An RNA Interference Tool to Silence Genes in Sarcoptes scabiei Eggs

In a quest for new interventions against scabies—a highly significant skin disease of mammals, caused by a parasitic mite Sarcoptes scabiei—we are focusing on finding new intervention targets. RNA interference (RNAi) could be an efficient functional genomics approach to identify such targets. The RNAi pathway is present in S. scabiei and operational in the female adult mite, but other developmental stages have not been assessed. Identifying potential intervention targets in the egg stage is particularly important because current treatments do not kill this latter stage. Here, we established an RNAi tool to silence single-copy genes in S. scabiei eggs. Using sodium hypochlorite pre-treatment, we succeeded in rendering the eggshell permeable to dsRNA without affecting larval hatching. We optimised the treatment of eggs with gene-specific dsRNAs to three single-copy target genes (designated Ss-Cof, Ss-Ddp, and Ss-Nan) which significantly and repeatedly suppressed transcription by ~66.6%, 74.3%, and 84.1%, respectively. Although no phenotypic alterations were detected in dsRNA-treated eggs for Ss-Cof and Ss-Nan, the silencing of Ss-Ddp resulted in a 38% reduction of larval hatching. This RNAi method is expected to provide a useful tool for larger-scale functional genomic investigations for the identification of essential genes as potential drug targets.

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A Computational Approach to Design Potential siRNA Molecules as a Prospective Tool for Silencing Nucleocapsid Phosphoprotein and Surface Glycoprotein Gene of SARS-CoV-2

10.1101/2020.04.10.036335 ◽

2020 ◽

Cited By ~ 1

Author(s):

Umar Faruq Chowdhury ◽

Mohammad Umer Sharif Shohan ◽

Kazi Injamamul Hoque ◽

Mirza Ashikul Beg ◽

Mohammad Kawsar Sharif Siam ◽

...

Keyword(s):

Free Energy ◽

Rna Interference ◽

Target Genes ◽

Antiviral Treatment ◽

Gc Content ◽

Surface Glycoprotein ◽

Rnai Pathway ◽

Viral Gene ◽

Conserved Sequence ◽

Glycoprotein Gene

ABSTRACTAn outbreak, caused by a RNA virus, SARS-CoV-2 named COVID-19 has become pandemic with a magnitude which is daunting to all public health institutions in the absence of specific antiviral treatment. Surface glycoprotein and nucleocapsid phosphoprotein are two important proteins of this virus facilitating its entry into host cell and genome replication. Small interfering RNA (siRNA) is a prospective tool of the RNA interference (RNAi) pathway for the control of human viral infections by suppressing viral gene expression through hybridization and neutralization of target complementary mRNA. So, in this study, the power of RNA interference technology was harnessed to develop siRNA molecules against specific target genes namely, nucleocapsid phosphoprotein gene and surface glycoprotein gene. Conserved sequence from 139 SARS-CoV-2 strains from around the globe was collected to construct 78 siRNA that can inactivate nucleocapsid phosphoprotein and surface glycoprotein genes. Finally, based on GC content, free energy of folding, free energy of binding, melting temperature and efficacy prediction process 8 siRNA molecules were selected which are proposed to exerts the best action. These predicted siRNAs should effectively silence the genes of SARS-CoV-2 during siRNA mediated treatment assisting in the response against SARS-CoV-2

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Cellular Fitness Phenotypes of Cancer Target Genes from Oncobiology to Cancer Therapeutics

Cells ◽

10.3390/cells10020433 ◽

2021 ◽

Vol 10 (2) ◽

pp. 433

Author(s):

Bijesh George ◽

P. Mukundan Pillai ◽

Aswathy Mary Paul ◽

Revikumar Amjesh ◽

Kim Leitzel ◽

...

