Selective control of parasitic nematodes using bioactivated nematicides.

Parasitic nematodes are a major threat to global food security, particularly as the world amasses 10 billion people amid limited arable land1-4. Most traditional nematicides have been banned owing to poor nematode selectivity, leaving farmers with inadequate means of pest control4-12. Here we use the model nematode Caenorhabditis elegans to identify a family of selective imidazothiazole nematicides, called selectivins, that undergo cytochrome-p450-mediated bioactivation in nematodes. At low parts-per-million concentrations, selectivins perform comparably well with commercial nematicides to control root infection by Meloidogyne incognita, a highly destructive plant-parasitic nematode. Tests against numerous phylogenetically diverse non-target systems demonstrate that selectivins are more nematode-selective than most marketed nematicides. Selectivins are first-in-class bioactivated nematode controls that provide efficacy and nematode selectivity.

Single-cell RNA-sequencing reveals pre-meiotic X-chromosome dosage compensation in Drosophila testis.

Dosage compensation equalizes X-linked expression between XY males and XX females. In male fruit flies, expression levels of the X-chromosome are increased approximately two-fold to compensate for their single X chromosome. In testis, dosage compensation is thought to cease during meiosis; however, the timing and degree of the resulting transcriptional suppression is difficult to separate from global meiotic downregulation of each chromosome. To address this, we analyzed testis single-cell RNA-sequencing (scRNA-seq) data from two Drosophila melanogaster strains. We found evidence that the X chromosome is equally transcriptionally active as autosomes in somatic and pre-meiotic cells, and less transcriptionally active than autosomes in meiotic and post-meiotic cells. In cells experiencing dosage compensation, close proximity to MSL (male-specific lethal) chromatin entry sites (CES) correlates with increased X chromosome transcription. We found low or undetectable levels of germline expression of most msl genes, mle, roX1 and roX2 via scRNA-seq and RNA-FISH, and no evidence of germline nuclear roX1/2 localization. Our results suggest that, although dosage compensation occurs in somatic and pre-meiotic germ cells in Drosophila testis, there might be non-canonical factors involved in the dosage compensation mechanism. The single-cell expression patterns and enrichment statistics of detected genes can be explored interactively in our database: https://zhao.labapps.rockefeller.edu/gene-expr/.

Diverse and pervasive subcellular distributions for both coding and long noncoding RNAs.

In a previous analysis of 2300 mRNAs via whole-mount fluorescent in situ hybridization in cellularizing Drosophila embryos, we found that 70% of the transcripts exhibited some form of subcellular localization. To see whether this prevalence is unique to early Drosophila embryos, we examined ∼8000 transcripts over the full course of embryogenesis and ∼800 transcripts in late third instar larval tissues. The numbers and varieties of new subcellular localization patterns are both striking and revealing. In the much larger cells of the third instar larva, virtually all transcripts observed showed subcellular localization in at least one tissue. We also examined the prevalence and variety of localization mechanisms for >100 long noncoding RNAs. All of these were also found to be expressed and subcellularly localized. Thus, subcellular RNA localization appears to be the norm rather than the exception for both coding and noncoding RNAs. These results, which have been annotated and made available on a recompiled database, provide a rich and unique resource for functional gene analyses, some examples of which are provided.

Characterizing the Protein Isoforms of foraging (for), the PKGI Ortholog in Drosophila melanogaster.

The foraging (for) gene of Drosophila melanogaster encodes a cGMP-dependent protein kinase (PKG), which is a major effector of the cGMP signaling pathway involved in the regulation of behaviour and metabolic traits. Despite being well studied at the transcript level, little is known about the for gene at the protein level. Here, we provide a detailed characterization of the for gene protein (FOR) products and present new tools for their study, including five isoform-specific antibodies and a transgenic strain that carries an HA-labelled for allele (forBAC::HA). Our results showed that multiple FOR isoforms were expressed in the larval and adult stages of D. melanogaster and that the majority of whole-body FOR expression arises from three (P1, P1α, and P3) of eight putative protein isoforms. We found that FOR expression differed between the larval and adult stages and between the dissected larval organs we analyzed, which included the central nervous system (CNS), fat body, carcass, and intestine. Moreover, we showed that the FOR expression differed between two allelic variants of the for gene, namely, fors (sitter) and forR (rover), that are known to differ in many food-related traits. Together, our in vivo identification of FOR isoforms and the existence of temporal, spatial, and genetic differences in their expression lay the groundwork for determining their functional significance.

Long Noncoding RNAs and Repetitive Elements: Junk or Intimate Evolutionary Partners?

