piRNA pathway evolution beyond gonad context: Perspectives from apicomplexa and trypanosomatids.

piRNAs function as genome defense mechanisms against transposable elements insertions within germ line cells. Recent studies have unraveled that piRNA pathways are not limited to germ cells as initially reckoned, but are instead also found in non-gonadal somatic contexts. Moreover, these pathways have also been reported in bacteria, mollusks and arthropods, associated with safeguard of genomes against transposable elements, regulation of gene expression and with direct consequences in axon regeneration and memory formation. In this Perspective we draw attention to early branching parasitic protozoa, whose genome preservation is an essential function as in late eukaryotes. However, little is known about the defense mechanisms of these genomes. We and others have described the presence of putative PIWI-related machinery members in protozoan parasites. We have described the presence of a PIWI-like protein in Trypanosoma cruzi, bound to small non-coding RNAs (sRNAs) as cargo of secreted extracellular vesicles relevant in intercellular communication and host infection. Herein, we put forward the presence of members related to Argonaute pathways in both Trypanosoma cruzi and Toxoplasma gondii. The presence of PIWI-like machinery in Trypansomatids and Apicomplexa, respectively, could be evidence of an ancestral piRNA machinery that evolved to become more sophisticated and complex in multicellular eukaryotes. We propose a model in which ancient PIWI proteins were expressed broadly and had functions independent of germline maintenance. A better understanding of current and ancestral PIWI/piRNAs will be relevant to better understand key mechanisms of genome integrity conservation during cell cycle progression and modulation of host defense mechanisms by protozoan parasites.

Hsa-miR-183-5p Modulates Cell Adhesion by Repression of ITGB1 Expression in Prostate Cancer.

Prostate cancer is a major health problem worldwide. MiR-183 is an oncomiR and a candidate biomarker in prostate cancer, affecting various pathways responsible for disease initiation and progression. We sought to discover the most relevant processes controlled by miR-183 through an unbiased transcriptomic approach using prostate cell lines and patient tissues to identify miR-183 responsive genes and pathways. Gain of function experiments, reporter gene assays, and transcript and protein measurements were conducted to validate predicted functional effects and protein mediators. A total of 135 candidate miR-183 target genes overrepresenting cell adhesion terms were inferred from the integrated transcriptomic analysis. Cell attachment, spreading assays and focal adhesion quantification of miR-183-overexpressing cells confirmed the predicted reduction in cell adhesion. ITGB1 was validated as a major target of repression by miR-183 as well as a mediator of cell adhesion in response to miR-183. The reporter gene assay and PAR-CLIP read mapping suggest that ITGB1 may be a direct target of miR-183. The negative correlation between miR-183 and ITGB1 expression in prostate cancer cohorts supports their interaction in the clinical set. Overall, cell adhesion was uncovered as a major pathway controlled by miR-183 in prostate cancer, and ITGB1 was identified as a relevant mediator of this effect.

Lost and Found: Piwi and Argonaute Pathways in Flatworms.

Platyhelminthes comprise one of the major phyla of invertebrate animals, inhabiting a wide range of ecosystems, and one of the most successful in adapting to parasitic life. Small non-coding RNAs have been implicated in regulating complex developmental transitions in model parasitic species. Notably, parasitic flatworms have lost Piwi RNA pathways but gained a novel Argonaute gene. Herein, we analyzed, contrasted and compared the conservation of small RNA pathways among several free-living species (a paraphyletic group traditionally known as 'turbellarians') and parasitic species (organized in the monophyletic clade Neodermata) to disentangle possible adaptations during the transition to parasitism. Our findings showed that complete miRNA and RNAi pathways are present in all analyzed free-living flatworms. Remarkably, whilst all 'turbellarians' have Piwi proteins, these were lost in parasitic Neodermantans. Moreover, two clusters of Piwi class Argonaute genes are present in all 'turbellarians'. Interestingly, we identified a divergent Piwi class Argonaute in free living flatworms exclusively, which we named 'Fliwi'. In addition, other key proteins of the Piwi pathways were conserved in 'turbellarians', while none of them were detected in Neodermatans. Besides Piwi and the canonical Argonaute proteins, a flatworm-specific class of Argonautes (FL-Ago) was identified in the analyzed species confirming its ancestrallity to all Platyhelminthes. Remarkably, this clade was expanded in parasitic Neodermatans, but not in free-living species. These phyla-specific Argonautes showed lower sequence conservation compared to other Argonaute proteins, suggesting that they might have been subjected to high evolutionary rates. However, key residues involved in the interaction with the small RNA and mRNA cleavage in the canonical Argonautes were more conserved in the FL-Agos than in the Piwi Argonautes. Whether this is related to specialized functions and adaptations to parasitism in Neodermatans remains unclear. In conclusion, differences detected in gene conservation, sequence and structure of the Argonaute family suggest tentative biological and evolutionary diversifications that are unique to Platyhelminthes. The remarkable divergencies in the small RNA pathways between free-living and parasitic flatworms indicate that they may have been involved in the adaptation to parasitism of Neodermatans.

