As an example of haploid transposon (HTP) screening, we apply this approach to identifying determinants of cancer drug toxicity and resistance. In a screen for 6-thioguanine resistance we recovered components of the DNA mismatch repair pathway, a known requirement for toxicity. In a further screen for resistance to the clinical poly(ADP-ribose) polymerase (PARP) inhibitor olaparib we recovered multiple Parp1 mutants. Our results show that olaparib toxicity to normal cells is mediated predominantly via Parp1, and suggest that the clinical side effects of olaparib may be on target. The transposon mutant libraries are stable and can be readily reused to screen other drugs. The screening protocol described has several advantages over other methods such as RNA interference: it is rapid and low cost, and mutations can be easily reverted to establish causality.Genetic perturbation screens have the potential to dissect a wide range of cellular phenotypes. Such screens have historically been difficult in diploid mammalian cells. The recent derivation of haploid embryonic stem cells provides an opportunity to cause loss of function mutants with a random mutagen in a mammalian cell with a normal genetic background. We describe an approach to genetic screens that exploits the highly active  C. elegans and Drosophila. In mammals, cell culture systems provide one approach to producing enough random mutants to adequately sample all mammalian genes. However screening in most mammalian cell lines has a major drawback compared to whole organisms: there is no breeding strategy available to produce homozygous mutants from randomly-generated heterozygotes. Therefore loss-of-function screens have always been challenging, as the diploid mammalian genome often ensures that any introduced mutation in a gene is compensated for by another copy of the gene on the homologous chromosome. Thus, RNA interference has remained the method of choice for cell culture screens, despite the development of effective mammalian transposons and their success in whole-organism screens Forward genetic screens, in which random mutations are generated and the resulting mutants screened for a phenotype of interest, are of great use in the analysis of gene function. These screens have been most productively applied in model organisms with short generation times, such as yeast, The recent rediscovery and stable culture of haploid mammalian cells promises to change this situation. Haploid mammalian cells have only one copy of each chromosome, so random mutations can directly cause loss-of-function phenotypes. Haploid mammalian cell lines include a near-haploid human chronic myeloid leukaemia cell line Parp1 itself is required for olaparib toxicity in wild type mouse ES cells, and depletion of PARP1 in human cells also caused olaparib resistance. Our results not only exemplify the potential of HTP screens but also support a mechanism of action of PARP inhibitors in which the inhibited PARP1 enzyme forms a toxic DNA lesion.Here, we have applied piggyBac transposon mutagenesis in haploid ES cells to generate large, stable libraries of mutants for genetic screens, a process we will refer to as haploid transposon (HTP) screening. We have tested the potential of this system by identifying known genetic determinants of resistance to the chemotherapeutic 6-thioguanine. A major application for genetic screening is in investigating genetic mechanisms of toxicity and resistance as part of drug development. We therefore applied HTP screening to investigate mechanisms of response to the Poly (ADP-Ribose) Polymerase (PARP) 1/2 inhibitor olaparib puroΔTK, driven by the mouse phosphoglycerate kinase promoter (PGK), which allows selection for insertion into the genome using puromycin. Mutagenesis is achieved by two pairs of splice-acceptor bearing exons at either end of the transposon cargo in opposite orientations, which should disrupt splicing if the transposon is inserted into an intron of a gene. The penultimate exon of each pair is engineered with several premature stop codons in each reading frame, which should cause degradation of fusion transcripts by the nonsense-mediated mRNA decay pathway, thus even insertions at the 3′ end of genes are likely to be mutagenic.We designed a workflow and methods  that facUsing conditions that give, on average, one transposon insertion per cell Msh2, Msh6 and Mlh1 (Pms2) from this library, including three different insertions in Msh2, indicating good genome coverage even in this sub-saturating library. This demonstrated that HTP screens can efficiently identify known mechanisms of drug toxicity.To test the extent of loss-of-function mutagenesis in the libraries, we used 6-thioguanine selection. This positive selection screen has been previously used as a benchmark for mutagenesis systems, as 6-thioguanine is known to require the DNA mismatch repair pathway for toxicity and Mlh1 . Importa5 wild type cells  (PAR), the product of PARP enzymatic activity, further suggesting that there is no active truncated protein expressed  in human CAL51 and DLD1 tumour cells also caused resistance to BMN 673 and olaparib  Parp6, Parp7, Parp9, Parp11, Parp12, Parp14 and Parp16; and the tankyrases Tnks (Parp5a) and Tnks2 (Parp5b) (This sequencing identified insertions in the PARP superfamily genes (Parp5b) . As thesPARP1 is the major genetic determinant of PARP1/2 inhibitor toxicity in normal cells suggests that PARP1 protein is a necessary component of the toxic DNA lesion. As autoparsylation of PARP1 is required for the release of PARP1 from sites of DNA damage The unexpected observation that Parp1 mutant cells did not  and large mutant libraries can be rapidly generated using freely available plasmids. The 6-thioguanine screen was conducted in one well of a 6-well cell culture plate, and thus required far fewer cells compared with previous ES cell screening methods and gave a similar result in terms of recovering relevant mismatch repair mutants Cadm2) that have a mild resistance phenotype and retained Parp1 expression may have acquired another mutation that promotes resistance; however since the phenotype is not linked to the transposon insertion this is more difficult to investigate. Screening using a weaker selective pressure may result in more mutants with weaker resistance phenotypes and thus allow minor mechanisms to be investigated.After transposition, mutant libraries are stable and can be used for multiple screens, allowing common genetic mechanisms of resistance to be identified for different drugs. The reversion of transposition events allows causality to be established, a complex issue in RNAi screens where off-target effects are a major concern As demonstrated here and elsewhere The high plating efficiency of ES cells at low density makes them ideal for positive selection screens as described here, but high-throughput sequencing methods for mapping transposon integrations HAP-3 cells 5 cells were plated on a six-well feeder plate (this high density precludes isolation of Hprt mutants in 6-thioguanine due to cross-killing) and drugs added the following day. Drug concentrations used were: 6-thioguanine, 2 µM; olaparib, 4 µM; puromycin, 3 µg/ml. After isolation of mutants, ES cells were cultured in conventional ES cell medium supplemented with 15% fetal calf serum (FCS) and LIF. For survival curves, 2,500 ES cells were seeded per well on gelatinised 96-well plates. Drug-containing medium was added the next day (day two) and replenished 48 hours later. The live cell proportion was estimated on day six using CellTiterGlo (Promega).To isolate resistant mutants, 5–10×10d-arabinofuranosyl)-5-iodouracil) or G418 (180 µg/ml). Untransfected cells were also plated in FIAU to ensure that the reversion was PBase-dependent and did not arise from spontaneous mutation or loss of the puΔTK selectable marker or from contaminating wild type cells in the culture.To isolate revertants, mutant cells were electroporated with the hyPBase expression plasmid and cultured for three days without selection. Cells were then plated in 200 nM FIAU , 2 mM L-glutamine and penicillin–streptomycin (Gibco). DLD1 cells were obtained from Horizon Discovery Ltd. and cultured in McCoy’s 5A medium with 10% FCS, 2 mM L-glutamine and penicillin–streptomycin. Table S1A list of insertion sites identified by sequencing libraries H3L1–H3L8.(XLSX)Click here for additional data file.Protocol S1A detailed protocol for carrying out drug resistance screens.(DOCX)Click here for additional data file.


