2,946 results on '"DSRNA"'
Search Results
2. Chicken hnRNPK suppresses interferon production, thereby enhancing IBDV replication
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Wang, Ke, Hu, Ying, Nie, Jiangjiang, Zeng, Qinghua, Hu, Yu, and Wu, Huansheng
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- 2025
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3. A bioinformatics framework for human health risk assessment of externally applied dsRNA-based biopesticides
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Devisetty, Upendra K., De Neef, Emma, Gordon, Eric R.L., Velásquez-Zapata, Valeria, Narva, Kenneth, Mézin, Laurent, Cahon, Peter Mc, Witwer, Kenneth W., and Sridharan, Krishnakumar
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- 2025
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4. Prevalence and species diversity of dsRNA mycoviruses from Beauveria bassiana strains in the China's Guniujiang nature
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Shi, Najie, Zhu, Qiuyan, Yang, Guogen, Wang, Ping, and Huang, Bo
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- 2024
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5. RNA Interference for Plant Disease Management: Updated Methods, Current Applications and Future Directions
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Singh, Nivedita, Attri, Tarushi, Rajina, Thakur, Renu, Sharma, Monica, Patra, Jayanta Kumar, Series Editor, Das, Gitishree, Series Editor, Chen, Jen-Tsung, editor, Khan, Masudulla, editor, and Parveen, Aiman, editor more...
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- 2025
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6. Advances in A-to-I RNA editing in cancer.
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Zhang, Yi, Li, Lvyuan, Mendoza, Juana Jessica, Wang, Dan, Yan, Qijia, Shi, Lei, Gong, Zhaojian, Zeng, Zhaoyang, Chen, Pan, and Xiong, Wei
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RNA modification & restriction , *LINCRNA , *DOUBLE-stranded RNA , *MEDICAL sciences , *CIRCULAR RNA - Abstract
RNA modifications are widespread throughout the mammalian transcriptome and play pivotal roles in regulating various cellular processes. These modifications are strongly linked to the development of many cancers. One of the most prevalent forms of RNA modifications in humans is adenosine-to-inosine (A-to-I) editing, catalyzed by the enzyme adenosine deaminase acting on RNA (ADAR) in double-stranded RNA (dsRNA). With advancements in RNA sequencing technologies, the role of A-to-I modification in cancer has garnered increasing attention. Research indicates that the levels and specific sites of A-to-I editing are significantly altered in many malignant tumors, correlating closely with tumor progression. This editing occurs in both coding and noncoding regions of RNA, influencing signaling pathways involved in cancer development. These modifications can either promote or suppress cancer progression through several mechanisms, including inducing non-synonymous amino acid mutations, altering the immunogenicity of dsRNAs, modulating mRNA interactions with microRNAs (miRNAs), and affecting the splicing of circular RNAs (circRNAs) as well as the function of long non-coding RNAs (lncRNAs). A comprehensive understanding of A-to-I RNA editing is crucial for advancing the diagnosis, treatment, and prognosis of human cancers. This review explores the regulatory mechanisms of A-to-I editing in cancers and examines their potential clinical applications. It also summarizes current research, identifies future directions, and highlights potential therapeutic implications. [ABSTRACT FROM AUTHOR] more...
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- 2024
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7. IRE1α silences dsRNA to prevent taxane-induced pyroptosis in triple-negative breast cancer.
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Xu, Longyong, Peng, Fanglue, Luo, Qin, Ding, Yao, Yuan, Fei, Zheng, Liting, He, Wei, Zhang, Sophie S., Fu, Xin, Liu, Jin, Mutlu, Ayse Sena, Wang, Shuyue, Nehring, Ralf Bernd, Li, Xingyu, Tang, Qianzi, Li, Catherine, Lv, Xiangdong, Dobrolecki, Lacey E., Zhang, Weijie, and Han, Dong more...
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TRIPLE-negative breast cancer , *IMMUNE checkpoint inhibitors , *DOUBLE-stranded RNA , *BREAST cancer , *CELL death - Abstract
Chemotherapy is often combined with immune checkpoint inhibitor (ICIs) to enhance immunotherapy responses. Despite the approval of chemo-immunotherapy in multiple human cancers, many immunologically cold tumors remain unresponsive. The mechanisms determining the immunogenicity of chemotherapy are elusive. Here, we identify the ER stress sensor IRE1α as a critical checkpoint that restricts the immunostimulatory effects of taxane chemotherapy and prevents the innate immune recognition of immunologically cold triple-negative breast cancer (TNBC). IRE1α RNase silences taxane-induced double-stranded RNA (dsRNA) through regulated IRE1-dependent decay (RIDD) to prevent NLRP3 inflammasome-dependent pyroptosis. Inhibition of IRE1α in Trp53 −/− TNBC allows taxane to induce extensive dsRNAs that are sensed by ZBP1, which in turn activates NLRP3-GSDMD-mediated pyroptosis. Consequently, IRE1α RNase inhibitor plus taxane converts PD-L1-negative, ICI-unresponsive TNBC tumors into PD-L1high immunogenic tumors that are hyper-sensitive to ICI. We reveal IRE1α as a cancer cell defense mechanism that prevents taxane-induced danger signal accumulation and pyroptotic cell death. [Display omitted] • ER stress sensor IRE1α silences taxane-induced dsRNA through RIDD • p53 and IRE1α are two layers of protection against taxane-induced dsRNAs in TNBC • IRE1α inhibition allows taxane to induce dsRNA and NLRP3-GSDMD-mediated pyroptosis • IRE1α inhibitor sensitizes PD-L1− TP53 -mutant cold TNBC to chemo-immunotherapy Mechanisms dictating the form of chemotherapy-induced cancer cell death are elusive. This study reports that immunologically cold triple-negative breast cancer (TNBC) hijacks an ancient ER stress response to silence taxane-based chemotherapy-induced danger signals and avoid pore-forming inflammatory pyroptosis to achieve immune evasion. [ABSTRACT FROM AUTHOR] more...
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- 2024
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8. Identification of pathogenicity determinants in ToLCNDV and their RNAi-based knockdown for disease management in Nicotiana benthamiana and tomato plants.
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Sarkar, Mehulee, Gupta, Dipinte, Singh, Oinam Washington, Paul, Samrat, Kumar, Ravinder, Mandal, Bikash, and Roy, Anirban
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PLANT viruses ,NUCLEAR proteins ,HAIRPIN (Genetics) ,PLANT diseases ,GENETIC transcription ,NICOTIANA benthamiana - Abstract
Begomovirus solanumdelhiense (tomato leaf curl New Delhi virus, ToLCNDV), is member of the genus Begomovirus , family Geminiviridae , is a prolific bipartite whitefly transmitted begomovirus in the Indian sub-continent has a wide host range, including solanaceous, cucurbitaceous and other plants. Recently, dsRNA-mediated non-transgenic approaches have been promising in managing plant viruses. Such an approach could be effective if the pathogenicity determinants of a virus are targeted. In the case of ToLCNDV, viral pathogenicity has been demonstrated with coat protein (AV1), pre-coat protein (AV2), transcription activator protein (AC2) and nuclear shuttle protein (NSP). In the present study, we investigated the involvement of the three RNA silencing suppressor proteins (AV2, AC2, AC4) encoded by ToLCNDV in pathogenicity determinants through transient overexpression and hairpin RNAi-based knockdown assays in Nicotiana benthamiana plants. Further, we showed that the transcripts of AV2, AC2, and AC4 genes can systemically move and express their proteins. Hairpin RNAi constructs targeting each pathogenicity determinant could effectively reduce symptom development and virus titer upon inoculation of ToLCNDV in N. benthamiana plants. Exogenous application of dsRNA individually (dsAV2/dsAC2/dsAC4) or together (cocktail dsRNA: dsCk) against the pathogenicity determinants showed a significant reduction of viral load and reduced severity of disease in plants treated with dsCk followed by dsAC4. The present report reconfirms that the RNA silencing suppressor proteins encoded by DNA-A genomic component of ToLCNDV, can also act as pathogenicity determinants. Further, we demonstrated for the first time that exogenous application of dsRNA targeting those pathogenicity determinants reduces ToLCNDV load and limits symptom development in tomato plants. [ABSTRACT FROM AUTHOR] more...
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- 2024
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9. Role of dsRNA-Based Insecticides in Agriculture: Current Scenario and Future Prospects.
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Das, Pratyush Kumar and Nanda, Satyabrata
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RNA interference ,SUSTAINABLE agriculture ,SMALL interfering RNA ,INSECT pest control ,PEST control - Abstract
Insect pests cause severe crop damage, resulting in substantial economic losses and threats to global food security. Conventional insecticides are low-cost chemical agents that kill the target insects and some non-specific beneficial organisms. Due to their toxic and non-biodegradable nature, these conventional insecticides persist in the environment, thus causing pollution and accumulating in the food chain. The development of novel insecticidal products based on double-stranded (dsRNA)-based RNA interference (RNAi) technology is a sustainable tool to effectively control insect pests. The dsRNA-based insecticides are known for their specificity, non-toxicity, and biodegradability. The current review introduces the dsRNA-based RNAi technique as a novel tool to control crop insect pests. The review highlights the mechanism behind dsRNA uptake into insect cells. Furthermore, it discusses the commercial aspects of different dsRNA-based products available in the market, their penetration rates, and public acceptance. The review details the latest developments in the field and the regulatory landscape regarding the technology. The advantages and limitations of dsRNA-based insecticides are discussed, and future research directions to overcome the potential challenges have been briefly suggested. The dsRNA-based insecticidal products may be a better alternative to conventional insecticides, thus delineating the resistance among insects and increasing agricultural productivity. [ABSTRACT FROM AUTHOR] more...
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- 2024
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10. Viral mimicry evasion: a new role for oncogenic KRAS mutations.
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Chen, Raymond, He, Aobo, and De Carvalho, Daniel D.
