IsomiR_Window: a system for analyzing small-RNA-seq data in an integrative and user-friendly manner.

BACKGROUND: IsomiRs are miRNA variants that vary in length and/or sequence when compared to their canonical forms. These variants display differences in length and/or sequence, including additions or deletions of one or more nucleotides (nts) at the 5' and/or 3' end, internal editings or untemplated 3' end additions. Most available tools for small RNA-seq data analysis do not allow the identification of isomiRs and often require advanced knowledge of bioinformatics. To overcome this, we have developed IsomiR Window, a platform that supports the systematic identification, quantification and functional exploration of isomiR expression in small RNA-seq datasets, accessible to users with no computational skills. METHODS: IsomiR Window enables the discovery of isomiRs and identification of all annotated non-coding RNAs in RNA-seq datasets from animals and plants. It comprises two main components: the IsomiR Window pipeline for data processing; and the IsomiR Window Browser interface. It integrates over ten third-party softwares for the analysis of small-RNA-seq data and holds a new algorithm that allows the detection of all possible types of isomiRs. These include 3' and 5'end isomiRs, 3' end tailings, isomiRs with single nucleotide polymorphisms (SNPs) or potential RNA editings, as well as all possible fuzzy combinations. IsomiR Window includes all required databases for analysis and annotation, and is freely distributed as a Linux virtual machine, including all required software. RESULTS: IsomiR Window processes several datasets in an automated manner, without restrictions of input file size. It generates high quality interactive figures and tables which can be exported into different formats. The performance of isomiR detection and quantification was assessed using simulated small-RNA-seq data. For correctly mapped reads, it identified different types of isomiRs with high confidence and 100% accuracy. The analysis of a small RNA-seq data from Basal Cell Carcinomas (BCCs) using isomiR Window confirmed that miR-183-5p is up-regulated in Nodular BCCs, but revealed that this effect was predominantly due to a novel 5'end variant. This variant displays a different seed region motif and 1756 isoform-exclusive mRNA targets that are significantly associated with disease pathways, underscoring the biological relevance of isomiR-focused analysis. IsomiR Window is available at https://isomir.fc.ul.pt/ .

Comparative analysis of completeness of death registration, adult mortality and life expectancy at birth in Brazil at the subnational level.

BACKGROUND: Estimates of completeness of death registration are crucial to produce estimates of life tables and population projections and to estimate the burden of disease. They are an important step in assessing the quality of data. In the case of subnational data analysis in Brazil, it is important to consider spatial and temporal variation in the quality of mortality data. There are two main sources of data quality evaluation in Brazil, but there are few comparative studies and how they evolve over time. The aim of the paper is to compare and discuss alternative estimates of completeness of death registration, adult mortality (45q15) and life expectancy estimates produced by the National Statistics Office (IBGE), Institute for Health Metrics and Evaluation (IHME), and estimates presented in Queiroz et al. (2017) and Schmertmann and Gonzaga (2018), for 1980 and 2010. METHODS: We provide a descriptive and comparative analysis of aforementioned estimates from four (4) sources of estimates at subnational level (26 states and one Federal District) in Brazil from two different points in time. RESULTS: We found significant differences in estimates that affect both levels and trends of completeness of adult mortality in Brazil and states. IHME and Queiroz et al. (2017) estimates converge by 2010, but there are large differences when compared to estimates from the National Statistics Office (IBGE). Larger differences are observed for less developed states. We have showed that the quality of mortality data in Brazil has improved steadily overtime, but with large regional variations. However, we have observed that IBGE estimates show the lowest levels of completeness for the Northern of the country compared to other estimates. Choice of methods and approaches might lead to very unexpected results. CONCLUSION: We produced a detailed comparative analysis of estimates of completeness of death registration from different sources and discuss the main results and possible explanations for these differences. We have also showed that new improved methods are still needed to study adult mortality in less developed countries and at a subnational level. More comparative studies are important in order to improve quality of estimates in Brazil.

Survival and development of bullfrog tadpoles in microcosms treated with abamectin.

As compared to other aquatic taxonomic groups, few studies have been conducted so far evaluating the potential risks of pesticides to amphibians. Furthermore, most existing studies with amphibians consist of acute laboratory toxicity tests that mostly only evaluated single peak pesticide exposure. In the present study, potential chronic effects of single and repeated abamectin applications on the survival and development of L. catesbeianus tadpoles under (semi-) field conditions were evaluated. To this end, tadpoles were housed in microcosms treated with single or repeated applications of abamectin (as the commercial product Vertimec® 18 EC). The single application level corresponded to the previously established laboratory 96 h LC50 of the test organism, whereas the repeated application was based on abamectin concentrations likely to occur in runoff water from agricultural areas where Vertimec® 18 EC is applied. Under semi-field conditions, toxicity after the single application was greater than would be expected from the laboratory toxicity value. Although the repeated application did not exert direct effects on tadpole survival, the observed delay in development may have pronounced effect on the fitness and survival of anuran populations in edge-of-field water bodies.

