Drosophila “photoshop” mutations, which increase expression of green fluorescent protein and other transgenes. Mapping and molecular analyses show that photoshop mutations are loss-of-function mutations in the Drosophila homologs of NMD genes Upf1, Upf2, and Smg1. We find that Upf1 and Upf2 are broadly active during development, and they are required for NMD as well as for proper expression of dozens of wild-type genes during development and for larval viability. Genetic mosaic analysis shows that Upf1 and Upf2 are required for growth and/or survival of imaginal cell clones, but this defect can be overcome if surrounding wild-type cells are eliminated. By contrast, we find that the PI3K-related kinase Smg1 potentiates but is not required for NMD or for viability, implying that the Upf1 phosphorylation cycle that is required for mammalian and Caenorhabditis elegans NMD has a more limited role during Drosophila development. Finally, we show that the SV40 3′ UTR, present in many Drosophila transgenes, targets the transgenes for regulation by the NMD pathway. The results establish that the Drosophila NMD pathway is broadly active and essential for development, and one critical function of the pathway is to endow proliferating imaginal cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells.Nonsense-mediated mRNA decay (NMD) is a cellular surveillance mechanism that degrades transcripts containing premature translation termination codons, and it also influences expression of certain wild-type transcripts. Although the biochemical mechanisms of NMD have been studied intensively, its developmental functions and importance are less clear. Here, we describe the isolation and characterization of  Drosophila, the authors identified a set of mutations they call “photoshop” mutations because they increase expression of green fluorescent protein transgenes such that cells expressing green fluorescent protein are more easily visualized. They found that the photoshop mutations are mutations in three different genes implicated in NMD. Using these mutations, they show that the NMD pathway not only degrades mutant mRNAs but also influences expression of many transgenes and dozens of endogenous genes during development and is essential for development beyond the larval stage. One important function of the pathway is to provide proliferating cells with a competitive growth advantage that prevents them from being overtaken by other proliferating cells during development. Thus, the Drosophila NMD pathway has critical cellular and developmental roles beyond the classical surveillance function of eliminating mutant transcripts.Cells possess a variety of surveillance mechanisms that detect and dispose of defective gene products. One such system is the nonsense-mediated mRNA decay (NMD) pathway, which degrades aberrant mRNAs containing nonsense mutations or other premature translation stop signals. In a genetic screen in  Drosophila mutants that affect NMD.Nonsense-mediated mRNA decay (NMD) is a cellular surveillance pathway in eukaryotes that recognizes and degrades transcripts with premature termination codons (PTCs). Such transcripts arise as a consequence of genomic mutation, as in numerous human genetic diseases ,2, and fup-frameshift suppressor [Upf] genes;  genes; ), and th] genes;  and cultla cells –10. Theriewed in ). Upf1 iP bodies . Upf1 isUpf1 undergoes a phosphorylation cycle  containing Smg5, Smg6, and/or Smg7, three similar proteins that are thought to recruit the phosphatase PPA2. The Upf1 phosphorylation cycle is apparently necessary for Upf1 and NMD activity at least in some organisms, because NMD function is abrogated when Smg1, Smg5, Smg6, or Smg7 activity is reduced  and PBac{WH}CG3044[f02328] as described in Thibault et al. [http://www.flybase.org. Flies were reared at 25 °C on cornmeal/dextrose medium.S system  drivers btl-GAL4 , e22c-GA22c-GAL4 , ppk-GALppk-GAL4 , and da-[da-GAL4 . GFP andt et al. . Marker 19Ay w FRT chromosome [19A/FMy w * FRT7 females to 19A, hsFLP122/Y; btl-GAL4, UAS-GFPgal80 FRT males. After a 45-min heat shock at 38 °C to induce FLP expression, embryos were returned to 25 °C to continue development. L3 larvae of genotype 19A/gal80 FRT19A, hsFLP122; btl-GAL4, UAS-GFP/+y w * FRT were identified by GFP mosaicism within the tracheal system and scored for the photoshop phenotype.The photoshop mutations were obtained by mutagenesis of an isogenized romosome  with 25 romosome  in a tra32APSmg1 chromosome (designated 32AP†) carried lethal mutations not associated with the photoshop phenotype. Lethals were removed by crossing 32AP†/19Ay w FRT females to w/Y; btl-GAL4, UAS-GFP males and identifying L3 larvae with enhanced GFP throughout their tracheal systems. These 32AP/Y; btl-GAL4, UAS-GFP/+Smg1 larvae developed into viable adult males. We also found a viable wing morphology mutation on 32AP† that is allelic to wavy. Existing wavy alleles do not show a photoshop phenotype, and the wing phenotype is separable from the photoshop phenotype, so wavy does not seem to contribute to the photoshop phenotype.The original 29AAUpf2 chromosome carries a linked lethal mutation. When recombined away from 29AA,Upf2 the mutation had no effect on tracheal development or reporter expression. However, we have not obtained a recombinant containing 29AAUpf2 without the extraneous mutation.The Upf2, we used genomic rescue transgenes located on the autosomes to generate males of genotype 14J v g f FRT19Ay w Upf2/+, Upf2+}/+Y; P{w and crossed these to 19A/FM7cy w * FRT females, where the asterisk indicates the tested mutation. Absence of Bar+, white-eyed female progeny indicated failure to complement. For complementation tests of Upf1, we used the Y-linked duplication +DpBSC1, y, which covers the Upf1 locus. Males of genotype 13DUpf1/+DpBSC1, y were crossed to 26AUpf1/FM7c females, and the absence of female Bar+ progeny indicated a failure to complement.For complementation tests of 19Ay w * FRT/19Asc cv ct v g f FRT females  to FM7c/Y males and scoring the viable male progeny for the visible markers to determine the lethal interval. To refine the map position, we collected male progeny in which a recombination event had occurred within the mapped interval and scored them for SNPs. For each mutation we typically scored 300–400 males for SNPs. For 26AUpf1 and 13D,Upf1 which map between v and g, we crossed the 19Ay w * FRT/19Asc cv ct v g f FRT females to males of genotype 1 sd1/Dpw+Df(1)64c18, g to distinguish + w cv+ ct+ v+ g fy sc recombinants, which we could not otherwise identify because of epistasis of w over v and g. We also crossed the 19Ay w * FRT/19Asc cv ct v g f FRT females to 19A/Ysc cv ct v g f FRT males to identify recombinant females, which were then tested for the photoshop phenotype in genetic mosaics to confirm that the lethality and photoshop phenotype mapped to the same interval. For the viable mutation 32AP, we followed a similar strategy as for the lethals, except we scored recombinant males for the presence or absence of 32AP by testing the enhancement of GFP in btl-GAL4, UAS-GFP transgenic animals.Identification of SNPs, construction of the SNP map of the X chromosome, and details of their use in mapping X chromosome mutations will be described elsewhere. Briefly, the location of the lethality associated with a photoshop mutation was mapped by crossing Upf2 genomic rescue construct, Drosophila BAC 24A2 [Upf2, was transformed into Escherichia coli strain EL250, which harbors heat-shock-inducible homologous recombination machinery [Upf2 coding sequence and UTRs based on the cDNA RE04053  tandemly into the Drosophila transformation vector pCaSpeR4 [Upf2 into pMM#200. The resultant plasmids were analyzed by restriction digestion, and one with the expected pattern (pMM#201) was used to establish transgenic lines on the second and third chromosomes by P-element transformation. Six lines were tested and all six rescued hemizygous male 14JUpf2 mutants to viability. The degree of rescue varied based on insertion site, but for the strongest lines +, Upf2+}11A(P{w and 24) rescued animals appeared indistinguishable from +Upf2 animals and were readily maintained as stocks of genotype 25G; P{w+, Upf2+}11AUpf2 or 24/+ or 14J; P{w+, Upf2+}11AUpf2 or 24/+.For the BAC 24A2 , which cachinery . We thenGFP reporter constructs, coding sequence of eGFP  was amplified by PCR using KpnI linker primers Kpn5GFP and Kpn3GFP  or pUAST-eGFP+I (to make pUAST.h-eGFP+I).The intron in the SV40 3′ UTR of om pGATB . The PCRDrosophila using standard microinjection techniques using the Δ2–3 helper plasmid as the transposase source.Constructs made using PCR were sequenced to confirm that mutations had not been introduced. Constructs were transformed into http://www.ambion.com). RNA concentration was determined spectrophotometrically and normalized before reverse transcription with MuMLV reverse transcriptase . Real-time quantitative PCR was done with a thermocycler (iCycler) and real-time PCR mix . Primers used in the qRT-PCR experiments were designed to amplify 60- to 100-bp fragments within single exons; amplification of Drosophila genomic DNA containing a UAS-GFP transgene showed that primers gave a linear amplification response at concentrations ranging over four orders magnitude. Control reactions performed on RNA without reverse transcription or with primers against nontranscribed regions of genomic DNA gave negligible signals compared to experimental reactions. Experimental reactions were carried out in duplicate (except EK161155 was done once) on two RNA samples that were derived independently from the RNA used for microarray analysis. Results were normalized to results with rp18LA transcript, a gene that is not developmentally regulated [L3 larvae of the appropriate genotype were identified by enhancement of GFP for mutant alleles or by sexing using gonad morphology for wild-type controls. Total larval RNA was prepared using Trizol reagent , and genomic DNA contamination was eliminated with DNAse  or AdhR2Fam (for capillary electrophoresis). PCR products were sequenced directly or digested with PvuII to distinguish the Adh+ and n4Adh alleles. Capillary electrophoresis was performed on an ABI 3730x1  and analyzed using GeneMapper v3.0 software (Applied Biosystems).For analysis of 25G/Y; btl-GAL4, UAS-GFP/+Upf2 and 19A/Y; btl-GAL4, UAS-GFP/+y w FRT L3 larvae using Trizol as described above. cDNA labeled with Cy3 or Cy5 was prepared from each RNA sample and hybridized to microarrays containing ~14,000 gene probes [http://genome-www5.stanford.edu). During analysis we noted that the 25GUpf2 mutants were delayed in development. To avoid confounding effects of changes in gene expression that result from developmental regulation rather than more direct effects of Upf2 loss of function, we used the available wild-type developmental gene expression time course [Upf2 allele, and thus provides only a lower estimate of genes regulated by NMD.RNA was isolated from e probes ,53. Hybre course  to filteTable S1(22 KB DOC)Click here for additional data file.Table S2(36 KB DOC)Click here for additional data file.Table S3(31 KB DOC)Click here for additional data file.http://flybase.net)/Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) accession numbers for the genes described in the text are Adh (CG3481/Gene I.D. 3771877), oda (CG16747), Smg1 (CG32743/Gene I.D. 31625), Upf1 (CG1559/Gene I.D. 32153), and Upf2 (CG2253/Gene I.D. 31724). Microarray data have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) and are accessible through accession number GSE5585.The FlyBase (