Keyword(s):

Drug Targets ◽

Target Genes ◽

Human Cancer ◽

Cancer Therapeutics ◽

Survival Duration ◽

Cancer Drug ◽

Targeted Therapeutics ◽

Cellular Targets ◽

Human Cancer Cells ◽

Cancer Types

To define the growing significance of cellular targets and/or effectors of cancer drugs, we examined the fitness dependency of cellular targets and effectors of cancer drug targets across human cancer cells from 19 cancer types. We observed that the deletion of 35 out of 47 cellular effectors and/or targets of oncology drugs did not result in the expected loss of cell fitness in appropriate cancer types for which drugs targeting or utilizing these molecules for their actions were approved. Additionally, our analysis recognized 43 cellular molecules as fitness genes in several cancer types in which these drugs were not approved, and thus, providing clues for repurposing certain approved oncology drugs in such cancer types. For example, we found a widespread upregulation and fitness dependency of several components of the mevalonate and purine biosynthesis pathways (currently targeted by bisphosphonates, statins, and pemetrexed in certain cancers) and an association between the overexpression of these molecules and reduction in the overall survival duration of patients with breast and other hard-to-treat cancers, for which such drugs are not approved. In brief, the present analysis raised cautions about off-target and undesirable effects of certain oncology drugs in a subset of cancers where the intended cellular effectors of drug might not be good fitness genes and that this study offers a potential rationale for repurposing certain approved oncology drugs for targeted therapeutics in additional cancer types.

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Identifying Personalized Metabolic Signatures in Breast Cancer

Metabolites ◽

10.3390/metabo11010020 ◽

2020 ◽

Vol 11 (1) ◽

pp. 20

Author(s):

Priyanka Baloni ◽

Wikum Dinalankara ◽

John C. Earls ◽

Theo A. Knijnenburg ◽

Donald Geman ◽

...

Keyword(s):

Drug Targets ◽

Metabolic Networks ◽

Target Genes ◽

Enrichment Analysis ◽

Metabolic Reprogramming ◽

The Cancer Genome Atlas ◽

Estrogen Metabolism ◽

Acid Oxidation ◽

A Genome ◽

Context Specific

Cancer cells are adept at reprogramming energy metabolism, and the precise manifestation of this metabolic reprogramming exhibits heterogeneity across individuals (and from cell to cell). In this study, we analyzed the metabolic differences between interpersonal heterogeneous cancer phenotypes. We used divergence analysis on gene expression data of 1156 breast normal and tumor samples from The Cancer Genome Atlas (TCGA) and integrated this information with a genome-scale reconstruction of human metabolism to generate personalized, context-specific metabolic networks. Using this approach, we classified the samples into four distinct groups based on their metabolic profiles. Enrichment analysis of the subsystems indicated that amino acid metabolism, fatty acid oxidation, citric acid cycle, androgen and estrogen metabolism, and reactive oxygen species (ROS) detoxification distinguished these four groups. Additionally, we developed a workflow to identify potential drugs that can selectively target genes associated with the reactions of interest. MG-132 (a proteasome inhibitor) and OSU-03012 (a celecoxib derivative) were the top-ranking drugs identified from our analysis and known to have anti-tumor activity. Our approach has the potential to provide mechanistic insights into cancer-specific metabolic dependencies, ultimately enabling the identification of potential drug targets for each patient independently, contributing to a rational personalized medicine approach.

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RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽

10.3390/insects12010041 ◽

2021 ◽

Vol 12 (1) ◽

pp. 41

Author(s):

Ya-Wen Chang ◽

Yu-Cheng Wang ◽

Xiao-Xiang Zhang ◽

Junaid Iqbal ◽

Yu-Zhou Du

Keyword(s):

Rna Interference ◽

Expression Analysis ◽

Target Genes ◽

Fluorescent Protein ◽

Control Strategies ◽

Vacuolar Atpase ◽

Invasive Pest ◽

Liriomyza Trifolii ◽

Horticultural Crops ◽

Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

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RNAi-Mediated Management of Whitefly Bemisia tabaci by Oral Delivery of Double-stranded RNAs

Proceedings ◽

10.3390/proceedings2019036011 ◽

2019 ◽

Vol 36 (1) ◽

pp. 11

Author(s):

