C. elegans and human pharmacopeia to identify and validate pharmacological targets that regulate C. elegans feeding rates.This paper takes advantage of similarities between the  C. elegans. This further raises the challenge of elucidating whether compound-target relationships found in model organisms are preserved in humans. To address these challenges we searched for compounds that affect feeding behavior in C. elegans and sought to identify their molecular mechanisms of action. Here, we applied predictive chemoinformatics to small molecules previously identified in a C. elegans phenotypic screen likely to be enriched for feeding regulatory compounds. Based on the predictions, 16 of these compounds were tested in vitro against 20 mammalian targets. Of these, nine were active, with affinities ranging from 9 nM to 10 µM. Four of these nine compounds were found to alter feeding. We then verified the in vitro findings in vivo through genetic knockdowns, the use of previously characterized compounds with high affinity for the four targets, and chemical genetic epistasis, which is the effect of combined chemical and genetic perturbations on a phenotype relative to that of each perturbation in isolation. Our findings reveal four previously unrecognized pathways that regulate feeding in C. elegans with strong parallels in mammals. Together, our study addresses three inherent challenges in phenotypic screening: the identification of the molecular targets from a phenotypic screen, the confirmation of the in vivo relevance of these targets, and the evolutionary conservation and relevance of these targets to their human orthologs.Phenotypic screens can identify molecules that are at once penetrant and active on the integrated circuitry of a whole cell or organism. These advantages are offset by the need to identify the targets underlying the phenotypes. Additionally, logistical considerations limit screening for certain physiological and behavioral phenotypes to organisms such as zebrafish and  a priori knowledge of their targets. This forward pharmacological approach has the advantage of directly identifying new pharmacological agents that are active on complex biological processes. However, because of experimental feasibility, systematic application of this approach is generally limited to small animals such as the roundworm C. elegans and zebrafish, raising the question of whether use of these animals could identify compounds that act on ortholgous mammalian targets. A significant challenge in addressing this question is the determination of the molecular identities of the compounds' targets responsible for the desired phenotypic outcomes. Here we describe a computational approach for target identification based on structural similarities of newly identified compounds to known ligand interactions with mostly mammalian targets. For several of the compounds emerging from a C. elegans phenotypic screen, we predict and confirm mammalian targets using in vitro binding assays. Using genetic and pharmacological assays, we then demonstrate that a subset of these compounds alter C. elegans feeding rates through the C. elegans counterparts of the predicted mammalian targets.Many beneficial pharmacological interventions were first discovered by observing the effects of perturbation of intact biological systems by small organic molecules without  Before the molecular biology era, pharmacological targets were typically classified by the effects of organic molecules on whole tissues Phenotypic compound screens return to this classical approach to capture some of the same advantages for the discovery of molecules with systemic activity. Such screens have generally relied on high content microscopy assays in cell-based systems C. elegans have been used in pharmacology-based phenotypic screens C. elegans feeds using peristaltic contractions of a muscular pharynx to aspirate microbes into the lumen of the intestine C. elegans' central nervous system integrates signals from external cues such as food availability, food quality, and internal nutritional status to regulate feeding behavior C. elegansC. elegans' feeding behavior is subject to regulation by some of the same physiological parameters and molecular components as those in mammals.Given their small size and ease of manipulation, C. elegansC. elegans has been challenged Feeding behavior is a relatively challenging read-out for a screen-scale phenotypic effort. Therefore, we focused on surrogate phenotypes that could potentially enrich for identification of feeding regulatory compounds. From a high-content microscopy assay, we previously discovered 84 compounds, which increased or decreased Nile Red in in vitro and subsequently tested orthologs of these targets in C. elegans by chemical-genetic epistasis. We then combined the compounds with the mutants to map epistasis relationships for feeding behavior, identifying four signaling pathways previously unassociated with C. elegans feeding regulation. Together, our findings reveal that chemical screens in C. elegans lead to molecules with activity toward known human targets and highlight the utility of C. elegans for unambiguous assessment of compound–target–phenotype relationships in the context of an intact organism.For all but one of the compounds, no biological targets were previously known. To predict targets for these compounds, we used chemoinformatic inference based on ligand patterns against mammalian receptors. For a subset of the predictions, we tested the compounds against the predicted mammalian targets C. elegans phenotypic screen c), between a query molecule and each target ligand. The ensemble of all pairwise Tc's for a target's ligand set to the query are summed and compared to an expectation of random association. Using statistical machinery similar to BLAST, an expectation value  quantifies the possibility that observed structural similarities could occur by chance. Because SEA relies on reported ligand–target interactions, it cannot predict associations for chemically novel molecules or for targets for which no ligands have been identified. Nevertheless, SEA provides a rapid and systematic approach for discovering the pharmacological relevance of C. elegans actives vis-à-vis mammalian targets with known ligands.Computational chemoinformatics methods have been used to query annotated ligand–target interactions to identify novel targets for known drugs E-value threshold of less than 10−5, 79 of the 84 active compounds were associated with at least one target in ChEMBL. Most compounds were predicted to have two or more targets, with 572 distinct targets predicted overall , we only tested compound–target interactions for which established assays were readily accessible. We tested 16 different compounds against 20 targets (21 ligand–target pairs) ranging from G-protein coupled receptors (GPCRs) and nuclear hormone receptors (NHRs) to kinases and phosphatases . This 43% in vitro hit rate resembles that observed for chemoinformatic linkage of human drugs to new To test the SEA predictions, we assayed selected molecules phatases . Nine cophatases . Signifiin vitro . A finalE-value and potency, since target affinity is not considered when calculating ligand similarities. In several cases, SEA successfully predicted the overall target family but not the exact isoform. For instance, H6 and B16 were predicted to antagonize tachykinin receptor–3 (Tkr-3) and metabotropic glutamate receptor 5 (mGluR-5), respectively, but instead antagonized Tkr-1 and mGluR-8  assayed as L4 animals exhibited similar percentage increases in feeding in response to B16, H6, F15, and D20 as animals that were allowed an extra day under treatment then assayed as adults. L4 animals do have a basal pumping rate that is lower than adults, and this is reflected in different absolute pump counts for the two stages  and 5-flurox (IC50 = 15 nM) C. elegans to regulate pharyngeal pumping.To further compare the pharmacological parallels between Flt-3 inhibitors and pharyngeal pumping, we tested a known Flt-3 receptor inhibitor   . The reaC. elegans even if they fail to modulate the predicted mammalian targets. This appears to be the case for K9, an analog of D20 also predicted to inhibit Flt-3 kinase  Based on the SEA and s of F15 . The oxyectively . Like D2C. elegans sequences with similarity to the human oxytocin receptor. These include an oxytocin/vasopressin-like receptor NTR-1 (T07D10.2) that modulates male mating behavior ntr-1 and the gnrr-1, -2, and -3 mutants  animals –treated animals were indeed insensitive to the pharyngeal pumping rate increases elicited by either SB222200 or H6 treatment  structurally distinct from B16  and VER-3 inhibitors  interact nonadditively and reciprocally with ver-3 and mgl-2 mutants, indicating that these gene products may act in a common pathway. However, the additive effect of B16/MMPIP versus the antagonistic phenotypic effect of D20/K9/5-flurox activity on gnrr-1 mutants is consistent with their targets being distinct entities. In addition, GNRR-1 antagonists (F15 and L-371257) interact nonadditively with ver-2 mutants and antagonistically with ver-3, recapitulating the antagonistic interaction of VER-3 inhibitors with ver-2 mutants , but was reduced by ver-2(RNAi) (Mgl-2(RNAi) interacted additively with gnrr-1 mutants to increase the pumping rate, and ver-3(RNAi) antagonized the feeding increasing effects of the gnrr-1 mutation (Ver-2(RNAi) interacted nonadditively with the gnrr-1 mutants and was antagonistic to mgl-2 mutants (gnrr-1 mutants and ver-3(RNAi), ver-2(RNAi), and mgl-2(RNAi) were observed by measuring pumping over longer intervals by time lapse microscopy  . Mgl-2 receptor, are expressed in the hypothalamus, and administration of PDGF-β depresses food intake and anti-PDGF-β antibodies elevate food intake in rats C. elegans receptor tyrosine kinase (ver-3) through either mutation or pharmacological inhibition elevates pharyngeal pumping. These targets may thus have ancient evolutionary origins in the regulation of feeding behavior.Whether the specific compounds identified from the in vivo function. Third, while for feeding, the in vitro and in vivo data provided a compelling case for mechanisms of actions of F15, H6, B16, and D20, it remains to be determined whether these compounds also act on as-of-yet undetermined molecular pathways to alter other biological processes in C. elegans. Finally, while the link between compounds, targets, and C. elegans phenotypes now seems strong for several of the active compounds, linkage between C. elegans and mammalian in vivo pharmacology remains to be drawn.Key weaknesses of our approach merit discussion. First, whereas it is comforting that we can predict targets for most biologically active synthetic compounds even in a “diversity” library, this also reflects the restricted chemical-target space in which the field is working. A library composed of genuinely novel chemotypes might be more likely to illuminate unprecedented targets, a widely desired goal of the field. On the other hand, any such truly diverse library risks missing that