Our recent ability to sequence entire genomes, along with all of their transcribed RNAs, has led to the surprising finding that only ∼1% of the human genome is used to encode proteins. This finding has led to vigorous debate over the functional importance of the transcribed but untranslated portions of the genome. Currently, scientists tend to assume coding genes are functional until proven not to be, while the opposite is true for noncoding genes. This review takes a new look at the evidence for and against widespread noncoding gene functionality. We focus in particular on long noncoding RNA (noncoding RNAs longer than 200 nucleotides) genes and their 'junk' associates, transposable elements, and satellite repeats. Taken together, the suggestion put forward is that more of this junk DNA may be functional than nonfunctional and that noncoding RNAs and transposable elements act symbiotically to drive evolution.

Correction: 15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

[This corrects the article DOI: 10.1371/journal.ppat.1007597.].

An optimized QF-binary expression system for use in zebrafish.

The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.

New and Prospective Roles for lncRNAs in Organelle Formation and Function.

The observation that long noncoding RNAs (lncRNAs) represent the majority of transcripts in humans has led to a rapid increase in interest and study. Most of this interest has focused on their roles in the nucleus. However, increasing evidence is beginning to reveal even more functions outside the nucleus, and even outside cells. Many of these roles are mediated by newly discovered properties, including the ability of lncRNAs to interact with lipids, membranes, and disordered protein domains, and to form differentially soluble RNA-protein sub-organelles. This review explores the possibilities enabled by these new properties and abilities, such as likely roles in exosome formation and function.

15-keto-prostaglandin E2 activates host peroxisome proliferator-activated receptor gamma (PPAR-γ) to promote Cryptococcus neoformans growth during infection.

Cryptococcus neoformans is one of the leading causes of invasive fungal infection in humans worldwide. C. neoformans uses macrophages as a proliferative niche to increase infective burden and avoid immune surveillance. However, the specific mechanisms by which C. neoformans manipulates host immunity to promote its growth during infection remain ill-defined. Here we demonstrate that eicosanoid lipid mediators manipulated and/or produced by C. neoformans play a key role in regulating pathogenesis. C. neoformans is known to secrete several eicosanoids that are highly similar to those found in vertebrate hosts. Using eicosanoid deficient cryptococcal mutants Δplb1 and Δlac1, we demonstrate that prostaglandin E2 is required by C. neoformans for proliferation within macrophages and in vivo during infection. Genetic and pharmacological disruption of host PGE2 synthesis is not required for promotion of cryptococcal growth by eicosanoid production. We find that PGE2 must be dehydrogenated into 15-keto-PGE2 to promote fungal growth, a finding that implicated the host nuclear receptor PPAR-γ. C. neoformans infection of macrophages activates host PPAR-γ and its inhibition is sufficient to abrogate the effect of 15-keto-PGE2 in promoting fungal growth during infection. Thus, we describe the first mechanism of reliance on pathogen-derived eicosanoids in fungal pathogenesis and the specific role of 15-keto-PGE2 and host PPAR-γ in cryptococcosis.

The New RNA World: Growing Evidence for Long Noncoding RNA Functionality.

The past decade has seen a major increase in the study of noncoding RNAs (ncRNAs). However, there remains a great deal of confusion and debate over the levels of functionality and mechanisms of action of the majority of these new transcripts. This Opinion article addresses several of these issues, focusing particularly on long ncRNAs (lncRNAs). We reemphasize the unique abilities of RNAs to form myriad structures as well as to interact with other RNAs, DNA, and proteins, which provide them with unique and powerful abilities. One of these, the ability to interact sequence specifically with DNA, has been largely overlooked. Accumulating evidence suggests that evolution has taken advantage of RNA's properties via the rapid acquisition of new noncoding genes in testes, with subsequent gains of function in other tissues. This amplification process appears to be one of the major forces driving metazoan evolution and diversity.

Toxicokinetics of Brominated Azo Dyes in the Early Life Stages of Zebrafish (Danio rerio) Is Prone to Aromatic Substituent Changes.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (Danio rerio). The bioconcentration factor of DV93 at 120 hpf is 6.2-fold lower than that of DB373. The nontarget analysis revealed distinct metabolism routes between DB373 and DV93 by reducing nitro groups to nitroso (DB373) or amine (DV93), despite their similar structures. NAD(P)H quinone oxidoreductase 1 (NQO1) and pyruvate dehydrogenase were predicted as the enzymes responsible for the reduction of DB373 and DV93 by correlating time courses of the metabolites and enzyme development. Further in vitro recombinant enzyme and in vivo inhibition results validated NQO1 as the enzyme specifically reducing DB373, but not DV93. Global proteome profiling revealed that the expression levels of proteins from the "apoptosis-induced DNA fragmentation" pathway were significantly upregulated by all three BADs, supporting the bioactivation of BADs to mutagenic aromatic amines. This study discovered the bioactivation of BADs via distinct eukaryotic enzymes, implying their potential health risks.

Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75.

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Here, using genetic and chemical manipulations, we show that nitric oxide is produced in the Drosophila prothoracic gland, where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates.

