Bathycoccus, Micromonas, and Ostreococcus are particularly important in coastal regions. By using a combination of cell sorting by flow cytometry, whole genome amplification (WGA), and 454 pyrosequencing, we obtained metagenomic data for two natural picophytoplankton populations from the coastal upwelling waters off central Chile. About 60% of the reads of each sample could be mapped to the genome of Bathycoccus strain from the Mediterranean Sea (RCC1105), representing a total of 9 Mbp (sample T142) and 13 Mbp (sample T149) of non-redundant Bathycoccus genome sequences. WGA did not amplify all regions uniformly, resulting in unequal coverage along a given chromosome and between chromosomes. The identity at the DNA level between the metagenomes and the cultured genome was very high . At least two to three different genotypes seemed to be present in each natural sample based on read mapping to Bathycoccus RCC1105 genome.Among small photosynthetic eukaryotes that play a key role in oceanic food webs, picoplanktonic Mamiellophyceae such as  These three genera have also been found to bloom sporadically in open ocean waters Small photosynthetic eukaryotes <3 µm in size In the last decade, metagenomics approaches have been developed for marine microbial communities Bathycoccus sequences, which display high identity to the recently sequenced genome of a Mediterranean strain In the present paper, we report metagenomic data from two natural picoplankton populations sampled in the Pacific upwelling off the Chile coast. The data are dominated by Micromonas and Ostreococcus dominated in sample T142, while Bathycoccus and Micromonas dominated in T149. In T149, sequences from Chrysophyceae and parasitic Syndiniales (Alveolata) were also recovered. The DNA from these samples was amplified by multiple displacement amplification (MDA). 18S rRNA gene clone libraries performed after MDA contained only Bathycoccus sequences, with the exception of one Acantharea sequence in T149 Photosynthetic picoeukaryotes from two samples collected in the nutrient-rich coastal upwelling waters off central Chile  but not for other Mamiellophyceae. In contrast, we found plastid and mitochondrial 16S rRNA gene signatures for two other Mamiellophyceae, Micromonas and Ostreococcus. The only other eukaryotic signatures came from a Chrysophyceae (plastid) in T142 and from a Dictyochophyceae in T149. Although eukaryotes were targeted during flow cytometry sorting, we also found several 16S rRNA genes from bacteria in both samples. All bacterial signatures were affiliated to clades that are typical of the marine environment, in particular the alpha-proteobacteria Candidatus Pelagibacter and Roseobacter, as well as several Sargasso Sea clades (SAR) In order to determine the taxonomic composition of the assemblage from which the metagenomic sequences originated, we began by searching for SSU rRNA gene within contigs . AlthougOstreococcus  and Micromonas . It should be noted that while we clearly identified Bathycoccus as a key eukaryote in both samples based on rRNA analysis, the number of reads attributed to this genus by BLASTN analysis was small because the genome of Bathycoccus was not available in public databases at the time of this analysis. The contribution of other eukaryotic groups expected to be encountered within picoeukaryotes Candidatus Pelagibacter (SAR11), confirming the results from the rRNA gene analysis. Very few reads were attributed to Archaea or viruses. The majority of reads (∼60%) could not be affiliated to any taxon either because they had no hits in GenBank or because the LCA of their hits was unresolved . MEGAN analysis of Geneious contigs provided similar results, with an increased fraction of contigs, when compared to reads, attributed to bacteria (18%), and therefore fewer contigs with unknown affiliation (50% vs. 60% for reads).In a second step, we subjected both contigs and reads to a global BLASTN search against GenBank and analyzed the results with MEGAN, which provides, when possible, a taxonomic affiliation for each sequence using the Last Common Ancestor (LCA) algorithm Bathycoccus was the dominant eukaryote in both samples. At the time of this analysis, we gained access to the genome sequence of a Bathycoccus prasinos strain (RCC1105) isolated from the Mediterranean Sea Bathycoccus, we constructed a protein database including proteins from nuclear genomes of Bathycoccus RCC1105 and unicellular eukaryotes , mitochondria and plastid genomes from selected eukaryotes, genomes from typical marine archaea and bacteria such as that of the bacterium Candidatus Pelagibacter, unicellular cyanobacteria or Thermococcus, as well as a few eukaryotic virus genomes. Contigs were searched with BLASTX against this database and results were filtered using an algorithm specifically designed to extract Mamiellophyceae sequences using fine-tuned identity and coverage thresholds for each orthologous protein sequence  could be assigned to Mamiellophyceae without a clear relationship to any of the three genera. Very few contigs could be attributed to the other eukaryotic genomes available. Among prokaryotes, only Candidatus Pelagibacter strain HTCC1062 genome recruited a significant number of contigs, in agreement with the MEGAN and rRNA gene analyses reported above. A few contigs (5 for T142 and 17 for T149) could be assigned to available large double stranded Mamiellophyceae viruses Key problems with the public databases are the over-representation of irrelevant sequences and the absence of relevant sequences. Analysis of the SSU rRNA gene both in clone libraries (see above) and in contigs  suggesteence see . Again, ence see . On averBathycoccus had an average amino-acid identity of 96.0 and 95.9%, respectively, with those from the Bathycoccus sequenced genome. This amino-acid identity is an overestimate because our taxonomic affiliation only retains genes with greater than 80% amino-acid identity . These genes were searched by BLASTN among contigs assembled with Newbler for T142 and T149 samples (bioB (biotin synthase), we observed some amino acid deletions, often within proteins containing stretches with repeated amino acids, in particular serine.First, we selected 153 genes from  samples . We iden samples . NucleicBathycoccus genome, we aligned the RCC1105 genome sequence and the T142 and T149 consensus sequences for the three largest chromosomes (1 to 3). We removed all regions that had a coverage depth below 10× for both metagenomes and obtained three alignments varying between 78 and 153 kbp  yielded maximum contig lengths around 40 kbp , or by the fact that some of the photosynthetic picoeukaryotes maybe mixotrophs and may have ingested bacteria Assigning reads and contigs to sequenced genomes is much less straightforward for eukaryotes than for prokaryotes because of the paucity of unicellular eukaryote genomes available. This is particularly true for marine photosynthetic microalgae, for which less than 15 genomes have been sequenced. In a first step, we used SSU rRNA genes for which a very large database is available Bathycoccus genome Bathycoccus  to O. tauri genome taken as a reference. In fact, it is