The Ahr2-Dependent wfikkn1 Gene Influences Zebrafish Transcriptome, Proteome, and Behavior.

The aryl hydrocarbon receptor (AHR) is required for vertebrate development and is also activated by exogenous chemicals, including polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). AHR activation is well-understood, but roles of downstream molecular signaling events are largely unknown. From previous transcriptomics in 48 h postfertilization (hpf) zebrafish exposed to several PAHs and TCDD, we found wfikkn1 was highly coexpressed with cyp1a (marker for AHR activation). Thus, we hypothesized wfikkn1's role in AHR signaling, and showed that wfikkn1 expression was Ahr2 (zebrafish ortholog of human AHR)-dependent in developing zebrafish exposed to TCDD. To functionally characterize wfikkn1, we made a CRISPR-Cas9 mutant line with a 16-bp deletion in wfikkn1's exon, and exposed wildtype and mutants to dimethyl sulfoxide or TCDD. 48-hpf mRNA sequencing revealed over 700 genes that were differentially expressed (p < .05, log2FC > 1) between each pair of treatment combinations, suggesting an important role for wfikkn1 in altering both the 48-hpf transcriptome and TCDD-induced expression changes. Mass spectrometry-based proteomics of 48-hpf wildtype and mutants revealed 325 significant differentially expressed proteins. Functional enrichment demonstrated wfikkn1 was involved in skeletal muscle development and played a role in neurological pathways after TCDD exposure. Mutant zebrafish appeared morphologically normal but had significant behavior deficiencies at all life stages, and absence of Wfikkn1 did not significantly alter TCDD-induced behavior effects at all life stages. In conclusion, wfikkn1 did not appear to be significantly involved in TCDD's overt toxicity but is likely a necessary functional member of the AHR signaling cascade.

Phenotypically Anchored mRNA and miRNA Expression Profiling in Zebrafish Reveals Flame Retardant Chemical Toxicity Networks.

The ubiquitous use of flame retardant chemicals (FRCs) in the manufacture of many consumer products leads to inevitable environmental releases and human exposures. Studying toxic effects of FRCs as a group is challenging since they widely differ in physicochemical properties. We previously used zebrafish as a model to screen 61 representative FRCs and showed that many induced behavioral and teratogenic effects, with aryl phosphates identified as the most active. In this study, we selected 10 FRCs belonging to diverse physicochemical classes and zebrafish toxicity profiles to identify the gene expression responses following exposures. For each FRC, we executed paired mRNA-micro-RNA (miR) sequencing, which enabled us to study mRNA expression patterns and investigate the role of miRs as posttranscriptional regulators of gene expression. We found widespread disruption of mRNA and miR expression across several FRCs. Neurodevelopment was a key disrupted biological process across multiple FRCs and was corroborated by behavioral deficits. Several mRNAs (e.g., osbpl2a) and miRs (e.g., mir-125b-5p), showed differential expression common to multiple FRCs (10 and 7 respectively). These common miRs were also predicted to regulate a network of differentially expressed genes with diverse functions, including apoptosis, neurodevelopment, lipid regulation and inflammation. Commonly disrupted transcription factors (TFs) such as retinoic acid receptor, retinoid X receptor, and vitamin D regulator were predicted to regulate a wide network of differentially expressed mRNAs across a majority of the FRCs. Many of the differential mRNA-TF and mRNA-miR pairs were predicted to play important roles in development as well as cancer signaling. Specific comparisons between TBBPA and its derivative TBBPA-DBPE showed contrasting gene expression patterns that corroborated with their phenotypic profiles. The newer generation FRCs such as IPP and TCEP produced distinct gene expression changes compared to the legacy FRC BDE-47. Our study is the first to establish a mRNA-miR-TF regulatory network across a large group of structurally diverse FRCs and diverse phenotypic responses. The purpose was to discover common and unique biological targets that will help us understand mechanisms of action for these important chemicals and establish this approach as an important tool for better understanding toxic effects of environmental contaminants.

