx.doi.org/10.1182/blood.v108.11.1838.1838.
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Abstract In the majority of Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (Ph-ALL) cases, the resulting BCR-ABL gene generates 190 kD active tyrosine kinase (p190) which is responsible for leukemogenesis and can be a molecular target for therapy. Although a series of ABL kinase inhibitors including imatinib, nilotinib and dasatinib reveal potent activities against Ph-ALL, acquired resistance caused by point mutations in the kinase domain such as T315I still remains to be overcome. That is why a novel strategy is desired in the treatment of Ph-ALL. We previously reported that lentiviral delivery of maxizyme targeting p190 specifically induced apoptosis of Ph-ALL cells (Blood 104:356, 2004). Since RNA interference proved to be a more powerful tool in selective gene silencing, we applied this technology to test whether specific and efficient killing of Ph-ALL cells could be achieved by down-regulation of p190. We designed a series of 21-mer and 27-mer small hairpin RNA (shRNA) targeting p190 mRNA and constructed plasmid vectors expressing these shRNA, which were screened by transfection of 293T/p190 cells to determine optimal target sites. As a result, three candidate sequences were identified; junctional 27-mer, junctional 21-mer and ABL 21-mer. Then, we inserted each of the shRNA expression cassettes into the lentiviral vector (HIV-U6/shRNA) and prepared high titer virus stock for infection of leukemia cells. shBCR-ABL/21, but not shBCR-ABL/27, induced significant and specific cell death of p190+ Ph-ALL cells in a time-dependent manner. shABL was more potent than shBCR-ABL/21 and also active against p210+ CML cells as well as 293 cells, but did not substantially affect Ph-negative leukemia cells. Both shABL and shBCR-ABL/21 completely inhibited growth of Ba/F3 cells harboring either wild-type or mutant p190 which renders those resistant to imatinib. Furthermore, both shRNA at low multiplicity of infection additively cooperated with imatinib in growth inhibition of Ba/F3-p190 cells. These data suggest that shRNA targeting p190 may become a therapeutic option in Ph-ALL by improvement of its delivery system like liposome. Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL
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Lavender, Helen, Kevin Brady, Frances Burden, Oona Delpuech-Adams, Hubert Denise, Amy Palmer, Hannah Perkins, et al. "In VitroCharacterization of the Activity of PF-05095808, a Novel Biological Agent for Hepatitis C Virus Therapy." Antimicrobial Agents and Chemotherapy 56, no. 3 (December 27, 2011): 1364–75. http://dx.doi.org/10.1128/aac.05357-11.
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ABSTRACTPF-05095808 is a novel biological agent for chronic hepatitis C virus (HCV) therapy. It comprises a recombinant adeno-associated virus (AAV) DNA vector packaged into an AAV serotype 8 capsid. The vector directs expression of three short hairpin RNAs (shRNAs) targeted to conserved regions of the HCV genome. These shRNAs are processed by the host cell into the small interfering RNAs which mediate sequence-specific cleavage of target regions. For small-molecule inhibitors the key screens needed to assessin vitroactivity are well defined; we developed new assays to assess this RNA interference agent and so to understand its therapeutic potential. Following administration of PF-05095808 or corresponding synthetic shRNAs, sequence-specific antiviral activity was observed in HCV replicon and infectious virus systems. To quantify the numbers of shRNA molecules required for antiviral activityin vitroand potentially alsoin vivo, a universal quantitative PCR (qPCR) assay was developed. The number of shRNA molecules needed to drive antiviral activity proved to be independent of the vector delivery system used for PF-05095808 administration. The emergence of resistant variants at the target site of one shRNA was characterized. A novel RNA cleavage assay was developed to confirm the spectrum of activity of PF-05095808 against common HCV clinical isolates. In summary, our data both support antiviral activity consistent with an RNA interference mechanism and demonstrate the potential of PF-05095808 as a therapeutic agent for chronic HCV infection.
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Hu, Wenfeng, Jingjing Wu, Ting Ye, Zhuo Chen, Jinhua Tao, Lijuan Tong, Kai Ma, Jie Wen, Hui Wang, and Chao Huang. "Farnesoid X Receptor-Mediated Cytoplasmic Translocation of CRTC2 Disrupts CREB-BDNF Signaling in Hippocampal CA1 and Leads to the Development of Depression-Like Behaviors in Mice." International Journal of Neuropsychopharmacology 23, no. 10 (May 26, 2020): 673–86. http://dx.doi.org/10.1093/ijnp/pyaa039.
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Abstract Background We recently identified neuronal expression of farnesoid X receptor (FXR), a bile acid receptor known to impair autophagy by inhibiting cyclic adenosine monophosphate response element-binding protein (CREB), a protein whose underfunctioning is linked to neuroplasticity and depression. In this study, we hypothesize that FXR may mediate depression via a CREB-dependent mechanism. Methods Depression was induced in male C57BL6/J mice via chronic unpredictable stress (CUS). Subjects underwent behavioral testing to identify depression-like behaviors. A variety of molecular biology techniques, including viral-mediated gene transfer, Western blot, co-immunoprecipitation, and immunofluorescence, were used to correlate depression-like behaviors with underlying molecular and physiological events. Results Overexpression of FXR, whose levels were upregulated by CUS in hippocampal CA1, induced or aggravated depression-like behaviors in stress-naïve and CUS-exposed mice, while FXR short hairpin RNA (shRNA) ameliorated such symptoms in CUS-exposed mice. The behavioral effects of FXR were found to be associated with changes in CREB-brain-derived neurotrophic factor (BDNF) signaling, as FXR overexpression aggravated CUS-induced reduction in BDNF levels while the use of FXR shRNA or disruption of FXR-CREB signaling reversed the CUS-induced reduction in the phosphorylated CREB and BDNF levels. Molecular analysis revealed that FXR shRNA prevented CUS-induced cytoplasmic translocation of CREB-regulated transcription coactivator 2 (CRTC2); CRTC2 overexpression and CRTC2 shRNA abrogated the regulatory effect of FXR overexpression or FXR shRNA on CUS-induced depression-like behaviors. Conclusions In stress conditions, increased FXR in the CA1 inhibits CREB by targeting CREB and driving the cytoplasmic translocation of CRTC2. Uncoupling of the FXR-CREB complex may be a novel strategy for depression treatment.
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Chaudhry, Sonica, Vibhor Joshi, Naveen Bojjireddy, Maikho Thoh, Santosh K. Sandur, and Gosukonda Subrahmanyam. "Silencing of type II phosphatidylinositol 4-kinase β stabilizes prostate apoptosis response-4 and induces apoptosis in cancer cells." Biochemical Journal 476, no. 2 (January 31, 2019): 405–19. http://dx.doi.org/10.1042/bcj20180732.