The canonical RdDM pathway mediates the control of seed germination timing under salinity.

Plants respond to adverse environmental cues by adjusting a wide variety of processes through highly regulated mechanisms to maintain plant homeostasis for survival. As a result of the sessile nature of plants, their response, adjustment and adaptation to the changing environment is intimately coordinated with their developmental programs through the crosstalk of regulatory networks. Germination is a critical process in the plant life cycle, and thus plants have evolved various strategies to control the timing of germination according to their local environment. The mechanisms involved in these adjustment responses are largely unknown, however. Here, we report that mutations in core elements of canonical RNA-directed DNA methylation (RdDM) affect the germination and post-germination growth of Arabidopsis seeds grown under salinity stress. Transcriptomic and whole-genome bisulfite sequencing (WGBS) analyses support the involvement of this pathway in the control of germination timing and post-germination growth under salinity stress by preventing the transcriptional activation of genes implicated in these processes. Subsequent transcriptional effects on genes that function in relation to these developmental events support this conclusion.

Factors Modulating Captures of Gravid Aedes aegypti Females.

To improve detection and assessment of Aedes aegypti abundance, we investigated whether microhabitat factors of the location of autocidal gravid ovitraps (AGO traps) influenced captures of gravid females in 2 locations in southern Puerto Rico. One location had been under vector control for several years using mass AGO trapping (intervention site), where Ae. aegypti abundance was several times lower than in the other study site without mosquito control (nonintervention site). We observed 10 environmental factors describing trap microhabitat location, and monitored water volume and minimum, maximum, and average temperature in AGO traps. Air temperature, relative humidity, and rainfall were recorded at each site. We conducted a hot-spot analysis of AGO traps to understand whether trap captures were influenced by the local abundance of mosquitoes rather than or in addition to trap microhabitat factors. AGO traps were classified using a 2-step cluster analysis based on attributes of trap microhabitats, water temperature, and water volume. Captures of female Ae. aegypti in each cluster per site were compared between resulting clusters to determine whether trap microhabitat factors defining the clusters were associated with trap captures. Trap captures in both study sites were mostly correlated with captures in nearby traps regardless of trap microhabitat factors, possibly reflecting the influence of the spatial aggregation of mosquitoes coming from nearby aquatic habitats or the concentration of dispersing adults. These results indicated that AGO traps can be located at places that can be easily reached during periodic inspections, such as in front of houses, without much regard to local microhabitat conditions.

Genome-wide analysis of AGO, DCL and RDR gene families reveals RNA-directed DNA methylation is involved in fruit abscission in Citrus sinensis.

BACKGROUND: Small RNAs regulate a wide variety of processes in plants, from organ development to both biotic and abiotic stress response. Being master regulators in genetic networks, their biogenesis and action is a fundamental aspect to characterize in order to understand plant growth and development. Three main gene families are critical components of RNA silencing: DICER-LIKE (DCL), ARGONAUTE (AGO) and RNA-DEPENDENT RNA POLYMERASE (RDR). Even though they have been characterized in other plant species, there is no information about these gene families in Citrus sinensis, one of the most important fruit species from both economical and nutritional reasons. While small RNAs have been implicated in the regulation of multiple aspects of plant growth and development, their role in the abscission process has not been characterized yet. RESULTS: Using genome-wide analysis and a phylogenetic approach, we identified a total of 13 AGO, 5 DCL and 7 RDR genes. We characterized their expression patterns in root, leaf, flesh, peel and embryo samples using RNA-seq data. Moreover, we studied their role in fruit abscission through gene expression analysis in fruit rind compared to abscission zone from samples obtained by laser capture microdissection. Interestingly, we determined that the expression of several RNA silencing factors are down-regulated in fruit abscission zone, being particularly represented gene components of the RNA-dependent DNA Methylation pathway, indicating that repression of this process is necessary for fruit abscission to take place in Citrus sinensis. CONCLUSIONS: The members of these 3 families present characteristic conserved domains and distinct expression patterns. We provide a detailed analysis of the members of these families and improved the annotation of some of these genes based on RNA-seq data. Our data suggests that the RNA-dependent DNA Methylation pathway is involved in the important fruit abscission process in C. sinensis.