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IMMUNE checkpoint inhibitors , *TRANSCRIPTION factors , *INTERFERON gamma , *VIRAL mutation , *DOUBLE-stranded RNA - Abstract
“Viral mimicry” refers to the induction of an innate immune response and interferon signaling by endogenous stimuli such as double‐stranded RNA (dsRNA). This response has been shown to have strong cancer therapeutic potential, including by enhancing the effectiveness of immune checkpoint inhibition (ICI) therapies, and may represent a tumor suppression mechanism that needs to be overcome for malignant transformation to proceed. In a recent study, Zhou and colleagues identify KRAS, a frequently mutated oncogene, as a negative regulator of dsRNA and viral mimicry in an ICI‐resistant colorectal cancer model. Oncogenic KRASG12D mutations downregulate the RNA‐binding protein DDX60 by activating the AKT signaling pathway, which inhibits STAT3, a critical transcription factor regulating DDX60 and other interferon‐stimulated genes. Overexpression of DDX60, which competitively binds to dsRNA to prevent RISC‐mediated degradation, or targeting of KRASG12D elevated dsRNA levels, resulting in viral mimicry activation and potentiation of ICI treatment. These results establish KRAS as a promising target to sensitize immune “cold” tumors to ICI therapy and demonstrate the potential role of oncogenic mutations in viral mimicry evasion during tumorigenesis. [ABSTRACT FROM AUTHOR] more...
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- 2024
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11. RNA Interference Applied to Crustacean Aquaculture.
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Fajardo, Carlos, De Donato, Marcos, Macedo, Marta, Charoonnart, Patai, Saksmerprome, Vanvimon, Yang, Luyao, Purton, Saul, Mancera, Juan Miguel, and Costas, Benjamin
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RNA interference , *SMALL interfering RNA , *DOUBLE-stranded RNA , *GENE expression , *SHRIMP industry - Abstract
RNA interference (RNAi) is a powerful tool that can be used to specifically knock-down gene expression using double-stranded RNA (dsRNA) effector molecules. This approach can be used in aquaculture as an investigation instrument and to improve the immune responses against viral pathogens, among other applications. Although this method was first described in shrimp in the mid-2000s, at present, no practical approach has been developed for the use of dsRNA in shrimp farms, as the limiting factor for farm-scale usage in the aquaculture sector is the lack of cost-effective and simple dsRNA synthesis and administration procedures. Despite these limitations, different RNAi-based approaches have been successfully tested at the laboratory level, with a particular focus on shrimp. The use of RNAi technology is particularly attractive for the shrimp industry because crustaceans do not have an adaptive immune system, making traditional vaccination methods unfeasible. This review summarizes recent studies and the state-of-the-art on the mechanism of action, design, use, and administration methods of dsRNA, as applied to shrimp. In addition, potential constraints that may hinder the deployment of RNAi-based methods in the crustacean aquaculture sector are considered. [ABSTRACT FROM AUTHOR] more...
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- 2024
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12. Detection of Double-Stranded RNA Intermediates During SARS-CoV-2 Infections of Syrian Golden Hamsters with Monoclonal Antibodies and Its Implications for Histopathological Evaluation of In Vivo Studies.
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Beythien, Georg, de le Roi, Madeleine, Stanelle-Bertram, Stephanie, Armando, Federico, Heydemann, Laura, Rosiak, Malgorzata, Becker, Svenja, Lamers, Mart M., Kaiser, Franziska K., Haagmans, Bart L., Ciurkiewicz, Malgorzata, Gabriel, Gülşah, Osterhaus, Albert D. M. E., and Baumgärtner, Wolfgang more...
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VIRAL antigens , *GOLDEN hamster , *VIRUS diseases , *BIOMARKERS , *DOUBLE-stranded RNA - Abstract
The SARS-CoV-2 pandemic has highlighted the challenges posed by the emergence and rapid global spread of previously unknown viruses. Early investigations on the pathogenesis of newly identified viruses are often hampered by a lack of appropriate sample material and conventional detection methods. In this study, viral replication within the lungs of SARS-CoV-2-infected Syrian golden hamsters was assessed by immunolabeling dsRNA intermediates with three different monoclonal antibodies in formalin-fixed, paraffin-embedded tissue samples. The presence of dsRNA was compared to viral antigen levels, viral titers, and genomic RNA replicates using three different variants of concern and an ancestral virus strain at a single time point and during the course of infection with an ancestral variant, and then validated using fluorescent 2-plex in situ hybridization. The results indicate that the detection of viral infection using anti-dsRNA antibodies is restricted to an early phase of infection with high viral replication activity. Additionally, the combined detection of dsRNA intermediates and viral antigens may help to bridge the interpretation gaps between viral antigen levels and viral titers at a single time point. Further testing in other viral infections or species is needed to assess the potential of dsRNA as an early marker for viral infections. [ABSTRACT FROM AUTHOR] more...
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- 2024
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13. Hydroxychloroquine attenuates double‐stranded RNA‐stimulated hyper‐phosphorylation of tristetraprolin/ZFP36 and AU‐rich mRNA stabilization.
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Hitti, Edward G., Muazzen, Zeyad, Moghrabi, Walid, Al‐Yahya, Suhad, and Khabar, Khalid S. A.
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INFLAMMATORY mediators , *DOUBLE-stranded RNA , *PROTEIN stability , *AUTOIMMUNE diseases , *CELLULAR signal transduction - Abstract
The human innate immune system recognizes dsRNA as a pathogen‐associated molecular pattern that induces a potent inflammatory response. The primary source of pathogenic dsRNA is cells infected with replicating viruses, but can also be released from uninfected necrotic cells. Here, we show that the dsRNA poly(I:C) challenge in human macrophages activates the p38 MAPK‐MK2 signalling pathway and subsequently the phosphorylation of tristetraprolin (TTP/ZFP36). The latter is an mRNA decay‐promoting protein that controls the stability of AU‐rich mRNAs (AREs) that code for many inflammatory mediators. Hydroxychloroquine (HCQ), a common anti‐malaria drug, is used to treat inflammatory and autoimmune disorders and, controversially, during acute COVID‐19 disease. We found that HCQ reduced the dsRNA‐dependent phosphorylation of p38 MAPK and its downstream kinase MK2. Subsequently, HCQ reduced the abundance and protein stability of the inactive (phosphorylated) form of TTP. HCQ reduced the levels and the mRNA stability of poly (I:C)‐induced cytokines and inflammatory mRNAs like TNF, IL‐6, COX‐2, and IL‐8 in THP‐1 and primary blood monocytes. Our results demonstrate a new mechanism of the anti‐inflammatory role of HCQ at post‐transcriptional level (TTP phosphorylation) in a model of dsRNA activation, which usually occurs in viral infections or RNA release from necrotic tissue. [ABSTRACT FROM AUTHOR] more...
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- 2024
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14. siRNA与dsRNA在白纹伊蚊体内的干扰效果.
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吴启星, 姜玉庭, 刘露, 郭晓霞, 郭思含, 张瑞香, 邢丹, 赵彤言, and 郭文峰
- Abstract
Copyright of China Tropical Medicine is the property of China Tropical Medicine Editorial Office and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.) more...
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- 2024
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15. Coxsackievirus B3-Induced m 6 A Modification of RNA Enhances Viral Replication via Suppression of YTHDF-Mediated Stress Granule Formation.
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Zhao, Guangze, Zhang, Huifang M., Chen, Yankuan T., Shi, Kerry, Aghakeshmiri, Sana, Yip, Fione, Luo, Honglin, McManus, Bruce, and Yang, Decheng
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RNA modification & restriction ,STRESS granules ,RNA replicase ,DOUBLE-stranded RNA ,GENETIC translation - Abstract
N6-methyladenosine (m
6 A) is the most prevalent internal RNA modification. Here, we demonstrate that coxsackievirus B3 (CVB3), a common causative agent of viral myocarditis, induces m6 A modification primarily at the stop codon and 3′ untranslated regions of its genome. As a positive-sense single-stranded RNA virus, CVB3 replicates exclusively in the cytoplasm through a cap-independent translation initiation mechanism. Our study shows that CVB3 modulates the expression and nucleo-cytoplasmic transport of the m6 A machinery components—METTL3, ALKBH5 and YTHDFs—resulting in increased m6 A modifications that enhance viral replication. Mechanistically, this enhancement is mediated through YTHDF-driven stress granule (SG) formation. We observed that YTHDF proteins co-localize with human antigen R (HuR), a protein facilitating cap-independent translation, in SGs during early infection. Later in infection, YTHDFs are cleaved, suppressing SG formation. Notably, for the first time, we identified that during early infection CVB3's RNA-dependent RNA polymerase (3D) and double-stranded RNA (dsRNA) are stored in SGs, co-localizing with HuR. This early-stage sequestration likely protects viral components for use in late-phase replication, when SGs are disrupted due to YTHDF cleavage. In summary, our findings reveal that CVB3-induced m6 A modifications enhance viral replication by regulating YTHDF-mediated SG dynamics. This study provides a potential therapeutic strategy for CVB3-induced myocarditis. [ABSTRACT FROM AUTHOR] more...- Published
- 2024
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16. Quality by design approach to improve quality and decrease cost of in vitro transcription of mRNA using design of experiments.
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Boman, Jimmy, Marušič, Tjaša, Seravalli, Tina Vodopivec, Skok, Janja, Pettersson, Fredrik, Nemec, Kristina Šprinzar, Widmark, Henrik, and Sekirnik, Rok
- Abstract
In vitro transcription (IVT) reaction is an RNA polymerase‐catalyzed production of messenger RNA (mRNA) from DNA template, and the unit operation with highest cost of goods in the mRNA drug substance production process. To decrease the cost of mRNA production, reagents should be optimally utilized. Due to the catalytic, multicomponent nature of the IVT reaction, optimization is a multi‐factorial problem, ideally suited to design‐of‐experiment approach for optimization and identification of design space. We derived a data‐driven model of the IVT reaction and explored factors that drive process yield (in g/L), including impact of nucleoside triphosphate (NTP) concentration and Mg:NTP ratio on reaction yield and how to optimize the main cost drivers RNA polymerase and DNA template, while minimizing dsRNA formation, a critical quality attribute in mRNA products. We report a methodological approach to derive an optimum reaction design, with which cost efficiency of the reaction was improved by 44%. We demonstrate the validity of the model on mRNA construct of different lengths. Finally, we maximized the yield of the IVT reaction to 24.9 ± 1.5 g/L in batch, thus doubling the highest ever reported IVT yield. [ABSTRACT FROM AUTHOR] more...