Jain ◽

Robinson ◽

Mitter

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Bemisia Tabaci ◽

Oral Delivery ◽

Target Genes ◽

Insect Pests ◽

Control Strategies ◽

Plant Viruses ◽

Potential Game ◽

Viable Approach

The whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) is a significant global pest of economically important vegetable, fibre, and ornamental crops. Whiteflies directly damage the plants by piercing and sucking essential nutrients, indirectly through honeydew secretion and by transmitting more than 200 plant viruses that cause millions of dollars in produce losses per year. Whitefly management is mostly reliant on the heavy use of chemical insecticides. However, this ultimately leads to increasing resistance development, detrimental effects on beneficial insects and biomagnification of ecologically harmful chemicals in the environment. Responding to consumer demands for more selective, less toxic, non-GM insect control strategies, RNA interference (RNAi) has emerged as a potential game-changing solution. The RNA interference (RNAi) is a homology-dependent mechanism of gene silencing that represents a feasible and sustainable technology for the management of insect pests. In the present study, twenty-two whitefly genes were selected based on their essential function in the insect and tested in artificial diet bioassays for mortality and gene silencing efficacy. The nine most effective dsRNA constructs showed moderate-to-high whitefly mortality as compared to negative controls six days post-feeding. qPCR analysis further demonstrated significant knockdown of target gene mRNA expression. Additionally, uptake and spread of fluorescently labelled dsRNA was evident beyond the midgut of the whitefly supporting the systemic spreading of RNAi effectors. Taken together, the oral delivery of dsRNA shows effective RNAi mediated gene silencing of target genes and offers a viable approach for the development of dsRNA biopesticides against hemipteran pest.

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Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

Scientific Reports ◽

10.1038/s41598-020-75682-6 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Nicolas Bensoussan ◽

Sameer Dixit ◽

Midori Tabara ◽

David Letwin ◽

Maja Milojevic ◽

...

Keyword(s):

Rna Interference ◽

Tetranychus Urticae ◽

Pest Control ◽

Spider Mite ◽

26S Proteasome ◽

Rnai Pathway ◽

Rnai Machinery ◽

Two Spotted Spider Mite ◽

Species Specific

Abstract Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.

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An atlas of dynamic chromatin landscapes in mouse fetal development

Nature ◽

10.1038/s41586-020-2093-3 ◽

2020 ◽

Vol 583 (7818) ◽

pp. 744-751 ◽

Cited By ~ 13

Author(s):

David U. Gorkin ◽

Iros Barozzi ◽

Yuan Zhao ◽

Yanxiao Zhang ◽

Hui Huang ◽

...

Keyword(s):

Fetal Development ◽

Target Genes ◽

Developmental Stages ◽

Chromatin Accessibility ◽

Chromatin State ◽

Mammalian Development ◽

Mouse Fetus ◽

Chromatin Dynamics ◽

Mouse Tissues ◽

Genomic Resource

AbstractThe Encyclopedia of DNA Elements (ENCODE) project has established a genomic resource for mammalian development, profiling a diverse panel of mouse tissues at 8 developmental stages from 10.5 days after conception until birth, including transcriptomes, methylomes and chromatin states. Here we systematically examined the state and accessibility of chromatin in the developing mouse fetus. In total we performed 1,128 chromatin immunoprecipitation with sequencing (ChIP–seq) assays for histone modifications and 132 assay for transposase-accessible chromatin using sequencing (ATAC–seq) assays for chromatin accessibility across 72 distinct tissue-stages. We used integrative analysis to develop a unified set of chromatin state annotations, infer the identities of dynamic enhancers and key transcriptional regulators, and characterize the relationship between chromatin state and accessibility during developmental gene regulation. We also leveraged these data to link enhancers to putative target genes and demonstrate tissue-specific enrichments of sequence variants associated with disease in humans. The mouse ENCODE data sets provide a compendium of resources for biomedical researchers and achieve, to our knowledge, the most comprehensive view of chromatin dynamics during mammalian fetal development to date.