Citywide Control of Aedes aegypti (Diptera: Culicidae) during the 2016 Zika Epidemic by Integrating Community Awareness, Education, Source Reduction, Larvicides, and Mass Mosquito Trapping.

This investigation was initiated to control Aedes aegypti and Zika virus transmission in Caguas City, Puerto Rico, during the 2016 epidemic using Integrated Vector Management (IVM), which included community awareness and education, source reduction, larviciding, and mass-trapping with autocidal gravid ovitraps (AGO). The epidemic peaked in August to October 2016 and waned after April 2017. There was a preintervention period in October/November 2016 and IVM lasted until August 2017. The area under treatment (23.1 km2) had 61,511 inhabitants and 25,363 buildings. The city was divided into eight even clusters and treated following a cluster randomized stepped-wedge design. We analyzed pools of female Ae. aegypti adults for RNA detection of dengue (DENV), chikungunya (CHIKV), and Zika (ZIKV) viruses using 360 surveillance AGO traps every week. Rainfall, temperature, and relative humidity were monitored in each cluster. Mosquito density significantly changed (generalized linear mixed model; F8, 14,588 = 296; P < 0.001) from 8.0 ± 0.1 females per trap per week before the intervention to 2.1 ± 0.04 after the percentage of buildings treated with traps was 60% and to 1.4 ± 0.04 when coverage was above 80%. Out of a total 12,081 mosquito pools, there were 1 DENV-, 7 CHIKV-, and 49 ZIKV-positive pools from October 2016 to March 2017. Afterward, we found only one positive pool of DENV in July 2017. This investigation demonstrated that it was possible to scale up effective Ae. aegypti control to a medium-size city through IVM that included mass trapping of gravid Ae. aegypti females.

Integrated vector control of Aedes aegypti mosquitoes around target houses.

BACKGROUND: The developing fetuses of pregnant women are at high risk of developing serious birth defects following Zika virus infections. We applied an Integrated Vector Control (IVC) approach using source reduction, larviciding, and mass trapping with non-insecticidal sticky traps to protect targeted houses by reducing the density of female Aedes aegypti mosquitoes. METHODS: We tested the hypothesis that Ae. aegypti density could be reduced to below three female mosquitoes/trap/week around a target house in the center of a circular area with a 150 m radius using IVC. Two non-adjacent areas within the same neighbourhood were selected and randomly designated as the treatment or control areas. Sentinel Autocidal Gravid Ovitraps (SAGO traps) were placed in each study area and were sampled weekly from May to November, during the 2016 Zika epidemic in Puerto Rico. The experimental design was longitudinal with pre-and post-IVC treatment observations between treatment and control areas, and a partial cross-over design, where IVC was applied to the original control area after 2 months to determine if Ae. aegypti density converged to levels observed in the treatment area. Pools of female Ae. aegypti mosquitoes were analyzed by RT-PCR to detect Zika, dengue and chikungunya virus RNA. RESULTS: Overall, pre-treatment mosquito densities in the inner (0-50 m; 15.6 mosquitoes/trap/week), intermediate (50-100 m; 18.1) and outer rings (100-150 m; 15.6) were reduced after treatment to 2.8, 4.1, and 4.3 in the inner, middle, and outer rings, respectively. Density at the target house in the treatment area changed from 27.7 mosquitoes/trap/week before IVC to 2.1 after IVC (92.4% reduction), whereas after treating the original control area (cross-over) density changed from 22.4 to 3.5 (84.3% reduction). Vector reductions were sustained in both areas after IVC. Zika virus was detected in Ae. aegypti, but the low incidence of the virus precluded assessing the impact of IVC on Zika transmission during the study. CONCLUSIONS: Applying IVC to circular areas that were surrounded by untreated areas significantly decreased the number of mosquitoes around target houses located in the center. Gravid Ae. aegypti females in the center of the 150 m areas fell below threshold levels that possibly protect against novel invading arboviruses, such as chikungunya and Zika.

Yellow Fever Virus Modulates the Expression of Key Proteins Related to the microRNA Pathway in the Human Hepatocarcinoma Cell Line HepG2.

