A novel method of demonstrating the molecular and functional equivalence between in vitro and plant-produced double-stranded RNA.

A biotechnology-derived corn variety, MON 87411, containing a suppression cassette that expresses an inverted repeat sequence that matches the sequence of western corn rootworm (WCR; Diabrotica virgifera virgifera) has been developed. The expression of the cassette results in the formation of a double-stranded RNA (dsRNA) transcript containing a 240 bp fragment of the WCR Snf7 gene (DvSnf7) that confers resistance to corn rootworm by suppressing levels of DvSnf7 mRNA in WCR after root feeding. Internationally accepted guidelines for the assessment of genetically modified crop products have been developed to ensure that these plants are as safe for food, feed, and environmental release as their non-modified counterparts (Codex, 2009). As part of these assessments MON 87411 must undergo an extensive environmental assessment that requires large quantities of DvSnf7 dsRNA that was produced by in vitro transcription (IVT). To determine if the IVT dsRNA is a suitable surrogate for the MON 87411-produced DvSnf7 dsRNA in regulatory studies, the nucleotide sequence, secondary structure, and functional activity of each were characterized and demonstrated to be comparable. This comprehensive characterization indicates that the IVT DvSnf7 dsRNA is equivalent to the MON 87411-produced DvSnf7 dsRNA and it is a suitable surrogate for regulatory studies.

Assessing the Causal Effects of Adipokines on Uric Acid and Gout: A Two-Sample Mendelian Randomization Study.

Previous observational studies have highlighted associations between adipokines and hyperuricemia, as well as gout, but the causality and direction of these associations are not clear. Therefore, we attempted to assess whether there are causal effects of specific adipokines (such as adiponectin (ADP) and soluble leptin receptors (sOB-R)) on uric acid (UA) or gout in a two-sample Mendelian randomization (MR) analysis, based on summary statistics from large genome-wide association studies. The inverse-variance weighted (IVW) method was performed as the primary analysis. Sensitivity analyses (including MR-Egger regression, weighted median, penalized weighted median, and MR pleiotropy residual sum and outlier methods) were also performed, to ensure reliable results. In the IVW models, no causal effect was found for sOB-R (odds ratios (OR), 1.002; 95% confidence intervals (CI), 0.999-1.004; p = 0.274) on UA, or ADP (OR, 1.198; 95% CI, 0.865-1.659; p = 0.277) or sOB-R (OR, 0.988; 95% CI, 0.940-1.037; p = 0.616) on gout. The results were confirmed in sensitivity analyses. There was no notable directional pleiotropy or heterogeneity. This study suggests that these specific adipokines may not play causal roles in UA or gout development.

The Association of High-Frequency Nut Intake With a Low Risk of Psychological Problems in Female Methamphetamine Users.

Background: Recent years have witnessed a gradual increase in the number of female methamphetamine users. Meanwhile, female methamphetamine users are more likely to have psychological problems than male methamphetamine users. The association between diet and psychological problems have been found among non-methamphetamine user. The present study aims to investigate the relationship between dietary intake frequency and psychological problems in female methamphetamine users. Materials and Methods: A total of 109 female methamphetamine users, collected from a Compulsory Isolated Drug Rehabilitation Centre in northern China, participated in the study. All participants completed the Symptom Checklist 90 (SCL-90) questionnaire to assess psychological status. The relation of dietary intake frequency with the SCL-90 score was tested in partial correlation analysis. Multivariable regression models were used to calculate odds ratios to evaluate the association of dietary intake frequency with psychological problems. Results: Of the current female methamphetamine population, 33 participants were diagnosed with psychological problems using SCL-90. In the terms of dietary intake frequency, the frequency of nut intake in the psychiatric symptom group was significantly lower than that in the asymptomatic group. However, there was no difference in the frequency of other food intakes between the two groups. The frequency of nut intake was negatively correlated with the total score of SCL-90 and 8 different symptom clusters of psychopathologies on SCL-90. Logistic regression analysis indicated that the increased frequency of nut intake was associated with a lower risk of psychological problems. Conclusion: In the female methamphetamine population, increasing the frequency of nut intake may reduce the risk of psychological problems for female methamphetamine users.

Environmental Fate and Dissipation of Applied dsRNA in Soil, Aquatic Systems, and Plants.

Two primary use patterns exist for dsRNA-based products for crop protection: in planta produced dsRNA such as in a genetically engineered (GE) crop; and topically applied dsRNA such as a spray application. To enable effective environmental risk assessments for these products, dsRNA must be successfully measured in relevant environmental compartments (soil, sediment, surface water) to provide information on potential exposure. This perspective reviews results from numerous environmental fate and degradation studies with topically applied unformulated dsRNAs to demonstrate the high lability of these molecules and low potential for persistence in the environment. Additionally, we report on results of a pilot study of topically applied dsRNA on soybean plants demonstrating similar rapid degradation under field conditions. Microbial degradation of nucleic acids in environmental compartments has been shown to be a key driver for this lack of persistence. In fact, the instability of dsRNA in the environment has posed a challenge for the development of commercial topically-applied products. Formulations or other approaches that mitigate environmental degradation may lead to development of commercially successful products but may change the known degradation kinetics of dsRNAs. The formulation of these products and the resultant impacts on the stability of the dsRNA in environmental compartments will need to be addressed using problem formulation and product formulation testing may be required on a case by case basis to ensure an effective risk assessment.