small part of chemical space that is relevant to terrestrial biology, the bias towards which is, after all, a pragmatic advantage of the current libraries C. elegans and mammals, some of them target-based, some biology-based, the targets and ligand networks for a substantial number of small molecules are conserved enough to allow target and phenotypic association across phyla. We envision that such chemical-genetic epistasis maps could be extended to saturation mapping of the pharmacological target networks underlying feeding regulation and other processes in C. elegans. The amenability of C. elegans to genetic manipulation and pharmacological screening may find broad utility as a means to identify new small molecules with interesting phenotypes and human-relevant targets.In summary, whereas chemoinformatic linkage retains important liabilities, its success rate here and in earlier studies I10, G7, A5, and L15 were purchased from Chembridge. A15, D20, K9, G6, L10, H6, and F14 were purchased from SPECS. B16, N10, and F15 were purchased from Princeton Biomolecular Research. J16 was purchased from TimTec. MMPIP hydrochloride, SB222200, 100 nM L-371257, and 5-fluoroindirubinoxime were purchased from Tocris Biosciences. All other chemicals were purchased from Sigma.http://www.ebi.ac.uk/chembl) using the Similarity Ensemble Approach (SEA) We computationally screened 84 phenotypically active compounds against molecular target panels from the ChEMBL database  and later moved to an updated 2,482 molecular target panel derived from ChEMBL_11 . We filtered reference ligands by molecular weight  and by reported affinity (≤10 µM), and then subjected all ligand structures to cleaning, standardization, and de-duplication as before Bacillus cereus was assayed by CEREP, Inc. using glycero-phosphatidyl ethanolamine as a substrate, monitoring diacyl glycerol production. Radioligand binding assays for PPAR-α, γ, δ and the androgen receptor were performed by CEREP, Inc. CARNA Biosciences  performed in vitro kinase activity assays using recombinant catalytic domains and measured phosphorylation of an Src-derived peptide for Flt-3 and phosphatidyl inositol for PI3KCA. Radioligand binding assays for the oxytocin receptor, D4 dopamine receptor, cannabinoid CB2 receptor, and angiotensin type I and II receptors were performed as described previously, as were cell-based activity assays for mGluR 1a, 2, 4, 5, 6, and 8 Tachykinin, ghrelin, and calcium-sensing receptor activity were measured using a cell-based Ca flux assay by Multispan, Inc. . Cholescystokinin A and B receptors were assayed for effects on cAMP production, and nicotinic acid receptor activity was measured in a cell-based assay of forskolin-stimulated cAMP production by Multispan, Inc. Calcium-sensing receptor, PP2A phosphatase activity assays were performed by CEREP  using PP2A from human erythrocytes. Phospholipase C from mgl-1(tm1811) X, mgl-2(tm355) I, gnrr-2(tm4867) V, and gnrr-3(tm4152) X were obtained from the National Bioresource Project for the Nematode courtesy of Dr. S. Mitani at Tokyo Women's Medical University School of Medicine. Strains containing gnrr-1(ok238) I, ntr-1(ok2780) I, egl-15(n484) X, ver-1(ok1738) III, ver-2(ok897) I, ver-3(ok891) X, and ver-4(ok1079) X were obtained from the C. elegans Genetics Center, which is funded by the NIH National Center for Research Resources (NCRR). N2 (Bristol) strain was utilized as a reference wild-type strain. Strains were outcrossed 4 times to the wild-type background. Unless described otherwise, strains were cultivated on NGM-agar plates seeded with E. coli OP-50 as described Strains containing C. elegans were cultured for 3 d at 20°C on a lawn of HT115 E. coli induced with IPTG to express double-stranded RNAi as described Synchronized first larval stage E. coli OP-50 (100 µl of a 3× concentrated overnight culture in LB broth) and absorbed as a single drop onto 3.5 cm plates containing 2.5 ml of NGM agar forming a well-defined lawn of bacteria. For developmental exposures, 20–30 synchronized first larval stage C. elegans derived from alkaline hypochlorite treatment of gravid adults were applied to the preseeded plates and cultured until assay at mid-L4 stage  or as day 1 gravid adults . For naïve adult exposures, synchronized first larval stage animals were cultured on NGM-agar plates seeded with E. coli OP-50 for 3 d at 20°C, then transferred to assay plates, and pumping was assayed 1 to 16 h later. Comparisons between vehicle-treated and compound-treated animals were always performed between animals at the same developmental stage and same exposure time. Pharyngeal pumping was counted by live observation at 115–200× magnification using a stereo microscope and recorded at 10 s intervals using a manually controlled digital cell counter. Alternatively, C. elegans pumping was recorded for longer intervals (30–60 s) using bright-field time-lapse microscopy at 120× magnification with a 20 images per second acquisition rate. The resulting movies were analyzed manually at a playback rate of 10 images per second.Compounds at 1,000× stock concentrations (0.1–10 mM) in DMSO or an equal volume of DMSO were diluted in a suspension of t test was used. To express data as a percentage of control, the pumping rates of compound-treated animals were divided by the mean pumping rate of the DMSO-treated wild-type animals measured in the same experiment, unless indicated otherwise. Error bars on the control samples indicate the variation around that mean, which is utilized in all statistical calculations.Significance was determined by one-way ANOVA applying a Dunnett's posttest when comparing multiple treatments to a single control and a Bonferroni posttest when comparing the more than two treatments against one another. For pairwise nonrepeated measured comparisons, a student's Figure S1Dose-response measurements that validate SEA predictions for GPCRs. (A) B16 inhibits rat mGluR-8 activity in CHO cells that were stimulated with 1 µM L-AP4 agonist. Error bars represent the s.e.m. of eight measurements. One-way ANOVA (Bonferonni) at concentrations of B16>3×10−6 M indicate the responses are significant (p<0.001). (B) Inhibition of 3H-oxytocin binding to the human oxytocin receptor by unlabeled oxytocin peptide (black squares) or F15 (red triangles). (C) H6 inhibits the calcium flux induced in cells expressing the human tachykinin-1 receptor, stimulated by neurokinin-1 peptide. Error bars represent the s.d. of three measurements. One-way ANOVA (Bonferroni) at concentrations >10−6.4 M indicate the responses are significant (p<0.01). (D) Inhibition of 3H-risperidone binding to the human dopamine D4 receptor by chlorpromazine (black squares) or G7 (red triangles) at concentrations of G7>10−6.4 M indicates the response is significant: p<0.001 ANOVA (Bonferroni).(TIF)Click here for additional data file.Figure S2Dose-dependent inhibitory activity for compounds predicted by SEA to inhibit kinases. (A–B) Varying concentrations of J6 and L10 were incubated with full-length recombinant human PI3KCA and ATP (50 µM) for 5 h and the phosphorylation level of the substrate phosphatidylinositol was measured. (A) L10 inhibition of PI3-kinase p110α is significant at concentrations >1×10−6 M . (B) J6 inhibition of PI3-kinase p110α is significant at concentrations >1×10−6 M . (C) D20 inhibition of flt-3 receptor catalytic domain activity. Varying concentrations of D20 were incubated with recombinant human Flt-3 receptor catalytic domain (amino acids 564–993), 100 µM ATP, and the phosphorylation of srctide peptide substrate was measured after 1 h at each concentration of D20. Inhibition at concentrations >1×10−7 M is significant . Error bars represent the s.d. of two replicates.(TIF)Click here for additional data file.Figure S3Dose-response relationships for compounds that bind nuclear hormone receptors. (A) 3H-Rosiglitazone binding to human PPAR-γ in the presence of varying concentrations of I10. (B) 3H-Mibolerone binding to the human androgen receptor in the presence of varying concentrations of G6. Error bars represent the standard deviation of two replicates.(TIF)Click here for additional data file.Figure S4Measurement of the pharyngeal pumping effects of the compounds by time lapse microscopy.C. elegans were cultured for 3 d from L1 larvae in the presence of either 10 µM B16, H6, D20, and F15 or 0.1% DMSO as the vehicle control. Pharyngeal pumping over 60 s intervals was measured in time lapse recordings of at least 60 s in duration. A comparison with the pumping rates measured over 10 s (6-fold extrapolated) by real-time direct observation of the same populations of animals is presented. Real-time, 10 s interval manual counting involves a systematic underestimation of pumping across all conditions, however the relative ratios are similar. Ten animals were measured per condition. Error bars represent the standard deviation. *p<0.001 one-way ANOVA, Bonferroni posttest.(TIF)Click here for additional data file.Figure S5Effect on pharyngeal pumping of combinations of compounds that target the same human receptor. (A) C. elegans were cultured with DMSO (0.2%), 10 µM D20, 1 µM 5-flurox, or a combination of 10 µM D20 and 1 µM 5-flurox. (B) C. elegans were cultured with DMSO (0.2%), 10 µM F15, 200 nM L-371257, or a combination of 10 µM F15 and 200 nM L-371257. (C) C. elegans were cultured with DMSO (0.2%), 10 µM H6, 200 nM SB222200, or a combination of 10 µM H6 and 200 nM SB222200. (D) C. elegans were cultured with DMSO (0.2%), 10 µM B16, 2 µM MMPIP, or a combination of 10 µM H6 and 2 µM MMPIP. In (A–D) 12 animals per condition were counted. Error bars represent the standard deviation. *p<0.001 ANOVA, Dunnett's posttest.(TIF)Click here for additional data file.Figure S6Genetic interactions of gnrr-1 mutants quantified by time lapse measurements. Wild-type and gnrr-1 mutants were cultured on bacteria expressing double-stranded RNA targeting mgl-2, ver-3, ver-2, or the RNAi expression vector control. Pharyngeal pumping of 10 animals per condition for 30 s intervals was recorded by time-lapse microscopy. The color scheme of the figure matches that of p>0.05, *p<0.05, **p<0.01 ANOVA, Bonferroni posttest.(TIF)Click here for additional data file.Movie S1C. elegans treated with 10 µM F15.(MP4)Click here for additional data file.Movie S2C. elegans treated with 10 µM B16.(MP4)Click here for additional data file.Movie S3C. elegans treated with 10 µM D20.(MP4)Click here for additional data file.Movie S4C. elegans treated with 10 µM H6.(MP4)Click here for additional data file.Movie S5C. elegans treated with 0.1% DMSO.(MP4)Click here for additional data file.Table S1Target predictions that could not be confirmed in vitro.(TIF)Click here for additional data file.Table S2Activity of 10 µM B16 on human metabotropic glutamate receptors.(DOCX)Click here for additional data file.Table S3Known expression patterns of C. elegans homologs used in the manuscript and their sequence comparisons to human targets.(DOCX)Click here for additional data file.Table S4Epistasis data used to construct interaction matrix in .(DOC)Click here for additional data file.