possible that all three genomes correspond to the same biological species, since an analysis of the full rRNA operon revealed nearly identical sequences in the ITS2 region  from flow cytometry sorted cells collected at the deep chlorophyll maximum (DCM) in the tropical Atlantic. We compared, using our custom BLASTX-based algorithm (see Bathycoccus RCC1105 genome was much lower (84.4%) than for our metagenomes , despite the fact that the SSU rRNA sequence of the DCM metagenome is 100% identical to that of RCC1105 (but it lacks the intron). This raises the intriguing possibility that the Bathycoccus genus could contain different ecotypes, some adapted to coastal waters, the others to pelagic/deep waters, in a manner similar to its sister genera OstreococcusMicromonasMonier et al. ithm see , the DCMB. prasinos may contain several ecotypes as well as different genotypes within natural coastal populations. The application of this approach, provided that higher and more uniform coverage can be obtained, may bring in the future unique information on uncultured small photosynthetic eukaryotes that appear to dominate in the more oligotrophic oceanic regions This work illustrates the power of coupling flow cytometry sorting to target specific populations followed by NGS to obtain sequence data on photosynthetic microbes as shown in recent papers Sampling was performed in December 2004 during the oceanographic cruise BIOSOPE Concentrated samples were analyzed on board using a FACSAria flow cytometer  equipped with a laser emitting at 488 nm and the normal filter setup. The signal was triggered on the red fluorescence from chlorophyll. Photosynthetic picoeukaryotes were discriminated based on side scatter, as well as orange and red fluorescence, and sorted in “purity” mode. Cells were collected into Eppendorf tubes and, after a quick centrifugation, the volume of sorted samples was adjusted to 250 µL by adding filtered seawater. Samples were deep frozen in liquid nitrogen.DNA from the sorted pico-eukaryote population was extracted using DNeasy blood and tissue kit (Qiagen), as recommended by the manufacturer. Multiple displacement amplification (MDA) was performed using the REPLI-g Mini kit (Qiagen) following the manufacturer’s protocol with modified buffers as described previously About 10 µg of DNA amplified were fragmented by nebulisation. Fragments between 500 and 800 bp were selected and purified by AMPure (Beckman Coulter Genomics). Libraries were prepared following 454 protocol . Libraries were quantified and libraries profiles were evaluated using a 2100 Bioanalyzer . Each library was sequenced using 1/2 Pico Titer Plate on 454 GSFlx instrument with Titanium chemistry . About 671,000 reads were obtained for each sample.http://www.geneious.com/). First, 454 reads were trimmed at both ends using a probability threshold of p = 0.01 and no ambiguity. Trimmed reads were assembled . Summary of assembly results are provided in ssembled  using thBathycoccus  using Geneious with the default “Medium Sensitivity” (see previous paragraph). Since analysis was performed on an initial draft version of the genome, the correspondence between the initial and the final naming of the chromosomes has been provided in the different tables  We performed a BLASTN search of the raw reads and Geneious contigs against a subset of nr GenBank database, after removing certain taxa and sequence sets that yielded non-relevant hits . Results from the BLASTN search were analyzed with MEGAN 4.0 Bathycoccus, which was not yet publicly available at the time of this analysis We performed a stringent BLASTX-based two-step approach. First, we used a reference protein database  containiI, and alignment length, L,  between all Mamiellophyceae genes and between Prasinovirus genes. For each protein having an orthologous gene, we identified minimum identity percent minI and coverage length minL within the genus or within the class. For specific genes, i.e. genes present in only one genome, we set up a minimum of 80% identity over 50 amino acids.We pre-computed the identity , O. ‘lucimarinus’ EF1-alpha gene has a 76% amino-acid identity over a 476 amino-acids alignment within the genus Ostreococcus and a 48% identity over a 434 amino-acid alignment within Mamiellophyceae. We would thus assign a contig to the genus Ostreococcus if it matched O. ‘lucimarinus’ EF1-alpha gene with an identity greater than 76% and an alignment length greater than the observed alignment length. If either the identity or the alignment lengths were smaller than these thresholds, but still larger than the thresholds within Mamiellophyceae, we would assign this contig to Mamiellophyceae. If a contig did not match any gene with pre-computed thresholds, it was assigned to the taxonomic affiliation of its BBH using a minimum identity threshold of 80% over 50 amino-acids. The corresponding C codes are available upon request from ET .For example, the O. ‘lucimarinus’ using a Mamiellophyceae protein database and a pre-computed identity and threshold matrix without this species. As expected, our approach significantly lowered the rate of false positive taxonomic affiliation from 25.1% to 2.1% for 500 bp sequences and decreases with sequence length . This subset was used in order to focus only in similarities against Bathycoccus genome. Contigs with a positive match were selected based on e-value (< e−100) and visual inspection of the alignment. Contigs providing best match were aligned against the reference gene and nucleotide similarity was determined.For specific groups of genes that are important for phytoplankton metabolism  we examined the similarity between the genome of the cultured Bathycoccus for areas with high coverage (≥10×). For the three larger chromosomes (1 to 3), the Bathycoccus sequences and the T142 and T149 consensus were aligned with the MAFTT plugin under Geneious. We discarded all regions from the alignment for which either T142 or T149 had a genome coverage below 10× based on the annotation made by Geneious. We computed the percent of identical bases across the three genomes and for each pair of genome. We then performed the same computation both on coding (CDS) and non-coding regions.We used Geneious to annotate assemblies of T142 and T149 against Assembly of small regions of a few selected individual single-copy genes was manually analyzed under Geneious to assess the minimum number of haplotypes present in the two samples. Briefly, reads were ordered based on their similarity/differences to the reference RCC1105. Several reads were considered to correspond to one genotype when they presented similar changes at least two positions  was used to search by BLASTN Geneious contigs containing the operon. The two contigs T142_109 and T149_486 were aligned with the RCC1105 rRNA operon using Geneious aligner . Then, iBathycoccus prasinos RCC 1105 have been deposited to EMBL/GenBank under accession numbers FO082258 (Mitochondrion), FO082259 (Chloroplast), FO082278 (Chromosome1), FO082277 (Chromosome 2), FO082276 (Chromosome 3), FO082275 (Chromosome 4), FO082274 (chromosome 5), FO082273 (Chromosome 6), FO082272 (Chromosome 7), FO082271 (Chromosome 8), FO082270 (Chromosome 9), FO082269 (Chromosome 10), FO082268 (Chromosome 11), FO082267 (Chromosome 12), FO082266 (Chromosome 13), FO082265 (Chromosome 14), FO082264 (Chromosome 15), FO082263 (Chromosome 16), FO082262 (Chromosome 17), FO082261 (Chromosome 18), FO082260 (Chromosome 19). The annotation of the genome can be found on the BOGAS web site (http://bioinformatics.psb.ugent.be/webtools/bogas/).Sequences of Geneious contigs are available from EMBL-EBI database under accession numbers CAFX01000001-CAFX01015049 (T142) and CAFY01000001-CAFY01022174 (T149). Sequence data for the genome of Figure S1Relationship between numbers of reads assigned to each B. prasinos RCC1105 chromosome based on BLASTX-based analysis of contigs (see (TIF)Click here for additional data file.Figure S2Structure of the rRNA operon. (A) Alignment of the B. prasinos RCC1105 rRNA operon (forward copy) with the two contigs T142_109 and T149_486 that contain it. Both contigs do not contain the 433 bp SSU rRNA gene intron that characterizes RCC1105. Contig T149_486 appears to contain a 468 bp intron at the end of the LSU rRNA gene. (B) Detail of the putative intron at the end of the LSU rRNA gene of T149_486.(TIF)Click here for additional data file.Figure S3Genotypic variability of the rRNA operon. T142 and T149 reads were directly assembled with Geneious to the B. prasinos RCC1105 rRNA operon. (A) Individual reads for sample T142 in the region of the SSU rRNA intron present in the genome of RCC 1105. The arrow points to a read with a sequence nearly identical to the intron sequence. (B) Individual reads for sample T142 at the end of the LSU rRNA gene. Ten out of 15 reads have a different sequence suggesting the presence of an intron. (C) Idem for sample T149 at the end of the LSU rRNA gene. Only one read has a sequence suggesting the presence of an intron which sequence is similar that found in sample T142.(TIF)Click here for additional data file.Figure S4Percent of false assignment of O. ‘lucimarinus’ proteins based on Best BLAST hit  or after filtration with gene-specific thresholds for identity and alignment length  as a function of read length (see (TIF)Click here for additional data file.Table S1SSU rRNA genes detected in Geneious contigs for samples T142 and T149.(PDF)Click here for additional data file.Table S2Assignment of reads and Geneious contigs for samples T142 and T149 to phylogenetic groups based on BLASTN search of a subset of the nr GenBank database, analyzed by MEGAN (see  The contribution of the best represented groups and of the eukaryotic groups expected to be present in the samples are detailed.(PDF)Click here for additional data file.Table S3Assignment of Geneious contigs for samples T142 and T149 to reference genomes of microalgae, bacteria and viruses based on a BLASTX-based algorithm (see (PDF)Click here for additional data file.Table S4Assignment of Geneious contigs for samples T142 and T149 to specific chromosomes of B. prasinos RCC1105 based on a BLASTX-based algorithm Click here for additional data file.Table S5Similarity between specific genes from selected gene classes of B. prasinos RCC1105 genome and T142 and T149 Newbler contigs (see (PDF)Click here for additional data file.Table S6Estimation of the number of major haplotypes and of their frequency for selected regions of genes with a high read coverage in both samples.(PDF)Click here for additional data file.Table S7Genotype variability within the 5,646 bp rRNA operon. Positions are given along chromosome 11. Localisation of helix 3 of ITS2 follows Marin and Melkonian (PDF)Click here for additional data file.Table S8Assignment of reads for Pacific picoeukaryote samples T142 to individual chromosomes of O.’ lucimarinus’ using Geneious Assembler (see (PDF)Click here for additional data file.Table S9List of reference genomes used for BLASTX analysis of contigs.(PDF)Click here for additional data file.


Since cardiac-related death is major cause of mortality in DMD, it is important to evaluate the effect of any novel treatment on the heart. Therefore, we evaluated the long-term impact of ARB on both the skeletal muscle and cardiac phenotype of the mdx mouse. Mdx mice received either losartan (0.6 g/L) (n = 8) or standard drinking water (n = 9) for two years, after which echocardiography was performed to assess cardiac function. Skeletal muscle weight, morphology, and function were assessed. Fibrosis was evaluated in the diaphragm and heart by Trichrome stain and by determination of tissue hydroxyproline content. By the study endpoint, 88% of treated mice were alive compared to only 44% of untreated (p = 0.05). No difference in skeletal muscle morphology, function, or fibrosis was noted in losartan-treated animals. Cardiac function was significantly preserved with losartan treatment, with a trend towards reduction in cardiac fibrosis. We saw no impact on the skeletal muscle disease progression, suggesting that other pathways that trigger fibrosis dominate over angiotensin II in skeletal muscle long term, unlike the situation in the heart. Our study suggests that ARB may be an important prophylactic treatment for DMD-associated cardiomyopathy, but will not impact skeletal muscle disease.Duchenne muscular dystrophy (DMD) is a degenerative disorder affecting skeletal and cardiac muscle for which there is no effective therapy. Angiotension receptor blockade (ARB) has excellent therapeutic potential in DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the  Since losartan is a widely used antihypertensive drug that is known to be safe in humans, this research has generated interest in using losartan as a treatment for patients with DMD mdx mice after two years of treatment.A recent study demonstrated that antagonism of TGF-β with losartan, an angiotensin II receptor blocker that is known to significantly reduce TGF-β activity in a number of disease models Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health . All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Pennsylvania (#802238). Male mdx mice were weaned at four weeks and randomized to receive standard drinking water (n = 9) or water supplemented with losartan (0.6 g/L) (n = 8) All mice were handled in compliance with the M-mode echocardiography was performed on mice two years following the inception of losartan treatment under ketamine/xylazine anesthesia using a 15-MHz phased-array probe connected to a Sonos 7500 echocardiographic machine . In brief, an M-mode cursor was positioned in the parasternal short-axis view perpendicular to the interventricular septum and posterior wall of the LV at the level of the papillary muscles, and M-mode images were obtained for measurement of LV end-diastolic and end-systolic dimension (LVDd and LVDs). The percentage of fractional shortening (%FS) was calculated from the equation%FS  =  [(LVDd – LVDs)/LVDd] ×100. The end diastolic and end systolic volumes, ejection fraction, cardiac output and stroke volume were calculated using the Teicholtz formulas 2, 5% CO2, and subjected to isolated mechanical measurements using a previously described apparatus  The contractile properties of the soleus, EDL, and diaphragm were measured. Mice were