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Bottlebrush-architectured poly(ethylene glycol) as an efficient vector for RNA interference in vivo

Science Advances ◽

10.1126/sciadv.aav9322 ◽

2019 ◽

Vol 5 (2) ◽

pp. eaav9322 ◽

Cited By ~ 13

Author(s):

Dali Wang ◽

Jiaqi Lin ◽

Fei Jia ◽

Xuyu Tan ◽

Yuyan Wang ◽

...

Keyword(s):

Rna Interference ◽

Ethylene Glycol ◽

Target Genes ◽

Area Under The Curve ◽

Fold Increase ◽

Small Interfering Rnas ◽

Poly Ethylene Glycol ◽

Linear Poly ◽

Poly Ethylene

Nonhepatic delivery of small interfering RNAs (siRNAs) remains a challenge for development of RNA interference–based therapeutics. We report a noncationic vector wherein linear poly(ethylene glycol) (PEG), a polymer generally considered as inert and safe biologically but ineffective as a vector, is transformed into a bottlebrush architecture. This topology provides covalently embedded siRNA with augmented nuclease stability and cellular uptake. Consisting almost entirely of PEG and siRNA, the conjugates exhibit a ~25-fold increase in blood elimination half-life and a ~19-fold increase in the area under the curve compared with unmodified siRNA. The improved pharmacokinetics results in greater tumor uptake and diminished liver capture. Despite the structural simplicity these conjugates efficiently knock down target genes in vivo without apparent toxic and immunogenic reactions. Given the benign biological nature of PEG and its widespread precedence in biopharmaceuticals, we anticipate the brush polymer–based technology to have a significant impact on siRNA therapeutics.

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Comprehensive Transcriptome of the Maize Stalk Borer, Busseola fusca, from Multiple Tissue Types, Developmental Stages, and Parasitoid Wasp Exposures

Genome Biology and Evolution ◽

10.1093/gbe/evaa195 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2554-2560

Author(s):

Keyword(s):

Target Genes ◽

Developmental Stages ◽

Management Strategies ◽

Busseola Fusca ◽

Parasitoid Wasp ◽

Sub Saharan Africa ◽

Biological Research ◽

Parasitoid Wasps ◽

Pest Species ◽

Stalk Borer

Abstract Busseola fusca (Fuller) (Lepidoptera: Noctuidae), the maize stalk borer, is a widespread crop pest in sub-Saharan Africa that has been the focus of biological research and intensive management strategies. Here, we present a comprehensive annotated transcriptome of B. fusca (originally collected in the Western Province of Kenya) based on ten pooled libraries including a wide array of developmental stages, tissue types, and exposures to parasitoid wasps. Parasitoid wasps have been used as a form of biocontrol to try and reduce crop losses with variable success, in part due to differential infectivities and immune responses among wasps and hosts. We identified a number of loci of interest for pest management, including genes potentially involved in chemoreception, immunity, and response to insecticides. The comprehensive sampling design used expands our current understanding of the transcriptome of this species and deepens the list of potential target genes for future crop loss mitigation, in addition to highlighting candidate loci for differential expression and functional genetic analyses in this important pest species.

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RNA interference as a gene silencing tool to controlTuta absolutain tomato (Solanum lycopersicum)

PeerJ ◽

10.7717/peerj.2673 ◽

2016 ◽

Vol 4 ◽

pp. e2673 ◽

Cited By ~ 21

Author(s):

Roberto A. Camargo ◽

Guilherme O. Barbosa ◽

Isabella Presotto Possignolo ◽

Lazaro E. P. Peres ◽

Eric Lam ◽

...

Keyword(s):

Rna Interference ◽

Gene Silencing ◽

Target Gene ◽

Target Genes ◽

Larval Mortality ◽

Tuta Absoluta ◽

Specific Gene ◽

Gene Transcript ◽

Pest Species ◽

In Planta

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer (Tuta absoluta), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-AandArginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet forT. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on “in planta-induced transient gene silencing” (PITGS), a well-established method for silencing plant genes, used here for the first time to deliverin planta-transcribed dsRNA to target insect genes.Tuta absolutalarvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic ‘Micro-Tom’ tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage byT. absolutain these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.

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