CRB1) gene is mutated in progressive types of autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. The severity of the phenotype due to human CRB1 or mouse Crb1 mutations is dependent on the genetic background. Mice on C57BL/6J background with Crb1 mutations show late onset of retinal spotting phenotype or no phenotype. Recently, we showed that conditional deletion of mouse Crb2 in the retina results in early retinal disorganization leading to severe and progressive retinal degeneration with concomitant visual loss that mimics retinitis pigmentosa due to mutations in the CRB1 gene. Recent studies in the fruit fly and zebrafish suggest roles of the Crumbs (CRB) complex members in the regulation of cellular signalling pathways including the Notch1, mechanistic target of rapamycin complex 1 (mTORC1) and the Hippo pathway. Here, we demonstrate that mice backcrossed to C57BL/6J background with loss of CRB2 in the retina show a progressive disorganization and degeneration phenotype during late retinal development. We used microarray gene profiling to study the transcriptome of retinas lacking CRB2 during late retinal development. Unexpectedly, the retinas of newborn mice lacking CRB2 showed no changes in the transcriptome during retinal development. These findings suggest that loss of CRB2 in the developing retina results in retinal disorganization and subsequent degeneration without major changes in the transcriptome of the retina. These mice might be an interesting model to study the onset of retinal degeneration upon loss of CRB proteins.In humans, the Crumbs homologue-1 ( Retinal cell generation and differentiation in the mouse occurs from embryonic day (E) 11 to postnatal day (P) 10. Six major neuronal and one glia cell type are generated from multipotential retinal progenitors in a characteristic sequence during development . Cell ad The apical CRB complex is located at the subapical region adjacent to adherens junctions between the retinal progenitor cells in the developing retina  and, aftCRB1 gene are responsible for retinal diseases such as Leber congenital amaurosis and retinitis pigmentosa [Crb gene family member Crb2, specifically in early progenitors, results in disorganization during late retinal development leading to severe and progressive retinal degeneration with concomitant visual loss that mimics retinitis pigmentosa due to mutations in the CRB1 gene [Crb1 knockout (Crb1-/-) and Crb1Crb2 conditional knockout mice [In humans, mutations in the gmentosa -21. The RB1 gene . Variatiout mice ,22,23.Crb1 mutant retinas from mixed to C57BL/6J genetic background strongly suppressed the morphological phenotype [Crb2 null retinas from mixed (50% OLA129 and 50% C57BL/6J) to 99.9% C57BL/6J background did not suppress the severe morphological phenotype. To elucidate the molecular events that precede and lead to the morphological phenotype in the Crb2 retina-specific conditional knockout mice on 99.9% C57BL/6J background, we applied microarray-based mRNA profiling in retinal tissue of postnatal stages P0, P3, P6, and P10. The morphological phenotype did however not result in a significantly altered transcriptome at any stage of retinal development analysed.Microarray analysis has been used to study changes in gene expression during retinal development -29. Manyhenotype . Here, wCrb2 conditional knockout mice were generated and genotyped as described in [pde6b) or Crb1 (rd8) genes. mT/mG reporter mice were obtained from the Jackson laboratory (Gt(ROSA)26Sortm4) [ad libitum. All procedures concerning animals were performed with permission of the animal experimentation committee (DEC) of the Royal Netherlands Academy of Arts and Sciences (KNAW), permit number NIN06-46. ,-EGFP)) . AnimalsCrb2F/+/Chx10Cre+/-) and five Crb2Chx10 cKO (Crb2F/F/Chx10Cre+/-) retinas per time point were used. The retinas were isolated, snap-frozen in liquid nitrogen immediately, and stored at -80°C until use. Pups were euthanized by decapitation at different time points: postnatal day (P) 0, P3, P6 and P10. Five control  and homogenized with a pellet pestle. Total RNA was isolated using RNeasy Mini Kit according to the manufacturer's instructions. RNA extract was eluted in RNase-free water (30 μL). RNA yield and purity were determined using a NanoDrop ND-1000 spectrophotometer . RNA integrity was determined by the RNA integrity number (RIN), as measured by the Agilent 2100 bioanalyser . Overall, the isolated RNA was of high integrity , 1 min in 0.06x SSPE/0.005% N-Lauroylsarcosine and 30 s in acetonitrile (Sigma-Aldrich), then dried in a nitrogen flow. Microarrays were scanned using an Agilent DNA Microarray Scanner at 5 µm resolution and 10% (low) and 100% (high) photomultiplier tube setting. Microarray scans were quantified using Agilent Feature Extraction software (version 9.5.3.1).For microarray analysis, Agilent 4x44K v2 whole mouse gene expression microarrays  were used. Sample labelling and microarray hybridization were performed according to manufacturer’s instructions. Briefly, for each sample, 200 ng of RNA were linearly amplified and fluorescently labelled with either Cy3-CTP or Cy5-CTP (Perkin Elmer) using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Prior to hybridization, equal amounts (825 ng) of Cy3- and Cy5-labelled RNA were hydrolyzed for 30 min at 60°C in 1x fragmentation buffer (Agilent Technologies). The fragmented targets were hybridized to a microarray by incubating for 17 h at 60°C in 1x target solution (Agilent Technologies) in a rotating hybridization chamber. Specifically, each hybridization consisted of two individual samples (one control and one knockout), one labelled with Cy3 and one with Cy5. None of the samples were pooled. A full description of all hybridizations can be found in 2log-transformed intensity measurements per sample were extracted from the normalized ratio data and used in all following analyses.Raw expression data were analyzed in R statistical processing software (version 2.6.0) using the LIMMA package  in BiocoCrb2 cKO and control retinas at each timepoint separately t-tests were used. The Benjamini–Hochberg method was used to correct for multiple testing. P-values < 0.05 after correction were considered significant. To detect genes with a significant interaction between age and genotype, we performed a 2-way analysis of variance (ANOVA) using age and genotype as grouping factors. To compare gene expression between 2 transformed and quantile normalized. To visualize the clustering of the samples, principal component analysis (PCA) was used. Differentially expressed genes were selected using a 2-way ANOVA using the factors genotype and age. The cutoff value for significantly expressed genes was a false discovery rate (FDR) [A second and independent analysis of the data was done using Partek version 6.6 . The Feature Extraction result text files were imported into Partek, logte (FDR) , of 0.05The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are Crb2 cKO, as indicated before. After genomic DNA degradation with RNase-free DNase I (New England Biolabs), 0.5 µg of total RNA was reverse transcribed into first-strand cDNA with Superscript III Plus RNase H-Reverse Transcriptase (Life Technologies) and 50 ng random hexamer primers, during 50 min at 50°C in a total volume of 20 µl. To the resulting cDNA sample, 14 µl of 10 mM Tris, 1 mM EDTA was added. From all samples, a 1:20 dilution was made and used for qPCR analysis. For this analysis, primer pairs were designed with a melting temperature of 60 °C, giving rise to an amplicon of 60–206 bp. Real-time qPCR was based on the real-time monitoring of SYBR Green I dye fluorescence on a ABI Prism 7300 Sequence Detection System . The PCR conditions were as follows: 12.5 µL SYBR Green PCR 2x mastermix (Applied Biosystems), 20 pmol of primers  and Crb2Chx10 cKO (Crb2F/F/Chx10Cre+/-) mice, were collected at different time points: P10, 1 month-old (1M), 3M (n=3-4/age/group). Eyes were enucleated and fixed at room temperature with 4% paraformaldehyde in PBS for 20 minutes. After fixation, the eyes were dehydrated for 30 minutes in 30%, 50%, 70%, 90% and 96% ethanol and embedded in Technovit 7100 , according to the manufacture instructions and sectioned (3 µm). Slides were dried, counterstained with 0.5% toluidine blue and mounted under cover slips using Entellan . Bright field digital images were generated by a Leica epifluorescence microscope (DMRD), using LAS AF v2.4.1 software.Eyes, from control  were enucleated and fixed during 20 min in 4% paraformaldehyde in PBS. Subsequently, the tissues were cryo-protected with 30% sucrose in PBS, embedded in Tissue-Tek O.C.T. Compound  and used for cryosectioning. Cryosections 7 µm) were rehydrated in PBS. Samples were blocked for 1 h using 10% goat or donkey serum, 0.4% Triton X-100 and 1% bovine serum albumin (BSA) in PBS. The following primary antibodies were used: β-Catenin , Catenin pp120 (P120) , N-Cadherin , Calretinin , APC-conjugated CD11b , PE-conjugated CD45 , Cone arrestin , CRB1 . Total number of cells was determined by manually counting of positive cells on digital images generated by a Leica epifluorescence microscope, using LAS AF v2.4.1 software. (n) Represents the number of individual sections.Müller glia cells were counted at P10 using a SOX9 antibody. Five retina sections from three P<0.05, **P<0.01, ***P<0.001 were considered to be statistically significant. Values are expressed as means ± SEM. Calculation were made using the SPSS statistical package version 17.0.Normality of the distribution was tested by Kolmogorov-Smirnov test. Statistical analysis by Student’s t test or by Mann Whitney U test in case of a non-normal distribution. Values of *Crb2 cKO mice, the animals were backcrossed from mixed to 99.9% C57BL/6J background. Variation in the genetic background may influence the gene expression, as described before for the Crb1 knockout (Crb1-/-) mice [The CRB2 protein is expressed during retinal development in radial glial progenitor cells, and after differentiation in cone and rod photoreceptors and Müller glia cells . Since t/-) mice ,42. ApprCrb2 retina-specific conditional knockout mice, retinal tissue was collected at postnatal day 0 (P0), P3, P6 as well as P10. Five different retinas from Crb2Chx10 cKO (Crb2F/F/Chx10Cre+/-) and the control (Crb2F/+/Chx10Cre+/-) from each time point were used to cover the onset and first stages of the morphological phenotype observed in the mutant retinas. Equal amounts (825 ng) of Cy3- and Cy5-labelled RNA were hydrolysed and hybridized to a microarray, the hybridization pairs are indicated in P-values < 0.05 after correction were considered significant. Although we observed substantial changes in gene expression between time points , our analysis did not reveal significant differences in gene expression between mutant and control retinas. A second analysis, where we used t-tests to compare gene expression between Crb2 cKO and control retinas at each time point separately, also did not reveal significant changes in gene expression. A description of the top 100 genes with the lowest P-values for each time point can be found in the  We used a whole mouse gene expression microarray that contains over 44,000 probes. To elucidate the molecular events that precede and lead to the morphological phenotype in the Crb2 cKO mice were constructed in such way that the full length CRB2 protein could not be produced in the homozygote mutant retinas. The 5’ loxP site was inserted in intron 9, the 3’ loxP site was inserted in exon 13 behind the stop codon. The deleted area contains part of the CRB2 extracellular domain, and the entire transmembrane domain and the 37 amino acids of the C-terminal intracellular domain. Cre mediated recombination deleted coding exons 10-13 and resulted in a nonsense mutation with premature truncation of the CRB2 protein at amino acid 871. The unique 60 base pair Crb2 probe (ID: 235570) in the array is located in between the 3’ loxP site and the polyadenylation sequence of the gene  using two different sets of primers, one located outside of the recombined area (exon 7) and another set in the recombined area (exons 11/12)  (P = 0.004) (Crb2 levels using the primers located outside the recombined area (exon 7)   . At P3, = 0.004) . However(exon 7) . The dat= 0.381) , what suWe previously showed using an antibody against the intracellular C-terminal 37 amino acids domain of CRB2 that the protein was removed from the outer limiting membrane from