anesthetized with ketamine/xylazine (80/10 mg/kg) and exsanguinated. Blood samples were collected, allowed to clot for 2 hours at room temperature, then centrifuged at 2000 x g for 20 minutes to isolate serum. Serum was stored at −80°C, and creatine kinase was measured later using the assay manufactured by Genzyme . The EDL, soleus, and diaphragm were then removed, bathed in an oxygenated Ringers solution gas-equilibrated with 95% OUpon completion of muscle function analysis, muscles were weighed, then rapidly frozen in OCT compound  in melting isopentane for morphological analysis. Ten micron thick sections were cut, and the resulting slides were stained using standard hematoxylin and eosin (H&E) and Trichrome stains .Cardiac and diaphragm fibrosis was assessed by quantifying tissue hydroxyproline content Samples obtained for Western blotting were snap-frozen in liquid nitrogen. Specimens were pulverized, homogenized in 10 volumes of triple-detergent lysis buffer , and centrifuged at 13,000 rpm for 5 minutes. Protein concentration of the supernatant was then determined using the BioRad Protein Assay , and a 50 ug aliquot of each sample was denatured using an equal volume of 2X sample loading buffer  at 100°C (5 min) then fractionated electrophoretically on a 4–20% SDS-polyacrylamide gel . Proteins were then electroblotted onto a polyvinylidene fluoride membrane  using the iBlot transfer apparatus . The membrane was subsequently blocked by incubating in Tris-buffered saline containing 5% nonfat dry milk and 0.05% Tween 20. Immunoblotting was performed to detect Nav1.5 ; connexin 40 ; and GAPDH . Detection was performed using the Super Signal West Pico Chemiluminescent Substrate kit .Mean values from each experimental group were compared using the two-tailed Student's t test or one-way ANOVA with Student-Neuman-Keuls post hoc analysis as appropriate. The Kaplan-Meier survival curve was generated using Graph-Pad Prism software with log-rank analysis .mdx mice were weaned at four weeks and randomized to receive either standard drinking water (n = 9) or drinking water supplemented with losartan (0.6 g/L) (n = 8) for two years Male The extensor digitorum longus (EDL), soleus, and diaphragm were analyzed at 2 years. There was no significant difference in mass, length, or cross sectional area in losartan-treated mice vs. untreated Figure 2Hematoxylin and eosin (H&E) and Trichrome staining was performed on the EDL, soleus, quadriceps, and diaphragm. No significant differences were noted in terms of the number of centrally located nuclei (H&E) (data not shown) or extent of fibrosis (Trichrome) (Serum creatine kinase (CK) was measured in losartan-treated and untreated mice, and no significant difference was noted between the two groups (data not shown).mdx mice exhibited left ventricular dilation  and systolic dysfunction  at two years, while mdx mice treated with losartan exhibited preserved cardiac geometry with normal systolic function  via losartan has been proposed to have therapeutic potential for DMD based on recent data demonstrating attenuation of skeletal muscle disease progression during 6–9 months of therapy in the mdx mice. Van Erp et al followed mdx mice from 3–18 months and characterized the development of cardiomyopathy during this period mdx mice should be performed in animals greater than 18 months old. A second group followed mdx mice for 20–22 months and evaluated the cardiac phenotype in untreated mdx mice and in mdx mice carrying a mini-dystrophin transgene mdx mice exhibit cardiac fibrosis, ECG abnormalities, systolic dysfunction, and decreased exercise performance, and they report that mini-dystrophin expression can partially improve this phenotype.Several recent studies have described the phenotype of aged mdx mice. Mouisel et al followed mdx mice for 18–24 months and found that the skeletal muscles of these old mice were characterized by decreased absolute force, atrophy, and decreased regenerative capacity compared to 5 month old mdx mice mdx mice and reported that this approach was effective not only in halting disease progression but also in improving muscle structure and function by 24 months, thus demonstrating that it is possible to improve the phenotype of aged mdx mice mdx mouse is the small animal model that most closely mimics the severe phenotype of Duchenne muscular dystrophy (DMD) and therefore is a superior model to the young mdx mouse when evaluating novel therapeutic strategies for DMD.Other studies have focused on analysis of the skeletal muscle phenotype of aged mdx mouse, and we chose 24 months as an endpoint. This endpoint seemed ideal based on the existing literature since it would allow us not only to evaluate cardiac function at a timepoint after which basal ventricular dysfunction had been previously reported In this study, our goal was to evaluate the effects of angiotensin receptor blockade (ARB) on both the cardiac and skeletal muscle phenotype of the aged It is unclear why no long term benefit of losartan was observed in skeletal muscle. It may be that fibrosis early in the disease process is driven largely by angiotensin II, but that over time, other pathways become dominant in skeletal muscle. This would allow the rate of fibrosis to increase despite ARB at a time when dystrophic satellite cells are experiencing a decline in regenerative capacity mdx mice Although we did not observe any benefit of losartan in skeletal muscle function, cardiac function and geometry was preserved, and mortality was decreased after two years of angiotensin receptor blockade. This result is in agreement with a recent study that demonstrated improved cardiac function, but not improved skeletal muscle function, following 6 months of losartan treatment in mdx heart mdx heart mdx mice mdx mice compared to healthy C57 mice. We also found that while ARB did not alter expression of Nav1.5, it did restore Cx40 expression to levels found in the healthy C57 heart. Therefore, normalization of the function of gap junctions may contribute to the observed improvement in cardiac function and reduced mortality in mdx mice treated with ARB. Future investigation in needed to determine the effect of ARB on arrythmogenesis and on the abnormal ECG findings in mdx mice Cardiac function may also have been improved by a mechanism independent from a reduction in fibrosis. Previous studies have reported that the sodium channel Nav1.5 is decreased in the 2+/calmodulin-dependent protein kinase II signaling mdx mice treated with losartan.It has also been reported that defects in calcium handling secondary to dysregulation of the sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and phospholamban (PLB) are present in the failing hearts of humans and animals mdx mouse preserves cardiac function and decreases mortality. No benefit in terms of skeletal muscle morphology or function was observed, suggesting that pathway(s) other than angiotensin II are dominant in driving skeletal muscle fibrosis in late stage disease. In addition, our study suggests that ARB has potential as a treatment for DMD-associated cardiomyopathy. Future investigation should be directed at evaluating losartan in large animal models and patients with DMD, as well as comparing effectiveness of ARB and angiotension converting enzyme (ACE) ACE inhibition.In summary, we report that two years of ARB in the


