Preferential expression of biotransformation enzymes in the olfactory organs of Drosophila melanogaster, the antennae

Biotransformation enzymes have been found in the olfactory epithelium of vertebrates. We now show that in Drosophila melanogaster, a UDP-glycosyltransferase (UGT), as well as a short chain dehydrogenase/reductase and a cytochrome P450 are expressed specifically or preferentially in the olfactory organs, the antennae. The evolutionarily conserved expression of biotransformation enzymes in olfactory organs suggests that they play an important role in olfaction. In addition, we describe five Drosophila UGTs belonging to two families. All five UGTs contain a putative transmembrane domain at their C terminus as is the case for vertebrate UGTs where it is required for enzymatic activity. The primary sequence of the C terminus, including part of the transmembrane domain, differs between the two families but is highly conserved not only within each Drosophila family, but also between the members of one of the Drosophila families and vertebrate UGTs. The partial overlap of the conserved primary sequence with the transmembrane domain suggests that this part of the protein is involved in specific interactions occurring at the membrane surface. The presence of different C termini in the two Drosophilafamilies suggests that they interact with different targets, one of which is conserved between Drosophila and vertebrates

Rare deleterious germline variants and risk of lung cancer

Recent studies suggest that rare variants exhibit stronger effect sizes and might play a crucial role in the etiology of lung cancers (LC). Whole exome plus targeted sequencing of germline DNA was performed on 1045 LC cases and 885 controls in the discovery set. To unveil the inherited causal variants, we focused on rare and predicted deleterious variants and small indels enriched in cases or controls. Promising candidates were further validated in a series of 26,803 LCs and 555,107 controls. During discovery, we identified 25 rare deleterious variants associated with LC susceptibility, including 13 reported in ClinVar. Of the five validated candidates, we discovered two pathogenic variants in known LC susceptibility loci, ATM p.V2716A (Odds Ratio [OR] 19.55, 95%CI 5.04–75.6) and MPZL2 p.I24M frameshift deletion (OR 3.88, 95%CI 1.71–8.8); and three in novel LC susceptibility genes, POMC c.*28delT at 3′ UTR (OR 4.33, 95%CI 2.03–9.24), STAU2 p.N364M frameshift deletion (OR 4.48, 95%CI 1.73–11.55), and MLNR p.Q334V frameshift deletion (OR 2.69, 95%CI 1.33–5.43). The potential cancer-promoting role of selected candidate genes and variants was further supported by endogenous DNA damage assays. Our analyses led to the identification of new rare deleterious variants with LC susceptibility. However, in-depth mechanistic studies are still needed to evaluate the pathogenic effects of these specific alleles

Large-scale association analysis identifies new lung cancer susceptibility loci and heterogeneity in genetic susceptibility across histological subtypes.

Although several lung cancer susceptibility loci have been identified, much of the heritability for lung cancer remains unexplained. Here 14,803 cases and 12,262 controls of European descent were genotyped on the OncoArray and combined with existing data for an aggregated genome-wide association study (GWAS) analysis of lung cancer in 29,266 cases and 56,450 controls. We identified 18 susceptibility loci achieving genome-wide significance, including 10 new loci. The new loci highlight the striking heterogeneity in genetic susceptibility across the histological subtypes of lung cancer, with four loci associated with lung cancer overall and six loci associated with lung adenocarcinoma. Gene expression quantitative trait locus (eQTL) analysis in 1,425 normal lung tissue samples highlights RNASET2, SECISBP2L and NRG1 as candidate genes. Other loci include genes such as a cholinergic nicotinic receptor, CHRNA2, and the telomere-related genes OFBC1 and RTEL1. Further exploration of the target genes will continue to provide new insights into the etiology of lung cancer

Members of a family of drosophila putative odorant-binding proteins are expressed in different subsets of olfactory hairs

International audienceA polymerase chain reaction-based method was used to generate a Drosophila melanogaster antennal cDNA library from which head cDNAs were subtracted. We identified five cDNAs that code for antennal proteins containing six cysteines in a conserved pattern shared with known moth antennal proteins, including pheromone-binding proteins. Another cDNA codes for a protein related to vertebrate brain proteins that bind hydrophobic ligands. In all, we describe seven antennal proteins which contain potential signal peptides, suggesting that, like pheromone-binding proteins, they may be secreted in the lumen of olfactory hairs. The expression patterns of these putative odorant-binding proteins define at least four different subsets of olfactory hairs and suggest that the Drosophila olfactory apparatus is functionally segregated

Calcium imaging reveals that <i>ppk25</i> cells respond specifically to female pheromones.