embryonic day 12.5 onwards . So, theCre expression in a subset of cells. To evaluate the mosaicism of the Chx10Cre mouse line we crossed the Chx10Cre transgenic mouse line with a mT/mG (membrane-targeted tandem-dimer-Tomato RFP / membrane-targeted Enhanced GFP) reporter mouse line [mT/mG retinas only red fluorescent protein could be detected  retinas expression of red and green fluorescent proteins was found . The qPCR data confirmed the data obtained by microarray analysis. We investigated by qRT-PCR the role of pathways . Only thCrb2 cKO and control retinas.Analysis of cluster gene expression was performed at distinct retinal development time points. Crb1 knockout (Crb1-/-) mice [Crb2 null retinas, retinas of P10, and 1- and 3 months-of-age were analysed.The phenotype of retinas lacking CRB2 from early retinal development was previously described . Those a/-) mice ,22. So,  The phenotype of retinas lacking CRB2 on 99.9% C57BL/6J genetic background appeared grossly the same as on 50% C57BL/6J genetic background  at the t Adult retinas on 99.9% or 50% C57BL/6J genetic background showed similar progression of the morphological phenotype. In 1 month-old retinas, protrusions of photoreceptors into the subretinal space were observed . Three m In conclusion, the CRB2 null retinas on 99.9% C57BL6/J background showed a similar progression of the phenotype than on 50% C57BL6/J background.As in CRB2 null retinas on mixed background, the CRB2 null retinas on 99.9% C57BL6/J background showed at postnatal day 10 a consistent disorganized morphology with many photoreceptors misplaced in the subretinal space adjacent to the retinal pigmented epithelium, thus we examined 10-days old retinae in detail using immunohistochemistry. At P10, the outer limiting membrane was disrupted throughout the mutant retina, and although CRB2 was lost in the entire retina , loss of In CRB2 null retinas several ectopic photoreceptor nuclei were present in the subretinal space immediately adjacent to the retinal pigment epithelium . Most of Some of the ectopic photoreceptor cells were not able to establish synapses in the outer plexiform layer, demonstrated by ectopic localization of PSD-95  and MPP4 Most of bipolar cells stained by anti-PKCα , amacrin Also, no misplaced SOX9-positive Müller glia nuclei were observed in the CRB2 null retinas , howeverCrb2Chx10 cKO retinas (99.9% C57BL/6J) showed an increase in the number of Müller glia cells . In Crb2Chx10 cKO retina, we observed ectopic protein expression of two microglia cell markers, CD45 and CD11b, in the misplaced photoreceptor cell layer , only a limited number of ﬁve genes showed changes in gene expression in 3-months-old white-light-exposed Crb1-/- mice [Pttg1), chromatid cohesion (Esco1), or chromosome stabilization (similar to histone H2b). Interestingly, downregulation of Pttg1 was also detected in white light exposed Crb1C249W/- retinas that expressed a CRB1 protein with an amino acid substitution in the extracellular domain [In previous studies using -/- mice . Three or domain .Crb2 null retinas, the CRB2 protein is lost from the entire subapical region. However, loss of CRB2 leads to loss of localization of other apical proteins, as for example members of the CRB, PAR and adherens junction complexes, only at foci where mislocalized cells were found. In In conclusion, the results suggest that loss of CRB2 in the developing retina had no major influence on the gene expression profile. Figure S1Principal component analysis (PCA) of all probes and all samples.PCA analysis separates samples on the age. Each data point/shape represents a sample, with all the arrays presented on the PCA plot. Crb2 conditional knockout (CKO) (red), and control (CONT) (blue) samples, are distinguished by color, and age P0 (piramide), P32 (tetrahedron), P6 (octahedron), and P10 (cube) are distinguished by shape. Given PC 1 describes the largest amount of data variance (34%), the aggregation of the samples is mostly accounted for the sample age. The percentage values in parentheses indicate the proportion of total variance described by each PC. PC 1 principal component 1 (X-axis); PC 2 principal component 2 (Y-axis); PC 3 principal component 3 (Z-axis).(TIF)Click here for additional data file.Figure S2Loss of CRB2 results retinal disorganization in mice C57BL/6J genetic background.Toluidine stained light microscopic pictures of retina sections at different ages, P10 - (A), 1M - (C), 3M - (D). Whole section figures, from the CRB2 null retinas, were mounted from individual pictures using stitching/MosaicJ pluging from ImageJA v1.45b. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar: 50 µm.(TIF)Click here for additional data file.Table S1Concentration and integrity of the RNA and cRNA samples used in the microarray (P0 and P3).(DOCX)Click here for additional data file.Table S2Concentration and integrity of the RNA and cRNA samples used in the microarray (P6 and P10).(DOCX)Click here for additional data file.Table S3Description of the hybridization pairs and distribution of the samples in the arrays.(DOCX)Click here for additional data file.Table S4qRT-PCR primers used in this study.(DOCX)Click here for additional data file.Table S5Differential gene expression between control and knockout neuroretinas in fold differences, at postnatal day 0. Top 100 genes ranked on their P value given by the students’ t-test  before applying Benjamini–Hochberg  method for correct to multiple testing. The expression value to the individual genes for control (CONT) and knockout (CKO) groups (log2 intensity), and the fold differences between control and knockout (FC) are also described in the table.(DOCX)Click here for additional data file.Table S6Differential gene expression between control and knockout neuroretinas in fold differences, at postnatal day 3. Top 100 genes ranked on their P value given by the students’ t-test  before applying Benjamini–Hochberg  method for correct to multiple testing. The expression value to the individual genes for control (CONT) and knockout (CKO) groups (log2 intensity), and the fold differences between control and knockout (FC) are also described in the table.(DOCX)Click here for additional data file.Table S7Differential gene expression between control and knockout neuroretinas in fold differences, at postnatal day 6. Top 100 genes ranked on their P value given by the students’ t-test  before applying Benjamini–Hochberg  method for correct to multiple testing. The expression value to the individual genes for control (CONT) and knockout (CKO) groups (log2 intensity), and the fold differences between control and knockout (FC) are also described in the table.(DOCX)Click here for additional data file.Table S8Differential gene expression between control and knockout neuroretinas in fold differences, at postnatal day 10. Top 100 genes ranked on their P value given by the students’ t-test  before applying Benjamini–Hochberg  method for correct to multiple testing. The expression value to the individual genes for control (CONT) and knockout (CKO) groups (log2 intensity), and the fold differences between control and knockout (FC) are also described in the table.(DOCX)Click here for additional data file.


To understand the evolutionary steps required for a virus to become virulent in a new host, a human influenza A virus (IAV), A/Hong Kong/1/68(H3N2) (HK-wt), was adapted to increased virulence in the mouse. Among eleven mutations selected in the NS1 gene, two mutations F103L and M106I had been previously detected in the highly virulent human H5N1 isolate, A/HK/156/97, suggesting a role for these mutations in virulence in mice and humans.in vitro (mouse and canine cells) and in vivo (BALB/c mouse lungs) as well as enhanced virulence in the mouse. Only the M106I NS1 mutation enhanced growth in human cells. Furthermore, these NS1 mutations enhanced early viral protein synthesis in MDCK cells and showed an increased ability to replicate in mouse interferon β (IFN-β) pre-treated mouse cells relative to rPR8-HK-NS-wt NS1. The double mutant, rPR8-HK-NS-F103L + M106I, demonstrated growth attenuation late in infection due to increased IFN-β induction in mouse cells. We then generated a rPR8 virus possessing the A/HK/156/97 NS gene that possesses 103L + 106I, and then rescued the L103F + I106M mutant. The 103L + 106I mutations increased virulence by >10 fold in BALB/c mice. We also inserted the avian A/Ck/Beijing/1/95 NS1 gene (the source lineage of the A/HK/156/97 NS1 gene) that possesses 103L + 106I, onto the A/WSN/33 backbone and then generated the L103F + I106M mutant. None of the H5N1 and H9N2 NS containing viruses resulted in increased IFN-β induction. The rWSN-A/Ck/Beijing/1/95-NS1 gene possessing 103L and 106I demonstrated 100 fold enhanced growth and >10 fold enhanced virulence that was associated with increased tropism for lung alveolar and bronchiolar tissues relative to the corresponding L103F and I106M mutant.To determine the selective advantage of these mutations, reverse genetics was used to rescue viruses containing each of the NS1 mouse adapted mutations into viruses possessing the HK-wt NS1 gene on the A/PR/8/34 genetic backbone. Both F103L and M106I NS1 mutations significantly enhanced growth The F103L and M106I NS1 mutations were adaptive genetic determinants of growth and virulence in both human and avian NS1 genes in the mouse model. IAV have now caused 4 pandemics in the past century, the most lethal being the 1918 Spanish Flu pandemic, where global mortality exceeded 20 million  and H5N10.8 fold [Increased virulence of avian HPAI H5 and H7 IAVs require the presence of a multi-basic amino acid HA cleavage site . Key mut0.8 fold . Here, w0.8 fold .NS1 is a multifunctional protein that binds both ssRNA and dsRNA ,12 and iWith respect to the role of the NS1 protein in virulence, previous studies have identified single mutations in the NS1 gene  as well as multiple mutations in the PDZ ligand domain that increased viral pathogenicity -30. FurtMadin-Darby canine kidney (MDCK) cells, human embryonic kidney cells (293T), mouse kidney epithelial cells (M1), and human lung epithelial cells (A549) were maintained in complete minimum essential medium or Dulbecco's Modified Eagle Medium  supplemented with L-glutamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and fetal bovine serum (to 10%).50 of >107.7 pfu/ml in mice was used to generate virulent mouse-adapted strains [The prototype A/HK/1/68 (H3N2) (HK-wt)  having an LD strains ,34.The NS cDNA of A/HK/1/68 wt was inserted into pLLB vector by ligation independent cloning  and PCR AF036360)  were cloned into the pHH21 and pLLB plasmids respectively by homologous recombination as described [The NS genes of A/Chicken/Beijing/1/95-H9N2 (Ck/Bj/95)  and A/Hong Kong/156/97-H5N1 (A/HK/156/97)  were mixed with lipofectamine 2000  according to the manufacturer's instructions and incubated for 30 min at RT. The plasmids and the transfection reagent were added into a monolayer of 293T cells, 16 h post transfection, the transfection mix was replaced by opti-mem  supplemented with TPCK trypsin (1 μg/ml) . Forty-eight hours post-transfection the cell supernatant was collected and inoculated in 10-day old embryonated specific pathogen free embryonated eggs  for virus propagation at 37°C for 48 hours. The allantoic fluid was collected and viral yield was assessed by plaque assay. The NS gene sequence of the rescued viruses was verified by sequence analysis. The H9N2-NS plasmid was rescued onto the A/WSN/33 backbone using the 12 plasmid system as described .Monolayers of MDCK and A549 cells were washed twice with 1 × PBS and infected with each of the four rPR8 viruses at an MOI of 0.001 in presence of TPCK trypsin (1 μg/ml) in triplicate. The supernatant was collected at 12, 24, 48 and 72 hours post infection (hpi) and viral titer was assessed by plaque assay in duplicate for each sample.Virus samples were serially diluted in PBS. Six well plates of confluent monolayers of MDCK cells were washed twice with PBS and then infected with 100 μl of the different virus dilutions in duplicate for each dilution. The plates were incubated at 37°C for 30 min to allow virus adsorption. Following adsorption, the cells were overlayed with 0.65% agarose , in complete MEM supplemented with TPCK trypsin (1 ug/ml). The plates were incubated at 37°C and 3 days post incubation plaques were fixed with Carnoy's fixative (three parts acetic acid to one part methanol v/v).3 pfu of each of the rPR8 viruses in