Dataset Information

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

ABSTRACT: Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (Spodoptera litura, Locusta migratoria, Periplaneta americana, and Zophobas atratus) from different orders were characterized using a modified assay method. The results revealed that all insect dsRNA degrading nucleases tested showed high activity in alkaline environments at optimal Mg2+ concentrations and elevated temperatures. We also found that enzymes from different insects varied in terms of their optimal reaction conditions and kinetic parameters. Whole body enzyme activity differed dramatically between insect species, although enzymes with higher substrate affinities (lower Km) were usually balanced by a smaller Vmax to maintain a proper level of degradative capacity. Furthermore, enzyme activities varied significantly between the four tested tissues (whole body, gut, hemolymph, and carcass) of the insect species. All the insects tested showed several hundred-fold higher dsRNA degrading activity in their gut than in other tissues. Reaction environment analysis demonstrated that physiological conditions in the prepared gut fluid and serum of different insects were not necessarily optimal for dsRNA degrading nuclease activity. Our data describe the biochemical characteristics and tissue distributions of dsRNA degrading activities in various insects, not only explaining why oral delivery of dsRNA often produces lower RNAi effects than injection of dsRNA, but also suggesting that dsRNA-degrading activities are regulated by physiological conditions. These results allow for a better understanding of the properties of dsRNA degrading nucleases, and will aid in the development of successful RNAi strategies in insects.

SUBMITTER: Peng Y

PROVIDER: S-EPMC5985623 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

Peng Yingchuan Y Wang Kangxu K Fu Wenxi W Sheng Chengwang C Han Zhaojun Z

Frontiers in physiology 20180528

Double stranded RNAs (dsRNA) degrading nuclease is responsible for the rapid degradation of dsRNA molecules, and thus accounts for variations in RNA interference (RNAi) efficacy among insect species. Here, the biochemical properties and tissue-specific activities of dsRNA degrading nucleases in four insects (<i>Spodoptera litura, Locusta migratoria, Periplaneta americana</i>, and <i>Zophobas atratus</i>) from different orders were characterized using a modified assay method. The results revealed ...[more]

PMID: 29892232

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Project description:Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)--mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs.

Project description:BackgroundThe COVID-19 outbreak and the measures to contain the global pandemic can have an impact on the well-being and mental health status of individuals. Parents of young children are particularly at risk for high levels of parental stress due to the current public health crisis, which can impact parenting behaviors and children's well-being. Although different initial scales have been developed to measure COVID-19-related anxiety, they have not yet been tested sufficiently in parent samples. A brief measure of COVID-19-related anxiety is necessary for both quick assessment in practice and in larger epidemiological studies of parents.ObjectiveThe purpose of this study is to compare the distributions, validities, and reliabilities of four different COVID-19 anxiety scales: Fear of COVID-19 Scale, Coronavirus Anxiety Scale, Pandemic Anxiety Scale, and one subscale of the COVID Stress Scales. Based on the psychometric properties of these scales, we aim to provide recommendations for a brief unidimensional inventory to assess COVID-19-related anxiety among parents.MethodsA cross-sectional web-based survey of 515 German-speaking parents (465 mothers, 90.3%) with at least one child aged 0-6 years was conducted during a 6-week period (June 29 to August 9, 2020). Half of the parents were recruited via Facebook parenting groups, while the other half were recruited through childcare centers. We psychometrically tested 25 items on COVID-19-related anxiety using the framework of classical test theory, including item analysis, correlational analysis of family variables, and exploratory factor analysis. Moreover, an item response theory approach was applied to estimate item discriminations and item difficulties.ResultsBased on the psychometric properties, three items of the Pandemic Anxiety Scale were identified as a single unidimensional factor. The adapted scale demonstrated acceptable internal consistency (α=.79), moderate to high item discrimination, strong positive intercorrelation with two other COVID-19 anxiety scales, and a small positive association with parenting stress. Mothers and fathers did not differ in total scores (t513=-0.79, P=.42).ConclusionsFactor analysis suggests that existing COVID-19-related anxiety scales measure different latent constructs of anxiety. Furthermore, all scales showed only small to moderate correlations with trait health anxiety, suggesting that COVID-19-related anxiety is distinct from general health anxiety. The adapted "disease anxiety" subscale of the Pandemic Anxiety Scale is an economical measure for assessing COVID-19-related anxiety in parents. Directions for future research are outlined.

Dataset Information

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

Publications

Biochemical Comparison of dsRNA Degrading Nucleases in Four Different Insects.

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