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- 2024
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17. Optimizing the production of dsRNA biocontrols in microbial systems using multiple transcriptional terminators.
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Ross, Sebastian J., Owen, Gareth R., Hough, James, Philips, Annelies, Maddelein, Wendy, Ray, John, Kilby, Peter M., and Dickman, Mark J.
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Crop pests and pathogens annually cause over $220 billion in global crop damage, with insects consuming 5%–20% of major grain crops. Current crop pest and disease control strategies rely on insecticidal and fungicidal sprays, plant genetic resistance, transgenes, and agricultural practices. Double‐stranded RNA (dsRNA) is emerging as a novel sustainable method of plant protection as an alternative to traditional chemical pesticides. Successful commercialization of dsRNA‐based biocontrols requires the economical production of large quantities of dsRNA combined with suitable delivery methods to ensure RNAi efficacy against the target pest. In this study, we have optimized the design of plasmid DNA constructs to produce dsRNA biocontrols in Escherichia coli, by employing a wide range of alternative synthetic transcriptional terminators before measurement of dsRNA yield. We demonstrate that a 7.8‐fold increase of dsRNA was achieved using triple synthetic transcriptional terminators within a dual T7 dsRNA production system compared to the absence of transcriptional terminators. Moreover, our data demonstrate that batch fermentation production dsRNA using multiple transcriptional terminators is scalable and generates significantly higher yields of dsRNA generated in the absence of transcriptional terminators at both small‐scale batch culture and large‐scale fermentation. In addition, we show that application of these dsRNA biocontrols expressed in E. coli cells results in increased insect mortality. Finally, novel mass spectrometry analysis was performed to determine the precise sites of transcriptional termination at the different transcriptional terminators providing important further mechanistic insight. [ABSTRACT FROM AUTHOR] more...
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- 2024
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18. A biocontrol perspective on mycoviruses in fungal pathogen management.
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Galli, Matteo, Sede, Ana, Heinlein, Manfred, and Kogel, Karl-Heinz
- Abstract
Mycoviruses, viruses that infect fungi, have been identified across nearly every fungal taxon. Despite their widespread presence, the ecological effects of mycoviruses remain poorly understood. They can influence the biology of their hosts in various ways, including altering growth, reproduction, and pathogenicity. Their ability to induce either fungal hyper- or hypovirulence and thus regulate general fungal fitness by increasing fungal aggressiveness or, conversely, in extreme cases, converting harmful fungi into beneficial ones, has attracted increasing attention in recent years as a potential means of protecting plants from fungal diseases and pests. Increasing difficulties in controlling fungal diseases, pests and weeds with synthetic chemical pesticides, exacerbated by the emergence of resistance or tolerance to certain active ingredients, and stricter regulatory requirements due to environmental and health concerns, have stimulated interest in alternative approaches. In parallel with the introduction of double-stranded (ds)RNA-based products for crop protection and the fundamental knowledge generated in this field in recent years, the potential use of mycoviruses to control pathogenic fungi appears to be within reach. This review highlights recent advances in the field and emphasizes the potential of mycoviruses as biological control agents (BCAs), with the emphasis on the utilization of mycovirus-induced fungal hypovirulence to control fungi that cause plant diseases and mycovirus-induced fungal hypervirulence to protect plants from fungal hosts such insect pests or weeds. [ABSTRACT FROM AUTHOR] more...
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- 2025
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19. Analysis of Expression Patterns of PcABCG5 Gene in Penaeus chinensis Under Saline-Alkali Stress
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Yalun LI, Yuying HE, Qiong WANG, Chenhui GUAN, Yujie ZHOU, and Zhaoxia LI
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penaeus chinensis ,pcabcg5 ,saline-alkali stress ,dsrna ,Aquaculture. Fisheries. Angling ,SH1-691 - Abstract
Penaeus chinensis is one of the most economically important species in northern China. Strong market demand requires the expansion of aquaculture production capacity of P. chinensis. Saline-alkali water covers approximately 46 million hectares in China alone, primarily distributed in the northeast, northwest, and coastal areas. Low salinity, high carbonate alkalinity, high pH, and complex ionic composition are characteristics of such waters, which cause stress to aquatic animals by interfering with physiological homeostasis. High carbonate alkalinity can directly damage the gill tissue of crustaceans. The inhabitation of ion-exchange can result in alkalosis. The understanding of response mechanisms to saline-alkali stress in P. chinensis will contribute to the sustainable development of the shrimp industry.ATP-binding cassette (ABC) transporters are one of the most prominent families of transmembrane proteins. ABC transporters can transport molecules, such as inorganic ions, sugars, amino acids, lipids, peptides, specialized metabolites, and xenobiotic agents, across membranes by binding and hydrolyzing ATP (adenosine triphosphate). Members of the ABCG subfamily consist of a single ABC cassette in the amino terminal followed by six putative transmembrane domains, and thus, are referred to as half-sized ABC transporters. Members of this family play an important role in the efflux transport of cholesterol. The ABCG subfamily participates in signal transduction, antiviral defense, and antigen presentation through hormone transport and lipid metabolism, thus helping plants adapt to changing environments. Several members of this gene family show different expression patterns in P. chinensis when exposed to saline-alkali stress. Therefore, we aimed to explore the ABC functions in resistance to alkalosis in P. chinensis.In this study, ABCG5 (GenBank accession number: OQ318160) was identified in P. chinensis and named PcABCG5. The full length of PcABCG5 was 1, 923 base pairs, encoding 640 amino acids. The estimated molecular mass was 71.39 kDa, and the theoretical isoelectric point was 9.12. Subcellular localization prediction showed that PcABCG5 was located in the endoplasmic reticulum. The PcABCG5 protein contained an NBD (nucleotide-binding domains) and a TMD domain (transmembrane domain) and had no signal peptide. Homology and phylogenetic analysis showed that PcABCG5 was highly conserved and that mature PcABCG5 shared 98.91% and 97.97% similarity with ABCG5 sequences from Penaeus monodon and Penaeus japonicus, respectively. PcABCG5 expression profiles were assessed by qPCR. PcABCG5 mRNA was detected in the eyestalk, gills, heart, muscle, intestine, hepatopancreas, stomach, and hemolymph. Our results showed that saline-alkali stress induced significant upregulation of PcABCG5 and that the expression of PcABCG5 was highest in the gills. This may be owing to the location of the gills between the external and internal environments. Osmotic pressure regulation by ABCG transporters in the gills is the primary mechanism by which P. chinensis copes with saline-alkali stress. To determine the function of PcABCG5, dsRNA against PcABCG5 was successfully injected and PcABCG5 was downregulated by 82.9% (P < 0.05). At 48 h after RNAi, we observed a 20% increase in mortality of PcABCG5 mRNA knockdown shrimp, which verified that PcABCG5 participated in the response and improved the survivability of P. chinensis under acute saline-alkali stress. Co-expressed ABCG5 and ABCG8 formed heteromeric dimers that participated in lipid transport. They also affected membrane permeability by regulating the asymmetric distribution of membrane lipids. It was also evident that saline-alkali stress induced ion and osmotic stress. We speculated that PcABCG5 may participate in the maintenance of osmotic homeostasis by hydrolyzing ATP to release energy and enhance the transport function.In conclusion, the full length cDNA of PcABCG5 was cloned in P. chinensis. PcABCG5 was upregulated under saline-alkali stress. Moreover, RNAi resulted in increased mortality of PcABCG5-silenced shrimp under saline-alkali stress. Therefore, it can be concluded that PcABCG5 is involved in the response to saline-alkali stress in P. chinenesis. Our findings provide information for further understanding the genetic basis of saline-alkali tolerance and exploring the molecular breeding of P. chinensis. more...
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- 2024
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20. Activation of PKR by a short-hairpin RNA
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Kyle A. Cottrell, Sua Ryu, Helen Donelick, Hung Mai, Addison A. Young, Jackson R. Pierce, Brenda L. Bass, and Jason D. Weber
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Double-stranded RNA ,dsRNA ,PKR ,RNA interference ,RNAi ,Medicine ,Science - Abstract
Abstract Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54’s potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation. more...
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- 2024
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21. Nanoparticle LDH enhances RNAi efficiency of dsRNA in piercing-sucking pests by promoting dsRNA stability and transport in plants
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Xiaoqin Cheng, Qi Zhou, Jiedan Xiao, Xueying Qin, Yuan Zhang, Xiaoxue Li, Weiwei Zheng, and Hongyu Zhang
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Piercing-sucking pests ,dsRNA ,Nanoparticles ,Mortality ,Biopesticide ,Biotechnology ,TP248.13-248.65 ,Medical technology ,R855-855.5 - Abstract
Abstract Piercing-sucking pests are the most notorious group of pests for global agriculture. RNAi-mediated crop protection by foliar application is a promising approach in field trials. However, the effect of this approach on piercing-sucking pests is far from satisfactory due to the limited uptake and transport of double strand RNA (dsRNA) in plants. Therefore, there is an urgent need for more feasible and biocompatible dsRNA delivery approaches to better control piercing-sucking pests. Here, we report that foliar application of layered double hydroxide (LDH)-loaded dsRNA can effectively disrupt Panonychus citri at multiple developmental stages. MgAl-LDH-dsRNA targeting Chitinase (Chit) gene significantly promoted the RNAi efficiency and then increased the mortality of P. citri nymphs by enhancing dsRNA stability in gut, promoting the adhesion of dsRNA onto leaf surface, facilitating dsRNA internalization into leaf cells, and delivering dsRNA from the stem to the leaf via the vascular system of pomelo plants. Finally, this delivery pathway based on other metal elements such as iron (MgFe-LDH) was also found to significantly improve the protection against P. citri and the nymphs or larvae of Diaphorina citri and Aphis gossypii, two other important piercing-sucking hemipeteran pests, indicating the universality of nanoparticles LDH in promoting the RNAi efficiency and mortality of piercing-sucking pests. Collectively, this study provides insights into the synergistic mechanism for nano-dsRNA systemic translocation in plants, and proposes a potential eco-friendly control strategy for piercing-sucking pests. Graphical Abstract more...
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- 2024
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22. The quest for the best target genes for RNAi‐mediated pest control.