a volume of 50 μl [Groups of eleven BALB/c mice  were infected intranasally under halothane anesthesia with 5 × 10of 50 μl . Mice we6 pfu of rPR8-HK-NS (NS-WT and variants) and 106 pfu for the recombinant rWSN viruses. Groups of 3 mice were infected intranasally with 104 pfu of the rPR8-H5N1-NS viruses. Mortality was monitored for 14 dpi. Body weight was recorded daily and the LD50 was calculated by the Karber-Spearman method [Groups of 5 BALB/c mice were infected intranasally with 5 × 10n method .The animal studies were carried out in compliance with the guidelines of the Canadian Council on Animal Care (CCAC) as outlined in the Care and Use of Experimental Animals, Vol.1, 2nd Edn. (1993), which are internationally recognized as "best-practices" by the International Council for Laboratory Animal Science (ICLAS). The animal study protocol was approved by the University of Ottawa Animal Care Committee (Protocol Number: BMI-85). Efforts were made to minimize suffering and mice were euthanized at humane end-points when infections resulted in greater than 25% body weight loss plus respiratory distress.5 PFU of each of the recombinant viruses and lungs were collected two dpi. Lungs were inflated and fixed with 3.7% formaldehyde as described earlier [BALB/c mice were infected intranasally with 10 earlier . Lung se earlier . Rabbit 35S cysteine and methionine for one hour in cysteine and methionine free media . Post labeling, the cell lysates were collected with 1 × sodium dodecyl sulfate (SDS) sample buffer. The rate of viral protein synthesis was then assessed by SDS page gel electrophoresis and autoradiography as previously described [Monolayers of MDCK were infected with the different rPR8 viruses at MOI = 3. Two, 4, 6 and 8 hpi, the cells were pulse-labeled with 80 μCi/ml escribed .Monolayers of M1 cells were infected with each of the rPR8 and rWSN viruses at an MOI of 2. Twenty-four hours post infection the cell supernatants were collected and tested for IFN-β production. Infection of M1 cells with the VSV Indiana Δ 51 mutant that has a defect in the ability to block IFN induction was performed as a positive control using identical conditions . Mouse I6 PFU per 35 mm dish) for 30 min at 37°C. Following infections, the cells were washed twice with 1 × PBS, and serum free media was applied with TPCK trypsin (1 μg/ml). The supernatant was then collected at 0, 8, 24 and 48 hpi and the viral titer was assessed by plaque assay . The same experiment was done in parallel but without prior IFN-β treatment.Monolayers of M1 cells were treated with 200 U/ml of murine IFN-β  for 24 hours in triplicate assays. Twenty-four hours post treatment, the cells were washed twice with 1 × PBS prior to infection with the rPR8 viruses at MOI = 1  using the parameters of equal variance and two-tailed significance (unless otherwise indicated) with a probability of ≤ 0.05 considered as statistically significant. Values were calculated as means +/- standard deviation for sample size > 2 and +/- standard error for sample sizes of 2 . Because the F103L and M106I mutations have been associated with increased IFN-β induction in reassortants of the A/Hong Kong/156/97(H5N1) NS1 gene on the A/Udorn/1/1972 (H3N2) backbone [In order to identify nonsynonymous NS1 gene mutations that control virulence we generated virulent mouse adapted variants of A/HK/1/68 and compared their NS1 sequence changes to those found in the virulent H5N1 variant A/HK/156/97. Sequence analysis of the NS1 genes of 42 mouse-adapted clones identified two groups of variants MA20-B, -C and -D ,34 as webackbone  we measuTo test the role of F103L and M106I mutations on growth, we first generated viruses possessing the HK-wt (106F + 106M), or F103L, M106I and F103L + M106I mutations on the A/PR/8/34 backbone. To compare the growth kinetics of viruses that differed due to specific change in their NS1 genes, rPR8-HK-NS-wt, rPR8-HK-NS F103L, rPR8-HK-NS-M106I, and rPR8-HK-NS-F103L + M106I were used to infect MDCK and A549 epithelial cells, in triplicate, at a low MOI (0.001) to initiate multicycle replication. Supernatants were collected at 12, 24, 48 and 72 hpi and were assayed for yield of infectious progeny virus as measured by plaque assay. All the mutants had a significantly increased ability to grow in MDCK cells compared to rPR8-HK-NS-wt with the rPR8-HK-NS-M106I and rPR8-HK-NS-F103L + M106I mutants growing to higher titers than the rPR8-HK-NS-F103L mutant Figure . Growth in vitro compared to the rPR8-HK-NS-wt in MDCK and, in the case of rPR8-HK-NS-M106I and rPR8-HK-NS-F103L + M106I mutants, in A549 cells and thus demonstrate their adaptive nature in both human and canine cells.In contrast, growth of the different mutants in A549 cells was comparable to rPR8-HKNS-wt at early time points (i.e. 12 and 24 hpi), whereas at later time points (48 and 72 hpi) there was a trend to increased yield for the NS1 mutants where the rPR8-HK-NS-M106I and rPR8-HK-NS-F103L + M106I mutants demonstrate statistically significant enhanced growth compared to rPR8-HK-NS-wt at 48 hpi (P < 0.05) Figure . Therefo3 pfu each via the intranasal route, and their lung growth properties in lung tissue were compared. rPR8-HK-NS-wt virus reached a peak titer of 3.7 × 104 pfu/g in mouse lungs at 2 dpi and was cleared from the lungs by 5 dpi .The three mutant rPR8 viruses along with the rPR8-HK-NS-wt were used to infect groups of 11 BALB/c mice with 5 × 10i Figure . All thei Figure . Thus ali Figure . This in6 pfu of the different recombinants and mortality was monitored for 14 days pi. Although the LD50 of the rPR8 virus was 103.5 pfu (data not shown), introduction of the HK-wt NS1 gene onto this backbone resulted in rPR8-HK-NS-wt that was avirulent in mice and did not result in mortality (LD50>107 pfu) indicating an important role for PR8 NS1 in virulence  and post-transcriptional factors (CPSF-30). To explore whether the enhanced growth and pathogenicity of the mutant viruses correlated with enhanced viral protein synthesis, the rate of viral protein synthesis was determined by 4 pfu/ml at 24 hpi that was comparable to each of the single mutants at that time, while each of the mutations (103L and 106I) enhanced growth at the earliest (8 hpi) (by > 6 fold and >4 fold for the F103L and M106I mutants respectively) and at the latest time point (48 hpi) compared to rPR8-HK-NS-wt (P < 0.01)  (>7 fold increase by F103L mutant). At later time points (24 and 48 hpi) only the single mutants 103L and 106I were able to enhance growth in the presence of IFN-β relative to rPR8-HK-NS-wt (P ≤ 0.01)  Figure . The dou) Figure . Thus, f4 pfu. The rPR8-H5N1-NS-103L+106I resulted in 100% mortality by 8 dpi indicating a LD50 of <103.5  NS gene, that possesses both 103L and 106I residues, on another mouse adapted backbone A/WSN/33 backbone. We tested this backbone because WSN NS1 inhibits IFN-β induction by a different mechanism that includes CPSF-30 binding in contrast to PR8 NS1 that does not bind CPSF-30    to grow in BALB/c mice was also determined by infecting groups of 8 mice intranasally at a lower infection dose of 5 × 10) Figure . The gro) Figure . Thus thWe also assessed the extent of lung infection using immunofluorescent staining of infected lung sections to show that the presence of L103F and I106M mutations in the Ck/Bj/95 H9N2-NS1 gene on the WSN backbone resulted in lung infection that was restricted to specific foci in the bronchiolar epithelium Figure . In contAs both the F103L and M106I mutations were associated with greater levels of growth than the double mutant at later times following IFN-β pretreatment, we assessed the amount of IFN-β production during infection, which could explain the differences in mutant gene functions among these mutants. To assess the roles of our NS mutants in controlling IFN-β production, monolayers of M1 cells were infected with each of the rPR8 viruses at MOI = 2. Twenty-four hours post infection, cell supernatants were collected and IFN-β production was assessed by ELISA. Infection with the HK-wt and the PR8-wt viruses resulted in low or undetectable levels of IFN-β respectively indicating that both NS1 (and possible roles of the NEP) proteins can suppress IFN-β induction on their respective genetic backbones. The rPR8-HK-NS-wt recombinant also induced low levels of IFN-β (22 pg/ml) whereas each of the recombinants rPR8-HK-NS-F103L or rPR8-HK-NS-M106I induced 2 and 3 fold more IFN-β respectively compared to rPR8-HK-NS-wt (40 and 68 pg/ml respectively) while the double mutations together had a cumulative effect on increasing IFN-β induction to 113 pg/ml (P < 0.001) Figure . The incThe levels of IFN-β induction in M1 cells following infection with the recombinants that possess the H5N1 NS and Ck/Bj/95 NS genes were also measured and showed minimal levels of IFN-β induction at 24 hpi. Interestingly, mutating the NS1 sites 103L and 106I to L103F and I106M in either of these NS1 genes resulted in viruses of low and comparable interferon induction indicating a role of other NS1 gene mutations in the regulation of IFN-β induction by these viruses. The effectiveness of the influenza NS1 mutants to inhibit IFN-β induction was shown in comparison with the VSV Δ 51mtuant that has a defect in its ability to block innate immunity and resulted in 1,306 pg/ml IFN-β. We can conclude that IFN-β induction is controlled by multiple genetic factors involving the nature of the NS1 gene as well as interactions with the genetic backbone.Here we demonstrated that both the IAV NS1 F103L and M106I mutations were adaptive mutations that increased replicative abilities in cells of multiple species as well as virulence in the mouse lung. The observation that both the NS1 F103L and M106I mutations were under positive selection and were therefore adaptive mutations was evident by their increased prevalence in mouse adapted populations; where they were found in 3 of 6  and 3 ofThe NS1 mouse adaptive mutations demonstrated an increased ability to antagonize IFN-β activity observed by the enhanced ability to grow in mouse epithelial cells following IFN-β pre-treatment when compared to rPR8-HK-NS-wt. Increased growth of these mutants was also greater at early times in MDCK and mouse M1 cells consistent with greater replication at early times before IFN-β had been induced in the infected cell cultures. Although CPSF binding has been shown to result in inhibition of IFN and other host gene expression , A/PR/8/The increased protein synthesis of the F103L NS1 mutant was clearly seen for the M1 and NS1 proteins at all times in MDCK cells whereas the M106I mutation increased protein synthesis at 2 hpi but not at later times, and where the double mutant was not significantly enhanced relative to rPR8-HK-NS-wt  NS genes as shown on multiple genetic backgrounds (A/WSN/1/33 as well as A/PR/8/34). Therefore, we conclude that the NS1 protein of IAV is a virulence determinant that in addition to controlling IFN-β induction mediates gain-of-function effects on both protein synthesis and viral replication due to the presence of 103L and 106I that increase the extent of lung tropism and infection.The authors declare that they have no competing interests.SKD participated in the conception and design of the study, data acquisition, analysis and interpretation of data as well as drafting the manuscript. SW participated in the acquisition, analysis and interpretation of data. JP participated in the acquisition, analysis and interpretation of data. NEF participated in the acquisition, analysis and interpretation of data. LK participated in the acquisition, analysis and interpretation of data. YL participated in the acquisition, analysis and interpretation of data. EGB contributed to the study's conception and design, data acquisition, analysis and interpretation of data, drafting the manuscript and general supervision of the research group. All the authors have read and approved the final manuscriptEGB is a professor in the University of Ottawa, Department Of Biochemistry, Microbiology and Immunology. SKD, SW and NEF are PhD students at the University of Ottawa, Department Of Biochemistry, Microbiology and Immunology, Ottawa, Ontario, Canada. JP and LK are post-doctoral fellows at the University of Ottawa, Department Of Biochemistry, Microbiology and Immunology, Ottawa, Ontario, Canada. YL is a MSc. graduate from the University of Ottawa, Department Of Biochemistry, Microbiology and Immunology and currently employed with Health Canada, Ottawa, Ontario, Canada.