Drosophila, their expression is dependent on HBP/SLBP that binds to the RNA hairpin element. We showed previously that these cis and trans acting regulators of histone gene expression are conserved in C. elegans. Here we report the results of an investigation of the histone mRNA 3' end structure and of histone gene expression during C. elegans development.Histone protein synthesis is essential for cell proliferation and required for the packaging of DNA into chromatin. In animals, histone proteins are provided by the expression of multicopy replication-dependent histone genes. Histone mRNAs that are processed by a histone-specific mechanism to end after a highly conserved RNA hairpin element, and lack a poly(A) tail. In vertebrates and C. elegans histone mRNA 3' end formation occurs at this polyadenylation signal and results in polyadenylated histone mRNA, we investigated the mRNA 3' end structure of histone mRNA. Using poly(A) selection, RNAse protection and sequencing of histone mRNA ends, we determined that a majority of C. elegans histone mRNAs lack a poly(A) tail and end three to six nucleotides after the hairpin structure, after an A or a U, and have a 3' OH group. RNAi knock down of CDL-1, the C. elegans HBP/SLBP, does not significantly affect histone mRNA levels but severely depletes histone protein levels. Histone gene expression varies during development and is reduced in L3 animals compared to L1 animals and adults. In adults, histone gene expression is restricted to the germ line, where cell division occurs.Sequence analysis of replication-dependent histone genes revealed the presence of several highly conserved sequence elements in the 3' untranslated region of histone pre-mRNAs, including an RNA hairpin element and a polyadenylation signal. To determine whether in C. elegans histone genes is subject to control mechanisms similar to the ones in other animals: the structure of C. elegans histone mRNA 3' ends is compatible with histone-specific mRNA 3' end processing; CDL-1 functions in post-transcriptional control of histone gene expression; and C. elegans histone mRNA levels are elevated at periods of active cell division, indicating that histone gene expression is linked to DNA replication.Our findings indicate that the expression of  Packaging of DNA into macromolecular nucleoprotein complexes is essential for cell division and the control of gene expression. In eukaryotes, histone proteins associate with DNA to form chromatin. Chromatin formation occurs during S phase and is dependent on the coordination of histone and DNA synthesis. In animals, replication-dependent histone genes are expressed during S phase and provide the protein building blocks for chromatin. They are the only genes known to produce mRNAs that lack polyadenylated (poly (A)) tails, and instead end with a highly conserved hairpin structure. Specialised histones, termed replacement variant histones, define specialised chromatin domains and are independently integrated into chromatin. The expression of replacement variant histone genes is not linked to DNA synthesis, and their mRNAs, which normally lack the hairpin element, are polyadenylated.Drosophila dSLBP is essential and is required for histone-specific mRNA 3' end processing [In animals, the four core histone proteins H2A, H2B, H3 and H4 and the linker histone H1 are normally encoded by multi copy replication-dependent histone genes (hereafter simply referred to as histone genes). These genes are clustered and have conserved histone specific elements in the 3' untranslated region (3'UTR), including the hairpin RNA element. The RNA binding protein termed hairpin-binding, or stem-loop binding protein (HBP/SLBP) specifically binds the RNA hairpin and is a key regulator of histone gene expression -7. An adocessing ,11. In docessing . HoweverC. elegans has multicopy H2A, H2B, H3 and H4 histone genes that have hallmarks of replication-dependent histone genes: they are clustered and have a conserved hairpin structure in the 3'UTR [C. elegans histone H1 genes are similar to replacement variant histone genes and do not have the conserved 3'UTR sequence elements [C. elegans HBP/SLBP CDL-1 [C. elegans hairpin RNA element [cdl-1 is essential and required for histone synthesis [he 3'UTR . This elhe 3'UTR , and theelements . We prevBP CDL-1 , and sho element . cdl-1 iynthesis ,17.C. elegans histone genes during development and elucidate the structure of C. elegans histone mRNA 3' ends. We observed that, as is the case in other metazoans, histone mRNAs end after the hairpin structure and not after the conserved polyadenylation signal, compatible with histone mRNA 3' end formation by histone-specific processing. We demonstrate that knockdown of cdl-1 severely inhibits histone gene expression, but does not have a significant effect on histone mRNA steady state levels, compatible with a role for cdl-1 in post-transcriptional control of histone mRNA. Finally we report that histone gene expression in C. elegans correlates with the presence of actively dividing cells, consistent with a need to generate histones during cell division.Here we investigate the expression of C. elegans histone gene 3'UTRs and flanking regions revealed that the conserved 16 nucleotide hairpin sequence, the core element of the binding site for CDL-1 [Drosophila, histone genes are organised in tandem repeats with intergenic regions that contain polyadenylation signals at various distances from the hairpin, and these signals are used for mRNA 3' end processing in a dSLBP mutant background [Drosophila histone mRNAs end shortly after the hairpin, similar to vertebrate histone mRNA [A survey of or CDL-1 . Thus, cckground . In the one mRNA -21.C. elegans GAPDH mRNA were enriched, indicating that the selection procedures were successful. As expected, human histone mRNAs, which are known to lack a poly(A) tail [C. elegans histone mRNAs were similarly lost during the procedure, indicating that the structure of C. elegans histone mRNA 3' ends is similar to human histone mRNA ends. An exception was the enrichment of a low mobility C. elegans histone H3 mRNA species that may represent polyadenylated transcripts of C. elegans replacement variant histone H3 genes [We wished to determine whether histone mRNA 3' end processing would result in mRNAs ending after the hairpin as in other metazoans, or whether 3' end formation would normally occur by cleavage and polyadenylation using the conserved polyadenylation signal, and result in polyadenylated mRNA. First, we exploited the fact that polyadenylated histone mRNA is enriched by selection with oligo-dT coupled to cellulose ,23. Tota(A) tail ,25 and d(A) tail ,27, wereH3 genes .his-62 and the histone H3 genes his-9, his-13 and his-42 ending after the polyadenylation signal or after the hairpin structure, respectively. The 3'UTR and 3' flanking regions of the his-62 H2B gene contains the highly conserved 32 nucleotide element encompassing the hairpin element, and AATCC and AATAAA elements, but differs in the non-conserved regions from other H2B genes. RNase protection with the his-62 probe revealed a series of protected fragments. Using a combination of DNA marker and single-nucleotide ladder produced by KOH treatment of 5' end labelled probe RNA to estimate the length of the protected fragments, we found that the longest protected fragment was ~50 nucleotides long. This is compatible with protection of the probe by hybridisation with his-62 mRNA ending after the hairpin. Shorter protected fragments were most likely due to partial protection of the probe by hybridisation to other histone transcripts. Fragments of ~32 to ~36 nucleotides length can be accounted for by hybridisation to the region containing the hairpin element conserved in the other H2B mRNAs and also in mRNAs from other histone gene types .Next, we performed RNase protection assays to further characterise the ends of selected histone mRNA species Fig. . Probes his-9 gene is identical to the 3'UTRs of the his-13 and his-42 genes, which are therefore also detected in RNase protection assays with the probe designed to determine the his-9 mRNA 3' end. RNase protection with total RNA prepared from N2 adults and the his-9 probe revealed protected histone mRNA fragments of ~81 nucleotides in length, indicating that the majority of transcripts from the his-9, his-13 and his-42 genes lacked the polyadenylation signal and ended after the hairpin. Thus, the findings obtained with two experimental approaches described – poly(A) selection and RNase protection – are compatible with the majority of C. elegans histone mRNAs ending after the hairpin structure and lacking a poly(A) tail.The 3'UTR of the C. elegans histone mRNA 3' ends. We exploited the fact that yeast poly(A) polymerase will add non-template encoded nucleotides to the 3' end of RNA molecules with a 3'OH group [Drosophila histone mRNAs that normally end a few nucleotides after the hairpin, 3' of an A and also have a 3'OH group [C. elegans histone mRNA ends – represented by sequences where inosine was inserted into a poly(A) tail – have poly(A) tails added after the hairpin structure in vivo that were then extended during the experiment by incorporation of first inosine and then adenosine. Polyadenylated histone mRNAs with a short poly(A) tail added immediately after the hairpin have been detected in Xenopus oocytes [C. elegans histone mRNA do not extend past the polyadenylation signal but end three to six nucleotides after the hairpin structure. This is compatible with histone mRNA 3' ends being formed by histone mRNA cleavage after the hairpin, as in other animals [To independently confirm this we decided to determine the sequence of OH group . We firsOH group . In concOH group ,20,29. W oocytes . The tai oocytes . However animals -21,29.C. elegans HBP/SLBP CDL-1 and reported that it is essential for histone gene expression and cell division [cdl-1(RNAi) and RNAi targeting histone H3 and H4 genes resulted both in sterility and early embryonic arrest, linked to defects on chromatin structure [cdl-1(RNAi) treated animals was linked to a reduction in steady state histone mRNA levels. Animals were subjected to cdl-1(RNAi) by feeding. Untreated animals and gfp(RNAi) animals were used as controls. As reported previously, depletion of cdl-1 expression using the same RNAi-by-feeding regime resulted in a protruding vulva (Pvl) phenotype [cdl-1(RNAi) animals, but not in gfp(RNAi) animals which were wild type in appearance  animals compared to N2 and gfp(RNAi) animals, as determined by semi-quantitative RT-PCR  and gfp(RNAi) on histone H2B and H3 gene expression. We expect, given the similarities of histone genes and examples from other species, that findings are transferable to the other replication-dependent histone genes. Histone H2B and H3 mRNA levels were not significantly different in animals subjected to the two RNAi treatments, and were similar to levels in untreated animals  treatment caused RNA 3' end formation to occur more frequently at polyadenylation signals, we compared the structure of his-62 mRNA in RNA prepared from N2 animals and from cdl-1(RNAi) animals  animals. Analysis of histone protein levels by Western blotting revealed that histone H2B and H3 levels were broadly similar in untreated and gfp(RNAi) N2 animals  and untreated N2 animals. However this effect is minor compared to the severe depletion of histone proteins in cdl-1(RNAi) animals. This effect of cdl-1(RNAi) on histone protein synthesis indicates that cdl-1, as is the case for its homologues in other animals, is mainly required for post-transcriptional control of histone gene expression.We then examined the effect of als Fig. . For comals Fig. . RelativC. elegans development. We examined the level of histone transcripts from wild-type (N2) and glp-4(bn2) hermaphrodites by Northern Blotting. Hermaphrodites homozygous for the temperature-sensitive glp-4(bn2) mutation almost completely lack germ line proliferation when grown at the restrictive temperature of 25°C [glp-4(bn2) adult hermaphrodites grown at the restrictive temperature  and Bristol (N2) worms at the first and third larval stages and in adults  at the permissive (16°C) and non-permissive (25°C) temperatures  worms grown at the permissive temperature did not differ from wild-type in their levels of histone gene expression  and wild type larvae grown at the restrictive temperature, consistent with the fact that most of the dividing cells in these developmental stages are somatic cells. In agreement with the results seen in Figure glp-4(bn2) adult worms raised at the restrictive temperature is negligible  mRNA using oligo-dT cellulose, similar to HeLa cell histone mRNA known to end after the hairpin and to lack a poly(A) tail  end after the hairpin signal. Finally, cloning and sequencing of synthetically poly(I/A) tailed histone mRNAs showed that none of the mRNAs included the putative HDE element or polyadenylation signal  results in the inhibition of histone synthesis [cdl-1(RNAi) does not significantly affect histone mRNA levels, but, as shown previously, severely inhibits histone protein expression  adult animals grown at 25 °C, which essentially lack germ line proliferation when grown at the restrictive temperature, shows that in adults most, if not all histone gene expression is confined to the germ line. Thus our findings indicate that the link between DNA synthesis and histone gene expression observed in mammals and Drosophila is also the case for C. elegans.We have shown that during ues Fig. . Thus, aC. elegans is a well established animal model to study development. In contrast, comparatively little is known about the control of gene expression in this animal. Here we show that histone gene expression is linked to cell proliferation, and is subject to similar post-transcriptional controls as histone gene expression in other animal species. Thus C. elegans will be an ideal model to investigate the regulation of histone gene expression during animal development.Histone gene 3'UTR sequences were downloaded from the Wormbase database  and aligC. elegans culturing techniques were used [glp-4 temperature sensitive (ts) mutant [Caenorhabditis Genetics Center. Strains were grown at 16°C and 25°C as indicated in the text.Standard ere used . N2 (Bri) mutant , which icdl-1(RNAi) construct was described earlier [RNAi by feeding was carried out as described previously . The cdl earlier . GFP RNA earlier , Addgenecdl-1 and act-1 RT-PCR was performed using primers CGTAAAGCACCACGAGGCCGTC and GACGACATCTTGGAGAAGTCAGTTG, and CGTGGTTACTCTTTCACCACCACCGCT and GGACTCGTCGTATTCTTGCTTGGAGAT respectively. 21, 22 and 23 PCR cycles were performed using an annealing temperature of 55°C, and extension time of 1 minute for all reactions. Products were analysed by agarose gel electrophoresis, visualised by staining with ethidium bromide and quantitated using a Molecular Imager Gel Doc XR System (Biorad) and analysed using AIDA Image Analyzer Software (Raytest). To determine the efficiency of cdl-1 knockdown we standardised the cdl-1signal with respect to the corresponding actin signal. The average of the measurements with N2 RNA was defined as 100%. The standard deviations were calculated from the measurements made for RNA from N2, gfp(RNAi) and cdl-1(RNAi) animals, respectively.To confirm gene knockdown by RT-PCR, 1 μg of total RNA was reverse transcribed using random primers  and subsequent PCR was performed using Taq DNA polymerase (Promega). glp-4(bn2) embryos were obtained from bleaching gravid adult hermaphrodites, aliquoted onto NGM-OP50 plates and incubated at 16°C or 25°C. Worms were harvested from plates by gentle washing with sterile M9 salts. L1 larvae were harvested after 15 hours and 30 hours at 25°C and 16°C, respectively; L3 larvae were harvested following 36 hours and 72 hours at 25°C and 16°C, respectively; and adult animals were harvested after 72 hours and 144 hours at 25°C and 16°C, respectively.N2 and 260 nm corresponds to 40 μg RNA.Pelleted worms were snap-frozen in 250 μl Trizol reagent (Invitrogen) in liquid nitrogen, thawed at 42°C and vortexed for 30 seconds. Following five cycles of freeze-thawing, 50 μl chloroform was added and mixture incubated at room temperature for 3 minutes, spun at 4°C for 20 minutes at 13200 rpm in a table top centrifuge and clear phase containing RNA removed. Total RNA was isopropanol precipitated and stored in 75% ethanol (nuclease free) until required. RNA concentration was determined using a 1 cm cuvette assuming 1 ODC.elegans or HeLa cells was subjected to poly(A) selection with oligo-dT cellulose (Poly(A)Purist, Ambion) as per manufacturer's instructions. Half of the poly(A) enriched fraction was subjected to a second round of selection. Poly(A) selected RNA was precipitated with ethanol and resuspended in 10 μl DEPC treated water.200 μg total RNA from EcoRI. The C. elegans histone H4 probe was prepared by PCR using a cloned his-10 gene as template and spans nucleotides 9 – 312 of the his-10 ORF. The C. elegans histone H3 probe was prepared by PCR using a cloned his-9 gene as template and spans nucleotides 1 – 398 of the his-9 ORF. Probes detecting histone H2A (his-30) and H2B (his-62) and actin (act-1) mRNA were prepared by RT-PCR from Bristol (N2) RNA and span nucleotides 1 – 372, 9 – 366 and 589–1096 of the respective open reading frames. The probe detecting C. elegans GAPDH (gdp-1) mRNA was amplified from genomic DNA and spans nucleotides 19–995 of the gpd-1 open reading frame. This probe has high sequence identity to GAPDH gpd-2,gpd-3 and gpd-4 genes and cannot distinguish between transcripts from these genes. Probes were labelled with 32P-dCTP using a random prime labelling kit (Roche). C. elegans 18S rRNA was detected using the oligonucleotide ACGTATCTAATCGCCTTCGTTCC 32P end labelled using T4 polynucleotide kinase and γ-32PATP. Data was analysed by autoradiography and using a Fuji Phosphoimager and AIDA Image Analyzer Software (Raytest).RNA was fractionated on 1.2 % or 1.5 % agarose gels and transferred to Hybond N membranes as described . Probes his-62 gene spans nucleotides 3 – 145 of the his-62 3' UTR. The template for transcription was amplified from genomic DNA using primers CATTGGCTGAAAACTAACC and TAATACGACTCACTATAGGGGAATCCAACGTTTCAAGAAAAGAC. This produced a template for T7 RNA polymerase transcription containing the T7 RNA polymerase promoter followed by 10 non-histone specific bases prior to sequence complementary to his-62 mRNA. The probe to detect his-9 mRNA spans from nucleotide 400 in the his-9 ORF to nucleotide 205 of the 3' UTR. The template for transcription was amplified from genomic DNA using the primers AGCGTGCTTAAATGCCTTCC and TAATACGACTCACTATAGGGCGAATCGTACGCTGATAGCTTATGTTCAAAAACTGAGTA, resulting in a product containing T7 RNA polymerase promoter followed by 20 non-histone specific nucleotides and 216 nucleotides his-9 sequence. The PCR product was inserted into pGEM-T easy and sequenced . For RNA synthesis, the fragment was re-amplified using the same primers and transcribed using the T7 RNA polymerase to produce a probe complementary to his-9 mRNA. Uniformly labelled RNA was prepared by in vitro transcription using T7 RNA polymerase and α32P-UTP. RNase protection assays were normally done with 12.5 μg RNA using RNases A and T1 . 32P-labelled pBR322 DNA/MspI marker was prepared by endlabelling using a 32P-dCTP and Klenow fragment. 5'32P-labelled his-62 probe RNA was prepared by treating non-labelled RNA with alkaline phosphatase, followed by end-labelling with T4 polynucleotide kinase and γ-32PATP. The KOH ladder was produced using 5' 32P end-labelled probe as described [The probe to detect the escribed . ReactioRNA tailing with yeast poly(A) polymerase was done essentially as described . 10 μg tRK carried out the sequencing of histone mRNA ends, the RNAi experiments and helped draft the manuscript. SW carried out sequence alignments, poly(A) selection and RNAse mapping of histone mRNA 3' ends. Both SW and RK carried out experiments analysing histone gene expression during development. JP participated in the conception and design of the study and helped draft the manuscript. BM conceived of the study, and participated in its design and coordination and drafted the manuscript. All authors read and approved the final manuscript.C. elegans histone mRNA 3'UTR sequences. Sequence alignments used to identify consensus 3'UTR elements of histone mRNAs shown in Figure Alignment of Click here for filecdl-1(RNAi) treatment on the 3' end of his-62 mRNAEffect of . Internally 32P labelled his-62 probe was hybridised with either total RNA from adult N2 animals, or total RNA from cdl-1(RNAi) adults, or yeast RNA and subjected to treatment with RNase A/T1 as described. Samples were analysed by denaturing PAGE and visualised by autoradiography. The asterisk marks probe protected by hybridisation to template DNA. Note that the protection patterns obtained with N2 and cdl-1(RNAi) treatment are identical and that cdl-1(RNAi) does not result in longer protected fragments diagnostic for end-formation using the polyadenylation signal.Click here for file


Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%–4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a  Huntington's Disease (HD) is a fatal inherited neurodegenerative disease, which typically begins in middle age and progresses with symptoms of severe uncontrolled movements and cognitive dysfunction. HD is uniformly fatal with death occurring ten to 15 years after onset of symptoms. There is currently no effective treatment for HD. The genetic mutation underlying HD causes a protein called huntingtin (Htt) to contain an abnormally long tract of the amino acid glutamine. This extended span of glutamines changes the shape of the Htt protein, which can cause it to interact in abnormal ways with other cellular proteins. In this study, we have identified a large number of new proteins that bind to normal and mutant forms of the Htt protein. To establish a potential role for these interacting proteins in HD, we show that changing the expression of many of these proteins can modulate the pathological effects of mutant Htt on fly neurons that deteriorate when they express mutant Htt. Identifying cellular proteins that bind to Htt and modulate its pathological activity may facilitate the discovery of an effective treatment for HD. Huntington's Disease (HD) is a member of a family of dominantly inherited neurodegenerative diseases caused by expansion in a glutamine-encoding CAG tract. HD occurs when the polyglutamine (polyQ) tract in huntingtin (Htt) expands beyond ~35 glutamine (Q) repeats and manifests with movement disorder, psychological disturbances, and cognitive dysfunction progressing over a period of about ten to 15 years until death. Currently there is no effective treatment or cure for HD.Caenorhabditis elegans, Drosophila, and mouse y were crossed to males from the mutant strains. In cases where the mutation was on the X chromosome, the cross was reversed. Crosses were incubated at 27 °C and 29 °C to provide two different phenotypic readouts. Strains modifying the eye phenotype were recrossed to verify the modification. Only genes that consistently showed modification at different temperatures or using different alleles were further analyzed. Potential modifiers behaving as enhancers were tested for possible nonspecific eye phenotypes by crossing them to control females of the genotype 1w118; GMR-GAL4/CyO.yFor the modifier screen, females of the genotype For scanning electron microscopy (SEM) images, flies were crossed at 29 °C and newly eclosed adults were aged for five days. Whole flies were dehydrated in ethanol, critical-point dried, and analyzed with a JEOL JSM 6100 microscope. For paraffin sections of enhancers, flies were crossed at 25 °C and adults were aged for five days . Adult heads and torsos were fixed in 4% formaldehyde/85% ethanol/5% acetic acid, dehydrated, embedded in paraffin for vertical semi-thin sections, and then stained with Hemathox.For the climbing and survival assays, females of the genotype Elav-GAL4; UAS:128QHtt[F27B] were crossed to males of the mutant strains. Climbing assays were performed on 30 age-matched adult virgin female flies raised at 27 °C as described . The flihttp://www.roche.com). Protein determination was carried out with the BCA method . Lysate  were precleared with mouse IgG beads  and immunoprecipitated with monoclonal Htt antibody  by incubating overnight at 4 °C and then with protein G . Beads were washed 5× with TBS/0.05% Tween, sample was eluted with 1× sample buffer  and then resolved using 4%–12% Bis-Tris precast gels (Invitrogen). Western blot was preformed, and blots were probed with rabbit antibody to USP9X , Cullin 2 , CACNA2D1 , Htt BKP1 (1:500), PARP , mouse monoclonal GAPDH , STX1A , SNAP25 , goat antibody PKM2 , and PSMC2 .Whole brains from wild-type or YAC128 mice were lysed in T-PER (Pierce) with protease inhibitors  and silver stained. The presence of glutathione S-transferase (GST) and Htt in the bands was confirmed by matrix-assisted laser desorption/ionisation-time of flight MS and Western blotting (not shown). The predicted size of the GST-Htt fusion product is 53 kDa. We were unable to determine the difference in the two GST-Htt bands by MS; they may represent expanded (48 Q) and wild-type (22 Q) Htt fragments. The band at 28 kDa represents GST and likely occurs from cleavage of the fusion product between the GST and the bait as we saw a band of this size with numerous heterologous purified baits.(575 KB PDF)Click here for additional data file.Figure S2Only searches with N-terminal baits  that gave at least one positive were included in the analysis. The x-axis indicates numbers of screens performed. The y-axis shows the novel discovery index for prey proteins . A peak near 525 searches corresponds to introduction of new prey libraries.(529 KB PDF)Click here for additional data file.Figure S3 (B) show a severe degenerative phenotype when compared to (A) GMR-GAL4 controls of the same age and cultured at the same temperature. The phenotype consists of a shortening (see arrow) and detachment of the retina, as well as the presence of vacuoles in the retina. The Htt-induced phenotype can be suppressed by (C) reduced levels of armadillo G0234; GMR-GAL4/+; UAS:128Qhtt[M64(P{lacW}arm]/+), (D) reduced levels of hu li tai shao k06121; UAS:128Qhtt[M64]/+),(GMR-GAL4/P{lacW}hts (E) reduced levels of M6 BG00390), reduced levels of kinesin heavy chain 1pr1Khc8;(GMR-GAL4/b UAS:128Qhtt[M64]/+), (G) reduced levels of peanut KG00478; UAS:128Qhtt[M64]/+),(GMR-GAL4/P{SUP pr P-}pnut (H) reduced levels of 14-3-3e j2b10), reduced levels of G protein I a-subunit 65A KG01907ry506), reduced levels of Itp-r83A 05616ry506), over-expression of Src oncogene at 42A EY08937; UAS:128Qhtt[M64]/+),(GMR- GAL4/P{EPgy2}Src42A (L) reduced levels of clathrin heavy chain 4/+ GMR-GAL4/+; UAS:128Qhtt[M64]/+),(Chc (M) reduced levels of soluble N-ethylmaleimide-sensitive factor attachment protein M4), reduced levels of STX1A 506P{PZ}Syx1A06737), reduced levels of Rpt1 05643cn1; UAS:128Qhtt[M64]/+),(GMR-GAL4/P{PZ}Rpt1 (P) reduced levels of Eip75B 07041), reduced levels of myocyte enhancing factor 2 X1; UAS:128Qhtt[M64]/+),(GMR-GAL4/Df(2R)X1,Mef2 (R) reduced levels of crooked legs (GMR-GAL4/P{EPgy2}crolEY08953), (S) reduced levels of Glycerol 3 phosphate dehydrogenase 1Gpdhn1–4; UAS:128Qhtt[M64]/+),(GMR-GAL4/Al (T) reduced levels of Pdsw k10101; UAS:128Qhtt[M64]/+),(GMR-GAL4/P{PlacZ}Pdsw and (U) reduced levels of porin k05123; UAS:128Qhtt[M64]/+).Click here for additional data file.Figure S4(GMR-GAL4/+).(A) Age-matched controls cultured at the same temperature  cultured at 25 °C show a degenerative phenotype. The phenotype consists of a shortening (arrow), vacuolization, and detachment of the retina. This phenotype can be enhanced by (C) reduced levels of KG00083; UAS:128Qhtt[M64]/+),CAP (GMR-GAL4/P{SUPor-P}CAP (D) reduced levels of KG06490; UAS:128Qhtt[M64]/+),CLIP-190 (GMR-GAL4/P{SUPor-P}CLIP-190 (E) reduced levels of G00158; UAS:128Qhtt[M64]/+),LaminC (GMR-GAL4/P{PTTor-GB}LamC (F) overexpression of EY07032),M6  reduced levels of 02957; UAS:128Qhtt[M64]/+),zipper (GMR-GAL4/P{PZ}zip (H) reduced levels of hs)}G13 shot3; UAS:128Qhtt[M64]/+),short stop (GMR-GAL4/P{FRT(w (I) overexpression of  Scer\UASc.Ca),14-3-3ɛ  overexpression of EY03325; UAS:128Qhtt[M64]/+),14-3-3ς (GMR-GAL4/ P{EPgy2}14-3-3ς (K) overexpression of EY10355),G-iα65A  overexpression of EY02522),Itp-r83A  reduced levels of G00044Lachesin , (N) reduced levels of k10108Src oncogene at 42A , (O) overexpression of soluble NSF-attachment protein , (P) overexpression of Syntaxin1A ,P{EP}Syx1A (Q) reduced levels of KG09881Aspartyl β-hydroxylase , (R) reduced levels of Dynein heavy chain 64C ,P{SUPor-P}Dhc64C (S) reduced levels of fat facets ,faf (T) overexpression of EP2153Rpt1 , (U) reduced levels of 04418; UAS:128Qhtt[M64]crooked legs (GMR-GAL4/P{PZ}crol/+), (V) reduced levels of nNC1; UAS:128Qhtt[M64]Phosphogluconate isomerase (GMR-GAL4/Pgi/+), (W) reduced levels of RhoGAP92B , (X) reduced levels of G0423a; GMR-GAL4/+; UAS:128Qhtt[M64]Unc-76 (P{lacW}Unc-76/+), and (Y) overexpression of EY09750; UAS:128Qhtt[M64]CG12455 (GMR-GAL4/P{EPgy2}CG12455/+). These mutations do not cause an abnormal eye phenotype in control flies carrying the GMR-GAL4 driver without the UAS:128Qhtt[M64] transgene (unpublished data). However, when combined with 128-Q htt, they lead to further decrease in retinal thickness and in some cases increased retinal detachment and vacuolization.(B) Retinal sections of day 5 flies expressing N-terminal 128-Q htt (2.8 MB PDF)Click here for additional data file.Table S1Y indicates peptides that were manually validated and confirmed by inspection of the MS spectra; A refers to ambiguous peptides that could not be conclusively identified by manual validation of the MS spectra.(3.4 MB XLS)Click here for additional data file.Table S2http://www.prolexys.com).*The total number of unique interacting proteins refers to the number of unique gene sequences identified in a database of positives from nearly >250,000 high-throughput random Y2H searches performed at Prolexys Pharmaceuticals ((580 KB DOC)Click here for additional data file.Table S3http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene). E_VALUE_EXP is the negative log of the E value produced by the highest scoring BLAST hit and has a maximum of 180 . Amino acid coordinates of HD baits are indicated relative to NP_002102. Q-length repeats are shown in parentheses. HD bait sequences may be represented multiple times if more than one search generated positives. High-throughput sequencing was performed unidirectionally for identification purposes and does not necessarily represent the entirety of the clone. *Search ID 14291 (HD bait 1116–1196) was identified in a search using a complex bait library rather than individual bait clone.Search ID is an identifier given each Y2H mating event see . Positiv(3.2 MB XLS)Click here for additional data file.Table S4(98 KB DOC)Click here for additional data file.Table S5Drosophila prior to statistical filtering; E, Enhancer; S, Suppressor*Genes tested in (73 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein) accession numbers for MS studies (RefSeq) are: NP_000302.1, NP_000382.3, NP_000524.3, NP_000602.1, NP_000708.1, NP_001019645, NP_001367.2, NP_001377.1, NP_001419.1, NP_001753.1, NP_001779.2, NP_001834.2, NP_001853.2, NP_001854.1, NP_001907.2, NP_001914.2, NP_001951.2, NP_001990.1, NP_002046.1, NP_002064.1, NP_002065.1, NP_002102.4, NP_002329.2, NP_002536.1, NP_003033.2, NP_003124.1, NP_003156.1, NP_003170.1, NP_003356.2, NP_003357.2, NP_003365.1, NP_003366.2, NP_003696.2, NP_004246.1, NP_004309.2, NP_004364.2, NP_004365.1, NP_004484.1, NP_004491.1, NP_004539.1, NP_004542.1, NP_004543.1, NP_004594.1, NP_004850.1, NP_004993.1, NP_004996.1, NP_004997.4, NP_005156.1, NP_005264.2, NP_005268.1, NP_005653.3, NP_005736.3, NP_005995.1, NP_006046.1, NP_006279.2, NP_006308.3, NP_006576.2, NP_006810.1, NP_006830.1, NP_008839.2, NP_009034.2, NP_009204.1, NP_031407.2, NP_031457.1, NP_031464.1, NP_031669.2, NP_031736.1, NP_031773.1, NP_031887.2, NP_031959.1, NP_032246.2, NP_032518.1, NP_032644.2, NP_033012.1, NP_033033.1, NP_033321.1, NP_033332.1, NP_033333.2, NP_033441.1, NP_033805.1, NP_033851.1, NP_033914.1, NP_034053.1, NP_034078.1, NP_034438.1, NP_034442.1, NP_034715.1, NP_034829.1, NP_034944.1, NP_035229.2, NP_035253.1, NP_035523.1, NP_035558.1, NP_035824.1, NP_035825.1, NP_036288.2, NP_036560.1, NP_036611.2, NP_038709.1, NP_057049.3, NP_057223.1, NP_057606.1, NP_058084.2, NP_060064.2, NP_061359.2, NP_062681.1, NP_065593.1, NP_066268.1, NP_067541.1, NP_075553.1, NP_077128.2, NP_077173.1, NP_077725.1, NP_077745.2, NP_079589.1, NP_079612.1, NP_079634.1, NP_079683.2, NP_080175.1, NP_080720.1, NP_080971.2, NP_080979.1, NP_084501.1, NP_114080.2, NP_149124.2, NP_443106.1, NP_444427.1, NP_536846.1, NP_536849.1, NP_542970.1, NP_570824.1, NP_598429.1, NP_613063.1, NP_619621.1, NP_659409.2, NP_663493.1, NP_663589.2, NP_766024.1, NP_776169.2, NP_796376.2, NP_849209.1, NP_976218.1, XP_128725.4, XP_131103.3, XP_203393.2, and XP_622887.1.The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) accession numbers for Y2H studies are: 120, 161, 323, 1315, 1387, 1499, 1759, 1778, 1785, 2597, 3064, 3092, 3093, 3275, 3329, 3338, 3839, 4209, 4361, 4790, 5033, 5295, 5296, 5315, 5468, 5493, 5710, 5753, 6670, 6721, 6829, 6867, 7430, 7529, 7644, 7692, 7704, 7802, 8065, 8239, 8453, 8462, 8503, 8539, 9093, 9330, 9611, 9638, 9810, 9818, 9901, 9938, 10010, 10133, 10422, 10456, 10458, 10464, 10540, 10580, 10906, 10915, 11177, 11193, 23116, 23328, 23332, 23348, 23360, 23380, 23609, 23613, 23641, 25764, 26578, 27068, 28969, 29062, 29072, 29993, 51061, 51322, 51586, 51593, 51667, 55219, 55660, 55704, 55735, 56254, 57489, 57509, 57522, 57616, 63908, 79027, 79813, 80254, 83478, 84936, 128866, 134218, 139818, 152789, and 171392.The NCBI (GeneID) (


The crystal structure is stabilized by intermolecular N—H⋯O hydrogen bonds. An intramolecular O—H⋯N hydrogen bond is also present.In the title compound, C Å                           b = 10.3967 (4) Å                           c = 14.2938 (6) Å                           V = 1922.86 (13) Å3                                                   Z = 8                           Kα radiationMo −1                        μ = 0.30 mmT = 173 K                           0.48 × 0.42 × 0.39 mm                                 Bruker SMART 1000 CCD diffractometerSADABS; Sheldrick, 2004T                           min = 0.869, T                           max = 0.891Absorption correction: multi-scan (11037 measured reflections1881 independent reflectionsI > 2σ(I)1706 reflections with R                           int = 0.027                                                            R[F                           2 > 2σ(F                           2)] = 0.054                           wR(F                           2) = 0.150                           S = 0.98                           1881 reflections129 parametersH-atom parameters constrainedmax = 1.20 e Å−3                        Δρmin = −0.33 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.Data collection: 10.1107/S160053680903164X/xu2563sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S160053680903164X/xu2563Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Its structure contains a 1.8797 (13) Å P—C bond and two intermolecular N—H⋯O=P hydrogen bonds, resulting in centrosymmetric dimers. An intramolecular N—H⋯O=P hydrogen bond is also present.The title compound, C Å                           b = 10.1375 (13) Å                           c = 15.5955 (19) Å                           β = 93.264 (2)°V = 1845.4 (4) Å3                                                   Z = 4                           Kα radiationMo −1                        μ = 0.34 mmT = 113 (2) K                           0.60 × 0.42 × 0.40 mm                                 Bruker SMART CCD area-detector diffractometerSADABS; Sheldrick, 2003T                           min = 0.820, T                           max = 0.874Absorption correction: multi-scan (20001 measured reflections4568 independent reflectionsI > 2σ(I)4027 reflections with R                           int = 0.022                                                            R[F                           2 > 2σ(F                           2)] = 0.029                           wR(F                           2) = 0.082                           S = 1.03                           4568 reflections246 parametersH-atom parameters constrainedmax = 0.34 e Å−3                        Δρmin = −0.33 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.Data collection: 10.1107/S1600536808020175/si2094sup1.cif            Crystal structure: contains datablocks global, I. DOI: 10.1107/S1600536808020175/si2094Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


The oxadiazole ring makes dihedral angles of 82.82 (7) and 9.92 (7)° with the 3,4-dichlorobenzene and the 3,4,5-trimethoxybenzene ring planes, respectively. The crystal structure is stabilized by intermolecular C—H⋯ O and C—H⋯ N hydrogen bonds. Intramolecular C—H⋯O and C—H⋯N hydrogen bonds are also present.The title compound, C Å                           b = 15.9516 (8) Å                           c = 15.7483 (8) Å                           β = 90.8940 (10)°V = 1927.63 (17) Å3                                                   Z = 4                           Kα radiationMo −1                        μ = 0.37 mmT = 173 (2) K                           0.47 × 0.39 × 0.32 mm                                 Bruker SMART 1000 CCD diffractometerSADABS; Sheldrick, 2003T                           min = 0.845, T                           max = 0.890Absorption correction: multi-scan (10032 measured reflections4159 independent reflectionsI > 2σ(I)3238 reflections with R                           int = 0.025                                                            R[F                           2 > 2σ(F                           2)] = 0.041                           wR(F                           2) = 0.125                           S = 1.04                           4159 reflections257 parametersH-atom parameters constrainedmax = 0.34 e Å−3                        Δρmin = −0.31 e Å−3                        Δρ                                 SMART  used to solve structure: SHELXTL (Sheldrick, 2008SHELXTL; molecular graphics: SHELXTL; software used to prepare material for publication: SHELXTL.Data collection: 10.1107/S1600536808020771/wn2269sup1.cif            Crystal structure: contains datablocks I, global. DOI: 10.1107/S1600536808020771/wn2269Isup2.hkl            Structure factors: contains datablocks I. DOI:  crystallographic information; 3D view; checkCIF report            Additional supplementary materials:


Autosomal dominant polycystic kidney disease (ADPKD) is responsible for 10% of cases of the end stage renal disease. Early diagnosis, especially of potential fast progressors would be of benefit for efficient planning of therapy. Urine excreted proteome has become a promising field of the search for marker patterns of renal diseases including ADPKD. Up to now however, only the low molecular weight fraction of ADPKD proteomic fingerprint was studied. The aim of our study was to characterize the higher molecular weight fraction of urinary proteome of ADPKD population in comparison to healthy controls as a part of a general effort aiming at exhaustive characterization of human urine proteome in health and disease, preceding establishment of clinically useful disease marker panel.We have analyzed the protein composition of urine retentate (>10 kDa cutoff) from 30 ADPKD patients and an appropriate healthy control group by means of a gel-free relative quantitation of a set of more than 1400 proteins. We have identified an ADPKD-characteristic footprint of 155 proteins significantly up- or downrepresented in the urine of ADPKD patients. We have found changes in proteins of complement system, apolipoproteins, serpins, several growth factors in addition to known collagens and extracellular matrix components. For a subset of these proteins we have confirmed the results using an alternative analytical technique.Obtained results provide basis for further characterization of pathomechanism underlying the observed differences and establishing the proteomic prognostic marker panel. Autosomal dominant polycystic kidney disease (ADPKD) is an inherited disorder affecting 1 in 1000 people and responsible for 10% of cases of the end stage renal disease (ESRD). Apart from renal manifestations, changes in other organs may be present, including a.o. liver cysts and intracranial aneurysms. The disease is divided into 2 types based on mutated gene . The type of the mutation has prognostic significance, as the average age of ESRD depends on the type of the disease and amounts to 53 years in type 1, and 69 years in type 2. The solution was applied to 18 cm IPG strip with 3–11 NL pH gradients  for isoelectrofocusing (IEF): 340 μl of sample/strip, corresponding to 400 μg protein. The IPG strip was rehydrated overnight in an IPG box . The next day, the strips were isoelectrofocused using a Ettan IPGphor 3 electrophoresis system  as follows. Two steps of electrophoresis were used. The first step consisted of a 5 h pre-run at 500 V. During this step, the conductivity decreases, and salts and other highly conductive compounds move towards the electrode (anode). Second, a long gradient focusing program was used: 1 h at 500 V, 9 h at 1000 V and 30 h at 8000 V .After focusing, the strip was removed from the tray and the overlay oil was blotted with a paper tissue. Strip was wrapped in a parafilm and stored at −80°C. The strip was placed on a tray cooled with dry ice and cut into sections of ca. 7 mm. The sections were transferred into individual 1.5-ml siliconized Eppendorf tubes. In all, the 18-cm long gel strips were sliced into 26 sections. Peptides were extracted from gel sections by two cycles of adding 60 μl 0.1%TFA, 2% acetonitrile and vortexing the tubes for 40 minutes at room temperature. Aliquots with extracted peptides were stored at −80°C for LC-MS/MS analysis.The peptide mixture (20 μl) was applied to the nanoACQUITY UPLC Trapping Column (Waters) using water containing 0.1% formic acid as the mobile phase and then transferred to the nanoACQUITY UPLC BEH C18 Column  using an acetonitrile gradient (3–33% acetonitrile over 150 minutes) in the presence of 0.1% formic acid with a flow rate of 250 nl/min. The column outlet was directly coupled to the electrospray ion source of the LTQ-Orbitrap Velos mass spectrometer (Thermo Scientific) working in the regime of data-dependent MS to MS/MS switch. HCD fragmentation was used. Other Orbitrap parameters were as follows: one MS scan was followed by max. 5 MS/MS scans, capillary voltage was 1,5 kV, data were acquired in positive polarity mode.q-value estimates for each peptide spectrum match (PSM) in the dataset. All PSMs with q-values > 0.01 were removed from further analysis. A protein was regarded as confidently identified if at least two peptides of this protein were found. Proteins identified by a subset of peptides from another protein were excluded from analysis. Proteins that exactly matched the same set of peptides were clustered into one group/cluster. MS/MS spectra of peptides meeting the above acceptance criteria were subjected to quantitative analysis step to obtain a list  of proteins differentially populated between a set of three CPS’s and three DPS’s.The acquired MS/MS data were pre-processed with Mascot Distiller . The database search of the data using MASCOT search engine was carried out in a three-step procedure  were included. In the first step, using MascotDistiller program iTRAQ reporter ion peaks were detected in the preprocessed MS/MS spectra; next, their intensities were corrected for isotope impurity using the information provided by the reagent manufacturer. For each spectrum a geometric mean of two reporter ion intensities belonging to one study group (CPS or DPS) were separately calculated. A ratio of these mean values (CPS mean divided by DPS mean) was reported as peptide ratio. If more than one spectrum was obtained for a peptide in a single LC-MS/MS experiment, median peptide ratio value from all spectra was used. Prior to the protein ratio calculations, peptide ratios were median-normalized to remove systematic bias. Proteins ratios were calculated as the median ratio of their peptide’s ratios. The statistical significance of a single protein ratio was assessed by an in house program Diffprot. In thisWe have selected a subset of proteins from the Differential Protein List shown in Table Finally a set of peptides, corresponding to nine proteins, used for further MRM analysis of individual ADPKD and control samples, consisted of 14 peptides:1. Antithrombin-III – peptide: TSDQIHFFFAK, peptide: FATTFYQHLADSK2. Cystatin-M - peptide: DLSPDDPQVQK, peptide: AQSQLVAGIK3. Transthyretin – peptide: AADDTWEPFASGK4. Retinol-binding protein 4 – peptide: YWGVASFLQK, peptide: DPNGLPPEAQK5. Proactivator polypeptide – peptide: EIVDSYLPVILDIIK, peptide: LVGYLDR6. Apolipoprotein A-IV – peptide: SELTQQLNALFQDK, peptide: LLPHANEVSQK7. Complement C3 – peptide:TIYTPGSTVLYR8. Histidine-rich glycoprotein – peptide: DGYLFQLLR9. Myocilin – peptide: YELNTETVKhttp://www.peptideatlas.org/, Institute for Systems Biology, Seattle, WA), and on specific criteria required in SIS peptides such as length and lack of amino acid modifications[13C615 N2-Lys (98% isotopic enrichment) or 13C615 N4-Arg (98% isotopic enrichment). MRM analysis was performed using a Waters Xevo TQ mass spectrometer  coupled to a Waters nanoAcquity UPLC via a Zspray Nanoflow source with a 10 μm SilicaTip PicoTip emitter . Mobile phase A was 0.1% formic acid (Sigma-Aldrich) in LC-MS grade water , and mobile phase B was LC-MS grade acetonitrile (J.T.Baker) with 0.1% formic acid. Peptides were loaded onto a Waters Symmetry C18 pre-column  and separated using a 40 min LC run, with a 27 min gradient of mobile phase B changing from 1 to 45% on a Waters nanoAcquity UPLC BEH130 C18 Column . Other MS instrument parameters included a capillary voltage of 3.1 kV, a purge gas flow of 100 L/h, cone gas flow of 5 L/h, NanoFlow gas set at 1.0 Bar, and a source temperature of 150°C. MRM parameters were empirically optimized using pure SIS peptides to generate the highest possible signal for each individual peptide and resulting ion fragments. The optimal charge state and optimal cone voltage were determined for each SIS peptide by injecting 1 pmol (in 0.1% formic acid) on-column and ramping the cone voltage from 20 to 70 V in 5 V steps while gating all the possible parent ion charge states  using the selected ion recording (SIR) function controlled by the Waters MassLynx V4.1 software. The daughter ions generating the highest possible signal and their individual, optimal collision energy (CE) voltages were determined empirically by injecting 1 pmol (in 0.1% formic acid) of SIS peptide on-column and ramping the CE voltage up and down five 2 V steps from that suggested by the Skyline Ver. 1.3 software  for the Waters Xevo instrument. All possible b- and y-series fragment ions for both 2+ and 3+ precursor ion charge states spanning a m/z range from 300 to 1500 were tested. MRM scans for optimization of MRM Q1/Q3 ion pairs were conducted with the optimized cone voltages with the Span setting set to 0 and with dwell times of 10 milliseconds for each transition. From this data, using the Skyline Ver. 1.3 software, the 5 transitions that produced the strongest signals were selected on a per-peptide basis, with a preference toward higher-mass y series ions if the abundances were similar. These top 5 transitions were then checked for signal interferences when present in a sample-digest background. The SIS peptide mix was analyzed by LC-MRM/MS using transitions for heavy (SIS) and natural (endogenous) peptides, both in buffer and in a sample digest. Identical MRM acquisition parameters were used for the heavy and natural forms of each peptide, while taking into account the Q1/Q3 mass differences due to the stable-isotope label. The transitions that maintained the same relative intensities in both the buffer and sample were considered as interference free. This analysis is also used to determine the retention time as well as confirm the identity of the ion signals observed for natural and heavy peptides, thus verifying the identity of the natural peptides which co-elute with the corresponding SIS peptides.Specific tryptic peptide sequences, to be used for SIS peptide synthesis, were selected for the nine proteins based on the number of observations in the PeptideAtlas MS/MS database , with an injection volume of 4 μl resulting in 2 μg of protein digest on-column. Samples were prepared basically as for iTRAQ pooling analysis, see Sample filtration section. First, from each sample, an aliquot of protein fraction containing 10 μg of total protein was transferred to silanized vials. Then the volume of each sample was brought to 30 μl using 100 mM solution of NH4HCO3. 100 mM DTT (Sigma Cat no D8161-5 G) was added to the samples to the final concentration of 10 mM and incubated at 56°C for 40 min. To block reduced cysteines 0.5 M iodoacetamide (Sigma Cat no I1149-5 G) to the final concentration of 50 mM was used and the sample was incubated at room temperature for 30 minutes in darkness. Trypsin (Promega cat. no V511A) was added to samples in 1:20 vol./vol. ratio and incubated at 37°C overnight. Finally, trifluoroacetic acid was added to digested protein samples to reduce pH to 2 and inactivate trypsin.MRM analysis was performed on a new set of 52 samples  peak to the integrated area of the corresponding standard (SIS) peptide.ADPKD: Autosomal Dominant Polycystic Kidney Disease; ESRD: End Stage Renal Disease; DPS: Disease Pooled Samples; CPS: Control Pooled Samples; DPL: Differential Protein List; SIS peptides: stable-isotope-labeled internal standard peptides.Magda Bakun and Mariusz Niemczyk contributed equally to this workThe authors declare that they have no competing interest.M.B. – optimized peptide IEF, carried out LC-MS experiments, analyzed LC-MS data. M.N. – did patients selection, characterization, enrollment to the study, sample collection, wrote the manuscript. S.N. - did patients selection, characterization, enrollment to the study. A.F. – optimized sample prep protocol, carried out LC-MS experiments, M.K. – provided statistical analysis of the data. D.D., R.J. – designed MRM experiments, selected peptides, optimized MRM conditions. A.P., A.B. – carried out MRM experiments, analyzed data. M.D. - designed the study, wrote manuscript. L.P. - designed the study, established exclusion/inclusion criteria, wrote manuscript. All authors read and approved the final manuscript.List of 1 700 proteins identified by at least two peptides.Click here for fileMass measurement error correction and identification estimation q-value.Click here for file