Acute and chronic sensitivity, avoidance behavior and sensitive life stages of bullfrog tadpoles exposed to the biopesticide abamectin.

As compared to other aquatic organism groups, relatively few studies have been conducted so far evaluating the toxicity of pesticides to amphibians. This may at least partly be due to the fact that regulations for registering pesticides usually do not require testing amphibians. The sensitivity of amphibians is generally considered to be covered by that based on toxicity tests with other aquatic organisms (e.g. fish) although the impact of a pesticide on amphibians may be very different. In the present study, acute and chronic laboratory tests were conducted to evaluate the acute and chronic toxicity of abamectin (as Vertimec(®) 18EC) to bullfrog (Lithobates catesbeianus) tadpoles. Acute tests were conducted at two tadpole stages (Gosner stage 21G and 25G) and avoidance tests were also conducted with stage Gosner stage 21G tadpoles. Calculated acute toxicity values were greater than those reported for standard fish test species, hence supporting the use of fish toxicity data as surrogates for amphibians in acute risk assessments. Given the limited number and extent of available amphibian toxicity studies, however, research needs to increase our understanding of pesticide toxicity to amphibians are discussed.

Preference and avoidance responses by tadpoles: the fungicide pyrimethanil as a habitat disturber.

Tadpoles of two amphibian species, the neotropical anuran Leptodactylus latrans and the North American bullfrog Lithobates catesbeianus, were used in experiments to assess their preferred spatial distribution along habitat gradients and, thus, to what extent contamination by the fungicide pyrimethanil could trigger active spatial avoidance. The tadpoles were tested in a non-confined multi-compartment static system with a pyrimethanil contamination gradient through which organisms could move freely. Two samples, with and without (reference) pyrimethanil contamination, taken from outdoor mesocosms, were assayed. Tadpoles showed to be able to detect and move to the most favorable environment by preferring compartments containing reference mesocosm water. Pyrimethanil concentrations from 0.2 to 1.4 mg L(-1) were below lethal levels, but acted as habitat disturber since spatial avoidance was triggered. Avoiders of L. latrans reached almost 50 % at 1.4 mg L(-1). The present data reinforces the hypothesis regarding the risk of plant protection products to act, not only as toxicants, but also as habitat disturber, potentially leading to avoidance-driven population decline of amphibians.

Field and laboratory studies to assess the effects of Vertimec(®) 18EC on Daphnia similis.

The present study aimed to evaluate the interactions of the pesticide Vertimec(®) 18EC in aquatic ecosystems. In this respect, soil plots were contaminated with Vertimec(®) 18EC at the concentration indicated for strawberry crops (0.125L of solution m(-2)). After the contamination, torrential rainfall was simulated and the surface runoff was collected and transferred to mesocosm tanks in five treatments, run in triplicate: (1) control-C; (2) runoff from an uncontaminated plot-UR; (3) runoff from the plot contaminated with Vertimec(®) 18EC-CR; (4) direct application of Vertimec(®) 18EC in the water-V and (5) water samples gathered randomly to verify whether there was contamination between the mesocosms-RS. Water samples from these tanks were also submitted to ecotoxicological tests with Daphnia similis and analyses to evaluate the limnological characteristics, in five collection periods over 10 days (240h). Physical and chemical differences were observed in the water samples, mainly related to increased turbidity, suspended solids and nutrients (nitrogen and phosphate forms). Acute toxicity was observed for the direct application treatment for the entire experimental period, and in some periods for the CR treatment (from 48h to 168h). The results obtained suggest that the pesticide did not fully degrade during the study period (10 days) in the direct application treatment, demonstrating that the presence of other substances in the commercial formulation contribute to the maintenance of toxicity. This represents a potential risk for aquatic ecosystems in areas adjacent to where the chemical is applied.

Toxicity of Vertimec® 18 EC (active ingredient abamectin) to the neotropical cladoceran Ceriodaphnia silvestrii.

The aim of the present study was to evaluate the toxicity of abamectin to the neotropical cladoceran Ceriodaphnia silvestrii. To this end, acute and chronic bioassays were conducted with the commercial formulation Vertimec® 18 EC. In addition, the toxicity of water samples taken from a microcosm experiment evaluating the effects of a single application (144µga.i./L) and two applications (2×36µga.i./L) of Vertimec® 18 EC, in the presence or absence of a tadpole species (Lithobates catesbeianus), was also assessed. The acute LC50-48h for immobilization was 1.47µga.i./L and chronic NOEC-8d for survival and fertility (number of neonates per female) were 169 and 84nga.i./L, respectively. Irrespective of the presence of tadpoles, water samples from the microcosms applied with the single concentration of 144µga.i./L remained toxic until the end of the experiment, even when samples were diluted 32 times with culture medium. Water in the repeated pesticide treatment showed a similar toxic response after both applications. Toxicity of water samples from the microcosms was lower than that expected based on the generated LC50 values, which is explained by a potential reduced bioavailability of the test compound resulting from absorbance to organic material. Potential side-effects on C. silvestrii related with the use of Vertimec® 18 EC in Brazil and the suitability of this species for tropical toxicity testing are discussed.