A cost-effective maize ear phenotyping platform enables rapid categorization and quantification of kernels.

High-throughput phenotyping systems are powerful, dramatically changing our ability to document, measure, and detect biological phenomena. Here, we describe a cost-effective combination of a custom-built imaging platform and deep-learning-based computer vision pipeline. A minimal version of the maize (Zea mays) ear scanner was built with low-cost and readily available parts. The scanner rotates a maize ear while a digital camera captures a video of the surface of the ear, which is then digitally flattened into a two-dimensional projection. Segregating GFP and anthocyanin kernel phenotypes are clearly distinguishable in ear projections and can be manually annotated and analyzed using image analysis software. Increased throughput was attained by designing and implementing an automated kernel counting system using transfer learning and a deep learning object detection model. The computer vision model was able to rapidly assess over 390 000 kernels, identifying male-specific transmission defects across a wide range of GFP-marked mutant alleles. This includes a previously undescribed defect putatively associated with mutation of Zm00001d002824, a gene predicted to encode a vacuolar processing enzyme. Thus, by using this system, the quantification of transmission data and other ear and kernel phenotypes can be accelerated and scaled to generate large datasets for robust analyses.

Microsurgical vascular bypass in the setting of pediatric limb length discrepancy.

Management of pediatric iatrogenic arterial occlusions can be challenging clinically, leading to chronic complications such as claudication and limb length discrepancy. We report the case of a 6-month-old female patient who had experienced iatrogenic right external iliac and common femoral arterial occlusion. At the age of 8 years, she had developed claudication and a limb length discrepancy of 3.2 cm. She underwent common iliac artery to superficial femoral artery and profunda artery bypass via a branched autologous reverse great saphenous vein using microsurgical techniques for the distal anastomoses. In the present report, we have focused on the musculoskeletal improvements, benefits of microsurgery in pediatric vessels, and maximization of epiphyseal perfusion.

Enterococcus faecalis Polymicrobial Interactions Facilitate Biofilm Formation, Antibiotic Recalcitrance, and Persistent Colonization of the Catheterized Urinary Tract.

Indwelling urinary catheters are common in health care settings and can lead to catheter-associated urinary tract infection (CAUTI). Long-term catheterization causes polymicrobial colonization of the catheter and urine, for which the clinical significance is poorly understood. Through prospective assessment of catheter urine colonization, we identified Enterococcus faecalis and Proteus mirabilis as the most prevalent and persistent co-colonizers. Clinical isolates of both species successfully co-colonized in a murine model of CAUTI, and they were observed to co-localize on catheter biofilms during infection. We further demonstrate that P. mirabilis preferentially adheres to E. faecalis during biofilm formation, and that contact-dependent interactions between E. faecalis and P. mirabilis facilitate establishment of a robust biofilm architecture that enhances antimicrobial resistance for both species. E. faecalis may therefore act as a pioneer species on urinary catheters, establishing an ideal surface for persistent colonization by more traditional pathogens such as P. mirabilis.

Experimental metatranscriptomics reveals the costs and benefits of dissolved organic matter photo-alteration for freshwater microbes.

Microbes and sunlight convert terrigenous dissolved organic matter (DOM) in surface waters to greenhouse gases. Prior studies show contrasting results about how biological and photochemical processes interact to contribute to the degradation of DOM. In this study, DOM leached from the organic layer of tundra soil was exposed to natural sunlight or kept in the dark, incubated in the dark with the natural microbial community, and analysed for gene expression and DOM chemical composition. Microbial gene expression (metatranscriptomics) in light and dark treatments diverged substantially after 4 h. Gene expression suggested that sunlight exposure of DOM initially stimulated microbial growth by (i) replacing the function of enzymes that degrade higher molecular weight DOM such as enzymes for aromatic carbon degradation, oxygenation, and decarboxylation, and (ii) releasing low molecular weight compounds and inorganic nutrients from DOM. However, growth stimulation following sunlight exposure of DOM came at a cost. Sunlight depleted the pool of aromatic compounds that supported microbial growth in the dark treatment, ultimately causing slower growth in the light treatment over 5 days. These first measurements of microbial metatranscriptomic responses to photo-alteration of DOM provide a mechanistic explanation for how sunlight exposure of terrigenous DOM alters microbial processing and respiration of DOM.