Mitochondrial DNA (mtDNA) mutations occur in head and neck squamous cell carcinoma (HNSCC) and are most frequently detected in the displacement-loop (D-loop) region. The D-loop is considered to be important because it controls mitochondrial gene expression and mtDNA replication. There is currently no evidence that mtDNA mutations can be used as prognostic or predictive biomarkers in HNSCC.We used denaturing high performance liquid chromatography to screen the entire mitochondrial genome of six oral squamous cell carcinoma-derived cell lines and then focused on detecting D-loop abnormalities in 34 HNSCC tissue samples.Mitochondrial DNA mutations are not ubiquitous in HNSCC because only half of the cell lines had detectable mtDNA abnormalities following screening of the entire mitochondrial genome and only 18% (6 of 34) of tissue samples had D-loop mutations. There was no correlation between D-loop mutations and determinates of clinical outcome; specifically, tumour stage and the expression of hypoxia-inducible genes included in a highly prognostic hypoxia metagene.Taken together, these data suggest that mtDNA D-loop mutations are stochastic events that may not significantly influence the biology of HNSCC and supports the hypothesis that mtDNA mutations in cancer represent bystander genotoxic damage as a consequence of tumour development and progression. Head and neck squamous cell carcinoma (HNSCC) is a major world health problem; estimates indicate that there are over 400 000 new cases diagnosed each year . TypicalSomatic mutations in mitochondrial DNA (mtDNA) have been increasingly observed in human cancers and have been proposed as important oncological biomarkers .The biological significance of mtDNA mutations in cancer remains unclear . RecentlIn the present study, we screened the entire mitochondrial genome of six oral squamous cell carcinoma-derived cell lines for mtDNA deletions and mutations using denaturing high performance liquid chromatography (DHPLC) and then focused on identifying D-loop abnormalities in a set of HNSCC tissue samples. For the first time, we examined the relationship between mtDNA mutations and the expression of hypoxia-inducible genes in HNSCC tissue samples.Six human oral squamous cell carcinoma-derived cell lines were used . Four ofThe tissue samples  were derived from patients with previously untreated primary HNSCC. The research was given a favourable opinion from the Newcastle and North Tyneside 2 Research Ethics Committee (Reference 09/H0907/58). The clinical features of the samples are summarised in μl−1 for use in downstream analysis.Genomic DNA was extracted from cell pellets using a standard laboratory kit . DNA was extracted from homogenised tissue samples using Tri Reagent  and cleaned using a standard laboratory kit (QIAGEN). Extracted DNA samples were diluted to 200 ng Genomic DNA from the cell lines were screened for large-scale mtDNA deletions using the Expand Long Template PCR System as previously described . In all, 15 of the 19 primer sets generated large amplicons that required digestion with restriction enzymes before heteroduplex formation. Heteroduplexed DNA was analysed by DHPLC on a Transgenomic Wave DNA Fragment Analysis System 3500 using Navigator software (Transgenomic Ltd).Genomic DNA samples were screened for small deletions, insertions, and point mutations using the Mitoscreen Assay System . The system comprises 19 overlapping primer sets that amplify the entire mitochondrial genome. Reactions were carried out according to the manufacturer's standard protocol. Briefly, PCRs were performed in 20–40 μl of water and reamplified using appropriate primer sets. The PCR products were resolved on a 2% low melting point agarose gel  containing ethidium bromide. The DNA band representing the amplicon was excised from the gel and the DNA was extracted using the QIAquick Gel Extraction Kit (QIAGEN). The purified DNA was eluted in 30 μl of Tris/EDTA buffer. The sequencing reaction was carried out using the GenomeLab Dye Terminator Cycle Sequencing Kit  according to the manufacturer's protocol. The products were run on a Beckman CEQ Genetic Analysis System 8000 (Beckman Coulter). Sequences were visualised using MacVector 7.0 software  and compared with the mtDNA revised Cambridge reference sequence . DNA was resuspended in 5 sequence .ΔCt− formula before statistical analysis as previously described  as described previously . Using DNA extracted from normal oral keratinocytes and HaCaT cells as a comparator, DHPLC profiles from the cell line DNA had a number of abnormalities. The abnormalities included broader wave profiles, peak shifts, and a change in the number of fragments in those samples subject to restriction enzyme digestion. Whole mitochondrial genome screening of the six cell lines generated 114 amplicons representing around 100 kb of DNA. A total of 17 mtDNA abnormalities were detected using this method and abnormalities were identified in all of the cell lines.Sequencing of ‘screen-detected’ mtDNA abnormalities in the cell lines alongside matched normal DNA demonstrated that around half of the abnormalities (8 of 17) were common to both samples and represented mtDNA polymorphisms. H357, H400, and H413 had polymorphic mtDNA, but no somatic mtDNA mutations. By contrast, H314 had a complex mtDNA genotype with nine homoplasmic point mutations located in two regions of the mitochondrial genome, namely cytochrome oxidase I and the D-loop . There wH764A and H764B, cell lines derived from the same patient representing a primary oral squamous cell carcinoma and a corresponding lymph node metastasis, respectively, had two sequence abnormalities not detected in the matched normal DNA. The first was a homoplasmic 50 bp deletion in the D-loop between nucleotide positions 298/306 and 348/356; the breakpoints were flanked by a 9-bp repeat sequence 5′-CCAAACCCC-3′ . The delTwenty-two abnormalities were detected following screening of 102 amplicons in 34 HNSCC with matched normal mucosal samples. Sequencing of the variant amplicons demonstrated that 15 of the samples contained known sequence variants (polymorphisms) that were identical in the normal and tumour DNA (data not shown). Eight somatic mutations were detected in seven samples; sample 85 contained two mutations . The majn=6) had lower mean gene expression levels when compared with those samples that had normal D-loop sequences (n=23), however, there was no statistically significant difference in hypoxia gene expression between the two groups  of the HNSCC tissue samples had D-loop mutations. Pooled data from nine studies that looked for D-loop mutations in over 400 HNSCCs indicate that around a third of HNSCCs harbour D-loop mutations; however, there is wide variation between different studies  that may not significantly influence the biology of HNSCC. Our data support the hypothesis that mtDNA mutations in cancer represent bystander genotoxic damage as a consequence of tumour development and progression .