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Abstract Type II phosphatidylinositol 4-kinase β (PtdIns 4-kinase II β) is an enigma among the phosphatidylinositol 4-kinase family. The role of PtdIns 4-kinase II β in MCF-7 cells was addressed with the help of short hairpin RNA (shRNA). PtdIns 4-kinase II β shRNA transfection increased pan-caspase activity and induced apoptosis in cancerous MCF-7 cells. Non-cancerous MCF-10A cells were resistant to PtdIns 4-kinase II β shRNA-induced apoptosis. Caspase 8 and 9 inhibitors rescued MCF-7 cells from apoptosis. Shotgun proteomic studies with Flag-tagged PtdIns 4-kinase II β immunoprecipitates showed tumor suppressor prostate apoptosis response-4 (Par-4) as one of the interacting proteins in HEK293 cells. In reciprocal experiments, Par-4 antibodies co-precipitated PtdIns 4-kinase II β from MCF-7 cells. Deletion of membrane localization motif (ΔCCPCC) or a mutation in ATP-binding region (D304A) of PtdIns 4-kinase II β did not affect its interaction with Par-4. Pull-down assays with GST-PtdIns 4-kinase II β-truncated mutants showed that the region between 101 and 215 amino acid residues is essential for interaction with Par-4. At molecular level, PtdIns 4-kinase II β shRNA transfection increased Par-4 stability, its nuclear localization and inhibition of NF-κB binding to target DNA. Knocking down of Par-4 with siRNA (small interfering RNA) rescued MCF-7 cells from PtdIns 4-kinase II β shRNA-induced apoptosis. These results suggest that PtdIns 4-kinase II β may be a novel regulator of Par-4 through protein–protein interactions. These studies have potential implications in cancer therapy.
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Müller-Tidow, C., E. Bulk, A. Hascher, B. Sargin, J. Vormoor, M. Hotfilder, W. E. Berdel, and H. Serve. "RNAi-based adjuvant therapy in a NSCLC mouse model prevents the development of distant metastasis." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 18089. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.18089.
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18089 Background: Surgery cures about 50% of the patients with early stage NSCLC. Although adjuvant chemotherapy improves outcome, a considerably fraction of patients still dies due to development of distant metastasis. Microarray analyses revealed that S100P expression is a strong adverse prognostic factor in early stage NSCLC. Methods: In a mouse NOD/SCID metastasis model based on subcutaneous xenotransplants of NSCLC cell lines we analyzed the effects of S100P expression on metastasis development after curative resection of the primary tumor. Tumors were resected 3–4 weeks after inoculation into the left flank after tumors reached a specified size. Adjuvant RNAi (shRNA based) therapy against S100P was i.v.-administered on the 5 days surrounding tumor resection. Results: S100P overexpression in HTB56 lung cancer cells led to more rapid tumor growth as xenografts. After tumor resection, metastasis developed in 14% (2/12) of the control mice but in 67% (8/12) of the S100P expressing tumors p<0.05. shRNA plasmids were developed and confirmed to be active in downregulating S100P. When these plasmids were i.v.-injected before resection, a significant decrease in S100P protein expression occurred. Control plasmid did not have any effect. The size of S100P-shRNA treated tumors decreased after 2 i.v.-applications. Metastasis developed in 69% (11/16) of the mice treated with the control plasmid. In S100P-shRNA treated mice only 28% (5/18) developed metastasis (p<0.05). After i.v.-injection of NSCLC cells with endogenous S100P expression, all mice (100% - 10/10) developed metastasis when adjuvant treatment was administered with control shRNA. However, when the mice were treated with S100P-shRNA in the first week after i.v.-injection of the tumor cells 33% of the mice (3/9) did not develop lung metastasis. Conclusion: Expression of S100P is a negative adverse prognostic marker in early stage NSCLC. S100P enhances metastasis formation in a mouse metastasis model. Adjuvant therapy with shRNA against S100P is feasible and effective in at least two murine NSCLC metastasis models. These findings suggest that S100P is a potentially important target to inhibit NSCLC metastasis and RNAi based therapy approaches might offer novel ways for targeted therapy in adjuvant situations. No significant financial relationships to disclose.
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Hope, Kristin J., Sonia Cellot, Stephen Ting, and Guy Sauvageau. "An RNA Interference Screen Identifies the Cell Fate Determinants Msi2 and Prox1 as Novel Regulators of Hematopoietic Stem Cell Self-Renewal." Blood 114, no. 22 (November 20, 2009): 394. http://dx.doi.org/10.1182/blood.v114.22.394.394.
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Abstract Abstract 394 Hematopoietic stem cells (HSC) can not yet be unambiguously prospectively identified, a fact which has made it difficult to determine whether a segregation of cell fate determinants underlies the asymmetric/symmetric self-renewal of these cells or whether deregulation of such determinants could contribute to the pathogenesis of hematopoietic malignancies by inducing constitutive symmetric self-renewal divisions. We have addressed these questions through a functional genetics approach taking advantage of systematic RNAi to evaluate the function of conserved polarity factors and cell fate determinants in HSCs. From a list of 72 of such factors identified in the literature, 30 murine homologues were chosen based on their differentially higher level of expression in HSC-enriched populations as measured by qRT-PCR. For each candidate we designed 3 unique short hairpin RNA (shRNA) encoding retroviral constructs also carrying EGFP for the purposes of following transduced cells. Primitive hematopoietic cells enriched for HSC were infected at high efficiency with the library in an arrayed 96-well format and their in vivo reconstituting potential was then evaluated through competitive repopulating unit assays. Genes for which shRNA vectors altered late transplant EGFP levels below or above thresholds as defined by a control shRNA to luciferase were considered as hits. Using this approach, we identified and comprehensively validated 4 genes, including the RNA binding protein Msi2, for which shRNA-mediated depletion dramatically impairs repopulation but does not induce cell death or a cell cycle block. Importantly, we show that the loss in the repopulating ability of these shRNA transduced cells is mediated at the stem cell level and is not due to progenitor or downstream cell toxicity or to any defect in the process of bone marrow homing. Subsequent expression profiling indicated that Msi2 is also upregulated in HOXB4-overexpressing symmetrically expanding HSC in line with our findings that it functions as a positive HSC regulator and further suggesting that it represents a potential novel HSC marker. As well as finding HSC agonists, the RNAi screen identified the homeodomain containing transcription factor Prox1 as a negative HSC regulator since its shRNA-mediated transcript loss consistently led to the dramatic in vivo accumulation of EGFP+ transduced cells. Grafts comprised of Prox1 shRNA-transduced cells did not exhibit any lineage skewing however, repeatedly contained an average of 10-fold more primitive Lin-Sca+CD150+48- cells as compared to non-transduced donor cells within the same recipient or to control shRNA-luciferase grafts indicating Prox1 knockdown leads to a significant in vivo expansion of phenotypic HSCs. Moreover, following a 7 day in vitro culture, cells infected with shRNAs to Prox1 were both morphologically and immunophenotypically more primitive than control cells and when transplanted at this time yielded a significantly enhanced engraftment level relative to control shRNAs (51+/-6% GFP vs 8+/-3% GFP). These results further suggest that Prox1 reduction by RNAi expands functional HSCs in vitro. Together these findings have identified conserved cell fate determinants as important and novel regulators of murine hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.