Yellow fever is a zoonotic disease caused by the yellow fever virus (YFV) and transmitted by mosquitoes of the family Culicidae. It is well known that cellular and viral microRNAs (miRNAs) are involved in modulation of viral and cellular gene expression, as well as immune response, and are considered by the scientific community as possible targets for an effective therapy against viral infections. This regulation may be involved in different levels of infection and clinical symptomatology. We used viral titration techniques, viral kinetics from 24 to 96 hours postinfection (hpi), and analyzed the expression of key proteins related to the miRNA pathway by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). The expression of Dicer was different when compared over the course of infection by the distinct YFV genotypes. Drosha expression was similar during infection by YFV genotype 1 or 2, with a decrease in their expression over time and a slight increase in 96 hpi. Ago1, Ago2, and Ago4 showed different levels of expression between the viral genotypes: for YFV genotype 1 infection, Ago1 presented a positive expression, while for YFV genotype 2, it showed a negative expression, when compared with negative controls. We conclude that YFV infection modulates the proteins involved in miRNA biogenesis, which can regulate both viral replication and cellular immune response.

The Class II Trehalose 6-phosphate Synthase Gene PvTPS9 Modulates Trehalose Metabolism in Phaseolus vulgaris Nodules.

Legumes form symbioses with rhizobia, producing nitrogen-fixing nodules on the roots of the plant host. The network of plant signaling pathways affecting carbon metabolism may determine the final number of nodules. The trehalose biosynthetic pathway regulates carbon metabolism and plays a fundamental role in plant growth and development, as well as in plant-microbe interactions. The expression of genes for trehalose synthesis during nodule development suggests that this metabolite may play a role in legume-rhizobia symbiosis. In this work, PvTPS9, which encodes a Class II trehalose-6-phosphate synthase (TPS) of common bean (Phaseolus vulgaris), was silenced by RNA interference in transgenic nodules. The silencing of PvTPS9 in root nodules resulted in a reduction of 85% (± 1%) of its transcript, which correlated with a 30% decrease in trehalose contents of transgenic nodules and in untransformed leaves. Composite transgenic plants with PvTPS9 silenced in the roots showed no changes in nodule number and nitrogen fixation, but a severe reduction in plant biomass and altered transcript profiles of all Class II TPS genes. Our data suggest that PvTPS9 plays a key role in modulating trehalose metabolism in the symbiotic nodule and, therefore, in the whole plant.

Genome-wide identification and in silico characterisation of microRNAs, their targets and processing pathway genes in Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L., Fabaceae) is a globally important staple crop, which is an important source of calories, protein and essential micronutrients. At the genomic level little is known regarding the small non-coding RNAs within the common bean genome. One of the most important classes of such small non-coding RNAs is microRNAs (miRNAs), which control mRNA and protein expression levels in many eukaryotes. Computational methods have been applied to identify putative miRNAs in the genomes of different organisms. In this study, our objective was to comprehensively identify and characterise miRNAs from the genome and transcriptome of P. vulgaris, including both mature and precursor miRNA forms. We also sought to identify the putative proteins involved in miRNA processing and the likely target genes of common bean miRNAs. We identified 221 mature miRNAs and 136 precursor miRNAs distributed across 52 different miRNA families in the P. vulgaris genome. Amongst these, we distinguished 129 novel mature miRNAs and 123 miRNA precursors belonging to 24 different miRNA families. We also identified 31 proteins predicted to participate in the miRNA-processing pathway in P. vulgaris. Finally, we also identified 483 predicted miRNA targets, including many which corroborate results from other species, suggesting that miRNA regulatory systems are evolutionarily conserved and important for plant development. Our results expand the study of miRNAs and their target genes in common bean, and provide new opportunities to understand their roles in the biology of this important staple crop.

Small molecule inhibitors of Ago2 decrease Venezuelan equine encephalitis virus replication.

Venezuelan equine encephalitis virus (VEEV) is classified as a Category B Select Agent and potential bioterror weapon for its severe disease course in humans and equines and its potential for aerosol transmission. There are no current FDA licensed vaccines or specific therapies against VEEV, making identification of potential therapeutic targets a priority. With this aim, our research focuses on the interactions of VEEV with host microRNA (miRNA) machinery. miRNAs are small non-coding RNAs that act as master regulators of gene expression by downregulating or degrading messenger RNA, thus suppressing production of the resultant proteins. Recent publications implicate miRNA interactions in the pathogenesis of various viral diseases. To test the importance of miRNA processing for VEEV replication, cells deficient in Ago2, an important component of the RNA-induced silencing complex (RISC), and cells treated with known Ago2 inhibitors, notably acriflavine (ACF), were utilized. Both conditions caused decreased viral replication and capsid expression. ACF treatment promoted increased survival of neuronal cells over a non-treated, infected control and reduced viral titers of fully virulent VEEV as well as Eastern and Western Equine Encephalitis Viruses and West Nile Virus, but not Vesicular Stomatitis Virus. ACF treatment of VEEV TC-83 infected mice resulted in increased in vivo survival, but did not affect survival or viral loads when mice were challenged with fully virulent VEEV TrD. These results suggest that inhibition of Ago2 results in decreased replication of encephalitic alphaviruses in vitro and this pathway may be an avenue to explore for future therapeutic development.