W69 and W106, two putative GPX genes, were obtained by de novo transcriptome sequencing of salt-treated wheat (Triticum aestivum) seedlings. The purified His-tag fusion proteins of W69 and W106 reduced H2O2 and t-butyl hydroperoxide (t-BHP) using glutathione (GSH) or thioredoxin (Trx) as an electron donor in vitro, showing their peroxidase activity toward H2O2 and toxic organic hydroperoxide. GFP fluorescence assays revealed that W69 and W106 are localized in chloroplasts. Quantitative real-time PCR (Q-RT-PCR) analysis showed that two GPXs were differentially responsive to salt, drought, H2O2, or ABA. Isolation of the W69 and W106 promoters revealed some cis-acting elements responding to abiotic stresses. Overexpression of W69 and W106 conferred strong tolerance to salt, H2O2, and ABA treatment in Arabidopsis. Moreover, the expression levels of key regulator genes  involved in salt, H2O2 and ABA signaling were altered in the transgenic plants. These findings suggest that W69 and W106 not only act as scavengers of H2O2 in controlling abiotic stress responses, but also play important roles in salt and ABA signaling.Oxidative stress caused by accumulation of reactive oxygen species (ROS) is capable of damaging effects on numerous cellular components. Glutathione peroxidases  are key enzymes of the antioxidant network in plants. In this study,  It is known that certain plant glutathione-S-transferase (GST) displayed also strong GPX activity (GST/GPX) toward organic hydroperoxides catalysing their reduction to the less toxic alcohols GPX gene was strongly expressed in normal tissues of high photosynthetic efficiency as well as in tissues stimulated by oxidative stresses GST/GPX gene confers salt and chilling tolerance due to enhanced ROS scavenging and reduces membrane damage in transgenic tobacco plants Arabidopsis GPX3 plays dual roles in H2O2 scavenging and ABA signal transduction Arabidopsis play a role in cross talk between photooxidative stress and immune responses Plant GPXs are involved in responses to many abiotic stresses and hormones Triticum aestivum L.) is one of the most important cereal crops in the world; however, ROS accumulation in cells induced by environmental stresses can cause severe oxidative damage to wheat growth and grain yield de novo transcriptome assembly technology. The isoenzymatic characteristics, subcellular localization, and transcript accumulation of the recombinant protein when transgenic plants were subjected to different stresses were investigated.Wheat (de novo assembly (unpublished data). Two up-regulated putative GPX cDNA fragments, designated as W69 (GenBank Accession No. KF031945) and W106 (GenBank Accession No. KF031946), were selected from the total transcript-derived assembled unigenes. Full-length cDNA of W69 and W106 were cloned successfully from wheat cDNA by reverse transcription PCR (RT-PCR) using special primers sets. The predicted W69 protein consists of 214 amino acids with a calculated molecular mass of 21.3 kD, and W106 protein consists of 211 amino acids and calculated molecular mass of 19.2 kD. Multiple sequence alignment of W69 and W106 with other reported GPXs from animals and plants showed that some motifs of GPXs were conserved among different species   . A numberomoters .W69 and W106 promoter regions have multiple abiotic stress-responsive elements . As shown in The elements . To asseW69 and W106 were rapidly activated to peak within 0.5 h after salt treatment, then declined to normal levels after 1 h, and were barely detectable within 24 h. Both were also rapidly induced by drought and ABA, reached a maximum level at 4 h and 1 h, respectively, and then declined. W69 was strongly induced by drought, but only weakly by ABA. In contrast, W106 was highly activated by ABA, but weakly by drought. In addition, W106 was immediately activated by H2O2 and absent after 6 h; however, the transcript of W69 was barely detectable over a 24 h treatment with H2O2. These results implied that the GPX subfamily may function mainly in oxidative responses induced by salt, H2O2 and ABA stresses, but with some divergence in roles between W69 and W106.Both E. coli BL21 cells was observed after IPTG induction. Samples of total proteins and purified soluble protein fractions were separated on SDS-PAGE. W69 (21 kDa) and W106 (19 kDa) were successfully expressed  program for predicting subcellular localization, indicated that both W69 and W106 with a chloroplastic N-terminal transit peptide would most probably localize in chloroplasts. To investigate the biological activity of the W69 and W106 proteins, the full-length cDNAs of W69 and W106 were fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into wheat mesophyll protoplasts. Fluorescence of both W69-GFP and W106-GFP was specifically detected in chloroplasts, whereas the control expressing hGFP alone showed fluorescence distributed throughout the protoplasts .W69 and W106 seedlings had similar phenotypes to wild-type seedlings under normal conditions. As expected, salt stress reduced the growth of both transgenic seedlings and wild-type seedlings to some extent . Therefore, overexpression of W69 and W106 resulted in enhanced early tolerance to high salt stress.Transgenic e extent . At 150 e extent . Furthertrations . In addiWe also examined tolerance to drought stress in the transgenic plants, but observed no detectable increases in tolerance (data not shown).Arabidopsis were transferred to MS medium plates supplemented with 1 mM H2O2. At 15 d, the seedlings of W69 and W106 grew vigorously and were larger than wild-type seedlings. At 1 mM H2O2, the transgenics had longer primary roots than wild-type plants. There were also significant increases in the number and total lengths of the lateral roots compared to wild-type  found in the promoters of W69 and W106 might be responsible for the responses of genes to ABA. The CRT/DRE and GT1GMSCAM4 elements present in the promoters of the two isoenzymes might be responsible for genetic responses to NaCl. More importantly, ARE, an antioxidant-responsive element in rat is also present in the promoters of the two isoenzymes, and might be related to genetic response to oxidative stress  seedlings grown hydroponically at 25°C for 10 d were subjected to various abiotic stresses. Seedlings were exposed to air on filter paper for rapid induction of water stress. To mimic salinity and ABA treatments, roots were submerged in 100 mM NaCl or 100 µM ABA, respectively. For hydrogen peroxide treatment, seedlings were transferred to solutions containing 0.1 mM H2O2. Materials were collected at 0, 1, 2, 4, 16, 12, and 24 h after various treatments. Harvested plants were dropped immediately into liquid nitrogen and stored at –80°C for RNA extraction.Wheat  method.GPX genes were measured by Quantitative real-time PCR (Q-RT-PCR), using the ABI Prism 7300 system . Total RNA was extracted from young leaves of 2-week-old wheat plants using Trizol reagent according to the manufacturer's protocol  and DNase I digestions were applied . First-strand cDNA was synthesized with AMV Reverse Transcriptase . Each PCR was repeated three times in total volumes of 20 μl containing primer (4 μM), cDNA (40 ng) and 9 μl RealMasterMix . Quantification of the target gene expression under each stress was carried out by the relative 2−△△CT method actin gene was used as an internal control for normalization of template cDNA. The gene-specific primers were as follows: W69, 5'- ACAGAGGATTCGGGAGGACT-3' and 5'- AGGAGGACCTTCCCCTTGTA-3'; W106, 5'-ACAAGGGGAAAGTCCTGCTT-3' and W106-RTR 5'-CTGGTTCCTGTCCAGCAAAT-3'; wheat actin, 5'- CTCCCTCACAACAACCGC-3' and 5'- TACCAGGAACTTCCATACCAAC-3'. Q-RT-PCR was also performed to analysis the expression of stress- and ABA-responsive genes, using 2-week-old transgenic and wild-type Arabidopsis seedlings. The actin gene was used as an internal control. The specific primers were as follows: RbohD (At5g47910), 5'-CCTCAACAACACCACCTCCT-3' and 5'-GTAAGAGGCCGTTGGAATCA-3'; ABI1 (At4g26080), 5'-TGAAGAAGCGTGTGAGATGG-3' and 5'-CTGTATCGCCAGCTTTGACA-3'ABI2 (At5g57050), 5’-TGCGGCGAGTAAAAGAAGAT-3' and 5'-TTCCTTTTTGCAAAGCCATC-3'; SOS1 (At2g01980): 5'-ACCGGCAGATCTAATGAACG-3'and 5'-CTCCGCTACTGTCGATGTCA-3'; Actin2 in Arabidopsis, 5'-GGTAACATTGTGCTCAGTGGTG-3' and 5'-C GACCTTAATCTTCATGCTGC-3'.Expression patterns for the two 5'-TACGGATCCATGGGGGCGTCCGAATCT-3' and 5'-TAGCTCGAGCTTCTGCGAGTCGGAAGATTCC-3') and W106-PET (5'-TACGGATCCAACATGGGTGCGGCAGAGT-3' and 5'-TAGCTCGAGAACCTCCAACAGCTTCTTGATG-3'). The PCR product was cut by BamHI and XhoI and then subcloned into corresponding sites of pET-28a (Novagen) with a Histidine-tag at the N-terminus. The fusion plasmids and empty vector (PET-28a) were transformed into E. coli BL21 (DE3) strain. E. coli cells carrying pET-W69, pET-W106, and PET-28a were grown in 40 ml LB medium containing 50 mg/ml kanamycin at 37°C to D600  = 0.5 and induced with 0.8 mM isopropyl-1-thio-bD-galactopyranoside (IPTG) at 22°C. Cells were extracted at 4 h and were collected by centrifugation at 3000 g for 10 min, resuspended in pre-chilled sodium phosphate buffer , and disrupted by sonication. His-tag fusion pET-W69/pET-W106/and/PET-28a proteins were purified using Ni-NTA Agarose  according to the manufacturer's instructions. Protein concentrations were quantified using the Bradford method. Purified proteins from each sample were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis.The two GPX cDNAs were amplified by reverse transcription PCR (RT-PCR) using the primer sets W69-PET . The reaction was started by addition of H2O2 or t-butyl hydroperoxide. The absorbance was immediately read at OD340 nm and again at 4 min using a Perkin-Elmer Lambda 35 UV/VIS spectrometer . The unit activity (U/L) is the amount of GPX that produces 1 μM of