Evidence that curtailing proactive policing can reduce major crime.

Governments employ police to prevent criminal acts. But it remains in dispute whether high rates of police stops, criminal summonses and aggressive low-level arrests reduce serious crime 1-7 . Police officers target their efforts at areas where crime is anticipated and/or where they expect enforcement will be most effective. Simultaneously, citizens decide to comply with the law or commit crime partly on the basis of police deployment and enforcement strategies. In other words, policing and crime are endogenous to unobservable strategic interaction, which frustrates causal analysis. Here, we resolve these challenges and present evidence that proactive policing-which involves systematic and aggressive enforcement of low-level violations-is positively related to reports of major crime. We examine a political shock that caused the New York Police Department (NYPD) to effectively halt proactive policing in late 2014 and early 2015. Analysing several years of unique data obtained from the NYPD, we find that civilian complaints of major crimes (such as burglary, felony assault and grand larceny) decreased during and shortly after sharp reductions in proactive policing. The results challenge prevailing scholarship as well as conventional wisdom on authority and legal compliance, as they imply that aggressively enforcing minor legal statutes incites more severe criminal acts.

Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs.

In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work.

The Caenorhabditis elegans RDE-10/RDE-11 complex regulates RNAi by promoting secondary siRNA amplification.

BACKGROUND: In nematodes, plants, and fungi, RNAi is remarkably potent and persistent due to the amplification of initial silencing signals by RNA-dependent RNA polymerases (RdRPs). In Caenorhabditis elegans (C. elegans), the interaction between the RNA-induced silencing complex (RISC) loaded with primary small interfering RNAs (siRNAs) and the target messenger RNA (mRNA) leads to the recruitment of RdRPs and synthesis of secondary siRNAs using the target mRNA as the template. The mechanism and genetic requirements for secondary siRNA accumulation are not well understood. RESULTS: From a forward genetic screen for C. elegans genes required for RNAi, we identified rde-10, and through proteomic analysis of RDE-10-interacting proteins, we identified a protein complex containing the new RNAi factor RDE-11, the known RNAi factors RSD-2 and ERGO-1, and other candidate RNAi factors. The RNAi defective genes rde-10 and rde-11 encode a novel protein and a RING-type zinc finger domain protein, respectively. Mutations in rde-10 and rde-11 genes cause dosage-sensitive RNAi deficiencies: these mutants are resistant to low dosage but sensitive to high dosage of double-stranded RNAs. We assessed the roles of rde-10, rde-11, and other dosage-sensitive RNAi-defective genes rsd-2, rsd-6, and haf-6 in both exogenous and endogenous small RNA pathways using high-throughput sequencing and qRT-PCR. These genes are required for the accumulation of secondary siRNAs in both exogenous and endogenous RNAi pathways. CONCLUSIONS: The RDE-10/RDE-11 complex is essential for the amplification of RNAi in C. elegans by promoting secondary siRNA accumulation.

The ERI-6/7 helicase acts at the first stage of an siRNA amplification pathway that targets recent gene duplications.

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7-dependent siRNAs require Dicer. We identify that the eri-6/7-dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3-4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7-dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA-associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation.

GENE-counter: a computational pipeline for the analysis of RNA-Seq data for gene expression differences.

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts.

Heterochromatin is required for normal distribution of Neurospora crassa CenH3.

