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      Gene silencing by RNA interference in Sarcoptes scabiei: a molecular tool to identify novel therapeutic targets

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          Abstract

          Background

          Scabies is one of the most common and widespread parasitic skin infections globally, affecting a large range of mammals including humans, yet the molecular biology of Sarcoptes scabiei is astonishingly understudied. Research has been hampered primarily due to the difficulty of sampling or culturing these obligatory parasitic mites. A further and major impediment to identify and functionally analyse potential therapeutic targets from the recently emerging molecular databases is the lack of appropriate molecular tools.

          Methods

          We performed standard BLAST based searches of the existing S. scabiei genome databases using sequences of genes described to be involved in RNA interference in Drosophila and the mite model organism Tetranychus urticae. Experimenting with the S. scabiei mu-class glutathione S-transferase ( SsGST-mu1) as a candidate gene we explored the feasibility of gene knockdown in S. scabiei by double-stranded RNA-interference (dsRNAi).

          Results

          We provide here an analysis of the existing S. scabiei draft genomes, confirming the presence of a double stranded RNA (dsRNA) - mediated silencing machinery. We report for the first time experimental gene silencing by RNA interference (RNAi) in S. scabiei. Non-invasive immersion of S. scabiei in dsRNA encoding an S. scabiei glutathione S-transferase mu-class 1 enzyme ( SsGST-mu1) resulted in a 35% reduction in the transcription of the target gene compared to controls.

          Conclusions

          A series of experiments identified the optimal conditions allowing systemic experimental RNAi without detrimental side effects on mite viability. This technique can now be used to address the key questions on the fundamental aspects of mite biology and pathogenesis, and to assess the potential therapeutic benefits of silencing S. scabiei target genes.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13071-017-2226-1) contains supplementary material, which is available to authorized users.

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          Most cited references67

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          Origins and Mechanisms of miRNAs and siRNAs.

          Over the last decade, approximately 20-30 nucleotide RNA molecules have emerged as critical regulators in the expression and function of eukaryotic genomes. Two primary categories of these small RNAs--short interfering RNAs (siRNAs) and microRNAs (miRNAs)--act in both somatic and germline lineages in a broad range of eukaryotic species to regulate endogenous genes and to defend the genome from invasive nucleic acids. Recent advances have revealed unexpected diversity in their biogenesis pathways and the regulatory mechanisms that they access. Our understanding of siRNA- and miRNA-based regulation has direct implications for fundamental biology as well as disease etiology and treatment.
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            Molecular basis for the recognition of primary microRNAs by the Drosha-DGCR8 complex.

            The Drosha-DGCR8 complex initiates microRNA maturation by precise cleavage of the stem loops that are embedded in primary transcripts (pri-miRNAs). Here we propose a model for this process that is based upon evidence from both computational and biochemical analyses. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction. Purified DGCR8, but not Drosha, interacts with pri-miRNAs both directly and specifically, and the flanking ssRNA segments are vital for this binding to occur. Thus, DGCR8 may function as the molecular anchor that measures the distance from the dsRNA-ssRNA junction. Our current study thus facilitates the prediction of novel microRNAs and will assist in the rational design of small hairpin RNAs for RNA interference.
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              The molecular basis of insecticide resistance in mosquitoes.

              Insecticide resistance is an inherited characteristic involving changes in one or more insect gene. The molecular basis of these changes are only now being fully determined, aided by the availability of the Drosophila melanogaster and Anopheles gambiae genome sequences. This paper reviews what is currently known about insecticide resistance conferred by metabolic or target site changes in mosquitoes.
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                Author and article information

                Contributors
                Deepani.Fernando@qimrberghofer.edu.au
                Edward.Marr@ed.ac.uk
                Martha.Zakrzewski@qimrberghofer.edu.au
                Simone.Reynolds@qimrberghofer.edu.au
                stewart.burgess@moredun.ac.uk
                Katja.Fischer@qimrberghofer.edu.au
                Journal
                Parasit Vectors
                Parasit Vectors
                Parasites & Vectors
                BioMed Central (London )
                1756-3305
                10 June 2017
                10 June 2017
                2017
                : 10
                : 289
                Affiliations
                [1 ]ISNI 0000 0001 2294 1395, GRID grid.1049.c, , QIMR Berghofer Medical Research Institute, Infectious Diseases Department, ; 300 Herston Road, Herston, Brisbane, 4006 Australia
                [2 ]ISNI 0000 0000 9320 7537, GRID grid.1003.2, School of Veterinary Sciences, , University of Queensland, ; Gatton, QLD 4343 Australia
                [3 ]ISNI 0000 0001 2186 0964, GRID grid.420013.4, , Parasitology Division, Moredun Research Institute, ; Pentlands Science Park, Bush Loan, Edinburgh, Midlothian, Scotland EH26 0PZ UK
                [4 ]ISNI 0000 0000 9816 8637, GRID grid.11139.3b, Department of Veterinary Pathobiology, Faculty of Veterinary Medicine and Animal Science, , University of Peradeniya, ; Peradeniya, Sri Lanka
                Article
                2226
                10.1186/s13071-017-2226-1
                5466799
                28601087
                3eadffb0-da9c-4113-8ca8-0f245818a2da
                © The Author(s). 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 16 March 2017
                : 1 June 2017
                Funding
                Funded by: Australian Government National Health and Medical Research Council
                Award ID: Project Grant ID 1067192
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/501100000923, Australian Research Council;
                Award ID: FT130101875
                Award Recipient :
                Funded by: QIMR Berghofer MRI
                Award ID: International PhD Scholarship
                Award Recipient :
                Categories
                Research
                Custom metadata
                © The Author(s) 2017

                Parasitology
                scabies mites,sarcoptes scabiei,rna interference,gene knockdown,glutathione s-transferase

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