oxidized glutathione (GSSG) per min at pH 7.6 and room temperature.Enzyme activity was measured spectrophotometrically using a Glutathione Peroxidase Assay Kit  with minor modifications. W69 and W106 genes were respectively cloned into 16318hGFP vectors, fused with the GFP reporter gene under the control of the 35S promoter W69-hGFP and W106-hGFP fusion plasmids were transformed to common wheat mesophyll protoplasts by the PEG-mediated method Green fluorescent protein (GFP)-fused GPXs, coexpression plasmids was constructed for the expression studies. The full open reading frames of the W69 and W106 were separately ligated into the modified pBI121 vector under control of the CaMV 35S promoter Arabidopsis transformants carrying W69 or W106 were generated using the vacuum infiltration method. Transformed plants were cultured on Murashige and Skoog (MS) medium containing 0.8% agar and 50 μM Kanamycin in a day/night regime of 16/8 h under white light (50 photons m−1s−1) at 22°C for 2 weeks and were then transferred to soil. The T3 generation seeds obtained were used for further analysis.Fragments of Arabidopsis plants were grown on 10×10 cm MS agar plates. They were routinely kept for 3 days in the dark at 4°C to break dormancy and transferred to a tissue culture room under a day/night of 16/8 h under white light (with 50 photons m−1s−1) at 22°C for 3 d.Seeds of transgenic (T3 generation) and wild-type 2O2 treatment, the plants were placed on MS agar plates supplemented with H2O2 (1–5 mM) for 7 days. For ABA treatment, the seeds of transgenic and wild-type Arabidopsis were grown on MS agar plates supplemented with 0.5–1 μM ABA for 7–14 days. Seeds were considered germinated when radicles had completely emerged from the seed coat.For salt treatment, 7-day-old seedlings were transferred to MS agar plates containing 150 mM NaCl for 7 days. For drought treatment, the seedlings were placed on MS agar plates with mannitol (100–200 mM) for 7 days. For HArabidopsis were measured with a ruler, and lateral roots were counted and measured with a dissection microscope. The fresh weight was measured on an analytical balance immediately after plant harvest. The samples used for dry weight determinations were measured after 48 h at 70°C.The root lengths of


Cydia pomonella (L.), an economically important fruit pest. Here we report the characterization and expression analysis of the first P450 gene, designated as CYP9A61, from this pest. The full-length cDNA sequence of CYP9A61 is 2071 bp long and its open reading frame (ORF) encodes 538 amino acids. Sequence analysis shows that CYP9A61 shares 51%–60% identity with other known CYP9s and contains the highly conserved substrate recognition site SRS1, SRS4 and SRS5. Quantitative real-time PCR showed that CYP9A61 were 67-fold higher in the fifth instar larvae than in the first instar, and more abundant in the silk gland and fat body than other tissues. Exposure of the 3rd instar larvae to 12.5 mg L−1 of chlorpyrifos-ethyl for 60 h and 0.19 mg L−1 of lambda-cyhalothrin for 36 h resulted in 2.20-and 3.47-fold induction of CYP9A61, respectively. Exposure of the 3rd instar larvae to these two insecticides also significantly enhanced the total P450 activity. The results suggested that CYP9A61 is an insecticide-detoxifying P450.Cytochrome P450 monooxygenases (CYPs or P450s) play paramount roles in detoxification of insecticides in a number of insect pests. However, little is known about the roles of P450s and their responses to insecticide exposure in the codling moth  Effective management of the codling moth mainly depends on chemical insecticides [The codling moth ltivated –6. The fcticides ,5. A majcticides –10 and scticides –13 in macticides ,10. In CHelicoverpa armigera (Hübner) [Aphis mellifera (L.) [Heliothis virescens (Fabricius) [Aedes aegypti (L.) [Cimex lectularius (L.) [Culex quinquefasciatus (Say) [Cytochrome P450 monooxygenases (CYPs or P450s) are a superfamily of enzymes responsible for detoxification of natural and synthetic xenobiotics including insecticides and plant toxins –19. In tera (L.) , Heliothbricius) , Aedes apti (L.) , Cimex lius (L.) , and Culus (Say) .C. pomonella, extensive studies have suggested that P450s may play a key role in detoxification of OP and SP due to the observed significantly higher total P450 enzyme activity in the resistant C. pomonella strains than in the susceptible laboratory strains [CYP9A61, from this species. The spatiotemporal and insecticide-induced expression profiles of CYP9A61 revealed by real-time quantitative PCR (RT-qPCR) suggest that it is a xenobiotic-metabolizing P450 gene.In  strains –5,9,10. 2.2.1.CYP9 genes  . It also shares a high degree of amino acids identity with the CYP9A members from B. mori , H. armigera , and Zygaena filipendulae (L.) . Moreover, CYP9A61 shares more than 50% identity with CYP9A38 from Cnaphalocrocis medinalis (Guenée) , CYP9A39 from Spodoptera litura (Fabricius) , CYP9A9 from Spodoptera exigua (Hübner) , CYP9A1 from H. virescens , and CYP9A5 from M. sexta .Amino acid sequence alignment indicates that CYP9A61 shares 58%–60% identity with the CYP9A members from C-terminal that is conserved in most of insect P450 enzymes. The typical aromatic motif PxxFxPxRF (meander) on the K-K′ loop is found at the position of 449–459 aa in this protein. The ERR-triad formed by the ExxR (residues 392–395) motif and PxRF (residues 454–457) motif may form a set of salt-bridge interactions to assure the cysteine pocket is at the right position to make sure the heme binds to protein [N-terminal in the C Helix may be involved in eliminating the charge of propionate side chains of the heme [N-terminal of β strand 1–4, and β strands 4-1 and 4-2 protrude into SRS6. The CYP9A61 was predicted to have no signal peptide in the protein N-terminal but contains a transmembrane domain (residues 2–24) at the N-terminus  CYP9 and M. sexta CYP9A5 with 48% bootstrap support, indicating CYP9A61 shared closest relationship with D. plexippus CYP9 and M. sexta CYP9A5. Although the Bombycidae’s CYP9s share the highest amino acid identity with CYP9A61 (57%–60%), they are located in another branch with the CYP9s from Noctuidae, Pyralidae and Zygaenidae. In this tree, the transcripts of some CYP9 members were observed to be induced by insecticides. For instance, deltamethrin increased the expression levels of CYP9A17 and CYP9A12 from H. armigera [CYP9A1 from H. virescens was increased through exposure to thiodicarb [To examine the relationship of CYP9A61 with members of the CYP9 family from Lepidoptera deposited in the GenBank database, a phylogenetic tree was constructed using the neighbor-joining method . The anaarmigera . The mRNiodicarb .2.3.CYP9A61 expression was observed in the first 48 h. The 60 h chlorpyrifos-ethyl exposure led to a 2.2-fold up-regulation of CYP9A61 at a concentration of 12.5 ng when compared with the control , and thereafter dropped to 2.60 fold after 60 h of lambda-cyhalothrin exposure . The gene expression level dropped at the pupal stage  and finally increased in adults . Interestingly, the expression of CYP9A61 was not different between the male and female codling moth in pupal and adult stages.To determine the relative expression patterns of her ages . The res2.5.CYP9A61 in the head, cuticle, silk gland, midgut, fat body, and Malpighian tubule dissected from third instar larvae were investigated  and fat body (384.00-fold higher than in the cuticle), while the gene has relative low expression in midgut, head and the Malpighian tubule, at 9.87-, 3.61-, and 12.58-fold of the cuticle  activity was observed after treatment with chlorpyrifos-ethyl  was observed for the experimental group at 48 h and 60 h, with the value of 6.75 and 6.66 pmole min−1 mg protein−1, respectively. Higher activity was detected for the longer time inductions (72 and 96 h), with 1.14 and 1.20-fold of PNOD activity increase when compared with the control.In order to determine whether chlorpyrifos-ethyl and lambda-cyhalothrin could influence the mixed-function oxidase (MFO) activity, the MFO activities using os-ethyl . The PNOFor the lambda-cyhalothrin exposed larvae, the main interaction effects for insecticide exposure and exposure time and insecticide exposure and exposure time were significant . Signifi3.CYP9A61 cloned from C. pomonella larvae in the present study represents the first P450 gene isolated from this species. Sequence alignment demonstrates that CYP9A61 shares 57% and 56% homology with H. armigera CYP9A12 and CYP9A17, respectively. In addition, the SRS1, SRS4 and SRS5 are highly conserved with the CYP9 members above  [CYP6Z1 from Anopheles gambiae [CYP9A61 expression levels in the whole body of the codling moth are higher in the active feeding stages (fourth and fifth instar larvae) than non-feeding stages (pupa and adult). This is somewhat consistent with CYP4 genes from fat body and midgut of M. sexta [CYP9A61 is potentially responsible for detoxification of xenobiotic compounds including insecticides or plant toxins suffered during feeding.We investigated the armigera , suggestasciatus . No signe female . Further(Meigen)  and CYP6 gambiae  where thM. sexta . In thatCYP9A61 in the tissues of the codling moth was determined. It has been reported that tissue-specific expression of detoxification related genes might illustrate some of the functions of their biological and physiological roles [CYP9A61 with NADH-cytochrome b5 reductase from H. armigera, but the tissue-specific expression pattern of CYP9A61 is inconsistent with the H. armigera NADH-cytochromeytochroreductase [CYP9A61 may be associated with detoxification of xenobiotic compounds. Silk glands, an organ which produce threads of silky material, where CYP9A61 is abundantly expressed, indicates that