Centromeres serve as platforms for the assembly of kinetochores and are essential for nuclear division. Here we identified Neurospora crassa centromeric DNA by chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) of DNA associated with tagged versions of the centromere foundation proteins CenH3 (CENP-A) and CEN-C (CENP-C) and the kinetochore protein CEN-T (CENP-T). On each chromosome we found an ∼150- to 300-kbp region of enrichment for all three proteins. These regions correspond to intervals predicted to be centromeric DNA by genetic mapping and DNA sequence analyses. By ChIP-seq we found extensive colocalization of CenH3, CEN-C, CEN-T, and histone H3K9 trimethylation (H3K9me3). In contrast, H3K4me2, which has been found at the cores of plant, fission yeast, Drosophila, and mammalian centromeres, was not enriched in Neurospora centromeric DNA. DNA methylation was most pronounced at the periphery of centromeric DNA. Mutation of dim-5, which encodes an H3K9 methyltransferase responsible for nearly all H3K9me3, resulted in altered distribution of CenH3-green fluorescent protein (GFP). Similarly, CenH3-GFP distribution was altered in the absence of HP1, the chromodomain protein that binds to H3K9me3. We conclude that eukaryotes with regional centromeres make use of different strategies for maintenance of CenH3 at centromeres, and we suggest a model in which centromere proteins nucleate at the core but require DIM-5 and HP1 for spreading.

mut-16 and other mutator class genes modulate 22G and 26G siRNA pathways in Caenorhabditis elegans.

Argonaute-associated siRNAs and Piwi-associated piRNAs have overlapping roles in silencing mobile genetic elements in animals. In Caenorhabditis elegans, mutator (mut) class genes mediate siRNA-guided repression of transposons as well as exogenous RNAi, but their roles in endogenous RNA silencing pathways are not well-understood. To characterize the endogenous small RNAs dependent on mut class genes, small RNA populations from a null allele of mut-16 as well as a regulatory mut-16(mg461) allele that disables only somatic RNAi were subjected to deep sequencing. Additionally, each of the mut class genes was tested for a requirement in 26G siRNA pathways. The results indicate that mut-16 is an essential factor in multiple endogenous germline and somatic siRNA pathways involving several distinct Argonautes and RNA-dependent RNA polymerases. The results also reveal essential roles for mut-2 and mut-7 in the ERGO-1 class 26G siRNA pathway and less critical roles for mut-8, mut-14, and mut-15. We show that transposons are hypersusceptible to mut-16-dependent silencing and identify a requirement for the siRNA machinery in piRNA biogenesis from Tc1 transposons. We also show that the soma-specific mut-16(mg461) mutant allele is present in multiple C. elegans laboratory strains.

Child abuse and the legal system: the orthopaedic surgeon's role in diagnosis.

BACKGROUND: Orthopaedic surgeons have unique training and experience in diagnosis of fractures, both accidental and nonaccidental. That experience is valuable in identifying physical child abuse and in avoiding false accusations or convictions. Both aspects are important to the welfare of children and their families. The events that follow a report of child abuse are outside the training and experience of most orthopaedic surgeons. QUESTIONS/PURPOSES: What process follows a report of suspected child abuse? What unexpected outcomes or results occur in this process? Are medical conclusions used in this process consistent with the state of our knowledge? METHODS: The child abuse legal process is described as experienced by one orthopaedic surgeon. Examples of unexpected problems that occurred in cases that went to trial are described. CONCLUSIONS: Inappropriate outcomes can result from incomplete or incorrectly applied information. The input of the orthopaedic surgeon is often needed to provide the best information available to ensure that the best interests of the child and the family are protected. Working within a hospital team is the preferred method, but direct courtroom testimony is sometimes necessary.

Phytophthora-ID.org: A Sequence-Based Phytophthora Identification Tool.

Contemporary species identification relies strongly on sequence-based identification, yet resources for identification of many fungal and oomycete pathogens are rare. We developed two web-based, searchable databases for rapid identification of Phytophthora spp. based on sequencing of the internal transcribed spacer (ITS) or the cytochrome oxidase (cox) 1 and 2 spacer region, followed by BLAST searching the databases. Both databases are highly selective. For ITS, only sequences associated with published Phytophthora spp. descriptions or classic Phytophthora phylogenetics references are included. For the cox spacer region, only data obtained by resequencing select isolates reported in published work were included. Novel taxa tentatively named are selectively included in the database and labeled as Phytophthora taxon "X"; as in, for example, P. taxon "asparagi". The database was validated with 700 Phytophthora isolates collected from nursery environments during 2006 to 2009. This resource, found at www.Phytophthora-ID.org , is a robust and validated tool for molecular identification of Phytophthora spp. and is regularly being updated.

