Experimental Acute Exposure to Thirdhand Smoke and Changes in the Human Nasal Epithelial Transcriptome: A Randomized Clinical Trial.

Importance:No previous studies have shown that acute inhalation of thirdhand smoke (THS) activates stress and survival pathways in the human nasal epithelium. Objective:To evaluate gene expression in the nasal epithelium of nonsmoking women following acute inhalation of clean air and THS. Design, Setting, and Participants:Nasal epithelium samples were obtained from participants in a randomized clinical trial (2011-2015) on the health effects of inhaled THS. In a crossover design, participants were exposed, head only, to THS and to conditioned, filtered air in a laboratory setting. The order of exposures was randomized and exposures were separated by at least 21 days. Ribonucleic acid was obtained from a subset of 4 healthy, nonsmoking women. Exposures:By chance, women in the subset were randomized to receive clean air exposure first and THS exposure second. Exposures lasted 3 hours. Main Outcomes and Measures:Differentially expressed genes were identified using RNA sequencing with a false-discovery rate less than 0.1. Results:Participants were 4 healthy, nonsmoking women aged 27 to 49 years (mean [SD] age, 42 [10.2] years) with no chronic diseases. A total of 389 differentially expressed genes were identified in nasal epithelium exposed to THS, while only 2 genes, which were not studied further, were affected by clean air. Enriched gene ontology terms associated with stress-induced mitochondrial hyperfusion were identified, such as respiratory electron transport chain (q = 2.84 × 10-3) and mitochondrial inner membrane (q = 7.21 × 10-6). Reactome pathway analysis identified terms associated with upregulation of DNA repair mechanisms, such as nucleotide excision repair (q = 1.05 × 10-2). Enrichment analyses using ingenuity pathway analysis identified canonical pathways related to stress-induced mitochondrial hyperfusion (eg, increased oxidative phosphorylation) (P = .001), oxidative stress (eg, glutathione depletion phase II reactions) (P = .04), and cell survival (z score = 5.026). Conclusions and Relevance:This study found that acute inhalation of THS caused cell stress that led to the activation of survival pathways. Some responses were consistent with stress-induced mitochondrial hyperfusion and similar to those demonstrated previously in vitro. These data may be valuable to physicians treating patients exposed to THS and may aid in formulating regulations for the remediation of THS-contaminated environments

Telomeric TART elements target the piRNA machinery in Drosophila.

Coevolution between transposable elements (TEs) and their hosts can be antagonistic, where TEs evolve to avoid silencing and the host responds by reestablishing TE suppression, or mutualistic, where TEs are co-opted to benefit their host. The TART-A TE functions as an important component of Drosophila telomeres but has also reportedly inserted into the Drosophila melanogaster nuclear export factor gene nxf2. We find that, rather than inserting into nxf2, TART-A has actually captured a portion of nxf2 sequence. We show that TART-A produces abundant Piwi-interacting small RNAs (piRNAs), some of which are antisense to the nxf2 transcript, and that the TART-like region of nxf2 is evolving rapidly. Furthermore, in D. melanogaster, TART-A is present at higher copy numbers, and nxf2 shows reduced expression, compared to the closely related species Drosophila simulans. We propose that capturing nxf2 sequence allowed TART-A to target the nxf2 gene for piRNA-mediated repression and that these 2 elements are engaged in antagonistic coevolution despite the fact that TART-A is serving a critical role for its host genome

Electrophoretic mobility shift assays.

<p>Nuclear proteins from cleaving sporangia or sporangia were mixed with ³²P-labeled double-stranded oligonucleotides containing M51, M75, and M58, and subjected to electrophoresis. Reactions were performed either with no competitor, or unlabeled competitors at 5×, 25×, and 125× the concentration of labeled probe. These were either a specific competitor (same as labeled probe), a nonspecific competitor containing unrelated sequences, or mutated competitors derived from the specific competitor but mutated for the motif. Stars indicate the specific bands, and FP stands for free probe.</p

Features of the 103 motifs from <i>P. infestans</i>.

<p>The heat map indicates <i>p</i>-values associated with over-representation in the hyphal (HY), sporangia (SP), cleaving sporangia (CL), swimming zoospore (ZO), and germinated cyst (GC)-induced promoter sets compared to total promoters. Also graphed are <i>p</i>-values in constitutive promoters (CON). For clarity, <i>p</i>-values below 10⁻⁶ are shown as 10⁻⁶; the original values are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003182#ppat.1003182.s002" target="_blank">Table S1</a>. The first column of ovals on the right of the heatmap indicates if motifs were evolutionarily conserved in <i>P. ramorum</i> or <i>P. sojae</i> (Evol. Con.), with black indicating yes in at least one species, white meaning no, and grey denoting ambiguous, <i>i.e.</i> motifs were found but in new locations. The next column indicates whether motifs were positionally biased (Pos. Bias), with black marking yes, white meaning no, and grey indicating ambiguous since the motifs did not occur enough in the relevant promoter database for trustworthy analysis. The column of ovals on the right indicates whether motifs had a significant (<i>p</i><10⁻²) orientation bias, with black meaning yes and white no. An asterisk next to the motif name indicates those that were subjected to functional testing in <i>P. infestans</i> transformants.</p