miRNA biogenesis: biological impact in the development of cancer.

microRNAs (miRNAs) are non coding RNAs with different biological functions and pathological implications. Given their role as post-transcriptional gene expression regulators, they are involved in several important physiological processes like development, cell differentiation and cell signaling. miRNAs act as modulators of gene expression programs in different diseases, particularly in cancer, where they act through the repression of genes which are critical for carcinogenesis. The expression level of mature miRNAs is the result of a fine mechanism of biogenesis, carried out by different enzymatic complexes that exert their function at transcriptional and post-transcriptional levels. In this review, we will focus our discussion on the alterations in the miRNA biogenesis machinery, and its impact on the establishment and development of cancer programs.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo.

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-ß superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes. Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

Involvement of microRNA-related regulatory pathways in the glucose-mediated control of Arabidopsis early seedling development.

In plants, sugars such as glucose act as signalling molecules that promote changes in gene expression programmes that impact on growth and development. Recent evidence has revealed the potential importance of controlling mRNA decay in some aspects of glucose-mediated regulatory responses suggesting a role of microRNAs (miRNAs) in these responses. In order to get a better understanding of glucose-mediated development modulation involving miRNA-related regulatory pathways, early seedling development of mutants impaired in miRNA biogenesis (hyl1-2 and dcl1-11) and miRNA activity (ago1-25) was evaluated. All mutants exhibited a glucose hyposensitive phenotype from germination up to seedling establishment, indicating that miRNA regulatory pathways are involved in the glucose-mediated delay of early seedling development. The expression profile of 200 miRNA primary transcripts (pri-miRs) was evaluated by large-scale quantitative real-time PCR profiling, which revealed that 38 pri-miRs were regulated by glucose. For several of them, the corresponding mature miRNAs are known to participate directly or indirectly in plant development, and their accumulation was shown to be co-regulated with the pri-miR by glucose. Furthermore, the expression of several miRNA target genes was found to be deregulated in response to glucose in the miRNA machinery mutants ago1-25, dcl1-11, and hyl1-2. Also, in these mutants, glucose promoted misexpression of genes for the three abscisic acid signalling elements ABI3, ABI4, and ABI5. Thus, miRNA regulatory pathways play a role in the adjustments of growth and development triggered by glucose signalling.

First record of Thylaeodus (Gastropoda: Vermetidae) from the Equatorial Atlantic Ocean, with the description of a new species

The vermetid Thylaeodus equatorialis sp. nov. is endemic to the São Pedro and São Paulo Archipelago, located at the mid equatorial Atlantic Ocean. The species is closely related to Thylaeodus rugulosus (Monterosato, 1878), as indicated by similar shell characters, coloration of the soft parts, and feeding tube scars. However, T. equatorialis sp. nov. mainly differs from T. rugulosus in the operculum/aperture diameter ratio (~79% versus 100%), by having well developed pedal tentacles and fewer egg capsules in brooding females. In addition, the new species has the following unique characteristics: size almost twice as large (shell, tube aperture, erect feeding tube, protoconch and egg capsules) as the other Atlantic species; unusual method of brooding egg capsules; radula with prominent and more numerous flanking cusps; and small pustules following the suture of the protoconch. A detailed discussion on the taxonomy and biology of vermetid Thylaeodus and allies is also presented.(AU)

First record of Thylaeodus (Gastropoda: Vermetidae) from the Equatorial Atlantic Ocean, with the description of a new species

The vermetid Thylaeodus equatorialis sp. nov. is endemic to the São Pedro and São Paulo Archipelago, located at the mid equatorial Atlantic Ocean. The species is closely related to Thylaeodus rugulosus (Monterosato, 1878), as indicated by similar shell characters, coloration of the soft parts, and feeding tube scars. However, T. equatorialis sp. nov. mainly differs from T. rugulosus in the operculum/aperture diameter ratio (~79% versus 100%), by having well developed pedal tentacles and fewer egg capsules in brooding females. In addition, the new species has the following unique characteristics: size almost twice as large (shell, tube aperture, erect feeding tube, protoconch and egg capsules) as the other Atlantic species; unusual method of brooding egg capsules; radula with prominent and more numerous flanking cusps; and small pustules following the suture of the protoconch. A detailed discussion on the taxonomy and biology of vermetid Thylaeodus and allies is also presented.