CYP9A61 has a potential role in protection of codling moth from mechanical injuries. However, to determine the accurate function of CYP9A61 in detoxification of xenobiotic compounds, the CYP9A61 expression level of specific-tissues of codling moth exposure to insecticides should be further investigated.To better understand the function of CYP9A61, the distribution of al roles . We founeductase . To our eductase ,34. In oH. virescens strain, the expression of CYP9A1 from whole body was 29- and 13-fold higher than in two susceptible strains [CYP9 mRNA transcripts for all developmental stages were observed in pesticide-exposed C. lectularius collected under repeated insecticide treatments in an apartment in Columbus, OH, USA [A. mellifera, CYP9Q3 in the midgut of 3-d-old worker bee was slightly induced (1.5-fold) by tau-fluvalinate [CYP9M8 (2.5-fold) and CYP9M9 (2-fold) in permethrin-exposed fourth instar A. aegypti larvae [H. armigera, deltamethrin induced CYP9A12 and CYP9A17 expression in the midgut whereas it repressed both CYP9A12 and CYP9A17 transcripts in fat body [CYP9A61 in the codling moth.In insects, substantial experimental effort is being focused on the P450s in response to plant allelochemicals and other secondary metabolites to elaborate plant-insect co-evolution interactions ,35. The  strains . Elevate OH, USA . In A. mvalinate . A simili larvae . Furtherfat body . Based oCYP9A61 to chlorpyrifos-ethyl and lambda-cyhalothrin exposure was investigated. The CYP9A61 transcript is significantly induced by lambda-cyhalothrin , a non-managed codling moth affected area. Low instar C. pomonella larvae were reared on the original collected whole apples. The higher instar larvae were removed from the apples and reared on flesh of the apple debris in the laboratory at 16:8 h light:dark photoperiod, 25 ± 1 °C and 60% ± 5% relative humidity [The apples with tunnels and frass produced in the skin after consumption by 4.2.et al. demonstrated that the LC50 of chlorpyrifos-ethyl and lambda-cyhalothrin on neonate codling moth were 157.65 mg/L and 0.35 mg/L [2 semi-artificial diet piece. In the present study, the topical application method [Chlorpyrifos-ethyl and lambda-cyhalothrin with purities of >99% were obtained from Aladdin Reagent . These insecticides were dissolved in acetone. Rodríguez .35 mg/L , respectn method  was usedn method  using an4.3.C. pomonella was extracted from five third instar larvae using the RNAiso Plus Kit based on the manufacturer’s instructions . The quality and concentration of extracted RNA was examined by agarose gel electrophoresis and spectrophotometer analysis . The RNA samples were then digested with DNase I (MBI) to remove the genomic DNA and for cDNA synthesis. For 3′-rapid amplification of cDNA ends (3′RACE), first strand cDNA was synthesized from 1 μg of total RNA using the SMART™ RACE cDNA Amplification Kit and MLV Reverse Transcriptase  as described by the manufacturer. For 5′-rapid amplification of cDNA ends (5′RACE), single-stranded cDNA was synthesized from 1 μg of total RNA by 5′-Full RACE Kit  according to the procedure recommended. The product was stored at −20 °C for future use.The total RNA of 4.4.CYP9A61 gene was amplified with specific primers P9F and P9R  to eliminate any potential error occurring by Taq DNA polymerase. The full-length cDNA sequence was subjected to the Open Reading Frame Finder  for open reading frame (ORF) prediction. The ORF of CYP9A61 cDNAs was amplified by using primer pairs P9ORF-F and P9ORF-R (Degenerate primers used for 3′ and 5′RACE amplification were designed  based on and P9R . The PCR P9ORF-R . The PCR4.5.pI) was also predicted. The deduced amino acid sequence of CYP9A61 was aligned with Manduca sexta CYP9A5 (GeneBank number: AAD51038.1), H. virescens CYP9A1 (GeneBank number: AAC25787.1) and Bombyx mori (L.) CYP9A20 (GeneBank number: NP_001077079.1) which have been reported as possibly induced by insecticide exposure [The CYP9A61 amino acid sequence was deduced from ORF cDNA sequence using the ExPASy Proteomics web tool . Using texposure  using thexposure . The secexposure . Motifs exposure  The presexposure . An un-rexposure  under th4.6.CYP9A61. The total RNA was extracted from collected tissues, the whole body of various developmental stages of C. pomonella, and insecticide and acetone (control) larvae. For developmental expression and insecticide induction expression assays, five individuals were used to isolate total RNA, using three independent extracts. For the tissue specific expression study, each tissue dissected from 30 third instar larvae was regarded as an independent specimen to isolate total RNA, using three independent extracts. The first strand cDNA of each RNA sample for qPCR was synthesized using 1.5 μg of total RNA using PrimeScript™ RT reagent Kit with gDNA Eraser  in a 20 μL reaction mixture as described by the manufacturer. The relative expression levels of CYP9A61 in different tissues, at each developmental stage, and under chlorpyrifos-ethyl and lambda-cyhalothrin exposure were assessed using the reference gene C. pomonella beta-actin  as a reference. In our previous study, CpActin was showed to be an excellent reference gene due to being expressed constantly in different tissues, developmental stages, and exposure to insecticides [CYP9A61 and CpActin were obtained using a linear gradient dilution (10-fold dilution) of cDNA as template. The amplification efficiencies, slopes, and correlation coefficients (R2) were 100.7%, −3.306 and 0.994 for CYP9A61 standard curve, and 95%, −3.334 and 0.995 for CpActin, respectively. The relative CYP9A61 mRNA expression was calculated according to the 2−ΔΔCt method [QPCR was carried out to study tissue distribution, stage specific and insecticide-induced expression of cticides . The prit method . A negat4.7.Whole bodies from insecticide-treated and acetone-treated third instar larvae were homogenized on ice for three replications of five individuals each in 2 mL ice-cold 0.1 M sodium phosphate buffer (pH 7.8) containing a final concentration of 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM ethylene diamine tetraacetic acid (EDTA) and 1mM dithiothreitol (DTT). The homogenates were centrifuged at 4 °C for 20 min at 12,000 g, and the supernatants were used as an enzymatic source for the MFO assay . The proet al. [p-nitroanisole, 10 μL of enzyme. After 5 min of incubation at 30 °C, the reaction was initiated by adding 10 μL of 9.6 mM reduced form of nicotinamide adenine dinucleotide phosphate (NADPH). A negative control reaction was conducted using heat inactivated enzyme (95 °C for 10 min) to eliminate any non-enzymatic activity. Moreover, a no-NADPH-added control was also performed. Pre-incubated sodium phosphate buffer was used for the blank. After 1 h of reaction at 30 °C, the absorbance was read in an Infinite M200 PRO Microplate Reader  at 405 nm. A standard curve was conducted with p-nitrophenol, and the result was expressed as pmole p-nitrophenol min−1 mg of protein−1. Each experiment contains three biological replicates and three replicates were conducted for each test and the average was used for statistical analysis.The MFO activities were assessed by measuring PNOD activity on Nunc 96-well transparent microplates  as described by Yang et al.  with sli4.8.p = 0.05) was used for statistical analysis to determine the significance of the difference in CYP9A61 mRNA expression for developmental stages and insecticide induction in the codling moth. The differences in CYP9A61 mRNA expression for tissue distribution was assessed by Student’s t-test . Factorial ANOVA and Student’s t-test were used to analyze the enzyme activity of C. pomonella treated with different insecticides and different durations of exposure to insecticide. The results were plotted using GraphPad Prism 5 .Statistical analysis was carried out by the SPSS statistics software . One-way analysis of variance (ANOVA, 5.C. pomonella. We can conclude that CYP9A61 is a xenobiotic-metabolizing P450 gene. This study advances our understanding of CYP9A61 function, and regulation mechanism, and the CYP9A61 response to insecticide exposure. Further studies of CYP9A61 detoxification of xenobiotic compounds in vitro are required for accurate assessments of the metabolic functional activity toward these chemicals.In summary, this is the first sequence of a P450 gene isolated from


Avian influenza virus A/chicken/Jiangsu/1001/2013(H5N2) was identified from a healthy chicken in an eastern China poultry market. Whole-genome analysis demonstrated that the H5N2 virus originated from a reassortance between a previous A/chicken/Hebei/1102/2010(H5N2) virus and an endemic H5N1 virus. The results indicated that continuing reassortance of H5N2 has been occurring in domestic poultry of China. From 2000 onwards, various reassortant viruses with the hemagglutinin (HA) gene from the influenza virus A/goose/Guangdong/1/96-like lineage and internal segments from other subtypes emerged in chickens, ducks, and geese . In AsiaIn this study, 20 feces samples from apparently healthy chickens in a live bird market in the Jiangsu province of eastern China were collected and used to screen for avian influenza virus infection with the primers and conditions described by Zhou et al. . The obtThe long-term endemicity of the highly pathogenic avian influenza H5N1 virus in poultry and the common practice of intermingling in poultry raising were thought to be possible reasons for the generation of reassortant H5 HPAI viruses with neuraminidase (NA) subtypes other than N1 . AlthougKF150631, KF150632, KF150633, KF150634, KF150635, KF150636, KF150637, and KF150638.The complete genome sequence of A/chicken/Jiangsu/1001/2013(H5N2) is available in GenBank under accession numbers