<p>A. Solutions (100 ng/µl in 10%hexane, 90% water) of 7,11-HD (HD), 7,11-ND (ND), 7T, cVA, a mixture of all pheromones (mix) or 10% hexane, 90% water solution alone (hex) were applied to single leg bristles of <i>ppk25-Gal4</i>, <i>20×UAS-GCaMP3</i> flies. “<i>wt</i>” flies contained one copy of the normal <i>ppk25</i> gene. <i>ppk25 null</i> mutants were heterozygous for two different deletions of the <i>ppk25</i> locus <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Lin1" target="_blank">[16]</a>, and “<i>ppk25</i> rescue” flies are <i>ppk25</i> mutants carrying <i>UAS-ppk25</i> and <i>ppk25-Gal4</i> transgenes to target <i>ppk25</i> expression to <i>ppk25</i> cells <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Starostina1" target="_blank">[10]</a>. “#” denotes pheromones that did not elicit responses significantly higher than hexane alone in “<i>wt</i>” flies and were not tested further. n = 7–10; Mean ± SEM; ttest to <i>wt</i>, *p<0.05, **p<0.01. B. The same pheromone solutions as in A were applied to single leg bristles of <i>ppk25</i> mutants carrying the <i>ppk23-Gal4</i> and <i>20×UAS-GCaMP3</i> transgenes. As previously observed in flies with normal <i>ppk25</i> genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>, one population of <i>ppk23</i> cells, the M cells, respond specifically to male pheromones. In contrast to <i>wt</i> males however, in <i>ppk25 null</i> mutants the second population of <i>ppk23</i> cells, corresponding to F cells does not respond to any pheromone. n = 8; Mean ± SEM; ttest to <i>wt</i>,*p<0.05,**p<0.01.</p

Female <i>ppk25-Gal4</i> neurons represent a subset of <i>ppk23-Gal4</i> neurons and are involved in female receptivity.

<p>A. Segment of female front legs showing expression of <i>ppk23-Gal4</i> (green) in each of the two <i>fru</i>-positive cells (<i>fru-LexA</i>, magenta) under each chemosensory bristle. Scale 10 µ<i>m</i>. B. Segment of female front legs showing expression of <i>ppk25-Gal4</i> (green) in only one of the two <i>fru</i>-positive cells (<i>fru-LexA</i>, magenta) present under each chemosensory bristle. Scale 10 µ<i>m</i>. C. Silencing of <i>ppk25-Gal4</i> or <i>ppk23-Gal4</i> neurons reduces female receptivity. Receptivity was calculated for females expressing active (<i>UAS-TNT</i>) or inactive (<i>UAS-IN-TNT</i>) forms of TNT under control of either <i>ppk23-Gal4</i> or <i>ppk25-Gal4</i>. Additional control females contained <i>UAS-TNT</i> driven by <i>Or22b-Gal4</i>, which is expressed in a neuronal subset not associated with mating behaviors <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Hallem1" target="_blank">[36]</a>. N = 30–35; ***p<0.001 (Fisher's exact test). D. Transient inactivation of <i>ppk25-Gal4</i> neurons inhibits female receptivity. Females expressing the temperature-sensitive dominant <i>Dynamin</i> allele <i>shi^ts</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Kitamoto1" target="_blank">[37]</a> under control of <i>ppk25-Gal4</i> were incubated at either permissive (23°C) or non-permissive (30°C) temperature for 20 minutes prior to, and during the test. Receptivity for females expressing GFP in <i>ppk25-Gal4</i> neurons or females with <i>UAS-Shi^(ts)</i> alone was measured under identical conditions. N = 30–45; ***p<0.001;**p<0.01;*p<0.05 (Fisher's exact test).</p

<i>ppk25</i> function in gustatory neurons is required for courtship stimulation by <i>Tai2</i> males or 7-pentacosene.