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Cedden, Doga and Bucher, Gregor
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RED flour beetle , *RNA interference , *SMALL interfering RNA , *PEST control , *GENE targeting - Abstract
RNA interference (RNAi) has emerged as an eco‐friendly alternative to classic pesticides for pest control. This review highlights the importance of identifying the best target genes for RNAi‐mediated pest control. We argue that the knowledge‐based approach to predicting effective targets is limited by our current gaps of knowledge, making unbiased screening a superior method for discovering the best target processes and genes. We emphasize the recent evidence that suggests targeting conserved basic cellular processes, such as protein degradation and translation, is more effective than targeting the classic pesticide target processes. We support these claims by comparing the efficacy of previously reported RNAi target genes and classic insecticide targets with data from our genome‐wide RNAi screen in the red flour beetle, Tribolium castaneum. Finally, we provide practical advice for identifying excellent target genes in other pests, where large‐scale RNAi screenings are typically challenging. [ABSTRACT FROM AUTHOR] more...
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- 2024
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23. Activation of PKR by a short-hairpin RNA.
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Cottrell, Kyle A., Ryu, Sua, Donelick, Helen, Mai, Hung, Young, Addison A., Pierce, Jackson R., Bass, Brenda L., and Weber, Jason D.
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RNA interference ,SMALL interfering RNA ,DOUBLE-stranded RNA ,CARRIER proteins ,VIRUS diseases - Abstract
Recognition of viral infection often relies on the detection of double-stranded RNA (dsRNA), a process that is conserved in many different organisms. In mammals, proteins such as MDA5, RIG-I, OAS, and PKR detect viral dsRNA, but struggle to differentiate between viral and endogenous dsRNA. This study investigates an shRNA targeting DDX54's potential to activate PKR, a key player in the immune response to dsRNA. Knockdown of DDX54 by a specific shRNA induced robust PKR activation in human cells, even when DDX54 is overexpressed, suggesting an off-target mechanism. Activation of PKR by the shRNA was enhanced by knockdown of ADAR1, a dsRNA binding protein that suppresses PKR activation, indicating a dsRNA-mediated mechanism. In vitro assays confirmed direct PKR activation by the shRNA. These findings emphasize the need for rigorous controls and alternative methods to validate gene function and minimize unintended immune pathway activation. [ABSTRACT FROM AUTHOR] more...
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- 2024
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24. Alu insertion-mediated dsRNA structure formation with pre-existing Alu elements as a disease-causing mechanism.
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Masson, Emmanuelle, Maestri, Sandrine, Bordeau, Valérie, Cooper, David N., Férec, Claude, and Chen, Jian-Min
- Subjects
- *
EXOCRINE pancreatic insufficiency , *GENE expression , *HUMAN genome , *GENETIC translation , *GENETIC disorders - Abstract
We previously identified a homozygous Alu insertion variant (Alu _Ins) in the 3′-untranslated region (3′-UTR) of SPINK1 as the cause of severe infantile isolated exocrine pancreatic insufficiency. Although we established that Alu _Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here, we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that, owing to its particular location, Alu _Ins could independently disrupt mRNA 3′ end formation and/or affect other post-transcriptional processes such as nuclear export and translation. However, employing a 3'-UTR luciferase reporter assay, Alu _Ins was found to result in only an ∼50% reduction in luciferase activity compared to wild type, which is insufficient to account for the severe pancreatic deficiency in the Alu _Ins homozygote. We then postulated that double-stranded RNA (dsRNA) structures formed between Alu elements, an upstream mechanism regulating gene expression, might be responsible. Using RepeatMasker, we identified two Alu elements within SPINK1 's third intron, both oriented oppositely to Alu _Ins. Through RNAfold predictions and full-length gene expression assays, we investigated orientation-dependent interactions between these Alu repeats. We provide compelling evidence to link the detrimental effect of Alu _Ins to extensive dsRNA structures formed between Alu _Ins and pre-existing intronic Alu sequences, including the restoration of SPINK1 mRNA expression by aligning all three Alu elements in the same orientation. Given the widespread presence of Alu elements in the human genome and the potential for new Alu insertions at almost any locus, our findings have important implications for detecting and interpreting Alu insertions in disease genes. It is well known that RNA secondary structures can influence splicing and that inverted Alu elements can form such structures. Here, we demonstrate that an Alu insertion variant causes a human genetic disease by creating extended double-stranded RNA structures with pre-existing Alu elements. [ABSTRACT FROM AUTHOR] more...
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- 2024
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25. Effective Synthesis of mRNA during In Vitro Transcription with Fewer Impurities Produced.
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He, Wei, Geng, Qi, Ji, Guiying, Li, Ji, Wang, Dan, He, Yucai, Jin, Qiuheng, and Ye, Jianren
- Subjects
- *
RNA polymerases , *GENETIC transcription , *DOUBLE-stranded RNA , *NUCLEOTIDES , *COVID-19 vaccines - Abstract
The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3′ loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant. [ABSTRACT FROM AUTHOR] more...
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- 2024
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26. The 5‐Fluorouracil RNA Expression Viewer (5‐FUR) Facilitates Interpreting the Effects of Drug Treatment and RRP6 Deletion on the Transcriptional Landscape in Yeast.
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Szachnowski, Ugo, Sallou, Oliver, Boudet, Mateo, Bretaudeau, Anthony, Wery, Maxime, Morillon, Antonin, and Primig, Michael
- Abstract
Saccharomyces cerevisiae is an excellent model to study the effect of external cues on cell division and stress response. 5‐Fluorocuracil (5‐FU) has been used to treat solid tumors since several decades. The drug was initially designed to interfere with DNA replication but was later found to exert its antiproliferative effect also via RNA‐dependent processes. Since 5‐FU inhibits the activity of the 3′−5′‐exoribonuclease Rrp6 in yeast and mammals, earlier work has compared the effect of 5‐FU treatment and RRP6 deletion at the transcriptome level in diploid synchronized yeast cells. To facilitate interpreting the expression data we have developed an improved 5‐Fluorouracil RNA (5‐FUR) expression viewer. Users can access information via genome coordinates and systematic or standard names for mRNAs and Xrn1‐dependent‐, stable‐, cryptic‐, and meiotic unannotated transcripts (XUTs, SUTs, CUTs, and MUTs). Normalized log2‐transformed or linear data can be displayed as filled diagrams, line graphs or color‐coded heatmaps. The expression data are useful for researchers interested in processes such as cell cycle regulation, mitotic repression of meiotic genes, the effect of 5‐FU treatment and Rrp6 deficiency on the transcriptome and expression profiles of sense/antisense loci that encode overlapping transcripts. The viewer is accessible at http://5fur.genouest.org. Take away: We present an improved 5‐Fluorouracil RNA (5‐FUR) expression viewerStrand‐specific RNA‐Seq data are available for mRNAs and different classes of lncRNAsNormalized data are displayed as filled diagrams, line graphs or heatmapsThe expression data are pertinent for regulation of mitotic and meiotic genesUsers can interpret the expression profiles of sense/antisense transcripts [ABSTRACT FROM AUTHOR] more...
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- 2024
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27. Characterization of an envelope protein 118L in invertebrate iridescent virus 6 (IIV6).
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Altun, Betul, Zengin, Kubra, Yayli Dabag, Sevde, Yesilyurt, Aydin, Nalcacioglu, Remziye, and Demirbag, Zihni
- Abstract
Invertebrate iridescent virus 6 (IIV6) is a nucleocytoplasmic insect virus and a member of the family Iridoviridae. The IIV6 genome consists of 212,482 bp of linear dsDNA with 215 non-overlapping and putative protein-encoding ORFs. The IIV6 118L ORF is conserved in all sequenced members of the Iridoviridae and encodes a 515 amino acid protein with three predicted transmembrane domains and several N-glycosylation/N-myristoylation sites. In this study, we characterized the 118L ORF by both deleting it from the viral genome and silencing its expression with dsRNA in infected insect cells. The homologous recombination method was used to replace 118L ORF with the green fluorescent protein (gfp) gene. Virus mutants in which the 118L gene sequence had been replaced with gfp were identified by fluorescence microscopy but could not be propagated separately from the wild-type virus in insect cells. Unsuccessful attempts to isolate the mutant virus with the 118L gene deletion suggested that the protein is essential for virus replication. To support this result, we used dsRNA to target the 118L gene and showed that treatment resulted in a 99% reduction in virus titer. Subsequently, we demonstrated that 118L-specific antibodies produced against the 118L protein expressed in the baculovirus vector system were able to neutralize the virus infection. All these results indicate that 118L is a viral envelope protein that is required for the initiation of virus replication. [ABSTRACT FROM AUTHOR] more...
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- 2024
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28. Molecular approaches for the management of papaya ringspot virus infecting papaya: a comprehensive review.
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Jyotika, R. K., Harish, S., Karthikeyan, G., Kumar, K. K., Murugan, M., Jayakanthan, M., and Chen, Tsung-Chi
- Abstract
Papaya ringspot virus (PRSV) is a catastrophic disease that causes huge yield losses in papaya cultivation around the world. Yield losses in severely infected plants can be upto 100%. Because of this disease, papaya cultivation has been shifted to other crops in some areas of the world. Many conventional methods and breeding approaches are used against this disease, which turns out to be less effective. Considering the yield loss caused by PRSV in papaya, it is high time to focus on alternative control methods. To implement effective management strategies, molecular approaches such as Marker Assisted Breeding (MAS) or transgenic methods involving post-transcriptional gene silencing targeting the genome viz., coat protein, replicase gene, or HC Pro can be pursued. However, the public's reluctance to widely accept the transgenic approach due to health and environmental concerns necessitates a consideration of non-transgenic alternatives. Prioritizing safety and ensuring efficient virus control, non-transgenic approaches which encompass cross-protection, genome editing, and topical applications of dsRNA to induce gene silencing within the host, can be adopted. This review aims to provide comprehensive insights of various molecular tools used in managing PRSV which in turn will help in sustainable agriculture. [ABSTRACT FROM AUTHOR] more...
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- 2024
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29. Nanoparticle LDH enhances RNAi efficiency of dsRNA in piercing-sucking pests by promoting dsRNA stability and transport in plants.