Tumor cell fusion with motile bone marrow-derived cells (BMDCs) has long been posited as a mechanism for cancer metastasis. While there is much support for this from cell culture and animal studies, it has yet to be confirmed in human cancer, as tumor and marrow-derived cells from the same patient cannot be easily distinguished genetically.We carried out genotyping of a metastatic melanoma to the brain that arose following allogeneic bone-marrow transplantation (BMT), using forensic short tandem repeat (STR) length-polymorphisms to distinguish donor and patient genomes. Tumor cells were isolated free of leucocytes by laser microdissection, and tumor and pre-transplant blood lymphocyte DNAs were analyzed for donor and patient alleles at 14 autosomal STR loci and the sex chromosomes.All alleles in the donor and patient pre-BMT lymphocytes were found in tumor cells. The alleles showed disproportionate relative abundances in similar patterns throughout the tumor, indicating the tumor was initiated by a clonal fusion event.Our results strongly support fusion between a BMDC and a tumor cell playing a role in the origin of this metastasis. Depending on the frequency of such events, the findings could have important implications for understanding the generation of metastases, including the origins of tumor initiating cells and the cancer epigenome. Tumor cell fusion with motile leucocytes such as myeloid lineage cells or stem cells has been put forward as a unifying explanation for metastasis However, fusion and genomic hybridization have yet to be proven on a genetic basis in human cancer, since genomic differences between cells from the same patient cannot be readily distinguished. To circumvent this problem we have analyzed secondary malignancies arising post allogeneic bone marrow transplants (BMT). In two previous reports we demonstrated donor alleles in patient cancer cells, but there was no provision to identify patient alleles and fusion could thus not be proven All samples used in this study were preexisting and de-identified before being received by the Yale research team. Exemption was granted under Yale IRB protocol #070900309 (JP and RL) from the Yale University Human Research Protection Program, Institutional Review Board.The patient was a 68-year-old man who received an allogeneic BMT from his brother for treatment of B-cell lymphoma. His last engraftment/chimerism profile was Recipient 3%; Donor 97%. Six years later the patient was diagnosed with metastatic melanoma involving lymph nodes, liver and brain, derived from an unknown primary tumor. We analyzed a brain metastasis (designated “MH3”) consisting of a 0.5×0.2×0.3 cm formalin-fixed paraffin-embedded (FFPE) tissue. The tumor was surgically removed, fixed in formalin, and embedded in paraffin by standard histological procedures. Pre-transplant donor and patient lymphocytes were stored at −90°C in the Yale-New Haven Hospital Stem Cell Bank and retrieved after the tumor analyses were completed.Handling and processing of tissue samples was carried out using ultraclean, DNA-free equipment. Five μ-thick histological sections were cut and immunostained for LCA/CD45  using an autostainer  at the Yale Dermatopathology Laboratories. The antibody was tested for staining efficiency as described in DNA extraction and STR analyses were by the Denver Police Department Crime Laboratory DNA Unit using standard forensic operating and valided procedures PCR was performed with the AmpFlSTR Identifiler PCR Amplification Kit . Generally, 1 ng of total DNA was targeted in each PCR amplification. In samples with less DNA (<0.1 ng/µl), samples were concentrated 5–20 fold using a Microcon centrifugal filter .Each STR locus was selected to be neutral with respect to other genetic linkage or associations with either Mendelian or non-Mendelian disorders. The loci were polymorphic and exhibited acceptable levels of heterozygosity, typically 70% or higher. They could be assayed together as a PCR multiplex and were robust for degraded DNA Allele signal peaks may overlap technical “stutter” positions from other alleles. Validation studies have established interpretation guidelines for forensic markers to distinguish true allele signals from stutter for any given allele at any locus Bayesian statistical models of fusion and donor cell contamination were fitted and compared using Markov chain Monte Carlo methods We performed extensive pathology analyses to ensure that laser dissected tumor samples were not contaminated by infiltrating donor leukocytes. To detect leucocytes we used an antibody to leucocyte common antigen (LCA/CD45), expressed on the surface of mature leukocytes and hemopoietic progenitor cells. In positive control experiments the antibody showed >99% staining efficiency against test cases of dermatitis and lymphoma  and DNA was extracted and amplified for alleles at 14 STR loci. All alleles found in the pre-transplant donor and patient blood lymphocytes were also detected in tumor cells. For each locus there was at least one allele common to both the donor and patient, consistent with the fraternal relationship. Eight loci exhibited donor- specific alleles and six of these exhibited both donor and patient specific-alleles. This indicated that the tumor cells were donor-patient hybrids , Table 1P<0.005 for contamination and P>0.3 for fusion  Our statistical analyses of the allelic patterns strongly favored BMDC-melanoma cell fusion over leucocyte contamination models.STR analyses of the tumor DNA revealed that donor and patient alleles were present together at multiple loci and that there were widespread allelic imbalances and aneuploidy. One drawback was the inability to perform STR analyses on individual tumor cells, but for this tumor the lower limit for DNA recovery for STR analyses was about 500 cells, even using the DNA extraction procedure for FFPE cells that improved DNA recovery. Thus, we could not definitively rule out that the patterns might be due to a chimeric mixture of patient tumor cells and donor BMDCs. However this is unlikely for the following reasons: 1) For a given locus the allelic ratios were similar throughout the tumor. It is difficult to explain these repeating patterns as due to chimeric mixtures as this would require that the different cell types existed together in the same ratios throughout the tumor. Notably, while most of the informative tumor loci had patient and shared alleles in greater relative abundance to donor-specific alleles, tumor locus D13S317 was reversed, with the patient-specific allele absent and the donor-specific allele in prominence. Since the initial dose of genomic DNA for each sample was determinative of PCR product intensity and varied widely between samples, given the consistency in allelic ratios from sample to sample the reversal of DNA dosage at locus D13S317 cannot be explained by preferential PCR or leukocyte contamination. 2) The tumor cells were dissected from regions free of LCA-positive cells . 3) It iIn stem cell biology both transdifferentiation and fusion appear to be operative in the transformation of stem cells into differentiated somatic cells in vitro between cancer cells, including melanoma, and normal epithelial cells or fibroblasts were generally suppressed in tumorigenicity and the expression of differentiated functions, leading to the discovery of tumor suppressor genes In earlier studies, experimental tumor hybrids generated A model for metastasis resulting from fusion of bone marrow-derived cells is diagramed schematically in File S1Figures S1, S2, S3, S4, S5 and Table S1.(PDF)Click here for additional data file.File S2Figures S6, S7, S8, S9 and Table S2.(PDF)Click here for additional data file.