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Sun, Zejin, Rikki Enzor, Paula Rio, D. Wade Clapp, and Helmut Hanenberg. "Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown." Blood 122, no. 21 (November 15, 2013): 2903. http://dx.doi.org/10.1182/blood.v122.21.2903.2903.
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Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.
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Jaako, Pekka, Johan Flygare, Karin Olsson, Axel Schambach, Christopher Baum, Jonas Larsson, David Bryder, and Stefan Karlsson. "A Novel Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia Locates the Erythroid Defect at CFU-E / Proerythroblast Transition." Blood 114, no. 22 (November 20, 2009): 178. http://dx.doi.org/10.1182/blood.v114.22.178.178.
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Abstract Abstract 178 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical malformations and predisposition to cancer. Presently, many different DBA disease genes are known that all encode for ribosomal proteins, suggesting that DBA is a disorder relating to ribosomal biogenesis or function. Among these genes, ribosomal protein S19 (RPS19) is the most frequently mutated (25 % of the patients). In order to study pathophysiology and to evaluate novel therapies, DBA animal models are needed. Since RNA interference -mediated RPS19 down regulation has been shown to result in a DBA phenotype in human cells in vitro, we decided to use the short hairpin RNA (shRNA) technology to create an RPS19-deficient mouse model for DBA. We designed miR30 -styled shRNAs against RPS19 and introduced them into mouse embryonic stem (ES) cells downstream of the collagen A1 locus using site-specific recombination. The resulting ES cell clones contain a single RPS19-targeting shRNA under the control of a doxycycline-responsive promoter. We have generated and characterized two mouse models expressing different RPS19-targeting shRNAs (shRNA-B and shRNA-D). In general, this system allows an inducible and dose-dependent regulation of shRNA expression providing an ideal tool to study conditions like DBA that are caused by haploinsufficient expression of a protein. To induce the expression of RPS19-targeting shRNAs mice were fed with doxycycline administered in drinking water. Induction of the shRNA-B construct had no effect on the erythrocyte level, hemoglobin concentration or hematocrit, although we saw a gradual elevation in mean corpuscular volume (MCV) and a decrease in platelet number. However, after 25–35 days of doxycycline treatment the mice homozygous for the shRNA-B underwent severe weight loss accompanied with a reduction in erythrocyte number and ultimately died. In contrast, shRNA-D mice exhibited decreased erythrocyte number, hemoglobin and hematocrit already after a 10 day doxycycline treatment. When the induction was kept on longer, the homozygous mice developed a more severe anemia and died around day 20, while the heterozygous mice were able to compensate the blood indices back to normal level. In spite of the differences in blood phenotypes, both models had a similar FACS phenotype revealing a profound decrease in the number of proerythroblasts, while the levels of erythroid and bipotential megakaryocytic-erythroid (MegE) progenitors were normal or increased. We also saw an accumulation of the late erythroid precursors. Proliferative potential of erythroid progenitors was evaluated at a clonal level in vitro. When MegE or CFU-E progenitors from induced mice were cultured in presence of doxycycline, the number and size of the erythroid clones were decreased compared to controls. However, when RPS19 expression was restored by culturing the cells without doxycycline, the observed proliferation defect of MegE clones was completely restored while the rescue of CFU-E clones was only partial. We also noticed that RPS19-deficient megakaryocytes appeared smaller in size compared to controls. Importantly, transduction of RPS19-deficient cells with a lentiviral vector overexpressing sequence-modified RPS19 cDNA rescued the proliferation and colony-forming defects in vitro demonstrating that the erythroid phenotype is specifically due to down regulation of RPS19. In summary, we have generated two novel mouse models for RPS19-deficient DBA that recapitulate the key erythroid phenotype seen in patients based on both FACS analysis and single-cell proliferation assays. These models will serve as a good tool to determine the molecular mechanisms responsible for DBA and also to test gene replacement therapies. Disclosures: No relevant conflicts of interest to declare.
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Ali, Nicole, Christine Karlsson, Marie Aspling, Guang Hu, Nir Hacohen, David T. Scadden, and Jonas Larsson. "Forward RNAi screens in primary human hematopoietic stem/progenitor cells." Blood 113, no. 16 (April 16, 2009): 3690–95. http://dx.doi.org/10.1182/blood-2008-10-176396.
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Abstract The mechanisms regulating key fate decisions such as self-renewal and differentiation in hematopoietic stem and progenitor cells (HSPC) remain poorly understood. We report here a screening strategy developed to assess modulators of human hematopoiesis using a lentiviral short hairpin RNA (shRNA) library transduced into cord blood-derived stem/progenitor cells. To screen for modifiers of self-renewal/differentiation, we used the limited persistence of HSPCs under ex vivo culture conditions as a baseline for functional selection of shRNAs conferring enhanced maintenance or expansion of the stem/progenitor potential. This approach enables complex, pooled screens in large numbers of cells. Functional selection identified novel specific gene targets (exostoses 1) or shRNA constructs capable of altering human hematopoietic progenitor differentiation or stem cell expansion, respectively, thereby demonstrating the potential of this forward screening approach in primary human stem cell populations.
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Jaako, Pekka, Shubhranshu Debnath, Karin Olsson, Axel Schambach, Christopher Baum, Johan Flygare, and Stefan Karlsson. "Gene Therapy Corrects the Lethal Bone Marrow Failure in a Mouse Model for RPS19-Deficient Diamond-Blackfan Anemia." Blood 120, no. 21 (November 16, 2012): 513. http://dx.doi.org/10.1182/blood.v120.21.513.513.
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Abstract Abstract 513 Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia associated with physical abnormalities and predisposition to cancer. Mutations in genes that encode ribosomal proteins have been identified in approximately 60–70 % of the patients. Among these genes, ribosomal protein S19 (RPS19) is the most common DBA gene (25 % of the cases). Current DBA therapies involve risks for serious side effects and a high proportion of deaths are treatment-related underscoring the need for novel therapies. We have previously demonstrated that enforced expression of RPS19 improves the proliferation, erythroid colony-forming potential and differentiation of patient derived RPS19-deficient hematopoietic progenitor cells in vitro (Hamaguchi, Blood 2002; Hamaguchi, Mol Ther 2003). Furthermore, RPS19 overexpression enhances the engraftment and erythroid differentiation of patient-derived hematopoietic stem and progenitor cells when transplanted into immunocompromised mice (Flygare, Exp Hematol 2008). Collectively these studies suggest the feasibility of gene therapy in the treatment of RPS19-deficient DBA. In the current project we have assessed the therapeutic efficacy of gene therapy using a mouse model for RPS19-deficient DBA (Jaako, Blood 2011; Jaako, Blood 2012). This model contains an Rps19-targeting shRNA (shRNA-D) that is expressed by a doxycycline-responsive promoter located downstream of Collagen A1 gene. Transgenic animals were bred either heterozygous or homozygous for the shRNA-D in order to generate two models with intermediate or severe Rps19 deficiency, respectively. Indeed, following transplantation, the administration of doxycycline to the recipients with homozygous shRNA-D bone marrow results in an acute and lethal bone marrow failure, while the heterozygous shRNA-D recipients develop a mild and chronic phenotype. We employed lentiviral vectors harboring a codon-optimized human RPS19 cDNA driven by the SFFV promoter, followed by IRES and GFP (SFFV-RPS19). A similar vector without the RPS19 cDNA was used as a control (SFFV-GFP). To assess the therapeutic potential of the SFFV-RPS19 vector in vivo, transduced c-Kit enriched bone marrow cells from control and homozygous shRNA-D mice were injected into lethally irradiated wild-type mice. Based on the percentage of GFP-positive cells, transduction efficiencies varied between 40 % and 60 %. Three months after transplantation, recipient mice were administered doxycycline in order to induce Rps19 deficiency. After two weeks of doxycycline administration, the recipients transplanted with SFFV-RPS19 or SFFV-GFP control cells showed no differences in blood cellularity. Remarkably, at the same time-point the recipients with SFFV-GFP homozygous shRNA-D bone marrow showed a dramatic decrease in blood cellularity that led to death, while the recipients with SFFV-RPS19 shRNA-D bone marrow showed nearly normal blood cellularity. These results demonstrate the potential of enforced expression of RPS19 to reverse the severe anemia and bone marrow failure in DBA. To assess the reconstitution advantage of transduced hematopoietic stem and progenitor cells with time, we performed similar experiments with heterozygous shRNA-D bone marrow cells. We monitored the percentage of GFP-positive myeloid cells in the peripheral blood, which provides a dynamic read-out for bone marrow activity. After four months of doxycycline administration, the mean percentage of GFP-positive cells in the recipients with SFFV-RPS19 heterozygous shRNA-D bone marrow increased to 97 %, while no similar advantage was observed in the recipients with SFFV-RPS19 or SFFV-GFP control bone marrow, or SFFV-GFP heterozygous shRNA-D bone marrow. Consistently, SFFV-RPS19 conferred a reconstitution advantage over the non-transduced cells in the bone marrow. Furthermore, SFFV-RPS19 reversed the hypocellular bone marrow observed in the SFFV-GFP heterozygous shRNA-D recipients. Taken together, using mouse models for RPS19-deficient DBA, we demonstrate that the enforced expression of RPS19 rescues the lethal bone marrow failure and confers a strong reconstitution advantage in vivo. These results provide a proof-of-principle for gene therapy in the treatment of RPS19-deficient DBA. Disclosures: No relevant conflicts of interest to declare.
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Lakshmikuttyamma, Ashakumary, Stuart Scott, C. Ronald Geyer, and John F. DeCoteau. "Decreased Expression of the Histone Methyltransferase SUV39H1 in AML Cells Reactivates Hypermethylated Tumor Suppressor p15INK4B in the Absence of Promoter Demethylation." Blood 110, no. 11 (November 16, 2007): 4150. http://dx.doi.org/10.1182/blood.v110.11.4150.4150.
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Abstract Re-expression of hypermethylated tumor suppressor genes using epigenetic modifiers, such as DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors, occurs by a mechanism whereby promoter demethylation is the dominant event. In support of this model, we found that the DNMT inhibitor 5-Aza-2-deoxycytidine (decitabine) induces expression of the tumor suppressor gene p15INK4B (p15) in AML cells with hypermethylated p15 promoters. Re-expression of p15 by decitabine is associated with decreases in p15 promoter methylation and histone H3 lysine 9 (H3K9) methylation and increases in H3K9 acetylation. DNA methylation is linked to H3K9 methylation through the DNA methyl binding protein MeCP2, which associates with DNMTs and H3K9 methyltransferases. Using chromatin immunoprecipitaton (ChIP) assays, we confirmed that MeCP2, DNMT1 and the H3K9 methylatransferase SUV39H1 interact with the methylated p15 promoter and that this interaction is reduced by decitabine. To determine whether promoter demethylation is also dominant to H3K9 demethylation, we monitored p15 re-expression in the presence of SUV39H1 shRNA alone and in combination with decitabine. SUV39H1 shRNA induces p15 expression and H3K9 demethylation, however it does not affect p15 promoter methylation. These results are in contrast to the HDAC inhibitor trichostatin A (TSA), which cannot induce p15 re-expression. SUV39H1 shRNA induced p15 expression and H3K9 demethylation are also enhanced by co-treatment with decitabine or TSA. Surprisingly, co-treatment with decitabine and SUV39H1 shRNA partially reverses decitabine induced promoter demethylation. Using ChIP assays we show that SUV39H1 shRNA increases the amount of the histone H3K9 dimethytransferase G9a and DNMT1 associated with the p15 promoter. Increased levels of G9a at the p15 promoter would enhance promoter methylation since G9a stimulates DNMT1 activity. Our results demonstrate that hypermethylated p15 can be reactivated in AML cells by an initial event that involves H3K9 demethylation. In addition, we found that the SUV39H1 inhibitor chaetocin induces p15 in AML cells with hypermethylated p15 promoters. Therefore, H3K9 methylatransferases represent novel therapeutic targets for developing inhibitors to reactivate the expression of hypermethylated genes.
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Milazzotto, Marcella Pecora, Marcelo Demarchi Goissis, Weber Beringui Feitosa, Leydson Ferreira Martins, Bryan Eric Strauss, Marcio Chaim Bajgelman, Mayra Elena Ortiz D'Ávila Assumpção, and Jose Antonio Visintin. "Myostatin gene knockdown through lentiviral-mediated delivery of shRNA for in vitro production of transgenic bovine embryos." Zygote 18, no. 4 (May 6, 2010): 339–44. http://dx.doi.org/10.1017/s0967199410000031.
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SummaryMyostatin is described as a negative regulator of the skeletal muscle growth. Genetic engineering, in order to produce animals with double the muscle mass and that can transmit the characteristic to future progeny, may be useful. In this context, the present study aimed to analyse the feasibility of lentiviral-mediated delivery of short hairpin RNA (shRNA) targeting of myostatin into in vitro produced transgenic bovine embryos. Lentiviral vectors were used to deliver a transgene that expressed green fluorescent protein (GFP) and an shRNA that targeted myostatin. Vector efficiency was verified through in vitro murine myoblast (C2C12) cell morphology after inductive differentiation and by means of real-time PCR. The lentiviral vector was microinjected into the perivitellinic space of in vitro matured oocytes. Non-microinjected oocytes were used as the control. After injection, oocytes were fertilized and cultured in vitro. Blastocysts were evaluated by epifluorescence microscopy. Results demonstrated that the vector was able to inhibit myostatin mRNA in C2C12 cells, as the transducted group had a less amount of myostatin mRNA after 72 h of differentiation (p < 0.05) and had less myotube formation than the non-transduced group (p < 0.05). There was no difference in cleavage and blastocyst rates between the microinjected and control groups. After hatching, 3.07% of the embryos exhibited GFP expression, indicating that they expressed shRNA targeting myostatin. In conclusion, we demonstrate that a lentiviral vector effectively performed shRNA myostatin gene knockdown and gene delivery into in vitro produced bovine embryos. Thus, this technique can be considered a novel option for the production of transgenic embryos and double muscle mass animals.
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Shin, Mi-Kyung, Roxana Mitrut, Chenjuan Gu, Lenise J. Kim, Bonnie H. Y. Yeung, Rachel Lee, Luu Pham, et al. "Pharmacological and Genetic Blockade of Trpm7 in the Carotid Body Treats Obesity-Induced Hypertension." Hypertension 78, no. 1 (July 2021): 104–14. http://dx.doi.org/10.1161/hypertensionaha.120.16527.
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Obesity increases levels of an adipocyte-produced hormone leptin, which activates the sympathetic nervous system leading to hypertension. We have recently reported that acute leptin infusion induces hypertension acting via the TRPM7 (transient receptor potential cation channel subfamily M member 7) cation channel in the carotid bodies. We hypothesize that this mechanism causes hypertension when leptin levels are elevated chronically as observed in diet-induced obesity. We have developed a novel extended release preparation, hydrogel, of a TRPM7 inhibitor FTY720, which was administered to the carotid body area bilaterally and compared with control hydrogel in (1) male lean C57BL/6J mice treated with subcutaneous infusions of leptin; (2) diet-induced obese male C57BL/6J with hyperleptinemia at baseline. In the experiment (3), diet-induced obese C57BL/6J mice, in which Trpm7 was silenced in the carotid body areas by transfection with Ad-Trpm7 shRNA , were compared with control mice transfected with Ad-CON-shRNA . All mice were implanted in the left femoral artery with telemetry before the experiments for continuous blood pressure monitoring. In lean mice, leptin increased 24 hours mean arterial pressure from 101.2±1.2 to 112±1.5 mm Hg; Trpm7 inhibitor abolished leptin-induced hypertension. Obese mice had elevated mean arterial pressure of 115.3±1.7 mm Hg, which was lowered by 8.7±1.0 mm Hg on week 2 after Trpm7 inhibitor treatment ( P <0.001), and this effect persisted by week 3. Trpm7 shRNA decreased blood pressure from 119.0±2.2 to 109.6±1.4 mm Hg ( P <0.01), whereas scrambled shRNA had no effect. In conclusion, our study has shown that inhibition of TRPM7 in carotid bodies abolished leptin-induced hypertension in obese mice.
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Konto-Ghiorghi, Yoan, Elena Bibikova, Bertil Glader, Anupama Narla, and Kathleen Sakamoto. "Transcriptional Profiling and Cytokine Signaling In The Pathogenesis Of Diamond-Blackfan Anemia." Blood 122, no. 21 (November 15, 2013): 2474. http://dx.doi.org/10.1182/blood.v122.21.2474.2474.
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Abstract Diamond-Blackfan Anemia (DBA) is a pediatric bone marrow failure syndrome, characterized by anemia and congenital abnormalities. Ribosomal protein S19 (RPS19) is mutated in approximately 25% of DBA patients, resulting in haploinsufficiency. Eighty percent of patients will initially respond to corticosteroids; however, this drug is associated with significant toxicity, including immunosuppression and growth delay. Identification of novel pathways could lead to new approaches to treat DBA that minimize the toxicities observed with current therapies. To recapitulate the effects of RPS19 deficiency, we transduced CD34+ cells purified from fetal liver or cord blood with two different shRNA lentiviral constructs against RPS19. GFP was used as a selection marker for cells transduced with RPS19 or control scrambled shRNA. RPS19 knockdown was confirmed by Western blot analysis and by quantitative real-time PCR (qRT-PCR) (73%, p< 0.01 for shRNA1, and 87%, p< 0.01 for shRNA2, respectively), compared with scrambled shRNA control. To further characterize the transcriptional landscape of RPS19-deficient cells, we performed RNA-sequencing analysis with mRNA from fetal liver CD34+ cells transduced with lentivirus RPS19 shRNA. Our results showed that genes involved in cytokine/chemokine signaling, including GDF15, CCL1, and CD70, are overexpressed in RPS19-deficient cells. We hypothesize that genes regulating the expression of these cytokines could contribute to red cell progenitor defects observed in DBA patients. qRT-PCR confirmed the three cytokines GDF15, CCL1, and CD70 to be overexpressed in both fetal liver CD34+ RPS19-deficient cells (more than 10-fold for the three genes, p< 0.01, using each of the two RPS19 shRNAs) and cord blood CD34+ RPS19-deficient cells (more than 10-fold for GDF15 and CCL1, and 3-fold for CD70, p< 0.01, using each of the two RPS19 shRNAs). To test whether GDF15 is a modulator of erythropoiesis, we transduced fetal liver CD34+ cells with lentiviral shRNA against GDF15 (50% knock-down, p<0.01) and assessed their erythroid differentiation potential in methylcellulose. Methylcellulose colony assays indicated that the numbers of erythroid colonies (BFU-E and CFU-E) are decreased by 60% in fetal liver CD34+ cells transduced with shRNA-GDF15 compared to fetal liver CD34+ cells transduced with a control scrambled shRNA, thus suggesting a role for GDF15 in erythroid differentiation. To understand the mechanisms that lead to GDF15, CCL1, and CD70 overexpression in RPS19-deficient CD34+ cells, we compared in silico the promoter sequences of these three cytokine genes. Similar putative transcription factor binding sites were identified in all three promoters, for regulators that have already been shown to play major roles in hematopoiesis: GATA1, GATA2, IKAROS1, IKAROS4, and E2F1. We then performed Western Blot analysis to measure the expression of GATA1, IKAROS1, and E2F1 at the protein level in RPS19-deficient CD34+ cells. These results showed that GATA1 expression was decreased by 50% in RPS19-deficient fetal liver CD34+ cells and by 25% in RPS19-deficient cord blood CD34+ cells. Likewise, IKAROS1 expression in Western Blot analysis was decreased by 20% in RPS19-deficient fetal liver CD34+ cells and by 15% in RPS19-deficient cord blood CD34+ cells. We are currently studying the mechanisms by which these pathways contribute to DBA pathogenesis and to identify potentially novel targets for DBA therapy. Disclosures: No relevant conflicts of interest to declare.