Transcription factors in light and circadian clock signaling networks revealed by genomewide mapping of direct targets for neurospora white collar complex.

Light signaling pathways and circadian clocks are inextricably linked and have profound effects on behavior in most organisms. Here, we used chromatin immunoprecipitation (ChIP) sequencing to uncover direct targets of the Neurospora crassa circadian regulator White Collar Complex (WCC). The WCC is a blue-light receptor and the key transcription factor of the circadian oscillator. It controls a transcriptional network that regulates ∼20% of all genes, generating daily rhythms and responses to light. We found that in response to light, WCC binds to hundreds of genomic regions, including the promoters of previously identified clock- and light-regulated genes. We show that WCC directly controls the expression of 24 transcription factor genes, including the clock-controlled adv-1 gene, which controls a circadian output pathway required for daily rhythms in development. Our findings provide links between the key circadian activator and effectors in downstream regulatory pathways.

Unique functionality of 22-nt miRNAs in triggering RDR6-dependent siRNA biogenesis from target transcripts in Arabidopsis.

RNA interference pathways can involve amplification of secondary siRNAs by RNA-dependent RNA polymerases. In plants, RDR6-dependent secondary siRNAs arise from transcripts targeted by some microRNAs (miRNAs). Here, Arabidopsis thaliana secondary siRNAs from mRNA as well as trans-acting siRNAs are shown to be triggered through initial targeting by a 22-nucleotide (nt) miRNA that associates with AGO1. In contrast to canonical 21-nt miRNAs, 22-nt miRNAs primarily arise from foldback precursors containing asymmetric bulges. Using artificial miRNA constructs, conversion of asymmetric foldbacks to symmetric foldbacks resulted in the production of 21-nt forms of miR173, miR472 and miR828. Both 21- and 22-nt forms associated with AGO1 and guided accurate slicer activity, but only 22-nt forms were competent to trigger RDR6-dependent siRNA production from target RNA. These data suggest that AGO1 functions differentially with 21- and 22-nt miRNAs to engage the RDR6-associated amplification apparatus.

MicroRNA gene evolution in Arabidopsis lyrata and Arabidopsis thaliana.

MicroRNAs (miRNAs) are short regulatory RNAs processed from partially self-complementary foldbacks within longer MIRNA primary transcripts. Several MIRNA families are conserved deeply through land plants, but many are present only in closely related species or are species specific. The finding of numerous evolutionarily young MIRNA, many with low expression and few if any targets, supports a rapid birth-death model for MIRNA evolution. A systematic analysis of MIRNA genes and families in the close relatives, Arabidopsis thaliana and Arabidopsis lyrata, was conducted using both whole-genome comparisons and high-throughput sequencing of small RNAs. Orthologs of 143 A. thaliana MIRNA genes were identified in A. lyrata, with nine having significant sequence or processing changes that likely alter function. In addition, at least 13% of MIRNA genes in each species are unique, despite their relatively recent speciation (approximately 10 million years ago). Alignment of MIRNA foldbacks to the Arabidopsis genomes revealed evidence for recent origins of 32 families by inverted or direct duplication of mostly protein-coding gene sequences, but less than half of these yield miRNA that are predicted to target transcripts from the originating gene family. miRNA nucleotide divergence between A. lyrata and A. thaliana orthologs was higher for young MIRNA genes, consistent with reduced purifying selection compared with deeply conserved MIRNA genes. Additionally, target sites of younger miRNA were lost more frequently than for deeply conserved families. In summary, our systematic analyses emphasize the dynamic nature of the MIRNA complement of plant genomes.

Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection.

Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins.