Drosophila to study the effects of PRL overexpression in a genetically controlled, organismal model. Our data support the paradigm that the normal cellular response to high levels of PRL is growth suppression and furthermore, that PRL can counter oncogenic activity of Src. The ability of PRL to inhibit growth under normal conditions is dependent on a CAAX motif that is required to localize PRL to the apical edge of the lateral membrane. However, PRL lacking the CAAX motif can still associate indiscriminately with the plasma membrane and retains its ability to inhibit Src function. We propose that PRL binds to other membrane-localized proteins that are effectors of Src or to Src itself. This first examination of PRL in a model organism demonstrates that PRL performs as a tumor suppressor and underscores the necessity of identifying the conditions that enable it to transform into an oncogene in cancer.Phosphatase of Regenerating Liver (PRL) family members have emerged as molecular markers that significantly correlate to the ability of many cancers to metastasize. However, contradictory cellular responses to PRL expression have been reported, including the inhibition of cell cycle progression. An obvious culprit for the discrepancy is the use of dozens of different cell lines, including many isolated from tumors or cultured cells selected for immortalization which may have missing or mutated modulators of PRL function. We created transgenic  In the past decade, Phosphatase of Regenerating Liver (PRL) family members have been touted as molecular markers that significantly correlate to the ability of cancers to metastasize et al.In vivo expression surveys support the notion that PRLs can contribute to growth arrest. For example, PRL-1 is highly expressed in differentiated intestinal cells relative to undifferentiated counterparts In contrast, a few studies failed to support a positive relationship between PRLs and cancer; one study found that PRL-3 levels did not affect outcomes of ovarian cancer All three PRL family members contain a consensus tyrosine phosphatase domain and a C-terminal prenylation, CAAX motif et al., (2008) could detect activation of Akt in response to PRL-3, but only transiently, until level of PRL-3 became highly elevated.Two signal transduction pathways that have been implicated as oncogenic effectors of PRLs are Src and PI3K signaling. PRL-3 activates Src signaling Drosophila is the first to examine overexpressed PRL in genetically controlled animal model. This system confirms that PRL can function as a growth inhibitor under normal and oncogenic conditions that can be dependent on submembrane distribution.Although there is a rapidly growing amount of literature on the mammalian family of PRL phosphatases, several studies have conflicting results. These studies each examine PRL in a different genetic environment, which may mean modulators and effectors of PRL localization or function are missing or mutated. Our study using Drosophila genome encodes a single PRL protein (dPRL-1), which is highly similar (74–76%) to all three human PRLs and contains the three domains shown to be required for PRL function in mammals: a Dual Specific Phosphatase (DSP) active site (HCxxGxxR) engrailed-Gal4  reduced the surface area by 20%  lead to an upward curvature, also indicative of a decrease in surface area  led to a smaller eye and head capsule  prevented larva growth; although the larvae consumed food, most stalled in the first instar (L1) of development  was cloned into pUAST via KpnI and XbaI restriction sites and sequenced, confirming a wild type sequence that encoded the expected protein of 176 amino acids (Accession #NP_609780). Transgenic animals were produced by BestGene . In order to construct a modified dPRL-1 lacking its CAAX motif, PCR primers were developed that amplified a truncated version of dPRL-1 (LD12894), while introducing restriction sites (EagI and KpnI) for subsequent cloning into pUAST. The primers sequences were forward primer: 5′-ATCGGCCGATGAGCATCACCATGCGTC-3′ reverse primer 5′- TAGGTACCCTATGAATTCTTATGACCATT-3′ . Following sequence confirmation that the CAAX motif had been deleted, transgenic animals were produced by GenetiVision . Other fly stocks were: w118; +; +, yw; enGal4; + (Bloomington stock #6356), w; apGal4/CyOGFP (Bloomington stock #3041), eyFLP (Bloomington stock #8205), w;+;act>cd2>Gal4, UASGFPNLSFull-length cDNA of 3. Tissues were mounted in a 1∶1 solution of PBS and Vectashield mounting medium with DAPI (Vector Labs). Microscopy was conducted using an Olympus BX40 Laboratory Microscope connected to a Lumen Dynamics X-Cite© Series 120Q Fluorescence Microscopy Illumination System and a ProgRes© C3 Jenoptik Digital Microscope Camera. Images were captured using the ProGres© Mac Capture Pro program. Confocal microscopy was conducted using a Nikon A1 Confocal Laser Microscope system. Images were captured and signal intensity quantified using the EZ-C1 and NIS-Elements Basic Research 3.10 software. Z-stacks were taken with step size 0.35 µm and pixel dwell 1.68 µs. Signal intensities were collected in flat portions of the dorsal  and ventral (control) compartments of wing discs. The average pixel intensity of two matched optical sections were examined per sample. High laser power coupled with minimal gain settings were used to achieve the strongest signal to noise ratios (SNR) in the absence of photo-bleaching.Polyclonal antibodies to dPRL-1 were generated in rabbits using a peptide consisting of amino acids 158–176 of dPRL-1 . Other primary antibodies used were: anti-cleaved Caspase , anti-DE-Cadherin (DHSB), anti-Src64CT and anti-pY343Src Images were prepared using Adobe Photoshop CS5 Extended.To compare tissue sizes in the posterior compartment, the Lasso and Histogram function  of Adobe Photoshop were used to quantify the surface area of tissue between the L5 vein and wing margin. As an internal control, anterior surface area was quantified between the L1 and L2 veins. Clones overexpressing PRL-1 were created by the Flp-out technique Crosses were set in embryo collection chambers, and adults were left to lay on grape plates for 2 hours. Using GFP as a marker for control (GFP+) and experimental (GFP−), L1 larvae were placed into vials and genotypes of pupae (for Ras assays) and adults (for Src assays) were tallied.Figure S1Quantification of growth inhibition following dPRL-1 expression in the wing. Comparison of surface area in the posterior (P) and anterior (A) compartments of adult wings of animals expressing dPRL-1  compared to control . dPRL-1 reduces the area of the posterior compartment by 20% (p = 003). The small reduction in the anterior compartment was not statistically significant (p = 0.24). Data is presented as average +/− standard error.(TIF)Click here for additional data file.


Aedes aegypti before and in response to blood feeding.The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families.We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding. Aedes aegypti, is the primary vector for dengue fever, several encephalitis viruses, yellow fever, and several types of filariasis The yellow fever mosquito, The fat body is the principal organ of intermediary metabolism, functioning as a storage unit for lipids, carbohydrates, and proteins in both mosquitoes and insects in general. The principal cell type of the fat body is a large polyploid cell referred to as the trophocyte, which is capable of synthesizing large amounts of protein and contains many ribosomes and oil droplets During the mosquito aquatic larval stage, the fat body accumulates and stores nutrients for use in the adult stage. Hexameric storage proteins of the arylphorin family are the major amino acid stores in mosquito larvae and are synthesized by the fat body and secreted into the hemolymph. These hexamerins are then reabsorbed later by the larval fat body via receptor-mediated endocytosis, stored in protein granula, and afterward hydrolyzed to deliver energy and building blocks required to drive metamorphosis into the adult insect In the adult stage the fat body continues to play a prominent role in energy metabolism by providing precursors for flight and yolk protein synthesis which is required by females for reproduction. It also assists in metabolizing potentially fatal amounts of excess ammonia after the female takes a blood meal Before a female mosquito takes a blood meal, her fat body is in a state-of-arrest with regards to the expression of genes involved in reproduction. When a blood meal is taken, a sequence of signals originating in other tissues affects the fat body, priming it for gene expression alterations. These signals include raised amino acid levels in the hemolymph, peptide hormones from the gut and the central nervous system Ae. aegypti fat body tissue following a blood meal. This was accomplished by comparing and contrasting read and contig libraries, obtained by 454 pyrosequencing, derived from fat body tissue of female non-blood fed (NBF) Ae. aegypti and 24 h post-blood meal (PBM) fat body tissue. This study describes a window of transcriptional alterations appearing in the Ae. aegypti fat body during the digestion of a blood meal and subsequent accumulation and utilization of nutrients. In particular we used an RNA-seq approach to observe transcript expression transitions from a resting fat body prepared for translational activity to a fat body producing transcripts involved in Ae. aegypti vitellogenesis.The adult fat body is a key organ in mosquito reproduction and immunity; therefore, it is important to understand fat body physiology on a molecular level. We now report on transcriptional alterations occurring in Ae. aegyptiTwenty four hours after a full blood meal, vitellogenesis, the production, secretion, and reuptake of yolk proteins, is at a peak in For practical reasons we isolated abdominal body walls with attached abdominal fat body tissue and processed them for library construction. This is a standard preparation for studying mosquito fat body physiology It has been shown previously that there is a large increase in RNA synthesis and accumulation in the PBM fat body compared to the NBF fat body Ae. aegypti fat body reads were generated in this study  and 7,625 (PBM) reference transcripts. The top 4 transcripts aligned with on a percentage basis from each sample are shown in Alignment of our NBF and PBM transcriptome sequencing reads to the Vectorbase Of these 9,984 distinct transcripts, 4,974 exhibited at least a 2-fold change in expression level. This is substantially lower than the number of differentially expressed genes previously reported  in NBF vs 24 hr PBM whole mosquitoes examined via microarray analysis Drosophila melanogaster fat body gene expression estimates (2261+ genes) When all isoforms of each transcript are counted as one, we identified 2,354 NBF and 2,746 PBM transcripts from the total of 6,019 and 7,625 distinct transcript isoforms. These numbers represent estimates of the number of genes expressed in the fat body under each physiological condition. They are comparable to previous The condition of the mosquito fat body before the female takes a blood meal has been described as a “previtellogenic state-of-arrest” during which yolk proteins are not expressed Cytosolic serine hydroxymethyltransferase is found in eukaryotes and prokaryotes. It is a central enzyme of the one-carbon metabolic pathway that catalyzes the production of the major one-carbon donors for the biosynthesis of thymidylate, purines, methionine and choline Ae. aegyptiThe strong presence of ribosomal protein transcripts in the NBF library was expected since these messages have been shown to accumulate in the fat body during the first days after eclosion of adult Our data support the finding of Niu and Fallon After a blood meal the fat body is activated and starts vitellogenic gene expression Vitellogenin is the principal yolk protein precursor in almost all oviparous vertebrates and invertebrates Ae. aegypti, vitellogenic cathepsin b (VCB) is secreted into the hemolymph as 44 kDa subunits. It is taken up by oocytes and processed during several stages of vitellogenesis. Finally, it becomes a 33 kDa form during embryogenesis. This is considered the active form and degrades vitellogenin Cathepsins are cysteine thiol proteases, usually located in the lysosomes, which are found in a wide variety of organisms ranging from humans, rats and cattle to papaya  to be heavily up-regulated in the PBM fat body and a fourth (AAEL009637) that appears to have a lesser degree of up-regulation . The chaAe. aegypti, vitellogenic carboxypeptidase (VCP) has been found to be transcribed and translated in the fat body and exported to the developing oocytes after a blood meal, with the peak of transcription at 24 hours post blood meal and very little transcription by 48 hours post blood meal We observed VCP to be one of the most up-regulated genes in the fat body after a blood meal. Serine carboxypeptidases are found in many different species, from invertebrates to humans, and in many cell types in those organisms. The described function for the serine carboxypeptidase family is the removal of one or more amino acids from the carboxy terminal end of an amino acid chain Ae. aegypti eggshells. We were surprised to find them highly expressed in the vitellogenic fat body, but we were able to confirm these results via RT-PCR repeatedly (data not shown). VMPs have been described as being exclusively secreted by the follicular epithelium Ae. aegypti fat body specific ESTs within the Unigene and dbEST database VMPs are an important part of the vitellogenic process, forming the inner layer of the Since the fat body is a key player in mosquito metabolism, efficient transport across its cell membrane is extremely important for homeostasis. The mosquito fat body must import nutrients derived from a blood meal for the rapid and energetically costly process of vitellogenesis. A wide variety of transporter families capable of transporting a plethora of substrates have been identified by our sequencing. We mapped reads to 54 out of 58 of the families represented by the list of transporters we obtained. The total number of transporter reads decreases PBM by approximately one third. This is approximately the number of reads taken up by yolk protein reads in the PBM library, which may reduce the number of reads against other transcripts.Ae. aegypti genome were identified, AAEL003512 (0 reads NBF/2 reads PBM), AAEL005001 (20 NBF/2 PBM) and AAEL005008 (34 NBF/8 PBM). While not significant by our statistical analyses, these numbers may show a trend which supports our previous findings that expression of these aquaporins decreases post-blood meal Three of the six aquaporins encoded in the As mentioned previously, the fat body is an important player in mosquito immunity. Synthesis of a variety of anti-microbial peptides to control and erradicate pathogens throughout the mosquito as a whole occurs within this organ Defensins are classically known as small (4 kDa) cationic peptides up regulated in response to bacterial challenge in insects. They are capable of forming ion channels into gram positive cells Ae. aegypti and A. gambiae defensins have been shown to respond to, and have an effect on, infections by certain stages of Plasmodium, the causative organism of malaria Cecropins are similar to defensins in size and function Ae. aegypti they have been shown to work in conjuction with defensins against bacterial and plasmodium infections Ae. aegypti's response to infection with filarial worms, the causative agent of filiariasis, a major health problem in many parts of the world and subject of a World Health Organization erradication program In order to verify differential expression of selected genes identified via 454 sequencing in the analysis of our libraries, we used quantitative real-time PCR directed at transcripts of interest. Transcripts and results are shown in Ae. aegypti or one of its organs have been reported on. Two projects have utilized microarrays to describe gene expression in whole Ae. aegypti mosquitoes Ae. aegypti vitellogenic fat body involved a very small number of randomly selected cDNA ESTs from 24 h PBM fat bodies To date, only a handful of projects describing the transcriptome of Ae. aegypti, an important disease vector and model organism. The two transcriptomes we compared were considerably different from each other in terms of mRNA expression and reflect the different physiological stages of the fat body in NBF versus PBM mosquitoes undergoing the process of vitellogenesis. This key tissue and its physiological functions are a prime target for novel vector control strategies and our results represent a first in-depth examination of a mosquito fat body transcriptome. In the future, we will extend this study by further analysis of the mosquito fat body transcriptome under different physiological and environmental conditions. One of our long term goals is to identify potential targets for the development of novel insecticides. We suggest that fat body proteins, especially transporters and ion channels, are rational targets for that.In female mosquitoes vitellogenesis involves specific cellular signaling events and transport of large quantities of nutrients in the fat body, an extremely important metabolic and reproductive-associated organ. The transition from a state-of-arrest to vitellogenic fat body is primed by blood feeding and culminates in the deposition of yolk protein into developing eggs. 454 pyrosequencing has enabled us to take a detailed look at the fat body transcriptome of the yellow fever mosquito The research plan used for this work involving animals was specifically approved by the Institutional Animal Care and Use Committee (IACUC) at New Mexico State University under approval ID #2008-034. All procedures and care are described in the New Mexico State University Animal Care Facility Standard Operating Procedure and on file in the IACUC office there. All persons involved in animal work successfully completed Animal Welfare Training at New Mexico State University and were specifically trained in protocols used in the research plan. All New Mexico State University IACUC care and protocols follow the NIH guidelines described in Guide for the Care and Use of Laboratory Animals: Eighth Edition, ISBN-10: 0-309-15400-6.Aedes aegypti Rockefeller strain eggs were obtained from MR4 (available as MRA-734) and used to start a laboratory colony Gallus gallus domesticus) on top of their cage for approximately 30 minutes.Mosquitoes were blood fed by placing a live chicken  and then had concentration measured as described in fat body dissection and RNA isolation.200 mosquito abdomens were dissected each from two groups of mosquitoes 72 hours post eclosion and 24 hours post-blood meal in groups of 10. Malpighian tubules, midgut, ovaries and crop were removed, then the abdomens were transferred to eppendorf tubes with 0.5 ml of Trizol reagent . They were then homogenized with pellet pestle and handheld homogenizer. Another 0.5 ml of Trizol was added to the cap afterwards and mixed by inversion. RNA was isolated and precipitated following the manufacturer's protocol. RNA pellets were dissolved in RNase-free water. Concentration was measured on a Thermo Scientific nanodrop 1000 (Thermo Scientific). RNA quality was checked using a Bioanalyzer 2100 (Agilent). mRNA isolation was accomplished by separation using poly-A tails with Oligotex solution  following the instructions of the manufacturer.(4)GT(9) CT(10)V N-3′5′-ATT CTA GAG ACC GAG GCG GCC GAC ATG T was used . An amplification step was performed using the Advantage 2 PCR kit (Invitrogen) with a modified 3′PCR primer: (4)GT CT(4)G TTC TGT(3) CT(4)V N-3′5′-ATT CTA GAG GCC GAG GCG GCC GAC ATG T. The first strand was synthesized by combining 3 ul of RNA, (>200 ng), 1 ul modified 3′ RT primer and 1 ul SmartIV oligo (Invitrogen). Tubes were incubated at 72°C for 2 min, cooled to room temperature for 2 min and then combined with 2 ul of 10× first strand buffer, 1 ul of DTT, 1 µl of dNTP mix, 1 ul of MMLV-reverse transcriptase, then incubated at 42°C for 90 minutes. 20 ul of water was added and first strand cDNA stored at −20°C. In a 0.2 ml PCR tube, 2 ul of first strand cDNA, 5 ul of 10× buffer, 1 ul of dNTP-Mix (10 mM), 1 ul of modified 3′ PCR primer (10 um), 1 ul of Advantage Polymerase and 39 ul of water were combined. In an Eppendorf MasterCycler, the following program was run: 94°C−2 min, 24 cycles: 94°C−20 sec , 65°C−20 sec, 68°C−6 min. 50 µg of cDNA was synthesized per experimental sample and purified by phenol/chloroform extraction.The cDNA libraries were constructed using the Clontech SMART cDNA Library Construction Kit (Invitrogen) following the manufacturers instructions with modifications de novo assembly of the reads using the assembler Mira de novo options were used to run the assembly software.The cDNA library samples described above were run on a 1/× picotitre plate and Roche 454 Genome Sequencer FLX instrument. Binary SFF files generated by the sequencing hardware were converted into fasta, fasta.qual and xml qual files by the sff_extract program Contig files from our assembly were analyzed using Blast2GO. This program uses a pipeline involving a BlastX step followed by gene ontology and annotation steps. For most contigs the default Blast options within Blast2GO were used with the exception of 7 contigs which required lower numbers of results returned due to the size of the results and the maximum size Blast2GO can report. Following Blastx, we performed a sanity check outside of Blast2GO on our contigs by analyzing the species of the best BlastX result for each contig. BlastX results then had gene ontology and annotation steps applied, and were grouped by GO term. Only contigs successfully passing all stages of the pipeline were used in GO term analysis. Level 2 GO terms for each contig were used for our analyses To identify potential new transcripts, we aligned our contigs against the reference transcripts available from Vectorbase, using the tool Blat Aligned sequences were generated by using Blat to align generated ESTs to reference transcript sequences from Vectorbase. The aligned output sequences were analyzed for statistically significant differential expression between samples using the R test Ae. aegypti membrane transporter genes based on predicted or known amino acid sequences. To this list we added an additional six genes for potential transporters, which we identified through searches of transcript information for Ae. aegypti at Vectorbase . We then searched for and identified matches in our aligned transcript data for the genes on this list.From membranetransport.org Ae. aegypti database at Vectorbase. Results obtained produced e-values typically less than 1e-50, with the majority of results being less than 1e-100, except where noted. Resulting AAEL numbers were then used to look up read numbers and differential expression statistics from our sequence alignment data.From our GO results, contigs with the term “immune system process” were selected and their sequences Blasted against the CTC GTT CCC GCT CTG GCA GC, reverse primer TGT AGC CGC GAC CAA TGT CGG, product length 282; Cathepsin b: Forward primer AGG GTG CAC AGC ACG TAG AGA, Reverse primer TGC CGG AGG TTT CGG GTT GC, Product length 308; Trypsin: Forward primer GCC AAG CTG CAA CGC TGT CC, Reverse primer GGC GCG CAA CAA CGT GTT CA, Product length 449; Arginase: Forward primer GCA ACA TGC TGC GCG GAA AAC A; Reverse primer GCC CAC ATC GCT GCA GTG CT, Product length 449; Serine-Type Endopeptidase: Forward primer AGG TGG CCC TTT TCG AGA CGG A , Reverse primer TGA TTT TCT TCC ACC CGG ATG CAA, Product length 450; Serine-Pyruvate Aminotransferase, Forward primer ACT ACTGAT GGG TCC AGG CCC A, Reverse primer AAG CGA GGC AAC CGT GTC CA, Product length 496; RPS7: Forward Primer TCA GTG TAC AAG AAG CTG ACC GGA, Reverse primer TTC CGC GCG CGC TCA CTT ATT AGA TT. A total of eight RNA samples  were prepared using the same method for the measurement of total RNA in fat bodies. Samples were then converted into cDNA using an Omniscript RT kit (Qiagen) following the manufacturer's supplied protocol.Gene-specific primers were developed using Primer BLAST Figure S1A and B Size distribution of EST library reads. (A) Library from fat bodies of NBF mosquitoes; (B) Library from fat bodies of mosquitoes 24 h PBM.(TIF)Click here for additional data file.Figure S2A and B. GC content of contigs from NBF (A) and PBM (B) samples.(TIF)Click here for additional data file.Figure S3A and B Blast2GO Blastx results broken down by species NBF(A) and PBM(B).(TIF)Click here for additional data file.Table S1Putative new genes.(XLS)Click here for additional data file.Table S2Percentage of the total number of unique read-transcript alignments (multiple isoforms counted as one transcript).(XLS)Click here for additional data file.Table S3Ae. aegypti reference transcripts from Vectorbase. R-value>9 is considered significant, meaning high likelihood of being differentially expressed between the two conditions.All reads aligned using blat to (XLS)Click here for additional data file.Table S4Transcripts reads aligned with using blat, fold change in expression between blood fed and not-blood fed state.(XLS)Click here for additional data file.


Recent high-throughput transcript discoveries have yielded a growing recognition of long intergenic non-coding RNAs (lincRNAs), a class of arbitrarily defined transcripts (>200 nt) that are primarily produced from the intergenic space. lincRNAs have been increasingly acknowledged for their expressional dynamics and likely functional associations with cancers. However, differential gene dosage of lincRNA genes between cancer genomes is less studied. By using the high-density Human Omni5-Quad BeadChips (Illumina), we investigated genomic copy number aberrations in a set of seven tumor-normal paired primary human mammary epithelial cells (HMECs) established from patients with invasive ductal carcinoma. This Beadchip platform includes a total of 2,435,915 SNP loci dispersed at an average interval of ~700 nt throughout the intergenic region of the human genome. We mapped annotated or putative lincRNA genes to a subset of 332,539 SNP loci, which were included in our analysis for lincRNA-associated copy number variations (CNV). We have identified 122 lincRNAs, which were affected by somatic CNV with overlapped aberrations ranging from 0.14% to 100% in length. lincRNA-associated aberrations were detected predominantly with copy number losses and preferential clustering to the ends of chromosomes. Interestingly, lincRNA genes appear to be less susceptible to CNV in comparison to both protein-coding and intergenic regions . In summary, our study established a novel approach utilizing high-resolution SNP array to identify lincRNA candidates, which could functionally link to tumorigenesis, and provide new strategies for the diagnosis and treatment of breast cancer. Long intergenic non-coding RNAs (lincRNAs) are intergenic region-derived, large transcripts (>200 nucleotides) that do not give rise to proteins. lincRNAs, together with other long non-coding RNAs (lncRNAs), such as antisense, interleaved, and protein-coding-gene-overlapped lncRNAs, are actively transcribed from the genome, and consist of a substantial fraction of the human transcriptome  at the Fox Chase Cancer Center (FCCC). The pathological histotypes of tumors included in this study were all invasive ductal carcinoma (IDC) at stage II or greater Table . Tumor cR ratios  and B allele frequencies , which provide information on copy number and genotyping.Genomic DNAs used in the global CNV analysis here were derived from paired normal-tumor primary human mammary epithelial cell lines (passage < 6) as previously described . Samples were loaded into Nexus by using a sample descriptor with matched pair design, where data from the normal sample are subtracted from that of the tumor sample to produce one result. Data from two paired replicate arrays were combined for normal samples and tumors, respectively. Probes mapping to the Y chromosome and SNPs with incorrect genotyping were filtered prior to importation to Nexus. Samples are subjected to quality assessment with a quality score below 0.02 . A linear correction was used to correct GC wave, and SNP-FASST2 segmentation algorithm was applied to generate CNV calls. A customized lincRNA annotation track was created on the basis of reference lincRNA (described below) to aid lincRNA specific CNV detection. Since the Nexus default gene track includes genes for known protein-coding as well as some of the non-coding genes, a customized protein-coding gene specific annotation track was also created to analyze CNV for the protein coding genes, which serves as an internal control for lincRNAs.in silico annotated using cDNA sequences (1) mapping to chromatin methylation sites outside protein coding loci, and (2) lacking protein-coding potentials (Ensembl). Given the dynamics of the number of lincRNAs in the database, other possibly relevant non-coding transcripts were also included to create a putative list. This list consisted of 6459 lincRNA and non-coding genes with starting and ending coordinates. Next, the intergenic SNPs from HumanOmni5 were subsetted and used to query the lincRNA putative list to remove any transcripts that were not present in intergenic regions to obtain a full probe list of 332,540 and a final reference list of 5800 lincRNA genes (Table S1). A protein-coding reference consisting of 22,088 genes was also obtained from Ensembl for the generation of a customized track in Nexus. Frequencies of CNV calls for lincRNA or genic regions from seven matched tumor-normal pairs were retrieved using the aggregate function of the Nexus package. Data were then formatted and input into Circos . Whole genome views of CNV frequencies for lincRNA or genic regions were created in PNG format.lincRNA reference is retrieved from the online genomic database Ensembl release 67, built on the Genome Reference Consortium release GRCh37 using the BioMart data management system. At Ensembl, lincRNA genes are −ΔΔCt method was also used to compare the results from a different analysis.Quantitative PCR (qPCR) was performed in the ABI 7900HT system using Power Syber® Green assays for targeted gene content and TaqMan assays for copy number references including alpha-satellite sequence and albumin both using a 5′ FAM/3′ BHQ labeled probe (Applied Biosystem). Primer and probe sequences are available upon request. Experiments were carried out using two levels of gDNA inputs for each sample in 384 microwell plates, and in triplicates for each level. Each reaction consisted of a 10 μL system containing 40 ng or 10 ng gDNA input in 5 ng/μL tRNA carrier (Roche Diagnostics), 5 μL Power Syber Green Master Mix® or TaqMan Master Mix, and 0.4 μL primer mixes. Standard curves for references and for target genes using 64, 16, 4, 1, 0.25, and 0.0625 ng of commercial gDNA from a healthy female (Promega) were performed in triplicate or quadruplicate with R square values of 0.99 or above according to a previous method . One hundred and twenty-two lincRNAs were affected by CNV genomic regions of over 86 Mb covering from 0.14% to 100% overlapping aberrations; the majority of events were seen in copy number losses (Tables S2 and S3).A total of 14 primary cell lines from 7 matched breast cancers and 7 surrounding healthy normal tissues were investigated by the HumanOmni5 Beadchips. Sample dataset outputs from GenomeStudio were quality-assessed by using log intensity plots between tumor and paired normal samples prior to importation into Nexus. CNV calls were initially generated as discordant logRRs that did not map to the HumanOmni5 reference derived from HapMap samples. These were subsequently improved by using the pair matched analysis where each tumor was adjusted by the corresponding matched normal sample since substantial noise was produced under the unpaired model. lincRNA-associated CNV calls were specifically identified as CNV regions that include or overlap reference lincRNA gene positions. Overall, 86 lincRNA-associated somatic aberration events, including copy gains/losses and allelic imbalances ranging in size from 1.2 Kb to greater than 18 Mb, were predicted among the seven tumor-normal paired samples  between lincRNA and genic region (percentage of CNV in length) across 7 samples  associated segments showed only 1.8% of the aberrations fell within the footprint of lincRNAs compared to 37.5% of protein coding genes and 60.6% of the non-lincRNA intergenic segments Figure . Obviouss Figure .per se, it is not surprising to find the heterogeneity in the somatic alterations in our samples. Some cancers carried many or large regions of changes while some exhibited few or none. For example, Figure There is tremendous evidence showing that cancer genomes are complicated and heterogeneous between individuals and even between tumors within an individual. With complicated nature of tumors Table S4).To further confirm that our identified lincRNAs are expressed in breast tissue even though in the context of cancer, since lincRNAs demonstrated high tissue-specific expression signatures, we queried our lincRNA list against a published expression database  the variable tumor purity , (2) the non-uniform nature of cancer genomes including intra-tumor and inter-tumor heterogeneity, (3) aneuploidy or polyploidy features between individuals and chromosomal regions, (4) extensive genomic rearrangements resulting in considerable variations in SNP array data, and (5) technical artifacts produced by SNP array For a close examination of individual samples, we found dramatically localized variations among seven primary cancer cell clones from either individual samples Table  or aggreIt has been shown that the expression signals of intergenic transcripts are generally ~10-fold lower than that of their protein-coding counterpart mRNAs (Cabili et al., In conclusion, we have described a novel approach for genomic copy number and LOH profiling for lincRNA genes in breast cancers using high resolution SNP array. We addressed a number of problems present in SNP-array–based cancer-associated lincRNA CNV analysis. lincRNA genes apparently manifest a relatively more stable manner than coding genes in terms of sensitivity to gene dosage. We identified a couple of lincRNA gene candidates in breast cancers with mostly copy number losses. Further expressional and functional studies on these lincRNAs would be worthwhile for a better understanding of their roles in breast cancer development.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cardinium hertigii, a member of the Bacteroidetes and symbiont of the parasitic wasp Encarsia pergandiella, is the only known bacterium other than the Alphaproteobacteria Wolbachia to cause CI. Here we report the genome sequence of Cardinium hertigii cEper1. Comparison with the genomes of CI–inducing Wolbachia pipientis strains wMel, wRi, and wPip provides a unique opportunity to pinpoint shared proteins mediating host cell interaction, including some candidate proteins for CI that have not previously been investigated. The genome of Cardinium lacks all major biosynthetic pathways but harbors a complete biotin biosynthesis pathway, suggesting a potential role for Cardinium in host nutrition. Cardinium lacks known protein secretion systems but encodes a putative phage-derived secretion system distantly related to the antifeeding prophage of the entomopathogen Serratia entomophila. Lastly, while Cardinium and Wolbachia genomes show only a functional overlap of proteins, they show no evidence of laterally transferred elements that would suggest common ancestry of CI in both lineages. Instead, comparative genomics suggests an independent evolution of CI in Cardinium and Wolbachia and provides a novel context for understanding the mechanistic basis of CI.Terrestrial arthropods are commonly infected with maternally inherited bacterial symbionts that cause cytoplasmic incompatibility (CI). In CI, the outcome of crosses between symbiont-infected males and uninfected females is reproductive failure, increasing the relative fitness of infected females and leading to spread of the symbiont in the host population. CI symbionts have profound impacts on host genetic structure and ecology and may lead to speciation and the rapid evolution of sex determination systems.  Cardinium hertigii, a symbiont of tiny parasitic wasps, is the only bacterial group other than the well-studied Wolbachia that is known to cause CI. Analysis of the Cardinium genome indicates that CI evolved independently in Wolbachia and Cardinium. However, a suite of shared proteins was likely involved in mediating host cell interactions, and CI shows functional overlap in both lineages. Our analysis suggests the presence of an unusual phage-derived, putative secretion system and reveals that Cardinium encodes biosynthetic pathways that suggest a potential role in host nutrition. Our findings provide a novel comparative context for understanding the mechanistic basis of CI and substantially increase our knowledge on reproductive manipulator symbionts that do not only severely affect population genetic structure of arthropods but may also serve as powerful tools in pest management.Many arthropods are infected with bacterial symbionts that are maternally transmitted and have a great impact on their hosts' biology, ecology, and evolution. One of the most common phenotypes of facultative symbionts appears to be cytoplasmic incompatibility (CI), a type of reproductive failure in which bacteria in males modify sperm in a way that reduces the reproductive success of uninfected female mates. In spite of considerable interest, the genetic basis for CI is largely unknown.  Bacterial symbionts of terrestrial arthropods are common, influential associates, known to affect fundamental aspects of the host life history, ecology, and evolution. These maternally inherited bacteria may, for example, provide essential nutrients supplementing their host's diet, confer protection against natural enemies, increase stress resistance, or influence host plant suitability Wolbachia pipientis, a member of the Alphaproteobacteria established in as many as 40% of terrestrial arthropod species Drosophila or the parasitic wasp Nasonia vitripennis, CI Wolbachia leads to asynchrony of the timing of maternal and paternal chromosome condensation and segregation during the first embryonic mitotic division, disrupting embryonic development The most common symbiont-induced reproductive manipulation, cytoplasmic incompatibility (CI), is also perhaps the most enigmatic. CI occurs, in the simplest case, when a symbiont-infected male host mates with an uninfected female. Affected host embryos die in early development. The symbiont spreads because of the decreased fitness of uninfected relative to infected female hosts Wolbachia strains have revealed a number of genes with a potential role in CI Wolbachia, and the absence of an independently evolved CI lineage with which to make comparisons has limited the progress in this area. Here we describe the genome of the only CI-inducing symbiont known that is distantly related to Wolbachia. Cardinium hertigii is a member of the Bacteroidetes, and the strain cEper1 infecting the parasitic wasp Encarsia pergandiella causes CI Drosophila spp.) lays eggs in whiteflies, and larval wasps develop at the whiteflies' expense, emerging as adults from the whitefly remains. Related Cardinium strains have also been found in the arthropod groups Hymenoptera, Hemiptera, Diptera, Protura, Acari and Araneae, and an estimated 6–7% of all arthropods are infected with these bacteria Candidatus Paenicardinium endonii’ within the Cardinium clade, and Cardinium as sister group to the Acanthamoeba endosymbiont Amoebophilus asiaticusCardinium/Amoebophilus clade is only distantly related to other known insect symbiont lineages within the Bacteroidetes.Genome analysis and expression studies of genes of diverse CI Cardinium hertigii cEper1 reveals a highly reduced genome, both in terms of genome size and metabolic pathways, and a 58 kb cryptic plasmid. Cardinium encodes a set of proteins with the potential to interfere with eukaryotic cell cycle regulation. These proteins, some of which also occur in CI-inducing Wolbachia strains, are good candidates for effectors mediating CI. Despite its metabolically restricted genome, Cardinium encodes a complete biotin biosynthesis pathway, which suggests a potential role of Cardinium in host nutrition. Lastly, several lines of evidence suggest that protists have served as hosts for the progenitor of Cardinium before its adaptation to insects.The genome sequence of Cardinium hertigii cEper1 consists of a single 887 kb chromosome and a 58 kb plasmid (pCher), with 841 protein coding genes (CDS)  symbionts of diverse insect hosts, which are typically highly reduced and range from 140 kb to 790 kb (Cardinium such as a low G+C content (36.6%) and a single (unlinked) set of rRNA genes are also common characteristics of intracellular bacterial symbionts. Cardinium differs from obligate symbionts in its abundance of transposable genetic elements  , Table 1o 790 kb  [29]––[56 species , suggestCardinium proteins contain characteristic TPR and ANK eukaryotic protein-protein interaction motifs , and, while ANK proteins are virtually absent in other sequenced Bacteroidetes genomes and the mutualist Wolbachia strain wBm . This overrepresentation of ANK proteins in CI-inducing but only distantly related Cardinium and Wolbachia strains suggests that this class of proteins comprises important mediators of host cell interaction possibly involved in CI. Indeed, it has been frequently suggested earlier that ANK proteins could play a role in Wolbachia CI ANK proteins play important roles in a variety of cellular processes in eukaryotes such as cell cycle regulation, cytoskeleton regulation, developmental and transcriptional regulation Cardinium encodes a DEAD box RNA helicase (CAHE_0677). Eukaryotic homologs of this protein promote chromosome segregation in concert with the RNA interference pathway Cardinium is conserved among five different Cardinium strains  coding for a cold shock DNA-binding protein that is also conserved in CI-inducing Wolbachia strains. strains , and shoCardinium encodes a protein with a putative RING domain ubiquitin ligase activity . These two features are commonly found in actin binding proteins, such as the Sca2 protein in Rickettsia of the spotted fever group, used for bacterial motility within the eukaryotic host cell Wolbachia. Other known virulence factors present in Cardinium include a patatin-like phospolipase (CAHE_0286) that is most similar to patatin-like proteins encoded in WO prophages in WolbachiaAlthough orthologs of some of these proteins were also detected in Cardinium strains that cause other phenotypes , they arCardinium shares a number of genome characteristics with its closest sequenced relative, the amoeba symbiont Amoebophilus. Sixty-seven percent of all CDSs (n = 561) show similarity with Amoebophilus proteins . Further, their metabolic pathways are similarly truncated, encode similar transporters for the import of host-derived metabolites, and contain a notably large fraction of transposases or remnants of IS elements compared to other bacteria. The similarity of these genome features between Cardinium and Amoebophilus is striking considering the low degree of 16S rRNA sequence similarity (91%) between these symbionts, indicative of a large evolutionary distance. Consistent with its smaller size (47% relative to Amoebophilus) the Cardinium genome represents a subset of the Amoebophilus genome, with fewer CDSs (841 versus 1557), a greater degree of truncation of metabolic pathways  and show truncated phospholipid biosynthesis pathways. While Amoebophilus still encodes the complete MreBCD complex, RodA, and IspA considered necessary for a rod-shaped morphology Cardinium lacks all of these genes with the exception of mreB (CAHE_0369) and indeed has a more coccoid appearance compared to Amoebophilus, a pattern also observed in other insect endosymbionts Cardinium genome represents a subset of the larger genome of the amoeba symbiont Amoebophilus. The large amount of inactivated transposase genes in the Cardinium genome suggests that it is undergoing further degradation and reduction.The reduction in the capabilities of the Cardinium genome, we identified 68 genes  that were possibly involved in past horizontal gene transfer (HGT) events  lived as a symbiont of amoebae or other protists, in which HGT with other amoeba-associated bacteria was facilitated. This notion is consistent with our observation that the Cardinium genome represents a subset of the genome of the sister lineage to Cardinium, the amoeba symbiont Amoebophilus. It is thus likely that the common ancestor of Cardinium and Amoebophilus lived as a symbiont of an amoeba or a protist. These unicellular eukaryotes are known to have contributed to the development of key features for survival in eukaryotic host cells by other intracellular bacteria In the ) events . A promikettsiae . HGT amoCardinium made the transition from amoebae to insect hosts and became a reproductive manipulator able to induce CI to facilitate its spread in host populations. Although Cardinium and Wolbachia share this phenotype, it is unknown whether the molecular mechanisms leading to CI are identical. If they were, and if the ability to cause CI originated in either one of the two groups and subsequently was acquired by the other through HGT during coinfection of the same host Cardinium and CI-inducing Wolbachia that likely mediate this phenotype and share a common evolutionary origin. Among the orthologous genes shared by Cardinium and Wolbachia there is not a single obvious case of a gene encoding a candidate effector involved in CI. Apart from the patatin-like phospolipase, which is considered a more general virulence factor, we identified only one orthologous gene (CAHE_0604) that was exclusive to Cardinium and some rickettsiae including the CI-inducing Wolbachia strains. This gene encodes a predicted integral membrane protein without any known functional domains and is thus unlikely to mediate CI. This suggests that there is no common evolutionary origin of CI in Cardinium and Wolbachia, and that the molecular mechanism of CI is either different in these two groups, or convergent.At some point during its evolutionary history, Wolbachia strains with the CI lineage of Cardinium revealed in both genomes a large number of proteins that contain eukaryotic domains and likely mediate host cell interaction and CI. These include a DEAD box RNA helicase, and many ANK and TPR proteins that are highly unusual in bacterial genomes and good candidates for CI effectors manipulating the eukaryotic cell cycle. Most of these proteins are highly divergent and show no sequence similarity beyond the presence of eukaryotic domains. This indicates an independent origin of genes involved in CI, most likely through independent HGT events and acquisition of host genes. This notion is further supported by the presence of ubiquitin modifying proteins in Cardinium, which might be involved in CI, and the absence of these in CI-inducing Wolbachia strains. Taken together, CI seems to be based on the exploitation of eukaryotic domains for host cell manipulation, and there is strong evidence for an independent emergence of the molecular mechanisms underlying CI in these two groups. In general, the Cardinium genome points to the utility of a comparative context for analysis of reproductive manipulation in symbiotic bacteria that are refractory to direct genetic manipulation, a fertile area for research in the coming years.It is striking, however, that comparison of the genomes of CI-inducing Cardinium hertigii. In an effort to create a convenient and consistent system, strains have been named in this study following the strain nomenclature of Wolbachia pipientiscEper1”, where “c” refers to Cardinium, “Eper” refers to the host Encarsia pergandiella, and “1” simply denotes the first named strain from this host.No strain nomenclature has previously been adopted for Cardinium hertigii cEper1 is a symbiont of the minute parasitoid wasp Encarsia pergandiella (∼18 µg) that attacks whiteflies Bemisia tabaci near Weslaco, Texas in October 2006, and kept in culture on B. tabaci on cowpea. Males of E. pergandiella develop as hyperparasites and were reared on another whitefly primary parasitoid, Eretmocerus eremicus. Prior to purification of Cardinium cells, wasps were reared on B. tabaci that were not infected with Rickettsia spp.Cardinium purification, wasps were reared on dozens of whitefly-infested plants. Approximately 8,000 adult wasps were collected from emergence jars. The Cardinium purification protocol was modified from 2) using a Dounce tissue grinder (Wheaton). The homogenate was transferred to a 1.5 ml centrifuge tube with an additional 1 ml of buffer A. Cellular debris was pelleted for 5 min at 80 g, 4°C. The supernatant was centrifuged for 5 min at 4000 g, 4°C. The resulting pellet was carefully resuspended in 1 ml of buffer A, then vortexed for 3 sec. Following a 5 min centrifugation at 300 g, the supernatant was loaded onto a 13 mm diameter filter cassette holder  containing a 0.8 to 8 µm pore size glass fiber prefilter (Millipore) and a strong protein binding 5 µm pore-size mixed cellulose ester membrane (Millipore). The supernatant was slowly pushed through the filter with a syringe. The filter cassette holder was washed with buffer A (without EDTA) until 1.5 ml of filtrate was obtained. The filtrate was centrifuged for 5 min at 5000 g, 4°C. Following resuspension of the pellet in buffer A (without EDTA), 10 units of DNase 1 (Roche) were added to the cell suspension and incubated for 30 min at 4°C to remove insect host DNA. The reaction was stopped with 100 µl 0.5 M EDTA. The tube was spun down for 5 min at 4100 g, 4°C, the pellet washed with 1 ml buffer A, then spun down again. The cell pellet was resuspended in 250 µl of TE buffer.For Cardinium cells were mixed with 675 µl of DNA extraction buffer  (w/v), 200 µg/ml proteinase K, pH 8.0; The purified Cardinium DNA was quantified using PicoGreen (Invitrogen), totaling approximately 2 ng, which was insufficient for library generation and sequencing, thus requiring amplification. To minimize bias, multiple displacement amplification (MDA) was performed on eight replicate reactions as follows. Approximately 0.1 ng of template DNA was denatured using alkaline solution and amplified using the Repli-g UltraFast Mini Kit (Qiagen) according to the manufacturer's instructions. MDA was performed overnight and the eight resulting MDA products were pooled prior to library generation and sequencing.The extracted http://www.jgi.doe.gov/. The Illumina GAii sequencing data was assembled with Velvet (http://genome.cshlp.org/content/18/5/821.short) with a hash length of 61 and with the following parameters -exp_cov 130 -cov_cutoff 1 -min_contig_lgth 100. The consensus sequences were shredded into 1.5 Kbp overlapped fake reads and assembled together with the 454 data. The velvet contig fake reads  and the 454 pyrosequencing reads (400.3 Mbp) were assembled using the Newbler assembler version 2.4 (Roche) using the parameters –ace -g –mi 98 -ml 80 –rip. The Newbler assembly consisted of 20,306 contigs in 1,154 scaffolds. Illumina reads were additionally used to correct potential base errors and increase consensus quality using the software SeqMan NGen from DNASTAR. One scaffold consisting of 78 contigs was identified as the Cardinium chromosome based on BLAST searches against the ribosomal rRNA database Silva (release_102); another scaffold (6 contigs) was identified representing the Cardinium plasmid based on BLAST searches against the non-redundant sequence dataset (nr) at GenBank/EMBL/DBBJ. The gaps in both scaffolds were closed by manual refinement of the assembly and by PCR and Sanger sequencing in house and by LGC Genomics .A combination of Illumina and 454 shotgun sequencing was performed on the pooled symbiont MDA DNA product. Two differing 454 standard libraries  were generated and sequenced totaling 300,490,911 bp. In addition, we generated and sequenced two 454 paired end libraries totaling 106,933,881 bp. An Illumina GAii shotgun library was constructed and sequenced (run mode 2×76 bp) generating 1,371,155,520 bp. All general aspects of library construction and sequencing can be found at −5, a minimum amino acid identity of 30%, and minimum alignment overlaps of 40% as threshold values, and by manual annotation of selected genes. The circular view of the genome (http://www.tcdb.org/index.php) Amoebophilus and then by using either blastp or psi-blast with proteins of the AFP from Serratia. Putative host cell interaction proteins were further analyzed using blastp; protein domains were predicted using PFAM Cardinium proteins against the non-redundant protein GenBank/EMBL/DDBJ sequence database (nr). Cardinium proteins with ten best blast hits to proteins from organisms outside the bacterial phylum Bacteroidetes were considered to potentially beinvolved in a past horizontal gene transfer (HGT) events. To further investigate this, the top 50 blast hits were used for amino acid sequence alignments with MUSCLE Cardinium was identified by the lowest number of internal nodes in the calculated trees. If there were several neighbors with the same number of nodes, the minimum sum of branch lengths was used as criterion. The sequences described in this paper have been deposited at GenBank/EMBL/DDBJ under accession numbers HE983995 (chromosome) and HE983996 (plasmid pCher). All contigs from the original Encarsia metagenome from which the Cardinium genome was reconstructed are also available at GenBank/EMBL/DDBJ.The genome was analyzed and automatically annotated using the Microbial Genome Analysis and Annotation Platform MaGe e genome  was genee genome  analysisEncarsia spp. cultures harboring different Cardinium hertigii strains, including the reference strain cEper1 were each collected in a 1.5 ml reaction tube, resuspended in 180 µl buffer ATL (QIAGEN DNeasy blood and tissue kit) and homogenized with a pellet pestle suitable for 1.5 ml microcentrifuge tubes. DNA from homogenized wasps was isolated with QIAGEN DNeasy blood and tissue kit as recommended in the manufacturer's protocol with the exception of the usage of 400 µg proteinase K (Roche) resuspended in 20 µl ddH2O instead of the proteinase K recommended by the manufacturer. A standard PCR cycling program with 35 cycles with primers specific for different Cardinium genes was used for the amplification . The samples were then placed in propylene oxide and embedded in Epon. Serial sections were cut with an RMC MT7000 ultra microtome. The grids were stained with saturated uranyl acetate and lead citrate and viewed under a Philips Electronic Instruments CM12 transmission electron microscope.Transmission electron microscopy of Figure S1Representation of clusters of orthologous gene (COG) categories in selected genomes of obligate and facultative bacterial symbionts.(PDF)Click here for additional data file.Figure S2Conservation of the RING-like domain encoded in CAHE_p0026. Comparison of the domain found in CAHE_p0026 with RING-like domains showing E3 ubiquitin ligase activity according to (PDF)Click here for additional data file.Figure S3Cardinium hertigii. An amino acid sequence alignment of the catalytic core domains of selected USPs is shown. The alignment was done with MAFFT http://www.ch.embnet.org/software/BOX_form.html). Data for important amino acid residues are taken from S. cerevisiae, CAA86791), UBP8_YEAST , UBP15_YEAST , Aasi_0770 , Aasi_1805 , USP_Cardinium .Multiple sequence alignment of selected ubiquitin-specific proteases (USPs) with CAHE_0028, the USP of (PDF)Click here for additional data file.Figure S4Cardinium hertigii and Amoebophilus asiaticus. Syntons comprising at least three genes are indicated by green lines if the orientation is conserved or by red lines in case of inversions. In total, 284 Cardinium CDSs are arranged in 106 syntons (larger than three genes) with Amoebophilus.Synteny between (PDF)Click here for additional data file.Figure S5Cardinium hertigii. Tree calculations were performed using the maximum likelihood algorithm (1000 bootstrap resamplings) with a concatenated dataset of six biotin synthesis proteins Click here for additional data file.Figure S6Cardinium hertigii and its putative donors/recipients. Only HGT candidates with a bootstrap value higher than 75% and a consistent grouping in both neighbor joining and maximum likelihood trees (shown in HGT-affected genes in shown in  were incshown in .(PDF)Click here for additional data file.Figure S7Cardinium hertigii. Phylogenetic trees are based on amino acid sequences and were calculated with MEGA using the neighbor-joining algorithm (NJ) with 2000× bootstrapping and maximum-likelihood algorithm (ML) with 100× bootstrapping. Bootstrap values are indicated at the respective nodes. GenBank accession numbers are indicated.Phylogenetic relationships of candidate HGT genes of (PDF)Click here for additional data file.Table S1Bacteroidetes are indicated by an asterisk; plasmids were not taken into account.Genome sizes of selected endosymbionts. Obligate (primary) symbionts are shaded in grey; obligate symbionts are indicated with a section sign; members of the (DOCX)Click here for additional data file.Table S2Cardinium hertigii.Transposases in the genome of (DOCX)Click here for additional data file.Table S3Cardinium hertigii proteins involved in biotin biosynthesis, glycolysis, peptidoglycan biosynthesis, and lipoate biosynthesis.(DOCX)Click here for additional data file.Table S4Cardinium hertigii.Transport proteins in the genome of (DOCX)Click here for additional data file.Table S5Amoebophilus asiaticus AFP-like gene cluster (as query) with the Cardinium hertigii AFP-like gene cluster and the Serratia entomophila AFP on the pADAP plasmid by blast. Blast results obtained using psi-blast are labeled with an asterisk; I, amino acid identity to best blast hit; E, E-value; n.d., not determined.Comparison of the (DOCX)Click here for additional data file.Table S6Serratia entomophila AFP gene cluster (as query) with the AFP-like gene cluster of Cardinium hertigii and Amoebophilus asiaticus by blastp. I, amino acid identity to best blast hit; E, E-value; n.d., not determined.Comparison of the (DOCX)Click here for additional data file.Table S7Cardinium hertigii strains, their Encarsia wasp hosts, and presence of selected genes detected by PCR. CI (cytoplasmic incompatibility inducing), PI (parthenogensis inducing).Phenotypes of different (DOCX)Click here for additional data file.Table S8Cardinium hertigii likely involved in host cell interaction. I, amino acid identity to best blast hit; E, E-value; n.a., not applicable; n.p., not present.Proteins of (DOCX)Click here for additional data file.Table S9Cardinium hertigii genes putatively involved in past horizontal gene transfer events. Nearest neighbors in phylogenetic trees are indicated Click here for additional data file.Table S10Cardinium hertigii strains (Primers used for the detection of putative host cell interaction genes in different  strains .(DOCX)Click here for additional data file.Table S11NCBI accession numbers of proteins from the biotin biosynthesis pathway used for a concatenated data set for the calculation of a phylogenetic tree with the maximum likelihood algorithm.(DOCX)Click here for additional data file.


It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology.The transcription factor  Danio rerio, is an excellent model organism for such studies due to embryo transparency, high fecundity, and fast development of organogenesis. In particular, the cardiovascular system is formed within one day of birth The cardiovasacular system, which includes the heart, vessels and blood, function together to deliver oxygen and nutrients to cells throughout the body and remove metabolic waste. Understanding the development of this system is instrumental to the advancement of both basic and clinical sciences. The zebrafish, ets related protein, etsrp, which was originally discovered through transcriptional profiling of the cardiovascular mutant line, cloche, that lacks blood and vasculature etsrp is both necessary and sufficient to induce both the vascular endothelial and primitive myelopoietic program in zebrafish ets variant 2, etv2, in mammals etsrp transgenic fish line has been useful to reveal the cellular events that establish the cranial vasculature and myelopoiesis etsrp might be associated with the initiation of the definitive hematopoietic program A critical transcription factor required for the development of vascular endothelial cells is a priori bias prompted us to re-examine the transcriptional profile of embryos overexpressing etsrp. In this study we present a panel of 39 more genes that are expressed in the developing zebrafish vasculature that were identified by this approach.The zebrafish genome is now in its ninth version and remains incompletely annotated. Nonetheless bioinformatic approaches have been used to identify important genes encoding transcription factors containing the ETS box DNA binding domain in hematopoietic and endothelial development etsrp results in the induction of vascular related genes during gastrulation stages of development, before the onset of angioblast specification etsrp RNA was injected into one cell flk1-gfp transgenic embryos and their transcriptome profiles were examined at later stages of gastrulation, 70–90% epiboly, when ectopic induction of flk1-gfp is detected  samples and the mapping results. About 43 million reads were uniquely mapped in total, which covered 77% of the unigene database . The expression level difference between the control and estrp oe samples were compared, resulting in the upregulation of 849 unigene entries above a 1.9-fold cutoff value in the etsrp oe group with a corrected p-value<0.05, while 726 entries were downregulated using the same parameters. A sample of the top 35 hits of annotated upregulated genes obtained in this dataset is listed in A total of 123 million single-end or paired-end reads were obtained and 35% of the reads were mapped onto the unigene transcript sequence database (Build #117) In order to determine the biological relevance of the gene pool obtained here, a gene ontology analysis was performed with the Database for Annotation, Visualization and Integrated Discovery Gene Ontology (DAVID GO) program, as demonstrated in the identification of novel genes expressed in myeloid cells in zebrafish xirp2a, which we found to have endothelial expression. These were noted as a subset of potential vascular genes in The top two clusters from the upregulated data contained genes associated with “vascular development”, “angiogenesis,” and other terms related to vascular endothelial cell biology. These data were combined resulting in 68 genes after duplicate deletion, and examined individually for involvement in vascular development. 18 genes were excluded because although several are expressed in mesoderm or heart, their specific expression or function within endothelial cells remains to be demonstrated. We did examine one of these, etsrp oe. They are lplastin, csf3r, cebp1, hsd3b7. This observation is consistent with what has been previously reported by our group In addition to endothelial specific genes, there were 4 genes of the myeloid lineage that were preferentially induced by etsrp (in vitro studies. 3. Has not been examined in developing vasculature. One of the top ranked genes (highest fold-induction), similar to Src homology 2 domain containing E, she, was used as a positive control since its expression in the vasculature has been reported previously etsrp overexpression in 49 of the 50 genes examined  at the 80% epiboly stages, when they are predicted to be upregulated by overexpressing etsrp . For genexamined . The higalidated . This suetsrp in the vasculature, we examined their expression in etsrp morphants, in which case the expression in the axial vasculature is prominently reduced as demonstrated in myo1F . Marked expression in the vasculature was noted for 39 of the 50 genes . To confin myo1F . Althougin myo1F , where t1F , is expressed in macrophages zgc:162941 has not been reported.The goose type lysozyme ortholog cdc42GAP1 catalyzes the hydrolysis of GTP bound by the Rho GTPases cdc42 and rac1, inactivating them cdc42GAP1 negatively modulates angiogenesis Originally cloned in mice, sperm protein antigen 17, spa17, and both contain a cAMP dependent protein kinase regulatory subunit domain and an IQ motif that binds proteins with EF-hand motifs, but zgc:153721/spa17 of zebrafish has a glutamine rich region between these motifs that is absent in spa17 in humans and mice. Although in mice it is expressed ubiquitously and is expressed in embryos, in rabbits, it is most highly expressed in the tissue from which it was originally identified, testis spa17 has been detected in esophageal, ovarian, and cervical cancers and has been used as a clinical marker for cancer in these tissues spa17 is also expressed in the pronephros , lymphocyte antigen 68 (ly68), and AA4.1. cd93 is a widely conserved member of the c type lectin family whose expression in endothelial and other cell types has been documented in mice and humans complement receptor C1qR-like (crl) has been used for a different gene in zebrafish, which is the likely homolog of c-type lectin domain family 14, member a (clec14a) clec14acd93 shown here, are vascular specific at the stage examined , and xirp2c, which has not been characterized. Functionally, xin repeat motifs cross-link f-actin with β-catenin at adherens junctions Xirp2a expression in the myoseptum, pronephric duct, heart, otic vesicle, head mesoderm, and liver has been demonstrated in developing zebrafish larvae ras association  domain family member 4, rassf4, is a member of Ras association domain family of proteins, which contain a Ras association domain and in the case of rassf4, a SARAH domain , with 95% gene identity to myl9 that also remains to be characterized.etsrp as predicted by RNA-seq and their confirmation by WISH reinforces the effectiveness of the methods used to interrogate transcriptional profiles in zebrafish. Of the genes selected from the dataset obtained, we identified a very high percentage of genes expressed in the vasculature of embryos, validating this approach to identify biologically relevant genes. However there may be non-vascular genes in this data that are induced due to the induction of off-target genes by the artificially elevated level of etsrp. This issue can be addressed by WISH using wild type embryos at a proper embryonic stage. Additionally, this method does not discriminate direct and indirect targets induced by etsrp. Further analysis by ChIP-Seq should reveal those genes that are transcriptionally regulated by etsrp.In this report we provide further support for the utility of combining the relatively unbiased approach of high throughput next generation RNA-sequencing to analyze whole embryo transcriptomes in zebrafish. The high correlation between genes induced by etsrp overexpression and down-regulation by etsrp deficiency in vascular endothelial and primitive myeloid cells highlights their genetic epistatic relationship to etsrp and show that etsrp is a potent transcriptional activator whose expression and activity must be tightly regulated. Although several genes exhibit basal levels of ubiquitous expression their level in the vasculature is still higher. These genes deserve as much attention as those only specific to the vascular lineage, as several studies have shown that the down-regulation of such genes resulted in the disruption of vascular development. For instance, knockdown of nrarp-a and nrarp-b1lpadep1a/dep1betsrp expression. Based on previous annotation and studies we can classify 6.2% of them as vascular genes. Experimental studies identified 39 more vascular genes and further increased the percentage to 10.8%. This leaves over 700 unigene entries in the dataset, some of which may be expressed and actively involved in the development of the zebrafish vasculature, and are potentially worth examining. The complete dataset is available in Both the ectopic induction of these genes by Flk1-gfp embryos were injected with 75 pg etsrp RNA at the one cell stage as described flk1-gfp expression is notable in injected embryos. Total RNA was extracted with the PerfectPure RNA Tissue kit (5 Prime). For pre-sequencing validation, cDNA was made with Oligo(dT) primers and Superscript II reverse transcriptase (Invitrogen). The following primers pairs were used for verification by RT-PCR: β-actin: 5′ -TGTTTTCCCCTCCATTGTTG-3′, 5′-ACATACATGGCAGGGGTGTT-3′; etsrp 3′UTR: 5′-GAGGAATTCTCGAAGGATTGG-3′;TGGTTTTCTAAAGGCACCTAG-3′; Fli1a: 5′-CCGAGGTCCTGCTCTCACAT-3′;5′-GGGACTGGTCAGCGTGAGAT-3′; scl: 5′-GGAGATGCGGAACAGTATGG-3′, 5′-GAAGGCACCGTTCACATTCT-3′. To test gene expression in etsrp morphants, flk1-gfp embryos were injected with a mixture of 4 ng each of translation blocking etsrp morpholinos as described in etsrp overexpression cDNA library at late stages of gastrulation (70%–90% epiboly). Primers used for amplification are noted in Different segments of the genes selected for evaluation were cloned into the pCRII Topo vector with the TOPO TA cloning kit  according to the manufacturer's instructions (Invitrogen). Amplicons were obtained from an In situ stained embryos were further processed by a serial dehydration in ethanol, followed by rehydration into 1XPBS. Embryos were imaged in 2% methylcellulose in depression microscope slides. Images were captured with a color digital CCD camera  mounted on an upright microscope  with Openlab 4.0.2 software . Serial images were combined, merged and processed with Adobe Photoshop CS5.mRNA-seq libraries were constructed with the Tru-Seq RNA prep kit (Illumina) according to manufacturer's instructions. After this project was initiated, the Illumina sequencer has gone through a series of improvements. As a result, we obtained both single-end and paired-end reads with different lengths  using the Illumina GA II sequencer and its earlier versions. The obtained reads were mapped to 51,481 Unigene transcript sequences  estrp oe sample. The number of uniquely mapped read-pairs for each gene in each sample was stored. The total number of mapped reads in each lane was normalized using the total mapped reads in each lane. Fisher's exact test was then performed using the above read counts for each gene. The resulting p-values were corrected via the Benjamini and Hochberg method as implemented in R. Finally, differentially expressed genes were defined as those with changes of at least 1.9-fold between a pair of samples at a false discovery rate (FDR) of 5%. estrp oe sample, while the 726 downregulated hits are tabulated in To determine expression levels of genes and exons, we used the variable RPKM (reads per kilobase of exon per million mapped reads) defined by Mortazavi et al. 2008 etsrp oe groups of embryos harvested at late gastrulation stages. Primers used are listed in qPCR was performed and analyzed exactly as stated in Figure S1Higher magnification of . The axial trunk vasculature of embryos displayed in etsrp morphant counterparts on the right for each gene. Embryos were positioned with anterior facing left.(TIF)Click here for additional data file.Table S1Total RNA-Seq reads obtained in control and estrp oe samples and mapping results.(XLS)Click here for additional data file.Table S2DAVID GO derived gene subset without clear vascular expression or not expressed in developing vasculature.(XLS)Click here for additional data file.Table S3Quantitative PCR of unigene hits predicted to be suppressed by etsrp oe via RNA-seq.(XLS)Click here for additional data file.Table S4Genes with double entries in Ensembl.(XLS)Click here for additional data file.Table S5Primers used for cloning to make WISH probes.(XLS)Click here for additional data file.Table S6Primers used to test downregulated gene set following etsrp overexpression by qPCR.(XLS)Click here for additional data file.Table S7Complete etsrp mRNA-seq dataset .(XLS)Click here for additional data file.Table S8Downregulated mRNA-seq dataset .(XLS)Click here for additional data file.Text S1Supplementary Text. A continuation of the results section that describes information regarding the genes examined in this study.(DOC)Click here for additional data file.


To examine whether fish and fish oil consumption across the lifespan is associated with a lower risk of prostate cancer.The study was nested among 2268 men aged 67–96 years in the AGES-Reykjavik cohort study. In 2002 to 2006, dietary habits were assessed, for early life, midlife and later life using a validated food frequency questionnaire. Participants were followed for prostate cancer diagnosis and mortality through 2009 via linkage to nationwide cancer- and mortality registers. Adjusting for potential confounders, we used regression models to estimate odds ratios (ORs) and hazard ratios (HRs) for prostate cancer according to fish and fish oil consumption.Among the 2268 men, we ascertained 214 prevalent and 133 incident prostate cancer cases, of which 63 had advanced disease. High fish consumption in early- and midlife was not associated with overall or advanced prostate cancer. High intake of salted or smoked fish was associated with a 2-fold increased risk of advanced prostate cancer both in early life  and in later life . Men consuming fish oil in later life had a lower risk of advanced prostate cancer [HR (95%CI): 0.43 ], no association was found for early life or midlife consumption.Salted or smoked fish may increase risk of advanced prostate cancer, whereas fish oil consumption may be protective against progression of prostate cancer in elderly men. In a setting with very high fish consumption, no association was found between overall fish consumption in early or midlife and prostate cancer risk. The association between fish consumption – and two important components in certain types of fish, namely long chain n−3 polyunsaturated fatty acids (PUFAs) and vitamin D – and prostate cancer have been investigated in several epidemiologic studies Most prior studies assessed diet only from midlife or later. However, we have previously shown the potential role of earlier life diet: a positive association between frequent milk consumption, and an inverse association between frequent rye bread consumption in adolescence and risk of advanced prostate cancer later in life The study protocol was approved by the Icelandic Ethical Review Board (VSNb2007120014/03-7) and the Icelandic Data Protection Authority and written informed consent was obtained from all study participants.The population-based, prospective Reykjavík Study comprises 8894 men aged 33 to 79 years, who resided in the Reykjavik capital area at enrolment (1967–1987). A random sample of 2424 of these men living in 2002 was enrolled in the AGES-Reykjavik study In the AGES-Reykjavik, 2268 (94%) men – including 214 men with a prostate cancer diagnosis prior to 2002- provided information in year 2002–2006 on dietary habits in early life (between the ages of 14 to 19), midlife (between the ages of 40–50) and current intake (between the ages of 67 to 96) using a food frequency questionnaire (FFQ) Total fish consumption was estimated by converting the weekly average estimates into daily estimates and combining the first two questions, on fish meals and fish as topping on bread and in salad, into one variable. Never became zero fish per day, less than once a week became 0.07 per day, 1–2 times per week became 0.21 per day, 3–4 times a week became 0.5 per day, 5–6 times per week became 0.79 per day, daily became 1 per day and more than once a day became 1.5 per day. The standard portion for a single fish meal was defined as 150 grams and fish on bread as 40 grams, based on average portions in a national nutrition survey th century http://www.matis.is/ISGEM/en/) the amount of eicosapentaenoic acid (EPA) is 0.75 grams (g) in 10 ml of cod liver oil and the amount of docosahexaenoic acid (DHA) is 1.0 g per 10 ml.Although the FFQ had no separate questions for each fish type, cod and haddock were most commonly consumed in the early 20Other food groups included in the FFQ were: meat, rye bread, blood sausage or liver sausage, potatoes, fruits, vegetables, milk and milk products and whole wheat bread (not included in the adolescent period).Our analysis includes men responding to questions on fish and fish oil consumption in early life (ranging from 2257 to 2266 respondents), midlife (ranging from 2257 to 2267 respondents), and current time with prevalent prostate cancer cases excluded (ranging from 2050 to 2055 respondents).P<0.001), but not for fish meals and fish toppings  The FFQ designed for the AGES-Reykjavik cohort has been validated with regard to midlife and current dietary habits in later life P<0.001) while the correlation coefficient for fish consumption was 0.26 (P = 0.037) For midlife dietary habits, retrospective food consumption of 56–72 year-old participants (n = 67) was estimated by comparing the results in the AGES-FFQ with detailed dietary data (an hour-long interview on dietary habits in the past 3 months) gathered from the same individuals 18–19 years previously. The strongest correlation was found for fish oil  and mortality through December 31, 2009. For incident cases, participants were followed from study entry into the AGES-Reykjavik (between 2002 and 2006) where they provided the dietary information, until diagnosis of prostate cancer, death or the end of the observation period . Because of computerized national roster that includes an individually unique national registration number for each person, follow-up is virtually complete Our analyses of early-life and mid-life diet included both incident and prevalent cancer, and thus we used logistic regression models to calculate odds ratios (ORs) and 95% confidence intervals (CIs). We compared men who were high (>4 portions per week), moderate (>2–4 portions per week) or low (≤2 portions per week) consumers of total fish. For advanced prostate cancer, we collapsed moderate and high intake groups given the small number of cases. Only six participants never consumed fish products in early life and two never in midlife.2), type 2 diabetes in midlife, education , family history of prostate disease, seeing a physician regularly, fish oil consumption (never vs. once a week or more); rye bread consumption (daily or more vs. less than daily); meat consumption (up to 2/week vs. 3+/week), and milk intake (daily or more vs. less than daily). Smoking habits and physical activity were excluded from the models because they did not affect the risk estimates.Information on potential confounders in midlife was retrieved from the questionnaire or health check-ups at entry to the Reykjavik Study. We adjusted in multivariate models for birth year (continuous), age at study entry in midlife (continuous), height (continuous), BMI  with less frequent consumers (≤3/month). Only 54 participants never consumed salted or smoked fish in early life, 82 in midlife and 340 in current time. We also divided salted or smoked fish intake in early life into three groups and explored the association with advanced prostate cancer risk: three times a month or less , 1–2 times per week (43.6% of participants) and four times per week or more (12.6%). We then employed a trend analysis using these three groups as a continuous variable. In the multivariate analysis we used the same covariates as for the total fish analyses. Lastly, the salted and smoked fish consumption in midlife and early life was pooled in one variable with four categories to assess potential effects of longitudinal consumption on advanced prostate cancer risk.When we analyzed later life consumption of fish oil and salted or smoked fish (1+ p/month vs. <1 p/month), 214 men with prevalent prostate cancer were excluded, and we used Cox proportional hazard regression models to calculate hazard ratios (HRs) and 95% CIs of total (n = 133) or advanced prostate cancer (n = 27), using the same covariates as in the early- and midlife analyses.www.spss.com).In all statistical analyses, we used SPSS software, version 19.0  was 46.8±6.9 years when the participants entered the Reykjavik Study and 76.6±5.3 years when they entered the AGES-Reykjavik component and provided the dietary information. P<0.001). Meat consumption was the only food item that showed negative correlation , suggesting a shift in consumption between regional areas and/or social groups during this study period. Dietary habits also changed markedly during the study period with high consumption of salted or smoked fish declining from 60% to 7% from 1907 to 2006. The highest category of total fish consumption, over 4 portions per week, also declined from 38% to 21%, whereas daily fish oil consumption increased from 30% to 61%, most probably due to greater access.Of the 2268 men, 347 had been or were diagnosed with prostate cancer during follow-up, 63 with advanced disease. The mean age (± SD) at cancer diagnosis was 74.8±6.5 years. After completion of the FFQ, 133 men were diagnosed with prostate cancer, of which 27 had advanced disease. When we analyzed current dietary habits assessed at entry to the AGES-Reykjavik study, the 214 prevalent cases were excluded. The mean follow-up time (± SD) from entry to the AGES-Reykjavik until diagnosis of prostate cancer, death or the end of the observation period was 5.1±1.6 years.Ptrend  = 0.03).High total fish consumption in early and midlife was not associated with total, or advanced prostate cancer . LikewisHigh intake of salted or smoked fish in midlife  showed nEarly or midlife consumption of fish oil was not statistically significant associated with total prostate cancer or with advanced disease . For curIn this population-based study of Icelandic men, consumption of total salted or smoked fish and intake of fish oil during different periods of life was not associated with risk of prostate cancer overall. In contrast, the risk of advanced prostate cancer was increased following high intake of smoked or salted fish during adolescence and late life, and substantially reduced among men who consumed fish oil at older age. Although our results for total fish intake in midlife are in line with a recent meta-analysis on 12 cohort studies To our knowledge only one other study (population-based case-control) assessed early life fish intake and prostate cancer risk, and found a marginally increased risk following frequent fish consumption Unexpectedly, we discovered a positive association between frequent salted or smoked fish consumption both in early- and late life and advanced prostate cancer. At least three case-control studies have reported findings on this subject. A study from China assessed intake five years prior to diagnosis and reported positive association between salted fish consumption and total prostate cancer th century was associated with increased risk of advanced prostate cancer We are not aware of studies that have examined smoked or salted fish consumption in early life in relation to prostate cancer, yet early life rural residency in Iceland (compared with early life city residency) examined in a larger cohort during the beginning and mid of 20We found no association between fish oil consumption in early-and midlife and prostate cancer, but late life consumption was inversely associated with advanced prostate cancer risk. This finding suggests a role in disease progression rather than etiology and fits with results on high prediagnostic plasma 25-hydroxyvitamin D predicting improved prostate cancer prognosis The ability to study fish and fish oil consumption across the life course is a particular strength of our study design. Other important strengths are the extensive background data allowing control for potential confounding factors and the complete follow-up. For analyses that include prevalent cases, our results are vulnerable to recall bias because men with prevalent prostate cancer may evaluate their past dietary consumption differently from men without prostate cancer. However, for salted or smoked fish consumption, we only found associations with advanced prostate cancer not total, and only for early life intake, a pattern of findings unlikely to arise due to different recall between cases and controls. Furthermore, findings on current diet in late life were based on incident cases only.The validation study on current food consumption in the AGES-Reykjavik did not show acceptable results for fish meals, possibly due to the inability of the 3-day food record used as a reference method, to adequately reflect individual intake of food items that are consumed 1–2 times per week or 3–4 times per week In summary, salted or smoked fish may increase risk of advanced prostate cancer, whereas in a setting with very high fish consumption no association was found between overall lean fish consumption in early life or midlife and prostate cancer risk. Potential exposure to carcinogens in salted or smoked fish needs further study. We observed reduced risk associated with fish oil consumption in late life, but not in early life or midlife, which may be an indication of a mechanism involving n-3 PUFAs and/or vitamin D and opens for studies on the potential protection of fish oil on the progression of prostate cancer. Improved understanding of potential dietary factors affecting prostate cancer risk, especially for advanced disease, could have a major public health impact.


Hepatitis B virus (HBV) infection is much more common in hemodialysis patients than the general population. These patients have an impaired immune response to HBV vaccination; to that end there are certain studies that have evaluated levamisole as an immunomodulator agent improving HBV vaccination response rate in hemodialysis patients.In the current review, we have assembled all of the results to determine whether lavamisole is of value as an adjuvant to HBV vaccination in hemodialysis patients.Science Direct (Elsevier), ProQuest, Springer, MD Consult, BMJ Journals, Pubmed and Wiley were searched for levamisole application to HBV vaccination in hemodialysis patients. All studies revealed a seroconversion response level between levamisole plus HBV vaccine versus HBV vaccine alone.From 10 relevant studies, 5 studies fulfilled our inclusion criteria. Three of them suggested the significant benefit of adding levamisole to the HBV vaccine to increase augment seroprotection level in hemodialysis patients. Another study reported a decrease in seroprotection level and another study showed no significant difference caused by levamisole administration.Due to the limited number of studies evaluated, it is challenging to perform a definite decision about routinely administering levamisole in addition to the HBV vaccine for all hemodialysis patients. However, it does seem reasonable to recommend administration of levamisole for impaired immune response patients. Hepatitis B virus (HBV) infection is a major global health issue which affects more than 2 billion people worldwide . ApproxiLevamisole (LMS), a synthetic and soluble phenylimidazothiazole, has been widely used as a pesticide for domestic animals for more than 50 years . NonetheIn our study we reviewed all these different results and with a view to sanctioning or not sanctioning lavamisole administration as an adjuvant to HBV vaccination in hemodialysis patients. The main point of interest is to evaluate the seroconversion response level to HBV vaccination in the study (HBV vaccination plus levamisole) and in control groups . Patients with serum antibody level more than 10 mIU/mL were considered as responders and the decision was made as to whether levamisole should be added as an adjuvant to HBVvaccination program in hemodialysis patients.In this paper we searched all available reliable electronic and published sources using Science Direct (Elsevier), ProQuest, Springer, MD Consult, BMJ Journals, Pubmed and Wiley. The key words ‘levamisole’, ‘Hepatitis B Vaccination’, ‘Hemodialysis’ and ‘Vaccine adjuvant’ were used. We included both invivo and invitro studies that were published in English and non-English from Jan. 1980 to Nov. 2011. A single investigator (M.K) extracted all relevant data and inserted them into an electronic database that was checked twice. All of the studies reviewed and used in this paper have been agreed upon by all authors.Inclusion criteria were the following:- Studies that compared the response level between levamisole plus HBV vaccination vs. HBV vaccination alone- Patients on maintenance hemodialysis- Studies based on plasma-derived or recombinant DNA hepatitis B vaccination- Studies that described dose schedules and route of vaccination- Seroprotection response level reported at least 1 month after the last vaccination dosage- Studies that enrolled adult patients- Controlled clinical trials/studies that provided both case and control groupsExclusion criteria were as the following:- Patients with positive HBs Ag serology- Patients with hepatitis B surface antibody (HBs Ab) titer higher than 10 mIU/M [responder patients]- Patients with a positive history of malignancy- Receiving immunosuppressive drugs - Patients with a positive human immunodeficiency virus antibody test (HIV Ab) or positive hepatitis C virus antibody (HCV Ab)- Studies that enrolled child patients- Studies that only published as abstracts or review articles Naive patients (who entered into primary vaccination program) and unresponsive patients (who do not respond to the vaccination program) were also eligible for our study.Among 604 citations, we identified 10 relevant studies in the literature review. One of the studies was excluded because it included child individuals . AnotherThe first study took place in Turkey by Deniz Ayli, et al. Sixty hemodialysis patients were randomized into four groups compared to 15 healthy controls. The patients received IM recombinant HBV vaccine at 0, 1st, 6th and 9th month. The first group received a single dose of vaccine (20μg). Second group had double dose (40 μg) whereas group 3 received a single dose plus 120 mg levamisole and group 4 received a double dose plus 120 mg levamisole. As a result, they discovered that there was no significant difference between a double dose and a single dose vaccination considering the antibody response and there was mention of a long term application of levamisole having salient effects when added to the HBV vaccination without any serious side effects .The second study included four groups of hemodialysis patients who received 40 μg of recombinant HBV vaccine at 0, 1st and 6th month. Group 1 received routine 3 doses of HBV vaccination. Group 2 received 3 doses of HBV vaccination plus 80mg levamisole whereas group 3 and 4 included unresponsive patients who did not respond to a routine vaccination program. Group 3 received 80mg levamisole plus HBV vaccine and group 4 only administered the HBV vaccine. Antibody response level in these four groups was 57%, 82%, 77% and 15%, respectively. Therefore, they had higher response level after using levamisole plus HBV vaccine (57% vs. 82%) and based on that significant difference, levamisol was recommended (77% vs. 15%) . One month after the last dose of vaccination, the seroconversion response level of Levamisole and placebo groups were 81.6%, 81.3%, respectively and there was no significant difference between these two groups .Despite other studies, an interesting article in 2011 declared a lower seroconversion response level of HBV vaccination when it was accompanied by levamisole supplementation. Of 36 hemodialysis patients, 18 patients received 40 μg HBV vaccination intra-muscularly on the 0,1st and 6th month plus 100 mg of levamisole and the rest received the same amount and method of vaccination plus placebo. The seroconversion response rate was evaluated on the 0, 2nd and 4th month. Following the last dose of vaccine, the antibody response level was significantly lower immediately after HBV vaccination. However, it did not last until the 2nd and 4th months after the last dose vaccine as shown in There have been various strategies to improve immune response level to HBV vaccination in hemodialysis patients which has included the use of immune-modulators such as zinc , γ-inter