Dissipation of DvSnf7 RNA from Late-Season Maize Tissue in Aquatic Microcosms.

The commercialization of RNA-based agricultural products requires robust ecological risk assessments. Ecological risk is operationally defined as a function of exposure and adverse effects. Information on the environmental fate of RNA-based plant-incorporated protectants is essential to define routes and duration of exposure to potentially sensitive nontarget organisms. Providing these details in problem formulation helps focus the ecological risk assessment on the relevant species of concern. Postharvest plant residue is often considered to be the most significant route of exposure for genetically modified crops to adjacent aquatic environments. Previous studies have shown that DvSnf7 RNA from SmartStax PRO maize dissipates rapidly in both terrestrial and aquatic environments. Although these studies suggest that direct exposure to DvSnf7 RNA is likely to be low, little is known regarding the fate of DvSnf7 RNA produced in plants after entering an aquatic environment. This exposure scenario is relevant to detritivorous aquatic invertebrates that process conditioned maize tissues that enter aquatic environments. To assess potential exposure to shredders, dissipation of DvSnf7 RNA expressed maize tissue was evaluated following immersion in microcosms containing sediment and water. Concentrations of DvSnf7 RNA in the tissue were measured over a duration of 21 d. The DvSnf7 RNA dissipated rapidly from immersed maize tissue and was undetectable in the tissues after 3 d. Concentrations of DvSnf7 RNA found in tissue as well as calculated water column concentrations were below levels known to elicit effects in a highly sensitive surrogate species, supporting the conclusion of minimal risk to aquatic nontarget organisms. Environ Toxicol Chem 2020;39:1032-1040. © 2020 SETAC.

No impact of DvSnf7 RNA on honey bee (Apis mellifera L.) adults and larvae in dietary feeding tests.

The honey bee (Apis mellifera L.) is the most important managed pollinator species worldwide and plays a critical role in the pollination of a diverse range of economically important crops. This species is important to agriculture and historically has been used as a surrogate species for pollinators to evaluate the potential adverse effects for conventional, biological, and microbial pesticides, as well as for genetically engineered plants that produce pesticidal products. As part of the ecological risk assessment of MON 87411 maize, which expresses a double-stranded RNA targeting the Snf7 ortholog (DvSnf7) in western corn rootworm (Diabrotica virgifera virgifera), dietary feeding studies with honey bee larvae and adults were conducted. Based on the mode of action of the DvSnf7 RNA in western corn rootworm, the present studies were designed to be of sufficient duration to evaluate the potential for adverse effects on larval survival and development through emergence and adult survival to a significant portion of the adult stage. Testing was conducted at concentrations of DvSnf7 RNA that greatly exceeded environmentally relevant exposure levels based on expression levels in maize pollen. No adverse effects were observed in either larval or adult honey bees at these high exposure levels, providing a large margin of safety between environmental exposure levels and no-observed-adverse-effect levels.

Quantification of transgene-derived double-stranded RNA in plants using the QuantiGene nucleic acid detection platform.

The expanding use of RNA interference (RNAi) in agricultural biotechnology necessitates tools for characterizing and quantifying double-stranded RNA (dsRNA)-containing transcripts that are expressed in transgenic plants. We sought to detect and quantify such transcripts in transgenic maize lines engineered to control western corn rootworm (Diabrotica virgifera virgifera LeConte) via overexpression of an inverted repeat sequence bearing a portion of the putative corn rootworm orthologue of yeast Snf7 (DvSnf7), an essential component of insect cell receptor sorting. A quantitative assay was developed to detect DvSnf7 sense strand-containing dsRNA transcripts that is based on the QuantiGene Plex 2.0 RNA assay platform from Affymetrix. The QuantiGene assay utilizes cooperative binding of multiple oligonucleotide probes with specificity for the target sequence resulting in exceptionally high assay specificity. Successful implementation of this assay required heat denaturation in the presence of the oligonucleotide probes prior to hybridization, presumably to dissociate primary transcripts carrying the duplex dsRNA structure. The dsRNA assay was validated using a strategy analogous to the rigorous enzyme-linked immunosorbent assay evaluations that are typically performed for foreign proteins expressed in transgenic plants. Validation studies indicated that the assay is sensitive (to 10 pg of dsRNA/g of fresh tissue), highly reproducible, and linear over ∼2.5 logs. The assay was validated using purified RNA from multiple maize tissue types, and studies indicate that the assay is also quantitative in crude tissue lysates. To the best of our knowledge, this is the first report of a non-polymerase chain reaction-based quantitative assay for dsRNA-containing transcripts, based on the use of the QuantiGene technology platform, and will broadly facilitate characterization of dsRNA in biological and environmental samples.