Genome-wide Prediction and Functional Validation of Promoter Motifs Regulating Gene Expression in Spore and Infection Stages of <em>Phytophthora infestans</em>

<div><p>Most eukaryotic pathogens have complex life cycles in which gene expression networks orchestrate the formation of cells specialized for dissemination or host colonization. In the oomycete <i>Phytophthora infestans</i>, the potato late blight pathogen, major shifts in mRNA profiles during developmental transitions were identified using microarrays. We used those data with search algorithms to discover about 100 motifs that are over-represented in promoters of genes up-regulated in hyphae, sporangia, sporangia undergoing zoosporogenesis, swimming zoospores, or germinated cysts forming appressoria (infection structures). Most of the putative stage-specific transcription factor binding sites (TFBSs) thus identified had features typical of TFBSs such as position or orientation bias, palindromy, and conservation in related species. Each of six motifs tested in <i>P. infestans</i> transformants using the GUS reporter gene conferred the expected stage-specific expression pattern, and several were shown to bind nuclear proteins in gel-shift assays. Motifs linked to the appressoria-forming stage, including a functionally validated TFBS, were over-represented in promoters of genes encoding effectors and other pathogenesis-related proteins. To understand how promoter and genome architecture influence expression, we also mapped transcription patterns to the <i>P. infestans</i> genome assembly. Adjacent genes were not typically induced in the same stage, including genes transcribed in opposite directions from small intergenic regions, but co-regulated gene pairs occurred more than expected by random chance. These data help illuminate the processes regulating development and pathogenesis, and will enable future attempts to purify the cognate transcription factors.</p> </div

Rethinking the evolution of eukaryotic metabolism: novel cellular partitioning of enzymes in stramenopiles links serine biosynthesis to glycolysis in mitochondria.

BackgroundAn important feature of eukaryotic evolution is metabolic compartmentalization, in which certain pathways are restricted to the cytosol or specific organelles. Glycolysis in eukaryotes is described as a cytosolic process. The universality of this canon has been challenged by recent genome data that suggest that some glycolytic enzymes made by stramenopiles bear mitochondrial targeting peptides.ResultsMining of oomycete, diatom, and brown algal genomes indicates that stramenopiles encode two forms of enzymes for the second half of glycolysis, one with and the other without mitochondrial targeting peptides. The predicted mitochondrial targeting was confirmed by using fluorescent tags to localize phosphoglycerate kinase, phosphoglycerate mutase, and pyruvate kinase in Phytophthora infestans, the oomycete that causes potato blight. A genome-wide search for other enzymes with atypical mitochondrial locations identified phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase, which form a pathway for generating serine from the glycolytic intermediate 3-phosphoglycerate. Fluorescent tags confirmed the delivery of these serine biosynthetic enzymes to P. infestans mitochondria. A cytosolic form of this serine biosynthetic pathway, which occurs in most eukaryotes, is missing from oomycetes and most other stramenopiles. The glycolysis and serine metabolism pathways of oomycetes appear to be mosaics of enzymes with different ancestries. While some of the noncanonical oomycete mitochondrial enzymes have the closest affinity in phylogenetic analyses with proteins from other stramenopiles, others cluster with bacterial, plant, or animal proteins. The genes encoding the mitochondrial phosphoglycerate kinase and serine-forming enzymes are physically linked on oomycete chromosomes, which suggests a shared origin.ConclusionsStramenopile metabolism appears to have been shaped through the acquisition of genes by descent and lateral or endosymbiotic gene transfer, along with the targeting of the proteins to locations that are novel compared to other eukaryotes. Colocalization of the glycolytic and serine biosynthesis enzymes in mitochondria is apparently necessary since they share a common intermediate. The results indicate that descriptions of metabolism in textbooks do not cover the full diversity of eukaryotic biology

Positional bias of representative motifs within promoter space.

<p>The relative frequency of each motif is mapped across 200-nt bins taken from the relevant stage-induced promoter set, normalized to a value of 1.0 for the most populous bin. Marked in each panel is the motif and promoter dataset used for analysis, using the abbreviations in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003182#ppat-1003182-g004" target="_blank">Figure 4</a>. The bottom row shows three controls, indicating the frequency of three motifs in 2000 randomly selected promoters. The actual number of hits for each motif in the stage-induced promoter sets are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003182#ppat.1003182.s002" target="_blank">Table S1</a>.</p

Expression pattern of genes containing M8.

<p>Expression pattern of genes containing M8.</p

Histochemical staining of GUS under control of stage-specific motifs in stable transformants of <i>P. infestans</i>.

<p>For cleavage-associated motif M51, shown are the staining patterns of sporangia kept at 22°C or incubated at 10°C for 1-hr, in a representative transformant containing pDEL187. No staining was seen in hyphae. Similar patterns were seen in transformants in which M51 alone was fused to the <i>NifS</i> minimal promoter (not shown). For the other motifs, the transformants contained fusions of the motifs to the minimal promoter, and the images presented illustrated when GUS expression was first detected. These were at early and late stages of sporulation (M39, M58, M64, M75) or in germinated cysts (M95). Labeled in the M95 panel are the cyst (c), germ tube (gt), and appressorium (a).</p