<p>A. <i>ppk25</i> function in cells defined by <i>ppk25-Gal4</i> expression is required for male courtship directed at <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for <i>ppk25 null</i> mutant males, control males with one wild-type copy of <i>ppk25</i>, and <i>ppk25</i> mutant males where <i>ppk25</i> expression had been restored in <i>ppk25</i> cells. N = 36–40; Mean ± SEM; ***p<0.001. B. Targeted RNAi knockdown of <i>ppk25</i> in all gustatory neurons reduces courtship toward <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for males expressing <i>ppk25</i> RNAi in gustatory neurons using <i>Poxn-Gal4</i>. Control males expressed eGFP or a control RNAi targeting <i>CG13895</i> under control of <i>Poxn-Gal4</i>. Error bars are SEM; N = 31–34; ***p<0.001. C. The number of wing extensions performed by males with normal or mutant copies of <i>ppk25</i>, or by <i>ppk25</i> mutant males in which <i>ppk25</i> function is restored in <i>ppk25</i> cells, was measured in the presence of oe- females painted either with solvent alone or with 7-Pentacosene. oe- females were pierced through the head with forceps, contributing to the low courtship background and assays were conducted in the light. n = 20–24; Mean ± SEM; ***p<0.001.</p

Male courtship of immature males requires <i>ppk25</i> function.

<p>A. <i>ppk25</i> expression in gustatory neurons defined by <i>ppk25-Gal4</i> is required for male courtship of immature males. CI, fraction of males initiating courtship toward decapitated immature males and Total Behavioral Index (TBI) were calculated for <i>ppk25</i> mutant males, control males with one <i>wt</i> copy of <i>ppk25</i> and for <i>ppk25 null</i> mutant males where <i>ppk25</i> expression has been restored in cells expressing <i>ppk25-Gal4</i>. N = 32–39; Mean ± SEM; ***p<0.001; **p<0.01 (Error bars indicate the SEM for the fraction of males initiating courtship and statistical significance was determined by Fisher's exact test. Error bars and statistical significance for TBI was determined as described previously for CI). Newly eclosed Canton-S males with light body pigmentation and unfurled wings were used as immature male targets. B. Targeted RNAi knockdown of <i>ppk25</i> in gustatory neurons with <i>Poxn-Gal4</i> reduces courtship toward <i>Tai2</i> males. CI, fraction of males initiating courtship toward decapitated <i>Tai2</i> males and TBI were calculated for males expressing <i>ppk25</i> RNAi in gustatory neurons. Control males expressed eGFP or a control RNAi targeting <i>CG13895</i>, a gene with no known involvement in mating behavior <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Benchabane1" target="_blank">[58]</a> in all gustatory neuron. N = 32–34; Mean ± SEM; ***p<0.001.</p

<i>ppk25</i> is required for courtship stimulation by 7,11HD but not for inhibition of courtship by 7-T.

<p>A. The number of wing extensions performed by males with normal or mutant copies of <i>ppk25</i>, or by <i>ppk25</i> mutant males in which <i>ppk25</i> function is restored specifically in <i>ppk25</i> cells, was measured in the presence of oe- females painted either with solvent alone or with a single female pheromone, 7,11HD. oe- females were pierced through the head with forceps, contributing to the low courtship background. These and all following assays involving perfumed oe- targets were conducted under normal laboratory lights <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>. n = 19–24; Mean ± SEM; ***p<0.001 (perfumed relative to solvent control). Error bars indicate the SEM for number of wing extensions and statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post hoc test. B. The number of wing extensions displayed by males with normal or mutant <i>ppk25</i> was measured in the presence of oe- males painted either with solvent alone or with a single male pheromone, 7T. In this experiment, males were raised in groups as this resulted in higher baseline courtship toward oe- targets, thereby allowing inhibition to be measured. n = 24–28; Mean ± SEM; **p<0.01; (Kruskal-Wallis test followed by Dunn's post hoc test to solvent control). C. The Courtship Index (CI, percentage of a ten minute observation time during which the male is courting <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Lin1" target="_blank">[16]</a>) was measured for control <i>w1118</i> males, males mutant for <i>ppk23</i>, and <i>ppk23</i> mutant males in which expression of <i>ppk23</i> is targeted to F cells using the <i>ppk25-Gal4</i> driver. Male-female courtship was measured in the presence of decapitated w1118 females to reduce behavioral feedback. Male-male courtship was performed with intact male targets in the light since <i>ppk23</i> mutants display robust male-male courtship under these conditions <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Thistle1" target="_blank">[13]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004238#pgen.1004238-Toda1" target="_blank">[14]</a>. n = 30–40; Mean ±SEM; ***p<0.001 to <i>wt</i>. Error bars indicate the SEM for CI and statistical significance was determined using the Kruskal-Wallis test followed by Dunn's post hoc test.</p