Rbm3, Eif4e2 and Elavl1. Given the fact that RBPs are considered as the main regulators of differential APA expression, we constructed a co-expression network between APAs and RBPs using the microarray data. Further incorporation of CLIP-seq data of selected RBPs showed that Nova2 represses and Mbnl1 promotes the polyadenylation of closest poly(A) sites respectively. Altogether, our study is the first microarray meta-analysis in a mammal on the regulation of APA by RBPs that integrated massive mRNA expression data under a wide-range of biological conditions. Finally, we present our results as a comprehensive resource in an online website for the research community.Alternative polyadenylation (APA) is a post-transcriptional mechanism to generate diverse mRNA transcripts with different 3′UTRs from the same gene. In this study, we systematically searched for the APA events with differential expression in public mouse microarray data. Hundreds of genes with over-represented differential APA events and the corresponding experiments were identified. We further revealed that global APA differential expression occurred prevalently in tissues such as brain comparing to peripheral tissues, and biological processes such as development, differentiation and immune responses. Interestingly, we also observed widespread differential APA events in RNA-binding protein (RBP) genes such as  In the last step of eukaryotic transcription, the mRNA transcripts are cleaved at the 3′ termini of the transcripts at so-called poly(A) site. Then poly(A) tails of 100–200 nt are added in a process named polyadenylation. This is a tightly regulated process involving the interplay between 3′UTRs that contain different cis-regulatory elements and RNA Binding Proteins (RBPs). RBPs such as cytoplasmic polyadenylation specific factor (CPSF) and cleavage stimulation factor (CstF) can recognize and bind to specific sequence motifs upstream and downstream of poly(A) site to enable the cleavage on the specific poly(A) site. Typically one or more alternative poly(A) sites exist for mammalian genes, resulting transcripts with different 3′UTRs for the same gene, in an event called alternative polyadenylation (APA). It can even produce new proteins with novel C-termini or lead to non-sense mediated decay (NMD) when acting upstream of a stop codon RBP and miRNA are known to interact with mRNA transcripts in their 3′UTRs. Transcript isoforms resulting from APA may have different expression levels due to different sets of RBP or miRNA binding sites in their 3′ UTRs. Although some poly(A) sites are more constitutively expressed than the others on the same gene, it has also been shown that the choices of poly(A) sites are tissue-specific and can change under different biological conditions. Zhang et al. examined possible poly(A) sites from existing mRNA and EST databases and found that the transcripts using different poly(A) sites express in a tissue specific manner DrosophilaRBPs have increasingly been recognized as important regulators of RNA metabolism at the post-transcriptional level. A large number of RBPs are known to bind the 3′UTRs of mRNA transcripts affecting their expression, stability and/or translation. In particular, RBPs play important role in APA and alternative splicing regulation. Furthermore, APA and alternative splicing are frequently coupled together and share common RBP regulators, such as U1 snRNP Rbm3), showed opposite expression changes during hypothermia and sleep-deprivation in mouse Rbm3 APA is still unclear. In this study, we hoped to search for the existence of more such differential expression among APA isoforms systematically in public microarray database. We identified new candidates including Eif4e2, Cd38, and Elavl1 in addition to Rbm3 that contain APAs with differential responses under a wide range of biological conditions. Furthermore, we found that global shift of differential APA expression is dependent on tissue or cell type, disease, and developmental states. Focusing on differential APA expression regulated by RBPs, we developed a co-expression analysis to predict the novel functions of RBPs in APA regulation. Finally, we demonstrated how to infer the functional link between APA differential expression and RBP regulation by incorporating the result of co-expression analysis and RNA binding data of selected RBPs.Previously, we observed that two APA isoforms of an RBP, RNA-binding protein motif 3 (The Known Gene (KG) annotations of mouse genome (Mm9) were extracted from UCSC genome browser. The information of 3′ ends of mRNA transcripts was integrated into poly(A) site database. We extracted 3′ UTRs from KG annotations and merged them into clusters if any overlaps occurred.Polya_DB2 is a comprehensive poly(A) database across different animals constructed by Lee et al. Data from a recent study using polyA-seq generated poly(A) site atlas for 24 tissues from human, rhesus, dog, mouse, and rat was downloaded from UCSC browser. We extracted poly(A) sites for mouse and merged them into clusters if the adjacent poly(A) sites are within the distance of 40 bps. This dataset gave rise to 126,028 poly(A) site clusters in total. We further required that one poly(A) cluster should be represented in at least two tissues and thus reduced the number of poly(A) site clusters to 39,710. Finally, we merged poly(A) sites from this dataset with the 3′ ends of KG genes and polyA_DB2 into the final poly(A) site database. The poly(A) sites within 40 bps were merged and the poly(A) clusters at the 3′-most sites were retained.We assigned each poly(A) site to the associated 3′ UTR cluster if it falls within the 3′UTR or at most 3,000 bps downstream. In the latter case, the 3′ UTR was also extended downstream to the poly(A) site. Then we defined sub-3′ UTR regions as the regions on 3′ UTRs partitioned by poly(A) sites. The pairs of sub-3′ UTRs from the same host gene were considered as potential APA events. We classified the type of APA event as either tandem UTR if its sub-UTRs are located on the same 3′ UTR or alternative UTRs if located on different exons. Thus we defined 42,735 tandem UTR and 17,756 alternative UTR events for a total of 12,250 mouse genes.To enable APA expression analysis using microarray data, we mapped all Affymetrix probes from Mouse4302 microarray onto Mm9 mouse genome by Bowtie software For the mock tandem UTR control, the remaining probes of Affymetrix probesets were equally divided into two adjacent genomic regions each containing at least four probes. Thus, we obtained 7,181 mock tandem UTR control events.ULI) for tandem UTR events by subtracting expression change of common UTR (com) from that of extended UTR (ext):To calculate differential expression of a specific APA event, we defined UTR Lengthening Index  as B value, i.e.r′ less than r and N = 7,181 is the total number of r′. In this case, the B value represents how significant the differential expression of APA is compared to random background. Similar to this, we have also normalized other statistical quantities such as Two-way ANOVA p values for APA events from their mock background distributions.Furthermore, for every microarray experiment we calculated Pearson's correlation All 1,365 mouse4302 microarray series (experiments) available until Sep. 2011 from Gene Expression Omnibus (GEO) were retrieved in the format of raw CEL files. The complete list of experiments can be found in Microarray normalization was carried out by RMA algorithm in R from Bioconductor package using our customized probeset annotation. To guarantee the statistical power of downstream analysis, we removed the experiments without sufficient biological replicates. Finally, 750 experiments in total were available for ANOVA tests and other downstream analysis. One-way ANOVA test was carried out for each probeset with the experimental conditions as the factor. Two-way ANOVA test was carried out on every APA event with experimental conditions and sub-3′UTR regions as two factors.Celf2 was previously reported as a bona fide target of NOVA2 by Licatalosi et al. Celf2 tandem 3′ UTR region on the genome.A tandem UTR event of Nova2 differential expression  from 750 microarray experiments. We then found significant differentially expressed APA events  in at least 40 experiments. For each individual experiment, we calculated Pearson correlations between APA ULIs to Nova2 expression values. For each APA event, we calculated the median value of all correlation coefficients across experiments to characterize the overall correlation between the APA and Nova2. Celf2 APA event appeared among the top list of our result, supporting the validity of our method.We further identified 101 experiments showing Celf2 APA showed differential expressions . For each RBP that was differentially expressed , we calculated Pearson correlation between its gene expression to ULI of Celf2 APA. We used the median value of correlation coefficients across experiments to denote the overall correlation strength. We can readily identify Nova2 in the top list of our result supporting its key regulatory role for Celf2 APA.To validate our result from the other perspective, we selected experiments from the microarrays in which r<−0.4, Two-way ANOVA p<0.01 and mock background corrected Two-way ANOVA p<0.01. We obtained the annotation of RBPs from RBPDB (version 1.2.2) that contained 557 mouse RBP genes We then applied our method to other significantly differentially expressed APA events. Specifically, the differential expressed APA in a given experiment is defined by Pearson correlation coefficient http://starbase.sysu.edu.cn/) for Nova2, Tardbp and Ago2 in mouse We downloaded the binding sites and unique CLIP tags derived from CLIP-seq data by starBase (website: p<0.01). The median values of correlation coefficients across all filtered experiments were used to characterize the overall correlation between individual APA event and the RBP in question.Next we integrated the gene expression changes for the RBPs from microarray studies with RBP binding site information. We defined RBP differential expression by one-way ANOVA test. The Pearson correlations between RBP expression and ULI of the targeted APA events that contain RBP binding sites were calculated. The APA differential expression was determined by two-way ANOVA  Statement for our analysis in We first obtained poly(A) site information from both polyA database polyA_DB2 We used the gene annotations from UCSC known genes and extracted their 3′ UTR regions. After incorporating genomic poly(A) sites, the 3′UTR regions can be divided into sub-UTR regions Data in , of whicTo systematically examine the expression of APA events under different biological conditions, we used all existing microarray data in Gene Expression Omnibus (GEO) database (until Sep. 2011) on Affymetrix Mouse4302 microarray platform . This typhosphoprotein and nucleotide binding category. RBPs are enriched in the top list by Fisher's exact test . This suggested that differential APA expression of RBPs might be a common phenomenon.The scheme of our meta-analysis pipeline is shown in Elavl1 was specifically expressed in brain tissues . This is consistent with the earlier studies Rbm3 showed differential expression in sleep deprivation and cold exposure in mouse Rbm3 is ranked top 22 among the differentially expressed APAs. We identified comprehensive biological conditions leading to Rbm3 differential APA expression including sleep deprivation , cold shock (GSE20645), hypoxia , and development. From these known cases of differential APA events in Elavl1 and Rbm3, it can be readily seen that our strategy is efficient to identify the biological conditions of differential APA events. We selected two genes with novel differential APAs for illustration in Eif4e2 ranked top 2 is a cap-binding protein and eIF4E homolog. It showed differential APA expression in different tissues  with short isoform preferred in liver, development (GSE20954) and spermatogenesis (GSE4193) (Cpsf6 ranked top 56 is itself a key component of poly(A) machinery. Its differential APA expression suggests its auto-regulation by APA mechanism. We found that the APA isoforms of the Cpsf6 resulted from tandem UTR were differentially expressed in embryonic development (GSE12454), cell differentiation (GSE8024), and different tissues   . Cpsf6 rSE23782) .To quantify the global 3′ UTR lengthening or shortening resulted from APA for each experimental treatment, we compared the global distributions of 3′UTR lengthening indices (ULI) across different conditions in a given microarray experiment . We obseTnrc6c|a|b, Hnrnpd, Rc3h1, Helz and Cpsf6. Among them, Tnrc6c|a|b are known to silence translation of target mRNAs and Helz is a RNA helicase. As a core component of poly(A) machinery, Cpsf6 enhances the recognition of weak poly(A) sites and was reported to regulate global UTR lengthening in human Elavl1, Ptbp1, Fubp1, Cstf2, and Ncbp2. Among them, Elavl1 is known to destabilize target mRNAs. Fubp1 is known to activate transcription. Cstf2 can stimulate mRNA cleavage at polyA sites and Ncbp2 can promote mRNA 3′-end processing. Taken together, many of the genes that we found using our approach are known to be either directly or indirectly involved in APA regulations.Several studies have shown that RBPs such as CPSF6 and PABPN1 in human play important roles in the regulation of global shift in APA patterns. To take advantage of large-scale microarray gene expression data in this study, we conducted a co-expression analysis between RBPs and APAs to search for the RBPs that are potentially linked to the regulation of global APA shift. We extracted gene expression for every RBP in a given microarray experiment and calculated their correlations to the global ULI in that experiment. We scored the RBPs by their median correlation coefficients across experiments   machinery, as a hub in the network. This finding is consistent with the result that Cpsf6 expression mainly correlates with global 3′ UTR lengthening. In addition, we identified other RBPs correlated with global APA shifts such as Tnrc6c, Helz as important hubs in the network.For a specific differential APA event, we sought to identify potential RBP regulators by co-expression analysis. We computed the correlations between the ULIs of differential APA events and RBPs. We applied this method to about 50 most significant differential APA events . The mosCelf2Nova2 expression was significantly correlated with Celf2 APA  sequencing binding data from either PAR-CLIP (photoactivatable ribonucleoside-enhanced CLIP) or CLIP experiments for four RBPs including Ago2, Tardbp, Nova2 from starBase p<0.01), we calculated the median Pearson correlation coefficients from Nova2 to ULIs of all differentially expressed APA events. We observed that the ULIs of APAs with Nova2 binding to the common UTR regions tend to be more positively correlated with Nova2 expression as compared to APAs with Nova2 binding to the extended UTR regions  sites within 200 bp flanking regions, containing 649 type II and 92 type III APA events. Among the type II APA events, Nova2 predominantly binds to the common UTR regions in 370 events as compared to the bindings in the extended regions in 202 events. From 101 microarray experiments where Nova2 was found significantly differentially expressed  sites, containing 571 type II and 82 type III APA events. Among the type II APA events, Mbnl1 also predominantly binds to the common UTR regions in 294 events as compared to the bindings in the extended UTR regions in 140 events. In microarray experiments, we identified 210 experiments in which Mbnl1 gene expression was significantly changed . Similarly, we calculated the median Pearson correlation coefficients from Mbnl1 to ULI of all differentially expressed APA events. Interestingly, ULIs of APAs with Mbnl1 binding to the common UTR regions tend to be more negatively correlated with Mbnl1 expression as compared to the APAs with Mbnl1 binding to the extended UTR regions  as a public resource. On this website, user can query and browse poly(A) sites and APA events for genes of interests on the embedded UCSC genome browser. The information such as the 3′UTR regions and microarray probes are also included. For a specific APA event, user can search for the microarray experiments in which it showed differential expression from APA query page. From the RBP query page, user can query an RBP to retrieve the correlated APA events. The global correlation coefficients between the RBP and the APA events were presented in a result table. Finally, the entire supplementary data in this paper can be downloaded from the download page.We have constructed an online website to present our integrated poly(A) site database and result of RBP-APA analysis at (Affymetrix microarray has been widely used for gene expression studies and was mainly designed to target genes in 3′ UTR region. It provided us the opportunity to detect subtle 3′ UTR expression changes representing the shift in APA usage among known poly(A) sites. In this study, we re-annotated mouse4302 microarray and developed a computational pipeline to systematically analyze APA differential expression for 1,365 mouse4302 microarray studies from NCBI GEO database. We revealed prevalent differential APA events and their related biological conditions. We found RBPs are enriched in differential APA list. We also observed that global shift in differential APA expression highly depends on biological conditions. While the microarray data can not be used to discover novel poly(A) sites as in high-throughput sequencing, the strength of our approach was the use of large volume of microarray data available in GEO database over a wide range of biological conditions.Several models have been proposed to describe the mechanisms how RBPs regulate APA expression. The core poly(A) factors, i.e. CPSF and CstF, can directly affect poly(A) site usage. For example, APA in IgM heavy chain in immune system results in two different protein isoforms that are either membrane bound or for excretion. Such APA selection is regulated by CstF-64 Interestingly, we found that RBPs themselves tend to contain APA events and may be subjected to their own or other RBPs' regulation. It has been observed that RBPs frequently regulate each other to form a complex regulatory network. There are already ample evidences that RBPs play important roles in the regulation of APA. Although other mechanisms such as uaRNAs Table S1Summarized poly(A) sites database from three Poly(A) data sources for mouse Mm9 genome.(XLSX)Click here for additional data file.Table S2Sub-UTR database for mouse Mm9 genome.(XLSX)Click here for additional data file.Table S3All GEO microarray experiments used in our meta-analysis.(XLSX)Click here for additional data file.Table S4APA events annotated for Affymetrix mouse4302 microarray platform.(XLSX)Click here for additional data file.Table S5The list of genes showing the most frequent differential APA events (full list). For each gene, the experiments in which its APA was differentially expressed are as listed.(XLSX)Click here for additional data file.Table S6The list of biological conditions that most significantly perturb global APA usage (full list).(XLSX)Click here for additional data file.Table S7The list of RBPs showing strong positive or negative association with the global shift in APA (full list). The median correlation coefficients between RBPs and gULIs across experiments are indicated in an individual column.(XLSX)Click here for additional data file.Table S8The RBP and APA co-expression network (full network). Median Pearson correlation coefficients between RBPs and APA targets were indicated.(XLSX)Click here for additional data file.Checklist S1PRISMA Checklist.(DOC)Click here for additional data file.


Older patients with acute myeloid leukemia (AML) experience short survival despite intensive chemotherapy. Azacitidine has promising activity in patients with low proliferating AML. The aim of this dose-finding part of this trial was to evaluate feasibility and safety of azacitidine combined with a cytarabine- and daunorubicin-based chemotherapy in older patients with AML.Prospective, randomised, open, phase II trial with parallel group design and fixed sample size.Patients aged 61 years or older, with untreated acute myeloid leukemia with a leukocyte count of <20,000/µl at the time of study entry and adequate organ function were eligible. Patients were randomised to receive azacitidine either 37.5 (dose level 1) or 75 mg/sqm (dose level 2) for five days before each cycle of induction (7+3 cytarabine plus daunorubicine) and consolidation (intermediate-dose cytarabine) therapy. Dose-limiting toxicity was the primary endpoint.Six patients each were randomised into each dose level and evaluable for analysis. No dose-limiting toxicity occurred in either dose level. Nine serious adverse events occurred in five patients  with two fatal outcomes. Two patients at the 37.5 mg/sqm dose level and four patients at the 75 mg/sqm level achieved a complete remission after induction therapy. Median overall survival was 266 days and median event-free survival 215 days after a median follow up of 616 days.The combination of azacitidine 75 mg/sqm with standard induction therapy is feasible in older patients with AML and was selected as an investigational arm in the randomised controlled part of this phase-II study, which is currently halted due to an increased cardiac toxicity observed in the experimental arm.NCT00915252).This trial is registered at clinical trials.gov (identifier:  Despite intensive treatment, acute myeloid leukemia (AML) in elderly patients still has a dismal outcome with the vast majority of patients succumbing to their disease within 2 years after diagnosis In vitro data suggest a synergistic effect of cytarabine and azacitidine when azacitidine is administered before cytarabine exposure While high-dose cytarabine has demonstrated strong clinical activity in vivo in cytarabine resistant primary AML cells, a phase I-study with 17 pediatric patients with relapsed acute lymphoblastic leukemia (ALL) after high-dose cytarabine treatment yielded a complete remission in two out of 9 evaluable patients after treatment with azacitidine followed by another course of high-dose cytarabine Even though no inactivation of dCK could be demonstrated The aim of this pilot trial was to establish the preliminary safety profile of azacitidine added to standard induction and consolidation therapy in older patients with newly diagnosed AML, and to determine the recommended dose for a subsequent controlled phase II trial.www.clinicaltrials.gov . The study was approved by the Ethics Committee of the University of Muenster and conducted in accordance with the Declaration of Helsinki. Written, informed consent was obtained from all patients before study enrolment.The protocol for this trial and supporting CONSORT checklist are available as supporting information; see Checklist S1 and Protocol S1. The dose finding trial reported herein was a prospective, randomised, open-label phase II trial with parallel group design and fixed sample size preceeding an open-label, randomised, controlled, multicenter phase II trial of the study alliance leukemia (SAL). Participating centers of the dose finding part of this trial were the University Hospitals of Muenster, Frankfurt am Main, Dresden and Essen, all located in Germany. The trial was registered at Patients with >60 years of age with untreated AML (excluding acute promyelocytic leukemia) were eligible. Exclusion criteria were: Known central nervous system manifestation of AML, heart failure class 3 or 4 according to New York Heart Association, unstable coronary artery disease, serious cardiac ventricular arrhythmias requiring anti-arrhythmic therapy beyond beta blockers or digoxin, inadequate renal (creatinin clearance <30 ml/min) or hepatic function , alanine aminotransferase (ALT) or aspartate aminotransferase (AST) ≥2.5×ULN not caused by leukemic infiltration), known human immunodeficiency virus and/or hepatitis C infection, evidence or history of severe non-leukemia associated bleeding diathesis or coagulopathy, evidence or recent history of CNS disease, including primary or metastatic brain tumors, seizure disorders, uncontrolled active infection, concurrent malignancies other than AML with an estimated life expectancy of less than two years, history of organ allograft, hypersensitivity to cytarabine (not including drug fever or exanthema), daunorubicin, azacitidine or mannitol and previous therapy with azacitidine (i.e. for an antecedent myelodysplastic syndrome). An exclusion criterion only for the dose finding part of this trial was a leukocytosis >20,000/µl at study entry. For the controlled part of the trial, those patients were eligible if the leukocyte count could be decreased to <20,000/µl with hydroxyurea.Patients were randomised 1∶1 between 37.5 (dose level 1) and 75 mg/sqm (dose level 2) of azacitidine without stratification. This parallel randomised evaluation allowed us a rapid evaluation of two different dose levels of azacitidine, and ensured six patients being treated with the recommended dose for the controlled part of the study. The random allocation sequence was generated using SAS software (proc plan). Randomisation lists were generated using block randomisation with a block size of 4. Randomisation was performed centrally providing allocation concealment. Patients were first screened for eligibility at clinical sites without having access to randomisation lists. Eligible patients were reported to a central randomisation unit by fax. Subsequently the intended intervention of each patient was reported back to clinical investigators.The treatment scheme is depicted in An early bone marrow evaluation was performed seven days after the end of the first induction therapy. A sufficient blast clearance in the early bone marrow evaluation was defined as <5% remaining leukemic blasts according to the revised recommendations of the International Working Group for AML The primary endpoint of this dose finding study was safety and toxicity of both dose levels. The recommended dose for the controlled part of the trial was defined as the highest dose level with one or less out of six evaluable patients experiencing a DLT. A separate dose level 0 with azacitidine 18 mg/sqm would have been evaluated in additional six patients in case of >1 DLT in each dose level. If >1 DLT would have been occurred also in this dose level 0, no azacitidine would have been administered preceeding the cytotoxic cycles in the controlled part of the trial. Adverse events (AE’s) and severe AE’s (SAE’s) were graded according to NCI common toxicity criteria (CTC) v3.0. CTCAE Grade 4 leukocytopenia, neutropenia, and thrombocytopenia as signs of active antileukemic treatment were assessed as an SAE only if persisting >42 days after the last application of cytotoxic chemotherapy in patients with blast clearance with (CR) or without (CRi) complete hematological recovery and then defined a DLT.Assessment of treatment response was performed seven days after the end of the first induction cycle as described above, after hematological recovery following induction therapy, but not later than 35 days after the start of the last induction cycle, and every three months after achievement of complete remission until disease progression. Patients were monitored with physical examination, complete blood count and chemistry profile at least once weekly during induction and consolidation, and then at least monthly for the first two years after treatment start during follow-up. Response to treatment was defined according to the revised recommendations of the International Working Group for AML NPM), fms-like tyrosine kinase 3 (FLT3), additional sex combs like 1 (ASXL1), DNA methyltransferase 3A (DNMT3A), ten-eleven translocation 2 (TET2), isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was performed as published TET2 were modified from Cytogenetic analysis was performed by classical G-banding as previously described Overall survival was measured from the first day of azacitidine treatment until death of any course. Event-free survival was measured from the first day of treatment until therapy failure, relapse or death of any course. Patients without an event were censored at last follow-up. Follow-up time was calculated using reverse censoring. These outcome parameters were determined only exploratory for the total cohort. Statistical analyses were performed using IBM SPSS Statistics 20 for Windows .Between September 2009 and February 2010, 12 patients were included in this pilot trial; four from two centers each, and three and one from the remaining centers, respectively. Data bank snapshot was taken on February 15th, 2012. The CONSORT statement of these patients is shown in No DLT occurred in either dose level. Nine severe adverse events occurred in five patients, two in the 75 mg/sqm dose level (two patients) and seven in the 37.5 mg/sqm dose level (three patients) . With thSeven patients received one and five patients received two induction courses. No patient had to be replaced due to hyperleukocytosis during the first azacitidine course as prespecified in the protocol. Reasons for applying only one induction course were complete blast clearance seven days after the end of the first induction cycle (four patients), early death due to pneumonia during the first course (one patient), progressive disease (one patient) and an SAE (one patient). Of all seven responding patients, four received both consolidation cycles. Two patients proceeded to allogeneic stem cell transplantation  in first complete remission.The treatment outcome is depicted in Of note is the outcome of patient no. 10. The patient with a complex karyotype AML received one induction course with azacitidine 37.5 mg/sqm followed by ‘7+3’. One week after commencement of the first induction cycle, the bone marrow evaluation showed persisting infiltration with 77% blasts. Due to massive hemolysis (most likely fluorochinolone-induced) and subsequent cardiac failure, both regarded as a SAE, trial treatment was stopped on day 31 of therapy. Despite no further treatment after termination of study participation, the patient achieved a complete remission on day 69 , while both SAE’s resolved. The patient remained in CR with good health and in the absence of further therapy for almost a year before relapse occurred.Outcome by ELN risk group revealed 2 out of 2 patients achieving a CR with good risk, 2/4 patients with intermediate-I risk, 2/3 patients with intermediate-II risk and 1/3 patients with high risk.We herein present data of a dose finding trial to evaluate safety, toxicity and feasibility of the addition of azacitidine to standard induction and consolidation therapy in older, medically fit patients with newly diagnosed AML. While intensive chemotherapy leads to a CR rate of 50% in this patient group, the probability of long-term survival is less than 10%, demonstrating the need for alternative and more efficacious treatment options. Azacitidine has a proven efficacy in patients with high-risk MDS and low-proliferating AML with 30% bone marrow blasts or less Since a combination of azacitidine with a cytarabine and anthracyclines has not been evaluated, we assessed two different dose levels of azacitidine to determine the maximal tolerated dose.The seven complete remissions seen in our pilot trial are in line with observed remission rates with intensive chemotherapy in this entity and age group, and the observed survival rates are encouraging.Recent reports found an association with response to therapy in patients carrying DNMT3A, ASXL1 or TET2 mutations Regarding toxicity, we observed three early deaths during induction therapy attributable to pneumonia, hepatorenal syndrome and apoplectic stroke, respectively. While the latter two toxicities are uncommon, the death rate of 25% after an intensive, cytarabine- and anthracycline-containing induction therapy in this patient population is in line with published results In our pilot trial, both dose levels could be safely administered prior to standard cytotoxic induction and consolidation therapy. Therefore, the independent Data Monitoring Committee (DMC) suggested 75 mg/sqm for 5 days as the recommended dose for the randomised controlled phase II part of this trial . Eligible patients in this open-label, controlled part of the trial are randomised 1∶1 between the experimental or the control arm. Patients in the experimental arm receive azacitidine 5×75 mg/sqm prior to each induction and, if responding, consolidation therapy cycles as described above, and responding patients in the experimental arm additionally receive a maintenance therapy after consolidation with azacitidine 75 mg/sqm over 5 days s.c. q28 days for up to one year after initiation of therapy. Patients in the control arm directly receive induction therapy, consisting of 7+3 induction, and responding patients receive consolidation therapy as described above. No placebo-controlled design was chosen because of ethical considerations (treatment of patients with untreated AML with placebo for 5 days before initiating induction therapy). Sample size of this controlled phase II part was initially planned for 216 subjects (108 each arm), with event-free survival being the primary endpoint. However, recruitment is currently halted due to an increased rate of cardiac toxicity observed in the experimental arm after recruitment of 213 patients. The DMC recently stated that there is no concern regarding a further conduct of the randomised phase II trial, and results of this controlled part will be published separately. Taken together, our results of the pilot trial reported herein demonstrate that it might be feasible to add azacitidine 75 mg/sqm for 5 days to standard induction chemotherapy. This is in line with a recently published phase I trial combining standard induction therapy with the hypomethylating agent decitabine, which also showed feasibility and promising CR rates Table S1Primer used for TET2 amplification and sequencing.(DOCX)Click here for additional data file.Table S2Mutational analysis for the genes ASXL1, DNMT3A, IDH1, IDH2 and TET2. Abbreviations: ASXL1, additional sex combs like 1; bp, base pair; chr., chromosome; IDH, isocitrate dehydrogenase; TET2, ten-eleven translocation 2. No aberrations in exons 15-23 of the DNMT3A coding sequence were detected.(DOCX)Click here for additional data file.Table S3List of all adverse events during study participation. All adverse events occurring from signing the informed consent until end of study participation are listed. Neutropenia, leukopenia and thrombocytopenia were not considered as an adverse event unless persisting >42 days with grade 4 after the last chemotherapy cycle in patients responding to therapy with a CR or CRi.(DOCX)Click here for additional data file.Checklist S1CONSORT checklist.(DOC)Click here for additional data file.Protocol S1Study protocol.(PDF)Click here for additional data file.


Because microbial abundance and diversity might differ in pregnancy, we sought to generate comparative metagenomic signatures across gestational age strata. DNA was isolated from the vagina  and the V5V3 region of bacterial 16S rRNA genes were sequenced (454FLX Titanium platform). Sixty-eight samples from 24 healthy gravidae (18 to 40 confirmed weeks) were compared with 301 non-pregnant controls (60 subjects). Generated sequence data were quality filtered, taxonomically binned, normalized, and organized by phylogeny and into operational taxonomic units (OTU); principal coordinates analysis (PCoA) of the resultant beta diversity measures were used for visualization and analysis in association with sample clinical metadata. Altogether, 1.4 gigabytes of data containing >2.5 million reads  were generated for computational analyses. Although gravidae were not excluded by virtue of a posterior fornix pH >4.5 at the time of screening, unique vaginal microbiome signature encompassing several specific OTUs and higher-level clades was nevertheless observed and confirmed using a combination of phylogenetic, non-phylogenetic, supervised, and unsupervised approaches. Both overall diversity and richness were reduced in pregnancy, with dominance of Lactobacillus species , Clostridiales, Bacteroidales, and Actinomycetales. This intergroup comparison using rigorous standardized sampling protocols and analytical methodologies provides robust initial evidence that the vaginal microbial 16S rRNA gene catalogue uniquely differs in pregnancy, with variance of taxa across vaginal subsite and gestational age.While current major national research efforts ( Such a view is seemingly in contrast to longstanding observations that humans serve as host to co-evolving microbes residing in highly plethoric communities. Indeed, microbiota are present from the time of birth, with up to 10-fold the number of microorganisms to adult human cells and a collective genome (the “metagenome”) which exceeds our human genome in terms of gene content by more than 100-fold i.e., the NIH Road Map initiative known as the Human Microbiome Project (HMP)) will enable sequence-based comprehensive characterization of the adult human microbiota and theoretically allow for cataloguing of the microbiota into core guilds, which can be thereafter interrogated for their associations with disease states what constitutes the adult human microbial guild, we will neither know how it is established nor whether it is dynamic during intervals in reproductive life when the next generation’s microbial community is being established.Current major national research efforts  gene sequence-based methodologies have enabled primary cataloguing of the bacterial composition of the human microbiome per se, significant variance in medication use with respect to vitamins and antacids were similarly observed. Recalling prospective subject enrollment, the majority of gravidae had uncomplicated pregnancy outcomes: mean gestational age at delivery exceeded 39 weeks and included appropriately grown infants (mean 3265 grams), with 2/24 (8%) of subjects delivering <37 weeks . The cesarean delivery rate was consistent with the regional population (33%). A single fetal comorbidity was observed among the cohort after enrollment , and a total of 3 subjects manifest 4 comorbidites common to the obstetrical population , followed by Clostridiales, Bacteroidales, and Actinomycetales. Distinctions between pregnant and non-pregnant communities by unweighted phylogenetic clustering are observed and annotated in the outermost circle  and likewise above 0.6 for the QIIME denoised data set (Tables S1 and S2). Pregnancy status was, in fact, the single feature best predicted by the microbiome , and sub-sites were not well distinguished . Using Boruta feature selection during RF analysis, 12 specific taxa were identified as discriminating between pregnant and non-pregnant cohorts at the genus level . In order to identify a taxonomic biomarker with high stringency, we additionally employed the LEfSe method Lactobacillus OTUs and potentially the Bifidobacteriaceae and Streptococcaceae. Our findings suggest that the composition of the microbial community in pregnancy represents a relatively diminished profile of species richness and diversity  exceeding 0.8 at OTU abundance thresholds of 100 or 500 for the AbundantOTU data set , to subject race or ethnicity, nor to readily identifiable clinical confounders. Interrogations of discrete contributors to community diversity revealed that the vaginal microbial community varied in pregnancy by gestational age and proximity to the cervix, but was less diverse and less rich overall  from a remote population of Amerindians et al have previously reported that the vaginal microbiome in healthy, reproductive-aged women occupies states dominated by Lactobacillus iners and Lactobacillus crispatus specifically in association with low vaginal pH per seLactobacillus . Given that <10% of screen failures  met such pH criteria for exclusion, we feel that this is an exceedingly unlikely potential confounder or bias. However, it cannot be formally excluded as such. To this end, in the recent publication of Ravel , , and As the number and robustness of computational approaches to analysis of metagenomics data increases, investigators are faced with distinct methodologic approaches to analyzing community profiles. In any emerging field of study, optimal measures of data analysis are not evident to investigators at the forefront and different methodologic approaches may yield variance in significance of findings Lactobacillus could be observed. While each denoising pipeline has its own strengths and limitations, one undeniable observation persists: the vaginal microbiome community is structured by pregnancy and varies with respect to richness, diversity, and specific microbial members.Similarly, for measurement of within community diversity  we employed variations in data filtering ranging from minimal removal with scant trimmed reads to well-described modest “denoising” and slaying of chimeras. We persistently observed less richness and diversity in pregnant communities when compared with parallel non-pregnant subject cohorts, regardless of computational pipeline, tool employed, or means of data projection 3. The lLactobacillus iners, Lactobacillus crispatus, Lactobacillus jensenii and Lactobacillus johnsonii. Although it is outside the scope of this initial manuscript to delve deeply into the species differentiation and clinical implications, these findings are of probable biologic significance nevertheless. For example, L. johnsonii encodes enzymes and transporters essential for the release bile salt hydrolase and is primarily found in the upper GI tract L. johnsonii production of Lactacin F both limits other lactobacillus as well as Enterococcus species in the GI tract. It’s notable increased dominance in the vagina in pregnancy may be important for establishing the neonatal upper GI microbiota upon delivery, or preserving the integrity of the community to reduce risk of ascending infection or preterm birth.Employing such robust analyses methods, we were able to detect species which are discriminately and specifically relative enriched in pregnancy . These include The vaginal microbiome signature in pregnancy is thus distinct from non-pregnant, and this distinction comprises both from lesser diversity and, to a lesser degree, from the absence and occasionally presence of unique taxa. Our reporting by gestational age and vaginal subsite now lays the foundations for further interrogations into microbial variance, including such presumed pathogen-related perinatal morbidities as preterm birth. Moreover, it lends to the growing understanding of the remarkable dynamic nature of our metagenome and its role in vertical transmission of the microbiota through subsequent generations.e.g., antibiotics, corticosteroids, cytokines, large doses of probiotics, etc.). Major dietary changes and history of moderate-high alcohol intake excluded individuals. Medications and dietary components that potentially might alter the human microbiome intentionally such as antibiotics and probiotics served as additional important exclusion criteria. Females were required to have a regular 21–35 day menstrual cycle or a history of regular cycles prior to beginning contraception or at the time of conception, and the use of specific contraceptives such as the combination hormone vaginal ring was exclusionary in non-gravid subjects. For gravidae, inclusion criteria additionally detailed best obstetrical dating for gestational age, defined as reliable last menstrual period consistent with <12 week sonogram or <10 week sonographic dating with unreliable last menstrual period. At the time of enrollment, all gravid subjects had presumed normal singleton gestations and were without known maternal comorbidities . However, given the prospective nature of the study with early second trimester enrollment, some subjects developed these comorbidities  bidities Table 1.http://www.hmpdacc.org/micro_analysis/microbiome_sampling.php]), it remains a formal possibility that our cohort comparison may be biased as posterior fornix pH was not an exclusionary criteria in our pregnant cohort.At the screening visit, the study and associated risks were explained and subjects documented informed consent by signing a study consent form. Investigators determined final study eligibility by taking a medical history and performing a review of systems, documenting concomitant medications, measuring vital signs, collecting urine for pregnancy testing (non-gravid) and either obtaining blood for serum HIV, HBV and HCV testing or reviewing previously obtained serologies . In addition, all subjects underwent targeted physical exams with attention to body site-specific exclusion criteria , and among non-gravid subjects measuring vaginal pH in females (pH>4.5 at posterior fornix was exclusionary). Gravid subjects did not undergo vaginal pH measurements as per study protocol limitations but were queried as to signs and symptoms of bacterial vaginosis and excluded if reported. Individuals who passed all screening criteria were eligible for enrollment and were sampled the first time within 30 days of the screening visit (non-gravid), or at the time of the prenatal visit (gravid). Although exclusion of non-gravidae for posterior fornix pH>4.5 at the time of screening was exceedingly rare  subjects. While in the index study protocol, two ethnically, racially, and socieconomically diverse U.S. cities  served as geographically distinct study sites for clinical sampling for this comparison. However, only subjects sampled at Baylor College of Medicine were considered in the comparison cohort. This served to limit potential additional variables, including personnel doing the sampling , site of microbial DNA extraction, and regional variability. To assure compliance of screening and sampling measures, a common set of human study protocols and consent forms were created, reviewed independently and subsequently approved at Baylor College of Medicine. An age range of 18–40 years was established to minimize variability due to childhood growth and development, aging, and hormonal influences during adolescence and menopause. Each of the enrolled non-gravid subjects agreed to primary and repeat (second) sampling so as many individuals as possible were sampled twice. A subset of non-gravid subjects was designated to undergo three samplings at each body site, at a priori designated intervals.For the non-gravid (non-pregnant) comparison cohort, of the 150 females from the HMP JumpStart initiative Specimen collection procedures involved minimal physical risk to subjects. As defined in 45 US Code of Federal Regulations (CFR) 46.102 (i), “Minimal risk” infers that the “probability and magnitude of harm or discomfort anticipated in the research are not greater in and of themselves than those ordinarily encountered in daily life or during the performance of routine physical or psychological examinations or tests.” Potential risks of participation as discussed with subjects included those associated with biologic sample collection and repository, and the unintentional release of protected health information; the protocol and informed consent form described precautions taken to reduce these risks. The study sites developed informed consent documents using NHGRI guidelines for genomics studies, which address the ethical, legal and social implications of such research. The Institutional Review Board reviewed and approved the protocol, informed consent and other study documents. Additional protections for subjects included Certificates of Confidentiality intended to protect against the forced disclosure of identifiable research data, coding of genomic and metagenomic specimens and sequence data, and use of controlled access databases for medical data and human genome sequence data. As part of the consent process, we informed subjects about data protection, including coding, use of controlled-access databases for human genomic data and controlled-access repositories for extracted metagenomic nucleic acids. If a subject withdrew consent after providing specimens, remaining specimens and extracted nucleic acids were to be destroyed; however, any metagenomic sequence data that was already published in open access databases could not be retracted.www.hmpdacc.org) established an internet data entry system for investigators to enter coded non-gravid subject information; gravid subject information was entered and maintained on a local database with secured protection but not released to the DACC. Clinical data elements included gender, race, ethnicity, age, place of birth, occupation, body mass index, vital signs, vaginal pH (non-gravid), date of last menstrual period and first sonogram, tobacco use, and both dental and health insurance status. A medical history, comprehensive obstetrical data, and targeted physical examination findings alongside medication history were also recorded for each subject.EMMES and the Data Analysis and Coordination Center for the Human Microbiome Project .Inclusion criteria included stringent requirements as otherwise detailed http://www.hmpdacc.org/doc/HMP_MDG_454_16S_Protocol_V4_2_102109.pdf).In this study protocol, vaginal specimens were coded, stored and processed for nucleic acid extraction at a single laboratory (J.P.) using a common protocol to reduce variability between samples. Genomic DNA was isolated on standard protocol with PowerSoil DNA Isolation Kit (MoBio) per HMP modifications  were mixed into individual wells in the 96 well reaction plate. The covered plate was spun and centrifuged at 2000 rpm to collect sample at the bottom of the wells. For the initial reaction, a 2 µL sample of DNA was added to the reaction wells. Barcoded primers (2 µM) from the primer plate were added to corresponding wells in the 96 well PCR plate. Two different PCRs were set up separately with a set of barcoded primers targeting the V3V1 region and V3V5 regions; this analysis examined V3V5 regions. The V3V5 regions of the 16S rRNA gene were amplified by PCR using bar-coded universal primers 354F and 926R (V3-V5) containing the A and B sequencing adaptors  obtained from Invitrogen. Primer sequences are as follows:B-354F; A-926R . Cycling conditions were 95°C for 2 min, followed by 30 cycles at 94°C for 20 s, 50°C for V3V5 primer sets. PCR products were cleaned using AmPure Beads Agencourt  using 1.8× volume beads. Beads were eluted with 25 µL 1× low TE, pH 8.0 and transferred to a new 96-well plate. PCR products were quantified using Quant-IT dsDNA high sensitivity assay (Invitrogen) according to the manufacturer’s specifications. All samples were diluted according to the sample that had the lowest concentration. Equal volumes of each (5–10 µL) sample were pooled and then concentrated using MinElute columns . DNA pool emulsion PCR amplification and 454 sequencing were performed at the BCM Human Genome Sequencing Center (HGSC) in Houston, TX. Sequencing was performed using the 454/Roche B sequencing primer kit in the Roche Genome Sequencer GS-FLX Titanium platform. Samples were combined in a single region of the picotiter plate such that approximately 20,000 to 40,000 sequences were obtained from each group with each primer set. Samples were isolated and quality-filtered from each multiplexed Standard Flowgram Format (SFF) file.In the gravid sample cohort, three subjects did not have valid sequences from a total of 4 body sites . In the non-pregnant cohort, 29 subjects were sequenced for all three vaginal subsites 1 time (29 of 301 samples), 21 subjects for all three subsites twice (42 of 301 samples), 6 subjects thrice (18 of 301 samples), and 2 subjects for all three subsites four times (8 of 301 samples).As a quality filtering step, each sample was preprocessed to remove sequences with length less than 200 nucleotides and sequences with minimum average quality less than 20. If they could be identified, reverse primers were also removed from sequences. In a first pass denoising, singletons and chimeric sequences (which occur as a byproduct of the PCR-amplification) were identified by ChimeraSlayer For second pass denoising, two programs were employed: AbundantOTU Unique reads were classified with the MSU RDP classifier v2.2 QIIME was utilized to produce OTU tables from the quality-filtered sequences as outlined above A custom in-house pipeline has been written to calculate alpha diversity measurements and plots using R packages Beta diversity analysis incorporated 9 binary non-phylogenetic , 14 non-phylogenetic , and 6 phylogenetic  beta diversity metrics i.e. UniFrac) in order to determine whether derived microbiome communities were significantly different as an estimate of the degree of divergence between different representative sequences Phylogenetic trees were produced as necessary input for the phylogenetic beta diversity metrics , exceeding the (still high) accuracy of other metadata predictions.(TIF)Click here for additional data file.Figure S2Supervised (machine) learning with definition by randomForest and confirmation by Boruta feature selection enables visualization of bacterial taxa contributing to clustering of vaginal communities in pregnancy. Bacterial taxa (leftmost column) were defined by randomForest (omForest  and conf(TIF)Click here for additional data file.Figure S3LEfSe analysis of binned taxonomy at discrete vaginal subsites. Bacterial taxa were selected as significantly differentially abundant between pregnant and non-pregnant communities by virtue of discrete sampling site and displayed by LDA Effect Size (LEfSe) algorithm. Taxa level projections are defined by pregnancy at each subsite, with specific Lactobacillus species detected consistently among pregnant individuals.(TIF)Click here for additional data file.Table S1i.e., contains the most discriminative features with least amount of noise). When describing estimated error rate per minimal row contribution, pregnancy was retained as the only significant clinical metadata category in the model simulation that had an acceptable level of estimated error .Estimated error rate of the randomForest simulation by virtue of potentially contributable clinical metadata  following abundant OTU pipeline for denoising of dataset. Top row header: Minimal row contribution cut off sum for each OTU to determine the best performing data set ((DOC)Click here for additional data file.Table S2i.e., contains the most discriminative features with least amount of noise). When describing estimated error rate per minimal row contribution, pregnancy was retained as the only significant clinical metadata category in the model simulation that had an acceptable level of estimated error .Estimated error rate of the randomForest simulation by virtue of potentially contributable clinical meta data  following QIIME pipeline for denoising of dataset. Top row header: Minimal row contribution cut off sum for each OTU to determine the best performing data set ((DOC)Click here for additional data file.Table S3The OTUs selected by Boruta feature selection are assigned to the species level by realigned to Greengenes database and BLAST with NCBI microbial database. Top OTUs identified by LEfSe are marked with an asterics.(DOC)Click here for additional data file.


Dengue virus causes ∼50–100 million infections per year and thus is considered one of the most aggressive arthropod-borne human pathogen worldwide. During its replication, dengue virus induces dramatic alterations in the intracellular membranes of infected cells. This phenomenon is observed both in human and vector-derived cells. Using high-resolution mass spectrometry of mosquito cells, we show that this membrane remodeling is directly linked to a unique lipid repertoire induced by dengue virus infection. Specifically, 15% of the metabolites detected were significantly different between DENV infected and uninfected cells while 85% of the metabolites detected were significantly different in isolated replication complex membranes. Furthermore, we demonstrate that intracellular lipid redistribution induced by the inhibition of fatty acid synthase, the rate-limiting enzyme in lipid biosynthesis, is sufficient for cell survival but is inhibitory to dengue virus replication. Lipids that have the capacity to destabilize and change the curvature of membranes as well as lipids that change the permeability of membranes are enriched in dengue virus infected cells. Several sphingolipids and other bioactive signaling molecules that are involved in controlling membrane fusion, fission, and trafficking as well as molecules that influence cytoskeletal reorganization are also up regulated during dengue infection. These observations shed light on the emerging role of lipids in shaping the membrane and protein environments during viral infections and suggest membrane-organizing principles that may influence virus-induced intracellular membrane architecture. Aedes aegyptii and Aedes albopictus mosquitoes and viral replication within the mosquito vector is required for transmission to a new human host. During this replication cycle, the virus causes significant changes to the membrane organization of infected cells. These virus-induced membrane alterations help to assemble arrays of viral replication factories and aid the virus to evade host antiviral defense mechanisms. Previously, much effort has been placed in trying to identify viral and cellular protein effectors that aid virus replication. In this study we have explored the role of lipids in the formation of these extensive membrane platforms in mosquito cells. Using high-resolution mass spectrometry we have profiled the lipid composition of dengue virus infected mosquito cells and compared it to uninfected cells. Through this we have identified several lipid classes that are differentially regulated during dengue virus replication. Using inhibitors of lipid biosynthesis we have also identified a lipid repertoire that is inhibitory to viral replication. Knowledge of how dengue virus utilizes cellular lipids and downstream signaling pathways to facilitate its replication will provide novel targets that could be utilized for developing effective antivirals. This study is also a forerunner for future comparative analyses of the human host and vector membrane environments required for viral replication.Dengue virus is one of the most aggressive human pathogens worldwide. It causes 50–100 million infections per year but there is no vaccine or antiviral that is currently effective against the disease. The virus is spread by  In the past 20 years, it has become increasingly evident that lipids are important bioactive molecules that mediate signalling cascades and regulatory events in the cell. The ability to synthesize lipids predisposes an organism to function as a host to parasites that have lost or lack this trait For most viruses that replicate in the cytoplasm of infected cells, lipids are essential for the replication of viral genomes. Both enveloped and non-enveloped viruses induce extensive ultrastructural changes in infected cells. Host-derived membranes are rearranged to provide extensive platforms that help to assemble arrays of replication factories In this study, we have focused on the importance of lipid biosynthesis on dengue virus (DENV) replication. DENV is one of the most aggressive re-emerging pathogens worldwide Previous electron microscopy studies on DENV infected mosquito cells have shown that virus-induced membrane structures similar to those observed in human cells are prevalent To investigate these possibilities, we used high-resolution mass spectrometry methods to profile the lipidome of DENV infected mosquito cells. We have identified several lipid classes that are regulated by DENV infection. Many of these lipids have characteristic roles in influencing membrane architecture as well as functioning in cellular signal transduction pathways. Specifically, we have identified differences induced in the lipid profile upon virus binding and entry alone compared to those induced by viral RNA replication, assembly and egress. We have also profiled the lipidome of cells treated with an inhibitor of de novo phospholipid biosynthesis. Through this we have identified a lipid environment that supports cell survival but is yet inhibitory to DENV replication.We had previously shown that fatty acid biosynthesis was a key target of DENV in human cells and that the rate limiting enzyme, fatty acid synthase (FAS), was both required and re-localized to sites of viral RNA replication during DENV infection Based on the observation that DENV induces significant rearrangements in the membrane architecture of infected cells, together with its distinct susceptibility to inhibitors of FAS, it was our hypothesis that lipid biosynthesis was important to DENV replication. Therefore, to investigate whether the intracellular lipid composition was altered during DENV infection, we carried out LC-MS-based analyses of the global lipidome of mosquito cells infected with DENV. To differentiate between changes to the lipid profile that occurred upon exposure to infectious virus versus that induced by virus entry alone, we included UV-inactivated DENV (UV-DENV) in the studies. This inactivated virus is only capable of binding, entry and initial rounds of viral RNA translation but does not have the ability to replicate its genome.Diptera species where PE is a predominant PL Based on the total number of lipids that were significantly regulated between groups  and subsequently structurally identified  in DENV and UV-DENV exposed cells is shown in In this whole cell analysis, the primary PLs that were significantly regulated (Anova p<0.05)  were mostly neutral or zwitterionic. PS was the only acidic lipid significantly regulated. At 36 hr post-infection, there was an ∼2 fold up-regulation of PC species in DENV-infected cells compared to UV-DENV and mock controls . At the Analysis of the overall fold change in PE at the 36 hr time point  does not2) and represent a PL that is missing an acyl chain Another group of PLs that were preferentially expressed in DENV-infected cells were the lysophospholipids (LPLs) . These l2-type enzymes, we investigated whether DENV infected cells displayed a higher activity of PLA2 compared to uninfected cells. Utilizing a fluorescent PC substrate (BODIPY-PC) we used mass spectrometry to monitor the hydrolysis of PC to LPC by intracellular PLA2. Essentially, we monitored the production of BODIPY-LPC (resulting from PLA2 mediated metabolism of PC) during a time course of DENV infection  above the mock control. Since there was a concurrent decrease in SM in UV-DENV exposed cells at 60 hr, the increased levels of CER in this case could be due to the degradation of SM resulting from increased cellular stress (resulting from incubation of the cells for 60 hr). Similar to other lipid species, CER also showed temporal regulation depending on the molecular species expressed  during DENV infection, selected molecular species were regulated only at the 36 hr time point while others were regulated at both time points. Representative examples for each lipid class are shown . These o14C-acetate to label newly synthesized lipids and the results are shown in 14C-acetate into lipids in the DENV infected cells compared to the mock control  enriched in viral replication components and a remaining cytoplasmic extract (CE)  control . This inTo identify the primary fraction containing viral induced membranes that included the viral replication complex, we carried out a comparison of the viral RNA genome copies to labeled lipid in the 16K and CE subcellular fractions . The res14C-acetate labeling studies above, the CE fraction showed an increased amount of newly synthesized lipids (compared to the 16K fraction at the 36 hr time point). In lipid biosynthesis pathways, acetate is a precursor to both phospholipid as well as sterol biosynthesis. Therefore, the labeled lipids (in CE) represent not only phospholipids in membranes, but also cholesterol and triglycerides that form lipid droplets Based on the Therefore, we focused our primary lipidomic analysis on lipids extracted from the 16K pellet at the 36 hr time point. Similar to the whole cell analysis, DENV infected cells displayed a unique expression profile compared to UV-DENV and mock infected cells . Over 85There was an up regulation of phospholipids, sphingolipids and several bioactive intermediates in DENV infected cells compared to UV-DENV and mock controls . AmongstAlthough the Blygh and Dyer method As shown in GlcCER were primarily detected in the 16K pellet of both DENV infected and UV-DENV exposed cells . HoweverThe FAS inhibitor, C75 is a potent inhibitor of DENV replication in both mosquito  and mammde novo synthesis of sphingolipids was also disrupted by C75. Key intermediates in this pathway such as N-palmitoylesphingosine and N-stearoylesphingosine were down regulated upon treatment of cells with C75 and up regulated in untreated DENV infected cells  As a second messenger, CER can also induce apoptosis and autophagy 22 in their life cycle Inverted cone-shaped lipids such as LPLs were also up regulated during DENV infection. Primarily, LPC was up regulated in the whole cell analysis. It results from the hydrolysis of PC by PLALPC also increases the permeability of membranes de novo lipid biosynthesis was up regulated during DENV infection It has been previously reported that Several intermediates in lipid catabolism were also detected in the replication complex membranes . Analysis of these intermediates suggested the up regulation of pathways for PC hydrolysis and triacylglycerol (TG) metabolism. The latter pathway is responsible for regulating lipid homeostasis and energy production in the cell through the metabolism of lipid droplets While several acidic phospholipid species were detected in the 16K membrane fraction very few were significantly regulated during infection. Previous studies in mammalian cells have indicated the importance of acidic lipids in DENV infection de novo phospholipid biosynthesis The most striking results were obtained by the comparison of DENV infected cells in the presence and absence of the FAS inhibitor, C75. It is well established that treatment of cells with this inhibitor disrupts In these studies we utilized UV-DENV as a control to identify lipid metabolic changes that occurred upon virus binding and entry alone. This virus is incapable of replication but immunofluorescence microscopy with anti-NS3 antibodies have indicated that there is a very low level of translation that occurs within the first 24 hr (data not shown). Analysis of the lipidome of UV-DENV exposed cells showed a significant difference in the phospholipid and sphingolipid content in comparison to DENV infected cells and the mock control. It is possible that binding, membrane fusion and entry alone, or the expression of viral proteins from the incoming viral RNA triggers the activation of lipases or lipid transport mechanisms that result in the differences observed in the lipidome of the cells. This has been shown for other viruses. Jan et al. have shown that binding and entry alone of Sindbis virus triggered the activation of sphingomyelinases that degraded membrane bound SM to Cer The most pronounced changes are those observed in the 16K pellet analyses where a significant decrease in phospholipids, sphingolipids and lipid intermediates are observed  cells (ATCC) were maintained in Minimal Essential Medium (MEM) supplemented with glutamine (2 mM), non-essential amino acids, 25 mM Hepes and 10% heat inactivated fetal calf serum. Dengue virus 2, strain 16681  stocks were amplified in C6/36 cells in MEM (supplemented as above) and 2% heat inactivated fetal calf serum. UV-inactivated DENV was obtained by exposing the same virus stock (used for infection) to UV-light for 3 hr and then conformation of inactivation by two blind passages of the virus on cells for 60 hr per passage. Lack of infectivity was confirmed by plaque assay and immunofluorescence assay.C6/36  was confirmed by immunofluorescence analyses. Each experiment included three biological replicates. Following infection, cells were harvested at 36 and 60 hr post-infection, pelleted and resuspended in 100 µl of 100 mM ammonium bicarbonate. Lipids were extracted from an equal number of cells using a modified Bligh and Dyer protocol. Briefly, a mixture of 2∶1 Chloroform∶methanol, 0.1% acetic acid and 0.01% butylated hydroxy toluene (BHT) were added to the cell suspension in ammonium bicarbonate such that there was a 4∶1 ratio of organic solvent to cells. Following lipid extraction, the organic phase was separated from the aqueous phase by centrifugation and dried down under a N2 stream in low retention microfuge tubes (Fisher). The dried lipids were resuspended in 75 ml of methanol and vortexed for 10 s. The samples were then centrifuged at 13,400× g for 5 min to remove any particulates.For infection, ∼5×10Lipid molecular species were profiled using a dual-column nanocapillary LC system equipped with 75 µm×65 cm columns, each packed with 3 µm Jupiter particles . The mobile phases were (A) 10 mM ammonium acetate in 50∶50 water/methanol (v/v) and (B) 10 mM ammonium acetate in 50∶50 methanol/acetonitrile (v/v). The LC system was equilibrated at 10,000 psi with mobile phase A prior to injecting 0.4 µL of sample. Exponential gradient elution was initiated 50 min after sample loading with an initial column flow of ∼300 nL/min. After 90 min of gradient separation, the mobile phase mixer was purged with 3 mL of mobile phase B, followed by a 5 min column wash. Finally, the mobile phase mixer was purged with 10 mL of mobile phase A, which represented the end of one separation cycle. While gradient elution was performed on one column, the other column was equilibrated with mobile phase A. The LC system was coupled to a hybrid linear ion-trap-Orbitrap mass spectrometer , and the capillary temperature and electrospray voltage were 200°C and +2.2 kV, respectively. The Orbitrap was used as the mass analyzer during MS survey scans over the m/z range 200–2000 with a duty cycle of ∼1.2 s. Data-dependant MS/MS was performed in the LTQ for the top 5 ions, with a normalized collision energy of ∼35%. Dynamic exclusion in the LTQ during data-dependant MS/MS experiments was enabled as follows: repeat count of 2, repeat duration of 30 s, exclusion list size of 250, and exclusion duration of 60 s.http://ncrr.pnl.gov/software/ and developed in-house. The first step involved deisotoping of the raw MS data to give the monoisotopic mass, charge state, and intensity of the major peaks in each mass spectrum using Decon2LS LC-MS datasets, defined as the data obtained from a single LC-MS analysis, were processed using the PRISM Data Analysis system Following chromatographic alignment and database matching, the abundances of all detected features (both AMT tag database matched and unmatched) were loaded into DAnTE 14C] (Perkin Elmer) was added to cell supernatants at 8 hr post-infection. At 12 hr post-infection, the label containing supernatant was removed and new medium was added to the cells. At 36 and 60 hr post-infection, cells were harvested by trypsinization and washed with buffer A . Cells were then osmotically shocked in buffer B  for 30 min on ice, homogenized and centrifuged at 1500× g, at 4°C for 5 min to remove nuclei. Post-nuclear supernatants were centrifuged at 16000× g for 30 min at 4°C to obtain a membrane pellet (referred to as the 16K pellet). The supernatant post nuclear and membrane fractionation is referred to as the cytoplasmic extract (CE). Total RNA was extracted from each fraction (16K and CE) using Trizol reagent (Sigma) according to the manufacturer's instructions. Following RNA extraction, total lipids were extracted using the Bligh and Dyer method mentioned above and counted using a Beckman LS 6000 scintillation counter. The viral RNA in each fraction was measured using the SuperScript III Platinum SYBR Green One-Step qPCR Kit with ROX and DENV specific primers; (forward) 5′ ACAAGTCGAACAACCTGGTCCAT 3′ and (reverse) 5′ GCCGCACCATTGGTCTTCTC 3′ on a Applied Biosystems 7300 Real Time PCR machine. The labeled lipid in subcellular fractions was standardized to the total RNA isolated from each fraction prior to determining the viral RNA genome copies per labeled lipid in subcellular fractions.Virus infection of C6/36 cells was carried out as described for the whole cell analyses. To label newly synthesized lipids, Acetic Acid, Sodium Salt, +; Protonated molecular ion, NET; normalized elution time, total carbon/double bond; corresponds to each lipid species detected, LM_ID; identification for each lipid as displayed in LIPID MAPS, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/Mock; fold change of average abundance from 4 replicates of each treatment. Identity abbreviations were made for phoshatidylcholine , phosphatidylethnolamine (PE), phosphatidylserine (PS), sphingomyelin (SM), ceramide (Cer), ceramide phosphoethanolamine (Cer-PE), lysophosphatidylcholine (LPC). The notation further indicates total number of carbons and double bonds however it does not discern redundancy associated with varying fatty acid composition for the same molecular weight. Accession numbers (LM_ID) were obtained from the Lipid Maps Gateway (lipidmaps.org).(PDF)Click here for additional data file.Table S2Select list of lipid species from the analysis of the 16K membrane fraction (isolated from mosquito cells treated with conditions described below) regulated across conditions (p<0.05). Conditions: Mock (uninfected cells), DENV , UV-DENV . A total of 484 features were detected in the mass spectrometry analysis following normalization of Mock, DENV and UV-DENV treated samples and 415 of these features were significantly regulated (p<0.05). Identification was successful for 68 lipids (ppm error <10). For each condition the following information is provided: pttest and pwilcox; p-values on minimum observation data (a measurement must be present in two of three replicates to be considered present), Exact mass; mass of each lipid from http://www.lipidmaps.org, M/Z; mass to charge ratio, RT; retention time, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/Mock; Fold UV-DENV/Mock; fold change of average abundance from 3 replicates of each treatment. Identity abbreviations were made for phoshatidylcholine (PC), phosphatidylethnolamine (PE), phosphatidylglycerol , sphingomyelin (SM), monoacylglycerol (MG), diacylglycerol (DG), phosphatidic acid (PA). Accession numbers were obtained from the Lipid Maps Gateway (lipidmaps.org) and the Human Metabolome Database (hmdb.ca).(PDF)Click here for additional data file.Table S3Select list of lipid species from the analysis of the 16K membrane fraction (isolated from mosquito cells treated with conditions described below) regulated across conditions (p<0.05). Conditions: DENV (cells infected with DENV in the presence of vehicle only), DENV C75 (cells infected with DENV and treated with the FAS inhibitor C75). A total of 366 features were detected in the mass spectrometry analysis following normalization of DENV and DENV C75 treated samples and 314 of these features were significantly regulated (p<0.05). Identification was successful for 27 lipids (ppm error <10). For each condition the following information is provided: pttest and pwilcox; p-values on minimum observation data (a measurement must be present in two of three replicates to be considered present), Exact mass; mass of each lipid from http://www.lipidmaps.org, M/Z; mass to charge ratio, RT; retention time, Formula, elemental composition of each lipid, PPM Error; difference in experimental mass compared to exact mass, Fold DENV/DENV C75; fold change of average abundance from 3 replicates of each treatment. Identity abbreviations were made for phoshatidylinositol (PI), phosphatidyllglycerol (PG), monoacylglycerol (MG), diacylglycerol (DG), phosphatidic acid (PA). Accession numbers were obtained from the Lipid Maps Gateway (lipidmaps.org) and the Human Metabolome Database (hmdb.ca).(PDF)Click here for additional data file.


Macrophage migration inhibitory factor (MIF) is a pro-inflammatory cytokine expressed by urothelial cells that mediates bladder inflammation. We investigated the effect of stimulation with thrombin, a Protease Activated Receptor-1 (PAR1) agonist, on MIF release and MIF mRNA upregulation in urothelial cells.in vitro) and in rats (in vivo). UROtsa cells were stimulated with thrombin, culture media were collected at different time points and MIF amounts were determined by ELISA. Pentobarbital anesthetized rats received intravesical saline (control), thrombin, or thrombin +2% lidocaine (to block nerve activity) for 1 hr, intraluminal fluid was collected and MIF amounts determined by ELISA. Bladder or UROtsa MIF mRNA was measured using real time RT-PCR.MIF and PAR1 expression was examined in normal human immortalized urothelial cells (UROtsa) using real-time RT-PCR, Western blotting and dual immunostaining. MIF and PAR1 immunostaining was also examined in rat urothelium. The effect of thrombin stimulation (100 nM) on urothelial MIF release was examined in UROtsa cells  and in vivo  while heat-inactivated thrombin had no effect. In rats, thrombin-induced MIF release was reduced but not abolished by intravesical lidocaine treatment. Thrombin also upregulated MIF mRNA in UROtsa cells (3.3-fold increase) and in the rat bladder (2-fold increase) where the effect was reduced (1.4-fold) by lidocaine treatment.UROtsa cells constitutively express MIF and PAR1 and immunostaining for both was observed in these cells and in the basal and intermediate layers of rat urothelium. Thrombin stimulation of urothelial cells resulted in a concentration- and time-dependent increase in MIF release both Urothelial cells express both MIF and PAR1. Activation of urothelial PAR1 receptors, either by locally generated thrombin or proteases present in the urine, may mediate bladder inflammation by inducing urothelial MIF release and upregulating urothelial MIF expression. Proteases clip and free the tethered ligand to bind to the receptor and mediate signal transduction in vitroin vitro and from rat urothelial cells in vivo and 4) whether thrombin stimulation elicits MIF upregulation in human urothelial cells in vitro and from rat urothelial cells in vivo.PAR receptors, although not studied in extensive detail in the urogenital tract Since MIF and/or PAR receptors had not been studied in UROtsa cells, we examined expression of these two proteins in UROtsa cells. RT-PCR showed that UROtsa cells constitutively expressed MIF  and PAR We tested three different PAR1 antibodies for their ability to detect PAR1 in formaldehyde-fixed rat bladder sections and compared the results to snap-frozen bladder cryostat sections that were post-fixed with acetone-PBS. We also compared the effects of formaldehyde fixation vs acetone-PBS fixation on PAR1 immunostaining in UROtsa cells. Of the three different antibodies tested, only one  gave positive results and only in acetone-fixed tissue. Consistently, formaldehyde fixation abolished PAR1 immunostaining in rat bladder when compared to acetone fixation. Formaldehyde fixation also reduced PAR immunostaining intensity in rat dorsal root ganglia We examined PAR1 and MIF immunostaining in UROtsa cells and rat urothelium using scanning laser confocal dual immunofluorescence. UROtsa cells showed MIF and PAR1 immunostaining. In rat urothelium, basal and intermediate cells showed MIF immunostaining, while superficial cells were lightly immunostained or devoid of MIF immunostaining  as reporWe examined release of MIF into the culture media by UROtsa cells in response to different concentrations of thrombin. Because a small amount of endotoxin was detected in the thrombin stock . Untreated cells (serving as controls) were exposed to media only (no thrombin treatment) for the duration of the experiment (180 min). Media collected from UROtsa cells after thrombin stimulation showed a statistically significant increase in the median amount of MIF at 15 minutes of exposure and reached a maximal effect at 60 minutes of exposure . Longer in vivo thrombin stimulation on MIF release from the rat bladder. Our results show that treatment with intraluminal thrombin elicited MIF release from the rat bladder  Thrombin stimulation also induces MIF upregulation in urothelial cells in vitro and in vivo. These results indicate that activation of PAR1 receptors mediates MIF release from urothelial cell which can then mediate MIF-mediated bladder inflammation, given MIF's pro-inflammatory role in the bladder The present study shows that: 1) urothelial cells (human and rat) express PAR1 receptors and MIF; 2) Thrombin stimulation of urothelial cells evokes MIF-release Expression of PAR1 and PAR2 receptors was described for normal human urothelial cells and an urothelial cancer cell line (RT4) in vitro (using UROtsa cells) or in vivo  results in MIF release, and this effect occurs quickly after thrombin application (15 min in vitro). Since thrombin contained a small amount of endotoxin and because endotoxin can elicit MIF release in vivo. Given that both human and rat urothelial cells were shown to express both MIF and PAR1 we consider it likely that thrombin stimulated PAR1 receptors on rat urothelial cells to elicit MIF release.We also document that thrombin stimulation of urothelial cells, whether Since PAR1 and MIF containing cells in rat urothelium are not located superficially, our findings that intravesical thrombin can induce MIF release from the rat bladder (presumably urothelium) suggest that intravesical thrombin was able to reach those cells to activate PAR1 receptors. Although local thrombin formation during inflammation is likely to occur in the suburothelial compartment and thus stimulate basal and intermediate cells to elicit MIF release, our findings suggest that proteases present in the urine may also be able to activate PAR1 receptors in the urothelium, elicit MIF release and thus contribute to the initiation or maintenance of cystitis. In fact, both mast cell tryptase and neutrophil elastase were documented to be increased in the urine of patients with interstitial cystitis Treatment with intravesical PAR1, PAR2 and PAR4 agonists induced inflammation in the mouse bladder We showed previously that substance P elicits MIF release from the bladder in general, and urothelium in particular, dependent on bladder nerve activation Animal experiments were approved by the Bay Pines VA Healthcare System Institutional Animal Care and Use Committee  and conformed to NIH guidelines for animal experimentation.Limulus Amebocyte lysate assay  according to the manufacturer's protocol. In order to establish that the effects of thrombin were due to thrombin and not to endotoxin, control experiments examined the effects of UROtsa stimulation with thrombin vs heat-inactivated thrombin  were anesthetized with halothane and placed on a heating pad. A ventral abdominal incision exposed the bladder and ureters. Both ureters were cut and allowed to drain to gauze while urine was removed from the bladder using a 30 ga needle and a hypodermic syringe. Rats were divided into three groups (n = 6/group) and 0.3 ml of either 1) sterile saline (vehicle control); 2) 100 nM thrombin; or 3) 2% lidocaine +100 nM thrombin were injected into the bladder lumen. After 1 hour the intraluminal fluid was collected and stored at −80ning see  below, tRNA was extracted from UROtsa cells and bladder tissues using TRIzol reagent (Invitrogen). One To determine the expression of PAR 1–4 receptors in UROtsa cells, approximately 2×in vitro experiments) was isolated by the direct addition of TRIzol reagent (Invitrogen). cDNA was diluted 1∶1 with nuclease-free water and 5 Thrombin induced upregulation of MIF expression in UROtsa cells was determined by real-time PCR . Total RNA from thrombin treated cells . The DNA in the resulting cell lysates was sheared by passing through a 25 ga needle. Equal volumes of the lysates were separated by 4–12% Bis-Tris SDS-PAGE (Invitrogen) and transferred to a polyvinylidene fluoride membrane. Blots were blocked with Odyssey blocking buffer  for 1 h at In vitro experiments were repeated three times and all assays were performed in duplicate.MIF amounts in the culture media of UROtsa cells exposed to thrombin were determined using a commercially available ELISA to detect human MIF, according to the manufacturer's protocol (R&D Systems). MIF amounts in rat intraluminal fluid were detected using a validated, custom ELISA protocol for detecting rat and mouse MIF developed in our laboratory and described previously UROtsa cells were grown in chamber slides, briefly rinsed in cold (4For the effect of different concentrations of thrombin on UROtsa MIF release, a concentration-response curve was fitted using log-logistic regression with 4-point parameter estimation (using R ogram R; . Real ti