The Drosophila DHR96 nuclear receptor binds cholesterol and regulates cholesterol homeostasis.

Cholesterol homeostasis is required to maintain normal cellular function and avoid the deleterious effects of hypercholesterolemia. Here we show that the Drosophila DHR96 nuclear receptor binds cholesterol and is required for the coordinate transcriptional response of genes that are regulated by cholesterol and involved in cholesterol uptake, trafficking, and storage. DHR96 mutants die when grown on low levels of cholesterol and accumulate excess cholesterol when maintained on a high-cholesterol diet. The cholesterol accumulation phenotype can be attributed to misregulation of npc1b, an ortholog of the mammalian Niemann-Pick C1-like 1 gene NPC1L1, which is essential for dietary cholesterol uptake. These studies define DHR96 as a central regulator of cholesterol homeostasis.

Spatially revealed roles for lncRNAs in Drosophila spermatogenesis, Y chromosome function and evolution.

Unlike coding genes, the number of lncRNA genes in organism genomes is relatively proportional to organism complexity. From plants to humans, the tissues with highest numbers and levels of lncRNA gene expression are the male reproductive organs. To learn why, we initiated a genome-wide analysis of Drosophila lncRNA spatial expression patterns in these tissues. The numbers of genes and levels of expression observed greatly exceed those previously reported, due largely to a preponderance of non-polyadenylated transcripts. In stark contrast to coding genes, the highest numbers of lncRNAs expressed are in post-meiotic spermatids. Correlations between expression levels, localization and previously performed genetic analyses indicate high levels of function and requirement. More focused analyses indicate that lncRNAs play major roles in evolution by controlling transposable element activities, Y chromosome gene expression and sperm construction. A new type of lncRNA-based particle found in seminal fluid may also contribute to reproductive outcomes.

Diindoles produced from commensal microbiota metabolites function as endogenous CAR/Nr1i3 ligands.

Numerous studies have demonstrated the correlation between human gut bacteria and host physiology, mediated primarily via nuclear receptors (NRs). Despite this body of work, the systematic identification and characterization of microbe-derived ligands that regulate NRs remain a considerable challenge. In this study, we discover a series of diindole molecules produced from commensal bacteria metabolites that act as specific agonists for the orphan constitutive androstane receptor (CAR). Using various biophysical analyses we show that their nanomolar affinities are comparable to those of synthetic CAR agonists, and that they can activate both rodent and human CAR orthologues, which established synthetic agonists cannot. We also find that the diindoles, diindolylmethane (DIM) and diindolylethane (DIE) selectively up-regulate bona fide CAR target genes in primary human hepatocytes and mouse liver without causing significant side effects. These findings provide new insights into the complex interplay between the gut microbiome and host physiology, as well as new tools for disease treatment.

Syndecan contributes to heart cell specification and lumen formation during Drosophila cardiogenesis.

The transmembrane proteoglycan Syndecan contributes to cell surface signaling of diverse ligands in mammals, yet in Drosophila, genetic evidence links Syndecan only to the Slit receptor Roundabout and to the receptor tyrosine phosphatase LAR. Here we characterize the requirement for syndecan in the determination and morphogenesis of the Drosophila heart, and reveal two phases of activity, indicating that Syndecan is a co-factor in at least two signaling events in this tissue. There is a stochastic failure to determine heart cell progenitors in a subset of abdominal hemisegments in embryos mutant for syndecan, and subsequent to Syndecan depletion by RNA interference. This phenotype is sensitive to gene dosage in the FGF receptor (Heartless), its ligand, Pyramus, as well as BMP-ligand Decapentaplegic (Dpp) and co-factor Sara. Syndecan is also required for lumen formation during assembly of the heart vessel, a phenotype shared with mutations in the Slit and Integrin signaling pathways. Phenotypic interactions of syndecan with slit and Integrin mutants suggest intersecting function, consistent with Syndecan acting as a co-receptor for Slit in the Drosophila heart.

Crystal structure of Fushi tarazu factor 1 ligand binding domain/Fushi tarazu peptide complex identifies new class of nuclear receptors.

The interaction between the orphan nuclear receptor FTZ-F1 (Fushi tarazu factor 1) and the segmentation gene protein FTZ is critical for specifying alternate parasegments in the Drosophila embryo. Here, we have determined the structure of the FTZ-F1 ligand-binding domain (LBD)·FTZ peptide complex using x-ray crystallography. Strikingly, the ligand-binding pocket of the FTZ-F1 LBD is completely occupied by helix 6 (H6) of the receptor, whereas the cofactor FTZ binds the co-activator cleft site of the FTZ-F1 LBD. Our findings suggest that H6 is essential for transcriptional activity of FTZ-F1; this is further supported by data from mutagenesis and activity assays. These data suggest that FTZ-F1 might belong to a novel class of ligand-independent nuclear receptors. Our findings are intriguing given that the highly homologous human steroidogenic factor-1 and liver receptor homolog-1 LBDs exhibit sizable ligand-binding pockets occupied by putative ligand molecules.

The structural basis of gas-responsive transcription by the human nuclear hormone receptor REV-ERBbeta.

Heme is a ligand for the human nuclear receptors (NR) REV-ERBalpha and REV-ERBbeta, which are transcriptional repressors that play important roles in circadian rhythm, lipid and glucose metabolism, and diseases such as diabetes, atherosclerosis, inflammation, and cancer. Here we show that transcription repression mediated by heme-bound REV-ERBs is reversed by the addition of nitric oxide (NO), and that the heme and NO effects are mediated by the C-terminal ligand-binding domain (LBD). A 1.9 A crystal structure of the REV-ERBbeta LBD, in complex with the oxidized Fe(III) form of heme, shows that heme binds in a prototypical NR ligand-binding pocket, where the heme iron is coordinately bound by histidine 568 and cysteine 384. Under reducing conditions, spectroscopic studies of the heme-REV-ERBbeta complex reveal that the Fe(II) form of the LBD transitions between penta-coordinated and hexa-coordinated structural states, neither of which possess the Cys384 bond observed in the oxidized state. In addition, the Fe(II) LBD is also able to bind either NO or CO, revealing a total of at least six structural states of the protein. The binding of known co-repressors is shown to be highly dependent upon these various liganded states. REV-ERBs are thus highly dynamic receptors that are responsive not only to heme, but also to redox and gas. Taken together, these findings suggest new mechanisms for the systemic coordination of molecular clocks and metabolism. They also raise the possibility for gas-based therapies for the many disorders associated with REV-ERB biological functions.

The omega-3 hydroxy fatty acid 7(S)-HDHA is a high-affinity PPARα ligand that regulates brain neuronal morphology.

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPARα) is emerging as an important target in the brain for the treatment or prevention of cognitive disorders. The identification of high-affinity ligands for brain PPARα may reveal the mechanisms underlying the synaptic effects of this receptor and facilitate drug development. Here, using an affinity purification-untargeted mass spectrometry (AP-UMS) approach, we identified an endogenous, selective PPARα ligand, 7(S)-hydroxy-docosahexaenoic acid [7(S)-HDHA]. Results from mass spectrometric detection of 7(S)-HDHA in mouse and rat brain tissues, time-resolved FRET analyses, and thermal shift assays collectively revealed that 7(S)-HDHA potently activated PPARα with an affinity greater than that of other ligands identified to date. We also found that 7(S)-HDHA activation of PPARα in cultured mouse cortical neurons stimulated neuronal growth and arborization, as well as the expression of genes associated with synaptic plasticity. The findings suggest that this DHA derivative supports and enhances neuronal synaptic capacity in the brain.

A Roadmap to the Structure-Related Metabolism Pathways of Per- and Polyfluoroalkyl Substances in the Early Life Stages of Zebrafish (Danio rerio).

BACKGROUND: Thousands of per- and polyfluoroalkyl substances (PFAS) with diverse structures have been detected in the ambient environment. Apart from a few well-studied PFAS, the structure-related toxicokinetics of a broader set of PFAS remain unclear. OBJECTIVES: To understand the toxicokinetics of PFAS, we attempted to characterize the metabolism pathways of 74 structurally diverse PFAS samples from the U.S. Environmental Protection Agency's PFAS screening library. METHODS: Using the early life stages of zebrafish (Danio rerio) as a model, we determined the bioconcentration factors and phenotypic toxicities of 74 PFAS. Then, we applied high-resolution mass spectrometry-based nontargeted analysis to identify metabolites of PFAS in zebrafish larvae after 5 d of exposure by incorporating retention time and mass spectra. In vitro enzymatic activity experiments with human recombinant liver carboxylesterase (hCES1) were employed to validate the structure-related hydrolysis of 11 selected PFAS. RESULTS: Our findings identified five structural categories of PFAS prone to metabolism. The metabolism pathways of PFAS were highly related to their structures as exemplified by fluorotelomer alcohols that the predominance of ß-oxidation or taurine conjugation pathways were primarily determined by the number of hydrocarbons. Hydrolysis was identified as a major metabolism pathway for diverse PFAS, and perfluoroalkyl carboxamides showed the highest in vivo hydrolysis rates, followed by carboxyesters and sulfonamides. The hydrolysis of PFAS was verified with recombinant hCES1, with strong substrate preferences toward perfluoroalkyl carboxamides. CONCLUSIONS: We suggest that the roadmap of the structure-related metabolism pathways of PFAS established in this study would provide a starting point to inform the potential health risks of other PFAS. https://doi.org/10.1289/EHP7169.