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Cheng, Xiaoqin, Zhou, Qi, Xiao, Jiedan, Qin, Xueying, Zhang, Yuan, Li, Xiaoxue, Zheng, Weiwei, and Zhang, Hongyu
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VASCULAR system of plants ,LAYERED double hydroxides ,COTTON aphid ,PLANT translocation ,PEST control - Abstract
Piercing-sucking pests are the most notorious group of pests for global agriculture. RNAi-mediated crop protection by foliar application is a promising approach in field trials. However, the effect of this approach on piercing-sucking pests is far from satisfactory due to the limited uptake and transport of double strand RNA (dsRNA) in plants. Therefore, there is an urgent need for more feasible and biocompatible dsRNA delivery approaches to better control piercing-sucking pests. Here, we report that foliar application of layered double hydroxide (LDH)-loaded dsRNA can effectively disrupt Panonychus citri at multiple developmental stages. MgAl-LDH-dsRNA targeting Chitinase (Chit) gene significantly promoted the RNAi efficiency and then increased the mortality of P. citri nymphs by enhancing dsRNA stability in gut, promoting the adhesion of dsRNA onto leaf surface, facilitating dsRNA internalization into leaf cells, and delivering dsRNA from the stem to the leaf via the vascular system of pomelo plants. Finally, this delivery pathway based on other metal elements such as iron (MgFe-LDH) was also found to significantly improve the protection against P. citri and the nymphs or larvae of Diaphorina citri and Aphis gossypii, two other important piercing-sucking hemipeteran pests, indicating the universality of nanoparticles LDH in promoting the RNAi efficiency and mortality of piercing-sucking pests. Collectively, this study provides insights into the synergistic mechanism for nano-dsRNA systemic translocation in plants, and proposes a potential eco-friendly control strategy for piercing-sucking pests. [ABSTRACT FROM AUTHOR] more...
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- 2024
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30. Influence of carboxylated and hydroxylated multi-walled carbon nanotubes on the development and reproductive abilities of Myzus persicae.
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Fu-Xiao Yu, Mobarak, Syed Husne, Mao-Fa Yang, Chao-Xing Hu, and Tong-Xian Liu
- Subjects
- *
GREEN peach aphid , *MULTIWALLED carbon nanotubes , *AGRICULTURAL pests , *CARBON nanotubes , *PEST control - Abstract
Extensive investigation has illustrated the potential of carbon nanotubes (CNTs) and their derivatives to boost crop growth and yield, bolster crop resistance to diseases and pests, enhance drought and salt tolerance, facilitate soil enhancement, and cater as carriers for fertilizers, pesticides, and nucleic acids to augment their efficacy. Consequently, a comprehensive assessment of the individual impact of CNTs on agricultural pests is imperative for guiding their largescale applications in agriculture. This study evaluates the potential effects of carboxylated and hydroxylated multi-walled carbon nanotubes (MWCNTs-COOH and MWCNTs-OH) at varying concentrations (0.004, 0.04 and 0.4 mg/ml) on the development and reproduction of tobacco aphid, Myzus persicae (Sulzer), using age-stage, two-sex life table analysis. Our results revealed a significant extension in the developmental and pre-reproductive periods of tobacco aphids with increasing concentrations of both nanotubes. Notably, the net reproduction rate (R0), intrinsic growth rate (r), and finite rate of increase (λ) were significantly lower under the two nanotubes treatments compared to the control. Furthermore, both nanotubes exhibited a substantial reduction in reproduction days and fecundity, particularly at higher concentrations, where these parameters reached their lowest values (6.23 days, 20.44 offspring for MWCNTs-COOH and 5.96 days, 17.13 offspring for MWCNTs-OH, respectively). Overall, both nanotubes demonstrated inhibitory effects on the development and reproductive capacity, with MWCNTs-OH generally exhibiting a more pronounced impact than MWCNTs-COOH. These findings offer a robust theoretical framework for the use of MWCNTs-COOH and MWCNTs-OH as potential pesticides and dsRNA carriers for the effective control of tobacco aphid. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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31. IBR1, a novel endogenous IFIH1‐binding dsRNA, governs IFIH1 activation and M1 macrophage polarisation in ARDS.
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Zhang, Shi, Huang, Wei, Wu, Xueling, Chen, Hanbing, Wang, Lu, Chao, Jie, Xie, Jianfeng, and Qiu, Haibo
- Subjects
- *
ADULT respiratory distress syndrome , *MACROPHAGE activation , *GENE knockout , *KNOCKOUT mice , *CELL analysis - Abstract
Background: Uncontrolled inflammation caused by macrophages and monocytes plays a crucial role in worsening acute respiratory distress syndrome (ARDS). Previous studies have highlighted the importance of IFIH1 in regulating macrophage polarisation in ARDS triggered by pneumonia. However, the mechanisms by which IFIH1 is activated in ARDS remain unclear. Methods: In this study, we utilised multiomics sequencing and molecular interaction experiments to explore the molecular mechanisms underlying IFIH1 activation in ARDS. Through the use of conditional gene knockout mice and primary cells, we demonstrated the significant role of these mechanisms in the development of ARDS. Additionally, we validated the associations between these mechanisms and ARDS by quantitative PCR analysis of CD14+ cells obtained from the peripheral blood of 140 ARDS patients. Results: Our investigation revealed that lipopolysaccharide, a critical component derived from Gram‐negative bacteria, activated IFIH1 by upregulating a novel transcript known as IFIH1‐binding RNA1 (IBR1) in monocytes and macrophages. Specifically, as an endogenous double‐stranded RNA, IBR1 bind to the helicase domain of IFIH1 because of its unique double‐stranded structure. Deletion of IBR1 significantly reduced the activation of IFIH1, M1 polarisation of macrophages, and inflammatory lung injury in ARDS. Moreover, IBR1 directly induced M1 polarisation of macrophages and ARDS, whereas deletion of IFIH1 inhibited IBR1‐induced macrophage M1 polarisation and inflammatory lung injury. Importantly, we observed a notable increase in IBR1 expression in ARDS patients with pneumonia caused by Gram‐negative bacteria. Furthermore, we demonstrated that the delivery of IFIH1 mutants through exosomes effectively counteracted IBR1, thereby reducing pulmonary inflammation and alleviating lung injury. Conclusions: This study revealed a novel mechanism involving IBR1, an endogenous double‐stranded RNA (dsRNA) that binds to IFIH1, shedding light on the complex process of macrophage polarisation in ARDS. The administration of IFIH1 variants has the potential to eliminate pulmonary dsRNA and alleviate inflammatory lung injury in ARDS. Highlights: In monocytes and macrophages, the endogenous double‐stranded RNA, IFIH1‐binding RNA 1 (IBR1), binds to the helicase domain of IFIH1 because of its unique double‐stranded structure.IBR1 plays a significant role in macrophage polarisation and the development of acute respiratory distress syndrome (ARDS) induced by Gram‐negative bacteria or lipopolysaccharide (LPS).Administration of IFIH1 variants has potential for eliminating pulmonary IBR1 and reducing inflammatory lung injury in ARDS patients. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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32. LINE-1 RNA triggers matrix formation in bone cells via a PKR-mediated inflammatory response.
- Author
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Mangiavacchi, Arianna, Morelli, Gabriele, Reppe, Sjur, Saera-Vila, Alfonso, Liu, Peng, Eggerschwiler, Benjamin, Zhang, Huoming, Bensaddek, Dalila, Casanova, Elisa A, Medina Gomez, Carolina, Prijatelj, Vid, Della Valle, Francesco, Atinbayeva, Nazerke, Izpisua Belmonte, Juan Carlos, Rivadeneira, Fernando, Cinelli, Paolo, Gautvik, Kaare Morten, and Orlando, Valerio more...
- Subjects
- *
GENE expression , *BONE density , *MESENCHYMAL stem cells , *BONE cells , *PROTEIN kinases - Abstract
Transposable elements (TEs) are mobile genetic modules of viral derivation that have been co-opted to become modulators of mammalian gene expression. TEs are a major source of endogenous dsRNAs, signaling molecules able to coordinate inflammatory responses in various physiological processes. Here, we provide evidence for a positive involvement of TEs in inflammation-driven bone repair and mineralization. In newly fractured mice bone, we observed an early transient upregulation of repeats occurring concurrently with the initiation of the inflammatory stage. In human bone biopsies, analysis revealed a significant correlation between repeats expression, mechanical stress and bone mineral density. We investigated a potential link between LINE-1 (L1) expression and bone mineralization by delivering a synthetic L1 RNA to osteoporotic patient-derived mesenchymal stem cells and observed a dsRNA-triggered protein kinase (PKR)-mediated stress response that led to strongly increased mineralization. This response was associated with a strong and transient inflammation, accompanied by a global translation attenuation induced by eIF2α phosphorylation. We demonstrated that L1 transfection reshaped the secretory profile of osteoblasts, triggering a paracrine activity that stimulated the mineralization of recipient cells. Synopsis: Transposable elements (TEs) are selfish genetic modules that have, in certain contexts, evolved into modulators of mammalian gene expression. This study provides evidence for a role of TEs in promoting an inflammatory response that is required for bone repair and mineralization via dsRNA sensing-dependent PKR activation. In mice, TE expression is transiently upregulated shortly after a bone fracture concurrently with the onset of the inflammatory process. In humans, mechanically loaded bones upregulate TE expression, which correlates with local bone mineral density. Transfection with LINE-1 (L1) TE RNA stimulates mineralization activity in human osteoblasts in vitro. Cytoplasmic L1 RNA accumulation in differentiating osteoblasts triggers PKR-dependent translational shutdown, inflammation, and reshaping of the secretome. The secretome of L1 RNA-primed osteoblasts induces the mineralization activity of recipient osteoblasts. Bone fracture is associated with upregulation of transient transposable element RNA expression triggering bone mineralization via a paracrine, secretome mediated process. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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33. Context‐dependent antisense transcription from a neighboring gene interferes with the expression of mNeonGreen as a functional in vivo fluorescent reporter in the chloroplast of Chlamydomonas reinhardtii.
- Author
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Navarrete, Axel and Pollak, Bernardo
- Subjects
- *
GENE expression , *ANTISENSE RNA , *GENETIC engineering , *GENETIC regulation , *CHLAMYDOMONAS reinhardtii - Abstract
SUMMARY: Advancing chloroplast genetic engineering in Chlamydomonas reinhardtii remains challenging, decades after its first successful transformation. This study introduces the development of a chloroplast‐optimized mNeonGreen fluorescent reporter, enabling in vivo observation through a sixfold increase in fluorescence via context‐aware construct engineering. Our research highlights the influence of transcriptional readthrough and antisense mRNA pairing on post‐transcriptional regulation, pointing to novel strategies for optimizing heterologous gene expression. We further demonstrate the applicability of these insights using an accessible experimentation system using glass‐bead transformation and reestablishment of photosynthesis using psbH mutants, focusing on the mitigation of transcriptional readthrough effects. By characterizing heterologous expression using regulatory elements such as PrrnS, 5′atpA, and 3′ rbcL in a sense‐transcriptional context, we further documented up to twofold improvement in fluorescence levels. Our findings contribute new tools for molecular biology research in the chloroplast and evidence fundamental gene regulation processes that could enable the development of more effective chloroplast engineering strategies. This work not only paves the way for more efficient genetic engineering of chloroplasts but also deepens our understanding of the regulatory mechanisms at play. Significance Statement: In this work, we develop a functional fluorescent reporter for Chlamydomonas chloroplast expression, and found construct context‐effects influencing gene expression through formation of antisense dsRNA. These findings will aid in developing more efficient chloroplast genetic strategies and illuminate aspects of chloroplast gene expression. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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34. Enhancing tomato disease resistance through endogenous antifungal proteins and introduced nematode‐targeting dsRNA of biocontrol agent Bacillus velezensisHS‐3.
- Author
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Han, Juan, Zhu, Jinchi, Liu, Shuyuan, Sun, Xuehan, Wang, Shunchang, and Miao, Guopeng
- Subjects
BACILLUS (Bacteria) ,DOUBLE-stranded RNA ,BIOLOGICAL pest control agents ,LIQUID chromatography-mass spectrometry ,FUNGAL cell walls ,RNA interference - Abstract
BACKGROUND: As a type of biological control agent (BCA), Bacillus velezensis possesses the efficacy of inhibiting pathogenic microorganisms, promoting plant growth, and overcoming continuous cropping obstacles (CCOs). However, there is limited reporting on the optimization of the cultivation conditions for such biocontrol agents and their role as double‐stranded RNA (dsRNA) delivery vectors. RESULTS: In this study, a Bacillus velezensis strain HS‐3 was isolated from the root zone of tomato plants with in vitro anti‐Botrytis cinerea activity. The investigation into active compounds revealed that HS‐3 predominantly employs proteins with molecular weights greater than 3 kDa for its antifungal activity. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis identified various proteases and chitosanase, further suggesting that HS‐3 most likely employs these enzymes to degrade fungal cell walls for its antifungal effect. To optimize the production of extracellular proteins, fermentation parameters for HS‐3 were systematically optimized, leading to an optimized medium (OP‐M). HS‐3 cultured in OP‐M demonstrated enhanced capacity to assist tomato plants in withstanding CCOs. However, the presence of excessive nematodes in diseased soil resulted in the disease severity index (DSI) remaining high. An RNA interference mechanism was further introduced to HS‐3, targeting the nematode tyrosine phosphatase (TP) gene. Ultimately, HS‐3 expressing dsRNA of TP in OP‐M effectively assisted tomatoes in mitigating CCOs, reducing DSI to 2.2% and 17.8% of the control after 45 and 90 days of growth, respectively. CONCLUSION: The advantages of Bacillus velezensis in crop disease management and the mitigation of CCOs become even more pronounced when utilizing both optimized levels of endogenous enzymes and introduced nematode‐targeting dsRNA. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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35. Development and Application of Automated Sandwich ELISA for Quantitating Residual dsRNA in mRNA Vaccines.
- Author
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Holland, David A., Acevedo-Skrip, Jillian, Barton, Joshua, Thompson, Rachel, Bowman, Amy, Dewar, Emily A., Miller, Danielle V., Zhao, Kaixi, Swartz, Andrew R., and Loughney, John W.
- Subjects
DOUBLE-stranded RNA ,ENZYME-linked immunosorbent assay ,GENETIC transcription ,VACCINE safety ,MESSENGER RNA - Abstract
The rise of mRNA as a novel vaccination strategy presents new opportunities to confront global disease. Double-stranded RNA (dsRNA) is an impurity byproduct of the in vitro transcription reaction used to manufacture mRNA that may affect the potency and safety of the mRNA vaccine in patients. Careful quantitation of dsRNA during manufacturing is critical to ensure that residual dsRNA is minimized in purified mRNA drug substances. In this work, we describe the development and implementation of a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) to quantitate nanogram quantities of residual dsRNA contaminants in mRNA process intermediates using readily available commercial reagents. This sandwich ELISA developed in this study follows a standard protocol and can be easily adapted to most research laboratory environments. Additionally, a liquid handler coupled with an automated robotics system was utilized to increase assay throughput, improve precision, and reduce the analyst time requirement. The final automated sandwich ELISA was able to measure <10 ng/mL of dsRNA with a specificity for dsRNA over 2000-fold higher than mRNA, a variability of <15%, and a throughput of 72 samples per day. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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- View/download PDF
36. Spray‐induced gene silencing to identify powdery mildew gene targets and processes for powdery mildew control
- Author
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McRae, Amanda G, Taneja, Jyoti, Yee, Kathleen, Shi, Xinyi, Haridas, Sajeet, LaButti, Kurt, Singan, Vasanth, Grigoriev, Igor V, and Wildermuth, Mary C
- Subjects
Plant Biology ,Biological Sciences ,Genetics ,Zero Hunger ,Arabidopsis ,Abscisic Acid ,Base Sequence ,Gene Silencing ,Plant Diseases ,Arabidopsis thaliana ,dsRNA ,Erysiphe necator ,Golovinomyces orontii ,grapevine ,powdery mildew ,spray-induced gene silencing ,Microbiology ,Crop and Pasture Production ,Plant Biology & Botany ,Evolutionary biology ,Plant biology - Abstract
Spray-induced gene silencing (SIGS) is an emerging tool for crop pest protection. It utilizes exogenously applied double-stranded RNA to specifically reduce pest target gene expression using endogenous RNA interference machinery. In this study, SIGS methods were developed and optimized for powdery mildew fungi, which are widespread obligate biotrophic fungi that infect agricultural crops, using the known azole-fungicide target cytochrome P450 51 (CYP51) in the Golovinomyces orontii-Arabidopsis thaliana pathosystem. Additional screening resulted in the identification of conserved gene targets and processes important to powdery mildew proliferation: apoptosis-antagonizing transcription factor in essential cellular metabolism and stress response; lipid catabolism genes lipase a, lipase 1, and acetyl-CoA oxidase in energy production; and genes involved in manipulation of the plant host via abscisic acid metabolism (9-cis-epoxycarotenoid dioxygenase, xanthoxin dehydrogenase, and a putative abscisic acid G-protein coupled receptor) and secretion of the effector protein, effector candidate 2. Powdery mildew is the dominant disease impacting grapes and extensive powdery mildew resistance to applied fungicides has been reported. We therefore developed SIGS for the Erysiphe necator-Vitis vinifera system and tested six successful targets identified using the G. orontii-A. thaliana system. For all targets tested, a similar reduction in powdery mildew disease was observed between systems. This indicates screening of broadly conserved targets in the G. orontii-A. thaliana pathosystem identifies targets and processes for the successful control of other powdery mildew fungi. The efficacy of SIGS on powdery mildew fungi makes SIGS an exciting prospect for commercial powdery mildew control. more...
- Published
- 2023
37. Exosomal lncRNAs as diagnostic and therapeutic targets in multiple myeloma
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Hong Yan, Nan Jiang, Xiaoying Li, Chenyang Lin, Fang Wang, Juan Zhang, Lijuan Chen, and Dan Li
- Subjects
lncRNA ,ceRNA ,exosomes ,multiple myeloma ,biological markers ,dsRNA ,Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,RC254-282 - Abstract
Multiple Myeloma (MM) is the second most common malignancy of the hematopoietic system, accounting for approximately 10% of all hematological malignancies, and currently, there is no complete cure. Existing research indicates that exosomal long non-coding RNAs (lncRNAs) play a crucial regulatory role in the initiation and progression of tumors, involving various interactions such as lncRNA-miRNA, lncRNA-mRNA, and lncRNA-RNA binding proteins (RBP). Despite the significant clinical application potential of exosomal lncRNAs, research in this area still faces challenges due to their low abundance and technical limitations. To our knowledge, this review is the first to comprehensively integrate and elucidate the three mechanisms of action of exosomal lncRNAs in MM, and to propose potential therapeutic targets and clinical cases based on these mechanisms. We highlight the latest advancements in the potential of exosomal lncRNAs as biomarkers and therapeutic targets, offering not only a comprehensive analysis of the role of exosomal lncRNAs in MM but also new perspectives and methods for future clinical diagnosis and treatment of multiple myeloma. more...
- Published
- 2025
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38. Non-specific effect of double-stranded RNAs on Egyptian broomrape (Phelipanche aegyptiaca) seed germination
- Author
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Nariman Zainali, Houshang Alizadeh, Hassan Alizadeh, and Philippe Delavault
- Subjects
broomrape ,CYP707A1 ,dsRNA ,germination ,nitrogen ,nucleotides ,Plant culture ,SB1-1110 - Abstract
Obligate root parasitic plants of the Orobanchaceae family exhibit an intricate germination behavior. The host-dependent germination process of these parasites has prompted extensive research into effective control methods. While the effect of biomaterials such as amino acids and microRNA-encoded peptides have been explored, the effect of double-stranded RNAs (dsRNAs) has remained unexamined during the germination process. In this study, we asked whether an exogenously applied dsRNA can inhibit the germination of a root parasite, P. aegyptiaca. To this end, a dsRNA was designed to target the CYP707A1 (dsCYP7), a marker gene of the chemically-dependent germination of broomrape seeds. Application of a concentrated dsCYP7 significantly reduced seed germination. However, two non-germination-specific dsRNAs designed to target mannose-6-phosphate reductase and green fluorescent protein brought about similar inhibitions. Moreover, applying rNTPs and dNTPs, which mimic nitrogenous bases of nucleic acids, also caused a similar reduction in germination, suggesting that the non-specific inhibitory effect of the dsRNAs might arise from their nucleotides. While dsRNA application inhibited seed germination, their non-specific effects may pose a challenge for their application in studying root parasites germination. This underscores the importance of finding solutions to minimize the non-specific effects of dsRNAs to improve the potential of dsRNA as a tool to study and control root parasitic plants. more...
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- 2025
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39. Identification of pathogenicity determinants in ToLCNDV and their RNAi-based knockdown for disease management in Nicotiana benthamiana and tomato plants
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Mehulee Sarkar, Dipinte Gupta, Oinam Washington Singh, Samrat Paul, Ravinder Kumar, Bikash Mandal, and Anirban Roy
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begomovirus ,dsRNA ,hairpin RNAi ,pathogenicity determinant ,tomato leaf curl New Delhi virus ,virus management ,Microbiology ,QR1-502 - Abstract
Begomovirus solanumdelhiense (tomato leaf curl New Delhi virus, ToLCNDV), is member of the genus Begomovirus, family Geminiviridae, is a prolific bipartite whitefly transmitted begomovirus in the Indian sub-continent has a wide host range, including solanaceous, cucurbitaceous and other plants. Recently, dsRNA-mediated non-transgenic approaches have been promising in managing plant viruses. Such an approach could be effective if the pathogenicity determinants of a virus are targeted. In the case of ToLCNDV, viral pathogenicity has been demonstrated with coat protein (AV1), pre-coat protein (AV2), transcription activator protein (AC2) and nuclear shuttle protein (NSP). In the present study, we investigated the involvement of the three RNA silencing suppressor proteins (AV2, AC2, AC4) encoded by ToLCNDV in pathogenicity determinants through transient overexpression and hairpin RNAi-based knockdown assays in Nicotiana benthamiana plants. Further, we showed that the transcripts of AV2, AC2, and AC4 genes can systemically move and express their proteins. Hairpin RNAi constructs targeting each pathogenicity determinant could effectively reduce symptom development and virus titer upon inoculation of ToLCNDV in N. benthamiana plants. Exogenous application of dsRNA individually (dsAV2/dsAC2/dsAC4) or together (cocktail dsRNA: dsCk) against the pathogenicity determinants showed a significant reduction of viral load and reduced severity of disease in plants treated with dsCk followed by dsAC4. The present report reconfirms that the RNA silencing suppressor proteins encoded by DNA-A genomic component of ToLCNDV, can also act as pathogenicity determinants. Further, we demonstrated for the first time that exogenous application of dsRNA targeting those pathogenicity determinants reduces ToLCNDV load and limits symptom development in tomato plants. more...
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- 2024
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40. Accessory viral protein, V, of Newcastle Disease Virus binds dsRNA to facilitate immune evasion
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Deval, Sunny, Nathan, Vaishnavi Senthil, Venkataraman, Sangita, Rao, P. L., Kar, Prajna Parimita, Srivastava, Anand, and Subbiah, Madhuri
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- 2025
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41. Using cationic liposomes as carriers for long dsRNA to trigger an antiviral response in rainbow trout cell lines: Delivery of long dsRNA by cationic liposomes
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Oberhoffner, Shayne J., Daniels, Dominique E., Cooper, Erin, Ijaz, Aizah, Richardson, Starla A., and DeWitte-Orr, Stephanie J.
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- 2025
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42. Gene silencing in the aedine cell lines C6/36 and U4.4 using long double-stranded RNA
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Bodunrin Omokungbe, Alejandra Centurión, Sabrina Stiehler, Antonia Morr, Andreas Vilcinskas, Antje Steinbrink, and Kornelia Hardes
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RNAi ,dsRNA ,Transfection reagents ,Aedes albopictus ,Vector ,Semliki Forest virus ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background RNA interference (RNAi) is a target-specific gene silencing method that can be used to determine gene functions and investigate host–pathogen interactions, as well as facilitating the development of ecofriendly pesticides. Commercially available transfection reagents (TRs) can improve the efficacy of RNAi. However, we currently lack a product and protocol for the transfection of insect cell lines with long double-stranded RNA (dsRNA). Methods We used agarose gel electrophoresis to determine the capacity of eight TRs to form complexes with long dsRNA. A CellTiter-Glo assay was then used to assess the cytotoxicity of the resulting lipoplexes. We also measured the cellular uptake of dsRNA by fluorescence microscopy using the fluorophore Cy3 as a label. Finally, we analyzed the TRs based on their transfection efficacy and compared the RNAi responses of Aedes albopictus C6/36 and U4.4 cells by knocking down an mCherry reporter Semliki Forest virus in both cell lines. Results The TRs from Biontex (K4, Metafectene Pro, and Metafectene SI+) showed the best complexing capacity and the lowest dsRNA:TR ratio needed for complete complex formation. Only HiPerFect was unable to complex the dsRNA completely, even at a ratio of 1:9. Most of the complexes containing mCherry-dsRNA were nontoxic at 2 ng/µL, but Lipofectamine 2000 was toxic at 1 ng/µL in U4.4 cells and at 2 ng/µL in C6/36 cells. The transfection of U4.4 cells with mCherry-dsRNA/TR complexes achieved significant knockdown of the virus reporter. Comparison of the RNAi response in C6/36 and U4.4 cells suggested that C6/36 cells lack the antiviral RNAi response because there was no significant knockdown of the virus reporter in any of the treatments. Conclusions C6/36 cells have an impaired RNAi response as previously reported. This investigation provides valuable information for future RNAi experiments by showing how to mitigate the adverse effects attributed to TRs. This will facilitate the judicious selection of TRs and transfection conditions conducive to RNAi research in mosquitoes. Graphical Abstract more...
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- 2024
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43. Knockdown of DJ-1 Resulted in a Coordinated Activation of the Innate Immune Antiviral Response in HEK293 Cell Line.
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Zohar, Keren and Linial, Michal
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RNA metabolism , *CELL lines , *IMMUNE response , *TYPE I interferons , *MITOCHONDRIAL RNA , *NON-coding RNA - Abstract
PARK7, also known as DJ-1, plays a critical role in protecting cells by functioning as a sensitive oxidation sensor and modulator of antioxidants. DJ-1 acts to maintain mitochondrial function and regulate transcription in response to different stressors. In this study, we showed that cell lines vary based on their antioxidation potential under basal conditions. The transcriptome of HEK293 cells was tested following knockdown (KD) of DJ-1 using siRNAs, which reduced the DJ-1 transcripts to only 12% of the original level. We compared the expression levels of 14k protein-coding transcripts and 4.2k non-coding RNAs relative to cells treated with non-specific siRNAs. Among the coding genes, approximately 200 upregulated differentially expressed genes (DEGs) signified a coordinated antiviral innate immune response. Most genes were associated with the regulation of type 1 interferons (IFN) and the induction of inflammatory cytokines. About a quarter of these genes were also induced in cells treated with non-specific siRNAs that were used as a negative control. Beyond the antiviral-like response, 114 genes were specific to the KD of DJ-1 with enrichment in RNA metabolism and mitochondrial functions. A smaller set of downregulated genes (58 genes) was associated with dysregulation in membrane structure, cell viability, and mitophagy. We propose that the KD DJ-1 perturbation diminishes the protective potency against oxidative stress. Thus, it renders the cells labile and responsive to the dsRNA signal by activating a large number of genes, many of which drive apoptosis, cell death, and inflammatory signatures. The KD of DJ-1 highlights its potency in regulating genes associated with antiviral responses, RNA metabolism, and mitochondrial functions, apparently through alteration in STAT activity and downstream signaling. Given that DJ-1 also acts as an oncogene in metastatic cancers, targeting DJ-1 could be a promising therapeutic strategy where manipulation of the DJ-1 level may reduce cancer cell viability and enhance the efficacy of cancer treatments. [ABSTRACT FROM AUTHOR] more...
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- 2024
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44. Functional canonical RNAi in mice expressing a truncated Dicer isoform and long dsRNA.
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Buccheri, Valeria, Pasulka, Josef, Malik, Radek, Loubalova, Zuzana, Taborska, Eliska, Horvat, Filip, Roos Kulmann, Marcos Iuri, Jenickova, Irena, Prochazka, Jan, Sedlacek, Radislav, and Svoboda, Petr
- Abstract
Canonical RNA interference (RNAi) is sequence-specific mRNA degradation guided by small interfering RNAs (siRNAs) made by RNase III Dicer from long double-stranded RNA (dsRNA). RNAi roles include gene regulation, antiviral immunity or defense against transposable elements. In mammals, RNAi is constrained by Dicer's adaptation to produce another small RNA class—microRNAs. However, a truncated Dicer isoform (ΔHEL1) supporting RNAi exists in mouse oocytes. A homozygous mutation to express only the truncated ΔHEL1 variant causes dysregulation of microRNAs and perinatal lethality in mice. Here, we report the phenotype and canonical RNAi activity in Dicer
ΔHEL1/wt mice, which are viable, show minimal miRNome changes, but their endogenous siRNA levels are an order of magnitude higher. We show that siRNA production in vivo is limited by available dsRNA, but not by Protein kinase R, a dsRNA sensor of innate immunity. dsRNA expression from a transgene yields sufficient siRNA levels to induce efficient RNAi in heart and muscle. DicerΔHEL1/wt mice with enhanced canonical RNAi offer a platform for examining potential and limits of mammalian RNAi in vivo. Synopsis: Modification of the Dicer gene in mice increases siRNA production in somatic organs, but dsRNA abundancy must be strongly increased for achieving efficient RNAi in vivo. Mice with enhanced RNAi will help to evaluate antiviral potential of mammalian RNAi in vivo. Mice tolerate the modification of one allele of Dicer to the highly active ΔHEL1 variant. DicerΔHEL1/wt mice produce higher levels of mirtrons but affect other miRNAs minimally when a full-length Dicer variant is present. DicerΔHEL1/wt mice show an order of magnitude higher siRNA production from long dsRNA but efficient RNAi requires siRNA levels comparable to those of highly abundant miRNAs. Modification of the Dicer gene in mice increases siRNA production in somatic organs, but dsRNA abundancy must be strongly increased for achieving efficient RNAi in vivo. Mice with enhanced RNAi will help to evaluate antiviral potential of mammalian RNAi in vivo. [ABSTRACT FROM AUTHOR] more...- Published
- 2024
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45. Effective target genes for RNA interference‐based management of the cabbage stem flea beetle.
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Cedden, Doga, Güney, Gözde, Debaisieux, Xavier, Scholten, Stefan, Rostás, Michael, and Bucher, Gregor
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FLEA beetles , *RNA interference , *SMALL interfering RNA , *RAPESEED , *NON-target organisms , *COLE crops - Abstract
The cabbage stem flea beetle (CSFB, Psylliodes chrysocephala) is a key pest of oilseed rape. The ban on neonicotinoids in the European Union due to environmental concerns and the emergence of pyrethroid‐resistant populations have made the control of CSFB extremely challenging. In search of a solution, we have recently shown that RNA interference (RNAi) has potential in the management of CSFB. However, the previously tested target genes for RNAi‐mediated pest control (subsequently called target genes) exhibited moderate and slow‐acting lethal effects. In this study, 27 double‐stranded RNAs (dsRNAs) were orally delivered to identify highly effective target genes in CSFB adults by leveraging the findings of a genome‐wide RNAi screen in Tribolium castaneum. Our screen using 500 ng of dsRNA identified 10 moderately effective (> 50% mortality) and 4 highly effective target genes (100% mortality in 8–13 days). The latter mainly included proteasome subunits. Gene expression measurements confirmed target gene silencing and dose–response studies revealed LD50 values as low as ~20 ng in 14 days following a single exposure to dsRNA. Four highly effective dsRNAs also inhibited leaf damage (up to ~75%) and one affected locomotion. The sequences of promising target genes were subjected to in silico target prediction in non‐target organisms, for example, beneficials such as honeybees, to design environmentally friendly dsRNAs. Overall, the study provides valuable insights for the development of dsRNA‐based insecticides against CSFB. [ABSTRACT FROM AUTHOR] more...
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- 2024
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46. Complete genome sequence and phylogenetic analysis of a newly discovered fusagra-like virus infecting Carica papaya in Ecuador.
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Cornejo-Franco, Juan F., Reyes-Proaño, Edison, Alvarez-Quinto, Robert A., Flores, Francisco J., and Quito-Avila, Diego F.
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A new fusagra-like virus infecting papaya (Carica papaya L.) was genetically characterized. The genome of the virus, provisionally named "papaya sticky fruit-associated virus" (PSFaV), is a single molecule of double-stranded RNA, 9,199 nucleotides (nt) in length, containing two discontinuous open reading frames. Pairwise sequence comparisons based on complete RNA-dependent-RNA-polymerase (RdRp) sequences revealed identity of 79.4% and 83.3% at the nt and amino acid (aa) level, respectively, to babaco meleira-like virus (BabMelV), an uncharacterized virus sequence discovered in babaco (Vasconcellea x heilbornii) in Ecuador. Additional plant-associated viruses with sequence identity in the 50% range included papaya meleira virus (PMeV) isolates from Brazil. Phylogenetic analysis based on the amino acid sequences of the capsid protein (CP), RdRp, and CP-RdRp fusion protein genes placed PSFaV in a group within a well-supported clade that shares a recent ancestor with Sclerotium rolfsii RNA virus 2 and Phlebiopsis gigantea mycovirus dsRNA 2, two fungus-associated fusagraviruses. Genomic features and phylogenetic relatedness suggest that PSFaV, along with its closest relative BabMelV, represent a species of novel plant-associated virus classified within the recently established family Fusagraviridae. [ABSTRACT FROM AUTHOR] more...
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- 2024
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47. Metatranscriptomic Sequencing of Sheath Blight-Associated Isolates of Rhizoctonia solani Revealed Multi-Infection by Diverse Groups of RNA Viruses.
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Urzo, Michael Louie R., Guinto, Timothy D., Eusebio-Cope, Ana, Budot, Bernard O., Yanoria, Mary Jeanie T., Jonson, Gilda B., Arakawa, Masao, Kondo, Hideki, and Suzuki, Nobuhiro
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RICE sheath blight , *RNA replicase , *WHOLE genome sequencing , *RHIZOCTONIA solani , *DOUBLE-stranded RNA , *NUCLEOTIDE sequencing - Abstract
Rice sheath blight, caused by the soil-borne fungus Rhizoctonia solani (teleomorph: Thanatephorus cucumeris, Basidiomycota), is one of the most devastating phytopathogenic fungal diseases and causes yield loss. Here, we report on a very high prevalence (100%) of potential virus-associated double-stranded RNA (dsRNA) elements for a collection of 39 fungal strains of R. solani from the rice sheath blight samples from at least four major rice-growing areas in the Philippines and a reference isolate from the International Rice Research Institute, showing different colony phenotypes. Their dsRNA profiles suggested the presence of multiple viral infections among these Philippine R. solani populations. Using next-generation sequencing, the viral sequences of the three representative R. solani strains (Ilo-Rs-6, Tar-Rs-3, and Tar-Rs-5) from different rice-growing areas revealed the presence of at least 36 viruses or virus-like agents, with the Tar-Rs-3 strain harboring the largest number of viruses (at least 20 in total). These mycoviruses or their candidates are believed to have single-stranded RNA or dsRNA genomes and they belong to or are associated with the orders Martellivirales, Hepelivirales, Durnavirales, Cryppavirales, Ourlivirales, and Ghabrivirales based on their coding-complete RNA-dependent RNA polymerase sequences. The complete genome sequences of two novel RNA viruses belonging to the proposed family Phlegiviridae and family Mitoviridae were determined. [ABSTRACT FROM AUTHOR] more...
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- 2024
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48. AMBRA1 promotes dsRNA- and virus-induced apoptosis through interacting with and stabilizing MAVS.
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Yuxia Lin, Changbai Huang, Huixin Gao, Xiaobo Li, Quanshi Lin, Shili Zhou, Zhiting Huo, Yanxia Huang, Chao Liu, and Ping Zhang
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APOPTOSIS , *SEMLIKI Forest virus , *MITOCHONDRIAL proteins , *GENOME editing , *ADAPTOR proteins , *VIRUS diseases - Abstract
Apoptosis is an important cellular response to viral infection. In this study, we identified activating molecule in Beclin1-regulated autophagy protein 1 (AMBRA1) as a positive regulator of apoptosis triggered by double-stranded (ds)RNA. Depletion of AMBRA1 by gene editing significantly reduced dsRNA-induced apoptosis, which was largely restored by trans-complementation of AMBRA1. Mechanistically, AMBRA1 interacts with mitochondrial antiviralsignaling protein (MAVS), a key mitochondrial adaptor in the apoptosis pathway induced by dsRNA and viral infection. Further co-immunoprecipitation analysis demonstrated that the mitochondrial localization of MAVS was essential for their interaction. The impact of AMBRA1 on dsRNA-induced apoptosis relied on the presence of MAVS and caspase-8. AMBRA1 was involved in the stabilization of MAVS through preventing its dsRNA-induced proteasomal degradation. Consistently, AMBRA1 upregulated the apoptosis induced by Semliki Forest virus infection. Taken together, our work illustrated a role for AMBRA1 in virus-induced apoptosis through interacting with and stabilizing MAVS. [ABSTRACT FROM AUTHOR] more...
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- 2024
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49. Recent advances in understanding of the mechanisms of RNA interference in insects.
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Koo, Jinmo and Palli, Subba Reddy
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RNA interference , *SMALL interfering RNA , *INSECTS , *LIVESTOCK productivity , *CROPS - Abstract
We highlight the recent 5 years of research that contributed to our understanding of the mechanisms of RNA interference (RNAi) in insects. Since its first discovery, RNAi has contributed enormously as a reverse genetic tool for functional genomic studies. RNAi is also being used in therapeutics, as well as agricultural crop and livestock production and protection. Yet, for the wider application of RNAi, improvement of its potency and delivery technologies is needed. A mechanistic understanding of every step of RNAi, from cellular uptake of RNAi trigger molecules to targeted mRNA degradation, is key for developing an efficient strategy to improve RNAi technology. Insects provide an excellent model for studying the mechanism of RNAi due to species‐specific variations in RNAi efficiency. This allows us to perform comparative studies in insect species with different RNAi sensitivity. Understanding the mechanisms of RNAi in different insects can lead to the development of better strategies to improve RNAi and its application to manage agriculturally and medically important insects. [ABSTRACT FROM AUTHOR] more...
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- 2024
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50. Current and future trends in the biocontrol of postharvest diseases.
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Li, Xiaojiao, Zeng, Shixian, Wisniewski, Michael, Droby, Samir, Yu, Longfeng, An, Fuquan, Leng, Yan, Wang, Chaowen, Li, Xiaojun, He, Min, Liao, Qinhong, Liu, Jia, Wang, Yong, and Sui, Yuan
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POSTHARVEST diseases , *DOUBLE-stranded RNA , *BIOLOGICAL pest control agents , *BIOLOGY - Abstract
Postharvest diseases of fruits and vegetables cause significant economic losses to producers and marketing firms. Many of these diseases are caused by necrotrophic fungal pathogens that require wounded or injured tissues to establish an infection. Biocontrol of postharvest diseases is an evolving science that has moved from the traditional paradigm of one organism controlling another organism to viewing biocontrol as a system involving the biocontrol agent, the pathogen, the host, the physical environment, and most recently the resident microflora. Thus, the paradigm has shifted from one of simplicity to complexity. The present review provides an overview of how the field of postharvest biocontrol has evolved over the past 40 years, a brief review of the biology of necrotrophic pathogens, the discovery of BCAs, their commercialization, and mechanisms of action. Most importantly, current research on the use of marker-assisted-selection, the fruit microbiome and its relationship to the pathobiome, and the use of double-stranded RNA as a biocontrol strategy is discussed. These latter subjects represent evolving trends in postharvest biocontrol research and suggestions for future research are presented. [ABSTRACT FROM AUTHOR] more...
- Published
- 2024
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