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Wang, Xiao-Yang, Yuzhi Yin, Hongyan Yuan, Toshiyuki Sakamaki, Hideyuki Okano, and Robert I. Glazer. "Musashi1 Modulates Mammary Progenitor Cell Expansion through Proliferin-Mediated Activation of the Wnt and Notch Pathways." Molecular and Cellular Biology 28, no. 11 (March 24, 2008): 3589–99. http://dx.doi.org/10.1128/mcb.00040-08.
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ABSTRACT The RNA-binding protein Musashi1 (Msi1) is a positive regulator of Notch-mediated transcription in Drosophila melanogaster and neural progenitor cells and has been identified as a putative human breast stem cell marker. Here we describe a novel functional role for Msi1: its ability to drive progenitor cell expansion along the luminal and myoepithelial lineages. Expression of Msi1 in mammary epithelial cells increases the abundance of CD24hi Sca-1+, CD24hi CD29+, CK19, CK6, and double-positive CK14/CK18 progenitor cells. Proliferation is associated with increased proliferin-1 (PLF1) and reduced Dickkopf-3 (DKK3) secretion into the conditioned medium from Msi-expressing cells, which is associated with increased colony formation and extracellular signal-regulated kinase (ERK) phosphorylation. Treatment with the MEK inhibitor U0126 inhibits ERK activation and decreases Notch and β-catenin/T-cell factor (TCF) reporter activity resulting from Msi1 expression. Reduction of DKK3 in control cells with a short hairpin RNA (shRNA) increases Notch and β-catenin/TCF activation, whereas reduction of PLF1 with a shRNA in Msi1-expressing cells inhibits these pathways. These results identify Msi1 as a key determinant of the mammary lineage through its ability to coordinate cell cycle entry and activate the Notch and Wnt pathways by a novel autocrine process involving PLF1 and DKK3.
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Mkwesha, Faith. "INTERVIEW WITH PETINA GAPPAH." Imbizo 7, no. 2 (May 26, 2017): 92–98. http://dx.doi.org/10.25159/2078-9785/1857.
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This interview was conducted on 16 May 2009 at Le Quartier Francais in Franschhoek, Cape Town, South Africa. Petina Gappah is the third generation of Zimbabwean writers writing from the diaspora. She was born in 1971 in Zambia, and grew up in Zimbabwe during the transitional moment from colonial Rhodesia to independence. She has law degrees from the University of Zimbabwe, the University of Cambridge, and the University of Graz. She writes in English and also draws on Shona, her first language. She has published a short story collection An Elegy for Easterly (2009), first novel The Book of Memory (2015), and another collection of short stories, Rotten Row (2016). Gappah’s collection of short stories An Elegy for Easterly (2009) was awarded The Guardian First Book Award in 2009, and was shortlisted for the Frank O’Connor International Short Story Award, the richest prize for the short story form. Gappah was working on her novel The Book of Memory at the time of this interview.
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Faber, J., A. Krivtsov, M. Stubbs, R. Wright, M. van den Heuvel-Eibrink, A. Kung, C. Zwaan, and S. Armstrong. "Suppression of HOX A cluster genes inhibits proliferation and induces cell death in human mixed-lineage leukemias." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 14020. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.14020.
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14020 Background: Leukemias harboring translocations of the mixed lineage leukemia locus (MLL) are generally associated with poor clinical prognosis. Using gene expression profiling we and others have previously shown that Homeobox (HOX) A cluster genes are highly expressed in leukemias with MLL rearrangements. Methods: Here we studied the role of aberrant HOXA9 expression in human MLL- rearranged and non-rearranged leukemias utilizing an shRNA mediated knockdown approach. Results: Three different shRNA constructs targeting human HOXA9 were synthesized and stably introduced into t(9;11) MOLM14 cells utilizing a lentiviral vector system. 75–80% HOXA9 RNA knockdown was confirmed by quantitative PCR and Western Blot analysis. In a panel of 17 AML/ALL cell lines (7 MLL rearranged, 10 non rearranged), HOXA9 directed shRNA inhibited cell proliferation starting as early as 48h after transduction, and induced apoptosis beginning at 72h. Interestingly, impaired cell proliferation and induction of apoptosis was significantly higher in the MLL rearranged cell lines (mean viability: 51.88%) than in the non-rearranged cells (mean viability: 90.98%; p=0.007) and also significantly correlated with the baseline HOXA9 mRNA expression before knockdown (R= 0.8, p=0.00017). We then further analyzed the effect of HOXA9 knockdown in MLL rearranged and non-rearranged primary human AML cells. Similar to our findings in cell lines, a marked induction of cell death was observed, which was significantly higher in leukemias with an MLL translocation (p=0.005) and also significantly correlated with the baseline HOXA9 mRNA expression (R= 0.8, p=0.001). Next, the in vivo effect of HOXA9 knockdown was assessed by transplanting luciferase-expressing SEMK2 (t4;11) cells into SCID-beige mice followed by in vivo bioluminescent imaging. Leukemia burden was significantly reduced in HOXA9 shRNA treated mice (n=10) with a peak difference at day 15 (p=0.000059) shortly before mice of the control group (n=10) succumbed from overt leukemia. At this point all mice of the HOXA9 shRNA treated group were still healthy with no signs of leukemia. Conclusions: Taken together our data implicates that depletion of HOXA9 might be a novel approach for targeted therapy in human MLL rearranged leukemias. No significant financial relationships to disclose.
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Roufayel, Rabih, and Seifedine Kadry. "Examination of the Role of miR-23a in the Development of Thermotolerance." Current Molecular Medicine 20, no. 3 (February 10, 2020): 194–201. http://dx.doi.org/10.2174/1566524019666191021111028.
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Background: Thermotolerance is an acquired state of increased heat resistance that occurs following exposure to non-lethal proteotoxic stress. A large body of evidences implicates that molecular chaperon members belonging to the heat shock protein family could be acting as potential mediators of the thermotolerant state. Objective: Recent evidence has demonstrated heat shock proteins HSP90, HSP70 and HSP27 have inhibited heat-induced cell death by intervening at various steps in stressinduced apoptotic pathways. Previous studies have shown that HSP70 prevented heatinduced apoptosis by preventing the NOXA dependent decrease in MCL-1 levels leading to both BAX activation and cytochrome c release from mitochondria. We have also demonstrated that HSP70 expressing cells have enhanced levels of miR-23a prevent heat-induced increase in NOXA levels and suppress apoptosis. Methods: Stably transfected cell lines expressing either a control shRNA or a miR-23a targeting shRNA are quantified using both RT-PCR and semi-quantitative RT-PCR to determine the effect of different hyperthermic exposure treatment on miR-23a and Noxa mRNA expression levels. Results: This study shows that thermotolerant-induced pre-heat shock treatment is capable of increasing miR-23a levels. Furthermore, stable cell clones expressing a miR- 23a targeting shRNA having reduced miR-23a levels are incapable of developing a thermotolerance state, leading to apoptosis. Conclusion: These results demonstrate the novel finding that miR-23a is an important factor in the development of the thermotolerant state.
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Sakuma, Keiichiro, Eiichi Sasaki, Kenya Kimura, Koji Komori, Yasuhiro Shimizu, Yasushi Yatabe, and Masahiro Aoki. "HNRNPLL, a newly identified colorectal cancer metastasis suppressor, modulates alternative splicing of CD44 during epithelial-mesenchymal transition." Gut 67, no. 6 (March 30, 2017): 1103–11. http://dx.doi.org/10.1136/gutjnl-2016-312927.
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ObjectiveDespite the recent advances in treatment of colon cancer, the prognosis is unfavourable for patients with distant metastases. The aim of this study was to identify targets for prevention and/or therapy of colon cancer metastasis.DesignCMT93 cells, a murine rectal cancer cell line with poor metastasising activity, were transduced with lentiviral shRNA library and transplanted into the rectum of syngeneic C57BL/6 mice. Genomic DNA was collected from metastatic lesions, and the integrated shRNA were retrieved by PCR for sequencing, followed by identification of the candidate genes targeted by the shRNA.ResultsThe genome-wide shRNA library screen identified Hnrnpll (heterogeneous nuclear ribonucleoprotein L-like) encoding a pre-mRNA splicing factor as a candidate metastasis suppressor gene. Knockdown of Hnrnpll enhanced matrigel invasion activity of colon cancer cells in vitro, as well as their metastatic ability in vivo. An RNA-immunoprecipitation analysis showed Hnrnpll-binding to Cd44 pre-mRNAs, and the level of Cd44 variable exon 6 (Cd44v6), a poor prognosis marker of colorectal cancer, was increased by knocking down Hnrnpll. A neutralising Cd44v6 antibody suppressed the matrigel invasion ability induced by Hnrnpll knockdown. HNRNPLL expression was downregulated when colon cancer cells were induced to undergo epithelial-mesenchymal transition (EMT). Immunohistochemistry of clinical samples indicated that colorectal cancer cells with low E-cadherin expression at the invasion front exhibited decreased HNRNPLL expression.ConclusionsHNRNPLL is a novel metastasis suppressor of colorectal cancer, and modulates alternative splicing of CD44 during EMT.
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Wu, S., S. Ren, L. Nguyen, J. S. Adams, and M. Hewison. "Splice Variants of the CYP27b1 Gene and the Regulation of 1,25-Dihydroxyvitamin D3 Production." Endocrinology 148, no. 7 (July 1, 2007): 3410–18. http://dx.doi.org/10.1210/en.2006-1388.
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The cytochrome P450 25-hydroxyvitamin D3-1α-hydroxylase (CYP27b1) plays a pivotal role in vitamin D physiology by catalyzing synthesis of active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. In common with other P450s, CYP27b1 is known to exhibit alternative splicing. Here we have cloned and sequenced several novel intron 2-containing, noncoding splice variant mRNAs for CYP27b1 in 1,25(OH)2D3-producing HKC-8 human proximal tubule and THP-1 monocytic cells. Regulation of 1,25(OH)2D3 synthesis in these cell lines by calciotropic and noncalciotropic factors was associated with altered expression of the CYP27b1 splice variants. To assess the functional significance of this, HKC-8 cells were transfected with short hairpin RNA (shRNA) to inhibit mRNAs containing sequences from intron 2. This resulted in a significant increase in the expression of CYP27b1 protein and synthesis of 1,25(OH)2D3 by HKC-8 cells compared with control cells for two different intron 2-containing shRNAs (both P < 0.001). shRNA to intron 2 had no significant effect on the levels of wild-type CYP27b1 mRNA, suggesting a posttranscriptional mechanism of action. By contrast, shRNA to wild-type CYP27b1 suppressed transcription and activity of the enzyme by 70 and 31%, respectively (both P < 0.01). These data indicate that noncoding splice variants of CYP27b1 are functionally active and may play a significant role in the regulation of 1,25(OH)2D3 synthesis during normal physiology.
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Zhao, Kai, Yan Liu, Zhe Xiong, Lian Hu, and Cheng-liang Xiong. "Tissue-specific inhibition of urokinase-type plasminogen activator expression in the testes of mice by inducible lentiviral RNA interference causes male infertility." Reproduction, Fertility and Development 29, no. 11 (2017): 2149. http://dx.doi.org/10.1071/rd16477.
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Urokinase-type plasminogen activator (uPA) is involved in many physiological processes, including male infertility. To explore the effects of uPA in male reproduction, we constructed an inducible uPA short hairpin RNA (shRNA) system expressed by lentiviral vectors. After proving inhibition of uPA expression in the mouse Sertoli cell line TM4 by 1 μg mL−1 doxycycline (Dox), two lentivirus (pLenti4-shRNA and pLenti6/TR) were co-microinjected into mouse testes to produce TetR&shuPA mice model. Though oral gavage by 0.75 mg mL−1 Dox each day for 1 week, the Plau mRNA expression, uPA protein level and uPA enzyme activity in mice testis decreased significantly in TetR&shuPA mice model. After Dox induction of 1 week, the TetR&shuPA mice mated with female mice. Our results show that the pregnancy rate was reduced by approximately 40% and the sperm motility also decreased significantly. These data indicated that downregulation of uPA could decrease the fertility of male mice, which may be caused by a reduction in sperm motility. To investigate the reversible effect and safety of the inducible uPA shRNA system, we withdraw Dox and found the mating rate and sperm motility gradually recovered after 2 weeks. The histopathology structure of the testis, epididymis, and main organs was not altered significantly. The results of the present study indicating that uPA may be regarded as a novel target for the regulation of male fertility.
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Liu, Chi-Jui, Shye-Jye Tang, Chun-Che Chou, Guang-Huan Sun, and Kuang-Hui Sun. "In Vivo Suppression of Autophagy via Lentiviral shRNA Targeting Atg5 Improves Lupus-Like Syndrome." BioMed Research International 2020 (May 2, 2020): 1–10. http://dx.doi.org/10.1155/2020/8959726.
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In both mouse models and clinical patients with lupus, autophagy levels were significantly elevated and correlated with disease activity. Furthermore, autophagy can promote the survival of B and T cells, plasma cell differentiation, and antibody production. These results suggest that autophagy may promote the progression of lupus by regulating the survival of autoreactive immune cells. Therefore, we aimed at studying whether suppressing autophagy can modulate lupus progression in vivo. First, we found that the autophagy levels in splenocytes and lymphocytes of peripheral blood (PB) were elevated and positively correlated with disease severity in lupus-prone mice. The shAtg5-lentivirus, which effectively inhibits autophagy in vitro, was then injected into the lupus-prone mice. Autophagy levels in lymph node cells and PB lymphocytes were reduced following Atg5 suppression. We also found that lymphadenopathy and the numbers of plasma cells, CD4-CD8-, and CD4+ T cells decreased in mice treated with the shAtg5-lentivirus. The mice treated with shAtg5-lentivirus exhibited lower levels of proteinuria, serum anti-dsDNA antibody, B-cell activating factor (BAFF), and glomerular immune complex deposition. Therefore, targeting autophagy to moderate overactivated autophagy in immune cells seems to be a novel strategy for combination therapy of lupus.
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Tan, Su-Fern, Xin Liu, Kathleen Broeg, Todd E. Fox, David J. Feith, and Thomas P. Loughran. "Acid Ceramidase Inhibition Impairs Tumor Progression in a Rat Model of LGL Leukemia." Blood 126, no. 23 (December 3, 2015): 1246. http://dx.doi.org/10.1182/blood.v126.23.1246.1246.
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Abstract Natural killer cell large granular lymphocytic (NK-LGL) leukemia is a disorder characterized by the expansion of clonal CD3-, CD56+ cells in the peripheral blood and marrow. NK-LGL leukemia could manifest in both aggressive and chronic forms. Currently, no curative treatment is available for patients with NK-LGL leukemia. The pathogenetic mechanisms of NK-LGL leukemia are also poorly understood, which highlights the need to increase our understanding of the disorder in order to develop novel therapeutic targets. Sphingolipid dysregulation has been shown to promote tumor growth and survival in several types of cancers, including LGL leukemia. In cancer cells, the accumulation of pro-survival sphingosine-1-phosphate (S1P) throws the S1P/ceramide rheostat off balance, which leads to tumor growth and activated signaling pathways. Our laboratory previously discovered that the reversal of sphingolipid dysregulation in NK-LGL leukemia, either through targeting sphingolipid enzymes or by the addition of antiproliferative ceramide, leads to programmed cell death in leukemic cells. Acid ceramidase, a cysteine hydrolase, catalyzes the breakdown of ceramide into sphingosine and fatty acid. We found that cells from chronic NK-LGL patients have lower pro-death ceramide levels and higher S1P levels when compared to normal NK donors. Furthermore, the mRNA expression and activity of acid ceramidase (AC) were elevated in leukemic NK cells compared to normal NK cells. We further demonstrate the importance of AC in NK-LGL leukemic cells through AC knockdown in human (NKL) and rat (RNK-16) NK-LGL leukemic cell lines. AC knockdown decreased cell viability and increased ceramide levels, significantly ceramide species C16 and C24 (p<0.05), showing that AC is essential for the growth of leukemic NK cells. We further confirm that the accumulation of both C16 and C24 ceramide mediated the decreased viability of NKL and RNK-16 cells by treating the cells with C16 and C24 ceramide (6.25µM, 12.5µM and 25µM) for 24 hours. As our data showed that AC mediates the survival of NK-LGL, we did further analysis on the effects of AC knockdown on survival signals. Protein expression of AC knockdown- NKL and RNK-16 cell lines showed that AC knockdown decreased survival signaling mediators, including Bcl-2, Mcl-1 and survivin. Conversely, pro-apoptotic puma increased with AC knockdown in both cell lines. We further extended our in vitro findings in an established in vivo NK-LGL Fischer rat model. RNK-16 cells stably expressing AC shRNA (RNK-16/shRNA) or stably expressing scrambled shRNA (RNK-16/scr) were transplanted into 6-week old male rats (7 per group). The rats engrafted with RNK-16/shRNA survived significantly longer than those engrafted with RNK-16/scr (Mantel-Cox test, p<0.05). Interestingly, RNK-16/shRNA rats did not exhibit organomegaly compared to their leukemic counterparts. Splenocytes isolated from RNK-16/shRNA engrafted animals showed 50 percent reduction in AC expression compared to splenocytes from RNK-16/scr. Lipid analysis on splenocytes isolated from both RNK-16/scr and RNK-16/shRNA showed that RNK-16/shRNA cells had significant increases in total ceramide, C16 and C24 ceramide species, and a significant decrease in S1P. Collectively, our data show that targeting AC slows the progression of NK-LGL leukemia by reversing sphingolipid dysregulation and warrants further investigation as a therapeutic target in this incurable disease. Disclosures No relevant conflicts of interest to declare.
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Shi, Da, Xiaobo Wang, Hongyan Shi, Jiyu Zhang, Yuru Han, Jianfei Chen, Xin Zhang, et al. "Significant Interference with Porcine Epidemic Diarrhea Virus Pandemic and Classical Strain Replication in Small-Intestine Epithelial Cells Using an shRNA Expression Vector." Vaccines 7, no. 4 (November 2, 2019): 173. http://dx.doi.org/10.3390/vaccines7040173.
Full textAbstract:
Porcine epidemic diarrhea (PED) re-emerged in China in 2010 and is now widespread. Evidence indicates that highly virulent porcine epidemic diarrhea virus (PEDV) strains belonging to genotype G2 caused a large-scale outbreak of diarrhea. Currently, vaccines derived from PEDV classical strains do not effectively prevent infection by virulent PEDV strains, and no specific drug is available to treat the disease. RNA interference (RNAi) is a novel and effective way to cure a wide range of viruses. We constructed three short hairpin RNA (shRNA)-expressing plasmids (shR-N307, shR-N463, and shR-N1071) directed against nucleocapsid (N) and determined their antiviral activities in intestine epithelial cells infected with a classical CV777 strain and LNCT2. We verified that shR-N307, shR-N463, and shR-N1071 effectively inhibited the expression of the transfected N gene in vitro, comparable to the control shRNA. We further demonstrated the shRNAs markedly reduced PEDV CV777 and LNCT2 replication upon downregulation of N production. Therefore, this study provides a new strategy for the design of antiviral methods against coronaviruses by targeting their processivity factors.
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Herault, Olivier, Kristin J. Hope, Eric Deneault, Nadine Mayotte, Jalila Chagraoui, Brian T. Wilhelm, Sonia Cellot, et al. "A role for GPx3 in activity of normal and leukemia stem cells." Journal of Experimental Medicine 209, no. 5 (April 16, 2012): 895–901. http://dx.doi.org/10.1084/jem.20102386.
Full textAbstract:
The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.