Lactococcus lactis, ssp lactis is reported in which emission intensities are used to follow and define metabolic activity during growth in nutrient broths. Optical density (OD) data were also acquired during L. lactis growth at 32°C and provided insight into the timing of the AE signals relative to the lag, logarithmic, and stationary growth phases of the bacteria. The inclusion of a metabolic inhibitor, NaN3, into the nutrient broth eliminated bacteria metabolic activity according to the OD data, the absence of which was confirmed using AE data acquisition. The OD and AE data were also acquired before and after the addition of Bacteriophage c2 in L. lactis containing nutrient broths during the early or middle logarithmic phase; c2 phage m.o.i. (Multiplicity of infection) was varied to help differentiate whether the detected AE was from bacteria cells during lysis or from the c2 phage during genome injection into the cells. It is proposed that AE measurements using piezoelectric sensors are sensitive enough to detect bacteria at the amount near 104 cfu/mL, to provide real time data on bacteria metabolic activity and to dynamically monitor phage infection of cells.The detection of acoustic emission (AE) from  Saccharomyces cerevisiae yeast cell walls was observed using atomic force microscopy (AFM) , , and  minutes after inoculation. Although the groups near 180 and 250 minutes could be associated with bacterial activity, because L. lactis under “normal” growth produced AE peaks as great as 278 minutes after inoculation  values of all AE peaks with timings after phage addition were small (~1.6 minutes). Their analyses required careful attention to avoid their removal by signal averaging when periods of three minutes or greater were used. In contrast, AE peaks associated with bacteria activity displayed in Figures L. lactis cell infection would be near 30 minutes [Durations of overall phage replication cycles are affected by four dependent variables: nutrient concentration; free or un-infected bacterial cell amount; free phage amount; and infected bacterial cell amount . For a p minutes . This vaThe presence in Furthermore, virus burst sizes are also known to affect the rate of cell lysis and phage-to-bacteria amount ratios –27. InteThe data presented herein were analyzed after completion of each test. However, the detection of phage genome injection into bacterial cells using AE techniques has the potential to be accomplished in real-time. This capacity would expand overall applicability of the AE technique for probing virus-cell interaction dynamics and for shedding insight into the prevention of viral infections. Additionally, the technique will serve as a novel way to study and analyze the critical steps of pathogenic life cycle in general, including the molecular basis of infection, the defensive mechanism by the host cell as well as host-pathogen interaction. Also, improved or new sensor-cell attachment mechanisms that optimize contact between the surfaces or immerse the sensor within nutrient media would help circumvent the AE transmittance losses at interfaces; for example, the loss of AE signal intensity at the broth-glass interface combined with at the glass-stainless steel sensor interface eliminated more than 75% of the intensity from the source of the acoustic wave. Nevertheless, the data presented herein show that AE signals from bacteria metabolic activity and phage genome injection were measurable and distinguishable, and the creation of a metabolically quiescent culture caused elimination of AE from the bacteria.Lactococcus lactis ssp. lactis C2 could be detected by measuring acoustic signals from cultures using piezoelectric sensing elements attached to the outside of bacteria growth flasks. The bacteria-related AE signals were evident when the bacterial amount attained ≥104 cfu/mL levels and only occurred during early or late periods of the logarithmic growth phase; these timings suggested a relationship between cell division and measurable acoustic emission, consistent with the higher metabolic activities during the steps of bacterial life cycle. It was also discovered that Bacteriophage c2 infection of L. lactis could be detected, and the origin of the ensuing AE signals was proposed to be a result of phage genome injection into the bacteria cells. This data has an important biological significance in terms of the real time study of phage mediated infection cycle and its consequences in the phage as well as host bacterium, at both molecular and cellular level. The multiplicity of the AE signals detected over ~250 minutes after phage addition to the bacteria culture contained timings that were in agreement with previous published c2 data during the infection of L. lactis and with the dynamics expected from virus-microbe assemblages under rapidly changing amounts. This multiplicity also suggested-extensive coordination of the timings of phage genome injection into bacteria cells. The acoustic detection of microbes could serve as a potentially valuable and real-time approach to address numerous basic and applied biological and/or clinical issues related to pathogenic microbial infections.It was discovered that the presence or absence of metabolic activity by bacteria


GJB2 may lead to various degrees of sensorineural hearing impairment and/or hyperproliferative epidermal disorders. So far studies of dominant GJB2 mutations were mostly limited to case reports of individual patients and families. In this study, we identified 7 families, 11 subjects with dominant GJB2 mutations by sequencing of GJB2 in 2168 Chinese Han probands with sensorineural hearing impairment and characterized the associated spectrum, de novo rate and genotype-phenotype correlation. We identified p.R75Q, p.R75W and p.R184Q as the most frequent dominant GJB2 mutations among Chinese Hans, which had a very high de novo rate (71% of probands). A majority (10/11) of subjects carrying dominant GJB2 mutations exhibited palmoplantar keratoderma in addition to hearing impairment. In two families segregated with additional c.235delC or p.V37I mutations of GJB2, family members with the compound heterozygous mutations exhibited more severe phenotype than those with single dominant GJB2 mutation. Our study suggested that the high de novo mutation rate gives rise to a significant portion of dominant GJB2 mutations. The severity of the hearing and epidermal phenotypes associated with dominant GJB2 mutations may be modified by additional recessive mutations of GJB2.Dominant mutations in  Numerous gap junction proteins, termed as connexins (Cx), are expressed in inner ear and epidermis. Formed by hexameric connexin hemichannels (connexon) between adjacent cells, those gap junctions play an important role in the potassium homeostasis of the inner ear as well as in the keratinocyte growth and differentiation of the epidermis GJB2 gene, encoding the gap-junction protein connexin 26 (Cx26), may lead to sensorineural hearing loss (HL) and hyperproliferative epidermal disorder. Recessive GJB2 mutations are predominantly associated with non-syndromic HL  and are the most common cause of hereditary HL GJB2 mutations may cause both non-syndromic  and syndromic HL GJB2 mutations includes keratitis-ichthyosis-deafness  syndrome, hystrix-like ichthyosis with deafness , palmoplantar keratoderma  with deafness, Vohwinkel syndrome (MIM124500) and Bart-Pumphrey syndrome (MIM149200). Major epidermal phenotypes associated with dominant GJB2 mutations include ichthyosis, pseudoainhum, palmoplantar hyperkeratosis, knuckle pads and nail abnormalities Mutations in the De novo mutation occurs in the germline during early embryogenesis or somatically in some overgrowth syndromes de novo mutations have nevertheless been reported in some disorders with lesser impact on fitness, including a number of de novo GJB2 mutations detected in sporadic patients with sensorineural HL and hyperproliferative epidermal disorder GJB2 mutations, for which the mutation spectrum and the genotype-phenotype correlation have been well characterized, previous studies of dominant GJB2 mutations were mostly limited to case reports of individual patients and families. In the present study, we aimed to systematically characterize the spectrum, de novo rate and genotype-phenotype correlation of dominant GJB2 mutations in Chinese Han patients with syndromic or non-syndromic HL.Unlike recessive GJB2 mutations. All subjects or their parents gave written, informed consent to participate in this study. This study was approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiao tong University School of Medicine.A total of 2168 Chinese Han probands with sensorineural HL were recruited from patients seeking genetic testing and counseling in Department of Otolaryngology - Head and Neck Surgery, Xinhua Hospital, Shanghai, China. Among them, 1904 (87.8%) probands had severe or profound HL, while the rest 264 (12.2%) had mild or moderate HL. Additional family members were recruited for those carrying known dominant GJB2 was performed by PCR amplification and bidirectional sequencing as previously described Genomic DNA was extracted from the whole blood samples using the Blood DNA kit . Mutation screening of all exons and flanking splicing sites of GJB2 mutations, physical examination was performed with special attention to hearing and epidermal abnormalities. Comprehensive auditory evaluations were performed including otoscope examination, tympanometry and pure-tone audiometry (PTA). Hearing threshold was calculated as the average of the hearing levels at 0.5, 1.0, 2.0, 4.0 KHz for the better ear. The severity of hearing impairment was defined as mild (20–40 dB), moderate (41–70 dB), severe (71–95 dB) and profound (>95 dB). Histological examination was performed in skin biopsies of patient C209-1.For subjects and family members carrying known dominant de novo mutations of GJB2, short tandem repeat (STR) analysis was performed in the affected individuals and their unaffected parents to confirm the parenthood using informative STRs D5S435, D5S629, D5S610, D5S351, D6S1714, D6S1573, D6S1344, D6S469, D3S1597, D3S3611, D3S3601 and D3S3589.For subjects carrying potential GJB2 in 2168 Chinese Han probands with sensorineural HL, we identified 7 (0.32%) probands carrying known dominant GJB2 mutations p.R75Q (n = 3), p.R75W (n = 2) and p.R184Q (n = 2). Four additional family members carrying p.R75Q (n = 2) and p.R75W (n = 2) mutations were also recruited in this study. All other GJB2 variants detected in this study were either previously reported recessive mutations or non-pathogenic polymorphisms. None of the three dominant mutations in GJB2 was seen in 100 ethnically-matched normal hearing controls.By mutation screening of GJB2 mutations. Except for subject D439-2, who had mild HL with delayed-onset, the majority of the subjects (10/11) had congenital, severe-to-profound HL. Ten of the 11 subjects had additional PPK-associated epidermal abnormalities in their hands and feet, including thickening and peeling of the skin, keratoderma, erythema, callus, deep fissures, brittle, thickened or spoon nails, knuckle pads and circular keratotic constriction bands (on bands .All subjects with the p.R75Q (n = 5) and p.R75W (n = 4) mutations had syndromic HL with PPK. C209-1 and D40-1, two subjects with the p.R184Q mutations, had syndromic and non-syndromic HL, respectively. Since the p.R184Q mutation has not been reported to be associated with epidermal abnormalities previously , we furtGJB2 mutations, the probands  and/or their first-degree relatives (D441-2 and D493-2) had unaffected parents. STR analysis of the trios confirmed the true parenthood (data not shown). Parental genotyping showed that the dominant GJB2 mutations detected in those 7 subjects were de novo  and Family D441 (D441-1 and D441-2), respectively. Interestingly, intrafamilial variations of the degree of HL and PPK were observed in both families and the severity of phenotype was apparently modified by the presence of additional GJB2, while the proband's half-sibling D439-2 had the p.R75Q heterozygous mutation only  was detected with known dominant GJB2 mutations. Though not as common as its recessive counterpart, dominant mutations in GJB2 still represent one of the most frequent causes for dominantly inherited HL. To date, more than 20 dominant mutations of GJB2 has been reported worldwide . The mutation spectrum revealed by the current study of Chinese Hans, however, evolved only three of them: p.R75Q (n = 3), p.R75W (n = 2) and p.R184Q (n = 2). Considering that all these three mutations have also been reported in sporadic cases with HL in Taiwan or mainland China  of the probands and two additional family members with the p.R75Q (n = 2), p.R75W (n = 3) and p.R184Q (n = 2) mutations (de novo mutations constitute transitions (purine↔purine or pyrimidine↔pyrimidine) rather than transversions (purine↔pyrimidine), and the mutation rate can be elevated at the CpG-rich region GG>CAG), p.R75W (CGG>TGG) and p.R184Q (CGG>CAG) mutations all fit in with this characteristics of de novo mutations, which may partly explain the relatively high frequency of these three dominant GJB2 mutations in Chinese Hans.One of the most interesting findings of our study was the very high utations . It has GJB2 mutations, family members with both mutations exhibited more severe HL and PPK phenotypes than those with the dominant GJB2 mutation only Click here for additional data file.Table S1Genotype-phenotype correlation of the p.R75Q, p.R75W and p.R184Q mutation in previous reports and the current study.(DOC)Click here for additional data file.


Samples were tested from 67 patients in the derivation cohort, 10 (15%) of whom had confirmed VAP. Using cycles to cross threshold (Ct) values as the result of the 16S PCR test, the area under the receiver operating characteristic (ROC) curve (AUROC) was 0.94 . Samples from 92 patients were available from the confirmation cohort, 26 (28%) of whom had confirmed VAP. The AUROC for Ct in this cohort was 0.89 . This study has derived and assessed the diagnostic accuracy of a novel application for 16S PCR. This suggests that 16S PCR in BAL could be used as a rapid test in suspected VAP and may allow better stewardship of antibiotics.Ventilator-associated pneumonia (VAP) remains a challenge to intensive care units, with secure diagnosis relying on microbiological cultures that take up to 72 hours to provide a result. We sought to derive and validate a novel, real-time 16S rRNA gene PCR for rapid exclusion of VAP. Bronchoalveolar lavage (BAL) was obtained from two independent cohorts of patients with suspected VAP. Patients were recruited in a 2-centre derivation cohort and a 12-centre confirmation cohort. Confirmed VAP was defined as growth of >10VAPRAPID trial ref NCT01972425. Ventilator-associated pneumonia (VAP) remains a significant problem in intensive care units (ICUs)The ubiquitous presence of a 16S ribosomal RNA gene in bacteria offers the possibility of detecting a wide range of bacteria in a single PCR.9/L; or mucopurulent sputum. In the derivation cohort patients were excluded if they had received new antibiotics within the 3 days prior to recruitment;4 colony forming units/mL (CFU/mL) of BAL fluid being defined as ‘VAP positive’ and growth <104 CFU/mL as ‘VAP negative’, these cut-offs being in line with established standards.Samples from two previously describedFull details of sample processing are described in the online 10.1136/thoraxjnl-2016-209065.supp1supplementary data2, 0.2 mM deoxynucleotide mix (dNTP), 0.25 µM primer 27-F, 0.75 µM primer 16S 1RR-B, 0.3 µM probe 514-S, nuclease-free water  and 10 µL nucleic acid extract to a final volume of 25 µL. Real-time PCR was carried out on the ABI 7500 instrument . This assay was used for samples from the derivation cohort, to establish proof in principle of the diagnostic utility of this approach, and was also used for samples from the confirmation cohort.The primer and probe sequences targeting the16S rRNA gene have been described previously.The primer and hybridisation probe sequences targeting the 16S rRNA gene have been described previously.2, 0.4 µM forward and reverse primers, 0.1 µM hybridisation probe, nuclease-free water  and 2 µL of target template for a final reaction volume of 10 µL. Real-time qPCR was carried out on a Light Cycler 480 instrument . This assay was used on samples from the confirmation cohort only.The final 16S PCR reaction mix contained 1X Platinum uracil DNA glycosylase Mastermix , 0.2 µM bovine serum albumin , a total of 4 mmol/L MgClt) as a measure of 16 s rRNA gene load and hence bacterial burden. A higher bacterial load will result in a lower time to cross threshold, that is, a lower Ct value. Details of statistical analyses used can be found in the online For the purposes of analysis, the metric was cycles to cross threshold  had ‘microbiologically confirmed VAP’. In the ‘confirmation’ cohort samples from 92 patients were available for analysis; 26 (28%) met the culture criteria for ‘microbiologically confirmed VAP’. The demographic details and organisms cultured are shown in the online t value, than those without VAP  and a ‘negative’ result would be a value above this cut-off .Samples from the confirmation cohort were also tested using 16S assay 2. As seen in analysis . Using tt values  patients were receiving antibiotics on the day of recruitment. In the confirmation cohort, 69 (75%) were receiving antibiotics and 14 (15%) had undergone a change of antibiotics within the past 3 days. Receipt of antibiotics and recent change in antibiotics were not associated with changes in 16S CFigure S2 shows the relationship between Ct values for the two 16S assays, demonstrating a non-linear association.To our knowledge, this is the first report of the use of real-time 16S PCR for diagnosing VAP. Although 16S rRNA gene sequencing has been used to explore the microbiome of ventilated patients, data on its diagnostic potential have been absent. In deriving and confirming a test, with a high agreement in test performance between the two cohorts, we demonstrate clear potential for the clinical utility of this test. Turnaround time is 4–6 hours; therefore, this test could impact on antibiotic use, which may otherwise only be rationalised following the results of conventional cultures at 48–72 hours.This study has a number of strengths. First, we were able to perform derivation and confirmation in two distinct cohorts, with confirmation in a cohort recruited from a diverse group of 12 ICUs. The results are therefore likely to be widely applicable; indeed, the microbiological spectrum found is similar to reports from other countries.A disadvantage of this study is that samples were obtained bronchoscopically, requiring resource and exposing patients to a small but definite risk, and the applicability of this test to other sample types cannot be inferred. The assays we describe here are also limited to bacterial detection. The differences between the two assays tested, and the use of stored samples, highlight the need for external prospective validation before this measure could be implemented in routine clinical practice. Further refinements of assays may also improve diagnostic performance. The reference standard of growth of organisms on conventional culture, remains imperfect, and indeed may well be influenced by intercurrent antibiotics generally, and recent changes in antibiotics specifically.In conclusion, we have derived and confirmed the diagnostic utility of a rapid laboratory test for VAP in a multicentre setting. We propose that this test has the potential to permit rapid decisions to direct antimicrobial therapy in patients with suspected VAP thus improving stewardship of antibiotics in the ICU.