Although highly active antiretroviral therapy (HAART) improves immunologic parameters, the relationship between DTH responsiveness and CD4 gains on HAART is not completely understood. We investigated CD4 reconstitution and the change in DTH responses from treatment baseline through 24 months of viral load (VL)-suppressive HAART in the U.S. Military HIV Natural History Study.Delayed-type hypersensitivity (DTH) testing is an n=302). DTH testing consisted of ≥3 recall antigens, and responses were classified by the number of positive skin tests: anergic (0–1) or non-anergic (≥2). Pre-HAART DTH results were compared for the outcome of CD4 reconstitution at 24 months of HAART. Improvement in DTH responses was also analyzed for those anergic before HAART initiation.Treatment-naïve subjects with VL <400 copies/mL after ≥24 months on HAART were included (p=0.104) and VL . Median CD4 gains 24 months after HAART initiation were similar between the non-anergic  and anergic groups . For individuals anergic before HAART initiation, DTH normalization occurred at 24 months post-HAART in the majority of participants . Normalization of DTH responses was not associated with CD4 count at HAART initiation  nor with AIDS diagnoses prior to HAART .Non-anergic responses were observed in 216 (72%) participants, while 86 (28%) individuals were anergic prior to HAART initiation. Demographically there were similar distributions of age at HIV diagnosis and HAART initiation, as well as gender and race or ethnicity. There were no significant differences between non-anergic and anergic participants in pre-HAART CD4 count  315–517 vs. 373 cells/μL, IQR 228–487; DTH responsiveness has been shown to predict HIV disease progression independent of CD4 count in untreated individuals. In the setting of HAART, pre-HAART anergy does not appear to impact CD4 gains or the ability to normalize DTH responses after 24 months of VL-suppressive HAART. The gDTH responsiveness is typically reduced in patients living with HIV compared to seronegative individuals , 6. For DTH responses have also been studied in patients receiving HAART. In addition to immune reconstitution measured clinically by CD4 cell gains, several studies demonstrated an improvement in DTH responses after initiation of HAART –14. HoweThe NHS is a prospective observational cohort of over 5500 military members, dependents, and beneficiaries who have been evaluated for HIV-1 positive status in the military healthcare system since 1986 . Study vThe NHS database was queried for participants on HAART who also received serial DTH testing for this retrospective analysis. Participants achieving a VL <400 copies/mL within six months of their initial HAART regimen and maintaining VL suppression for ≥24 months were included. The analysis was restricted to those achieving VL suppression because the outcome of immune reconstitution during HAART was the primary focus of this study and maximal CD4 gains occur when VL is fully suppressed. The association between pre-HAART DTH response and the outcome of VL suppression was not analyzed because data for some confounders, including HIV genotype and HAART adherence, were not available. Individuals were required to have DTH testing within six months prior to HI and repeat DTH testing performed at 24 months on HAART. DTH testing was performed according to standardized protocols, as previously described , 8, 13. p-values were calculated using t-tests or Wilcoxon tests for continuous variables depending on normality, and chi-squared tests or Fisher's exact tests for categorical variables depending on sparse cells. To estimate the impact of DTH status at HI on post-HAART immune function, we modelled CD4 gains at 24 months after HI with multiple linear regression while adjusting for other relevant covariates. Multiple logistic regression was used to understand factors associated with post-HAART DTH normalization among those who were anergic at HI.Baseline characteristic p=0.015). Median CD4 count at diagnosis was significantly higher in the non-anergic group  360–656) compared to the anergic group ; however, VL was no different .A total of 302 participants met inclusion criteria, of which 216 (72%) were non-anergic and 86 (28%) were anergic by DTH testing prior to HI . Demograp=0.104). VL at HI was also similar between groups. AIDS diagnoses prior to HI were more common in the anergic group  compared to the non-anergic group . The initial HAART regimen most commonly used was protease inhibitor (PI) based  compared to non-nucleoside reverse transcriptase inhibitor (NNRTI) based  and other regimens that typically contained both PIs and NNRTIs . For DTH test outcomes, there was a trend toward greater PI-based regimen use in the anergic group  compared to the non-anergic group .The year of HI was typically in the early HAART era (1998 for both groups). The median CD4 at HI was higher for non-anergic  versus anergic  participants, but this was not statistically significant (p=0.498). Linear regression was performed to investigate the factors associated with CD4 gains (p<0.001). Other variables, including diagnosis of AIDS prior to HI or type of HAART regimen, were not significantly associated with CD4 gains at 24 months.The median CD4 cell gains after HI were similar for non-anergic compared to anergic subjects, even when stratified by pre-HAART CD4 count ≤400 or >400 cells/μL . At 24 mD4 gains . Non-anen=86) was studied for factors associated with normalization of DTH responses at 24 months of HAART (p=0.129) nor AIDS prior to HI  was associated, with DTH normalization. Race or ethnicity was associated with DTH status at 24 months with African Americans  and Hispanic/others  more likely to become non-anergic than European Americans. Those whose first HAART regimen was NNRTI based compared to PI based were less likely to become non-anergic , but no other covariates were associated with DTH normalization.The subset of participants who were anergic at HI  jiroveci pneumonia prophylaxis was previously recommended in patients with CD4 >200 cells/μL combined with anergy [in vivo, DTH results can provide information about immune function in addition to the measurement of CD4 counts. DTH anergy has also been suggested as an indicator for initiating HAART in resource-limited settings [The utility of DTH testing has been demonstrated in many studies and was previously recommended in the evaluation and disease staging of patients living with HIV. For example, DTH responsiveness has been shown to be an independent predictor of opportunistic infections and death , 6, 8, 9h anergy . Since Dsettings , 11. AltDTH responsiveness to recall antigens in HIV-infected patients is associated with the magnitude of the CD4 count at the time of testing. For example, one study in our military population found that 60% of treatment-naïve patients with CD4 counts less than 200 cells/μL were completely anergic, whereas 86% with >400 cells/μL were non-anergic . Althouget al.[et al., 40% of participants did not achieve VL suppression on HAART, whereas our study included only VL-suppressed individuals [HAART has been previously shown to enhance DTH responsiveness in patients living with HIV who are anergic. In a study by Carr et al., DTH reset al., 22. A set al.. In the ividuals . In contividuals . We previviduals . Thus, iin vitro loss of T-cell function in patients living with HIV was progressive and sequential, beginning with impairments to antigen recall responses, then alloantigen responses, and ultimately mitogen-induced responses [One interesting observation in this study was that DTH normalization occurred in approximately 60% of anergic participants regardless of pre-HAART CD4 cell count and prior AIDS diagnoses. A previous study showed that esponses . Since rn=14) compared to PI-based HAART (n=64). Alternatively, this finding may also be related to race because four of the five participants on NNRTI-based regimens who failed to normalize DTH responses were European Americans. Further studies are necessary to investigate the impact of racial and genetic background on DTH responsiveness and immune recovery during HAART.Patients in the NHS cohort have free access to healthcare and medications, which mitigates social and economic issues that can confound analyses related to race and ethnicity in other cohorts. Multiple logistic regression analyses in the current study revealed that European Americans were more likely to be anergic before HI and less likely to normalize DTH responses during HAART. Since this finding was not attributable to low CD4 counts or the presence of AIDS diagnoses, other immunologic and/or genetic aspects may have contributed to the lack of DTH responses in this group. For example, CCL3L1-CCR5 GRG status, a genetic characteristic, has been associated with differences in DTH responsiveness . DTH norIn this study, the overall number of DTH responses was recorded, although detail about specific antigen responses was not available. Another potential limitation is that treatment occurred in the early HAART era. However, this was mitigated by the requirement of VL suppression for all participants included in this study. Other factors that may influence DTH responsiveness, such as vitamin deficiencies and nutritional status, were not measured, and some comparisons may have been limited by the relatively small sample size. The overall proportion of participants who were anergic at HI was lower (30%) compared to other studies, but this is likely due to relatively preserved pre-HAART CD4 counts (median 400 cells/μL) and the younger age (median 35 years) at the time of HI.DTH testing is a clinical tool that can be used to assess CMI. Although DTH responsiveness can predict HIV disease progression independent of CD4 count, pre-HAART DTH status does not appear to be predictive of CD4 gains after 24 months of VL-suppressive HAART. In participants with cutaneous anergy prior to HAART, a normalization of DTH responses occurred in approximately 60% of subjects after 24 months of suppressive HAART. In those with cutaneous anergy and advanced HIV disease, as evidenced by low CD4 cell counts and AIDS diagnoses, the capacity for both CD4 reconstitution and normalization of DTH responses during HAART appears to be similar to that of those without advanced disease.


The anemia of chronic disease  is an acquired disorder of iron homeostasis associated with infection, malignancy, organ failure, trauma, or other causes of inflammation. It is now widely accepted that induction of hepcidin expression in response to inflammation might explain the characteristic hypoferremia associated with this condition. To determine the role of hepcidin in acute inflammation and the regulation of its receptor, the iron exporter, ferroportin, wild-type, heterozygote and hepcidin knockout mice (Hepc−/−) were challenged with sublethal doses of lipopolysaccharide (LPS). Six hours after injection, ferroportin mRNA and protein levels were assessed in the duodenum and the spleen and plasma iron was determined. Our results demonstrate that hepcidin is crucial, though not the sole mediator of LPS-mediated acute hypoferremia, and also that hepcidin major contribution relies on decreased ferroportin protein levels found in the spleen. Furthermore, we establish that LPS-mediated repression of the membrane iron transporter DMT1 and oxidoreductase Dcytb in the duodenum is independent of hepcidin. Finally, our results in the hepc+/− mice indicate that elevated hepcidin gene expression is not a prerequisite for the setting of hypoferremia during early inflammatory response, and they highlight the intimate crosstalk between inflammatory and iron-responsive pathways for the control of hepcidin. One of the most potent pathogen-derived inflammatory signals is the Gram-negative bacterial cell wall component lipopolysaccharide, LPS, which induces a massive release of cytokines and other inflammatory mediators in the infected host. This release is critical for the integration of the innate immune response, but requires tight regulation to prevent subsequent inflammation, which may lead to endotoxic shock, a condition that is still one of the most frequent causes of mortality in the hospital intensive care unit. It is now well established that LPS mediates its effects through a member of the highly conserved Toll-like receptor (TLR) family, TLR4, for the induction of proinflammatory genes Mice were cared for in accordance with the European convention for the protection of laboratory animals. Animal studies received approval from the Regional Ethics Committee for Animal Experimentation of University Paris Descartes. Animals were given free access to tap water and a standard laboratory mouse chow diet . Age-matched wild-type (WT), heterozygote (Hepc +/−) and homozygote (Hepc−/−) male mice of 8-13-week-old on a C57BL/6 background were used in this study. Plasma iron was determined as previously described E.coli O111:B4; 2 mg/kg). Control mice were injected with sterile saline solution. Mice were sacrificed 6 hours later. Results are presented for a series of n = 3 animals per group and has been reproduced at least twice on different set of animals.Inflammation was induced by intra-peritoneal injection of LPS . P values less than .05 were considered statistically significant.Statistical analysis was performed using Student To study the LPS response in Hepc−/− mice, we first determined whether the acute phase response to LPS was altered in these mutant mice. For that, we measured the mRNA levels of the pro-inflammatory cytokines Tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL6) that are induced in response to LPS to trigger the expression of acute phase genes. Regardless the genotype, expression of TNF-α and IL6 was dramatically increased in response to LPS in both liver and spleen . SimilarThe effect of LPS on ferroportin level was analyzed in the two critical sites contributing to circulating iron, the duodenum (site of dietary iron absorption) and the spleen (containing tissue macrophages dedicated to iron recycling from hemoglobin catabolism). After 6h of LPS treatment, ferroportin mRNA levels were found decreased in both tissues, regardless the genotype . In the Second, our results establish that in the duodenum, ferroportin protein levels seem highly sensitive to acute inflammation. In this tissue, hepcidin deficiency is unable to counteract the inflammatory signals, i.e. to upregulate ferroportin levels, suggesting that the inflammatory signals are dominant over the lack of hepcidin in the regulation of ferroportin levels. In contrast, the amount of membrane ferroportin protein in the spleen is dependent on hepcidin in the setting of acute inflammation, hepcidin deficiency leading to reduce ferroportin degradation as compared to WT mice (i.e. acute suppression of ferroportin protein by LPS being efficient only in WT mice and not in Hepc−/− mice).The next step was to evaluate the physiological consequence of hepcidin/ferroportin changes after LPS treatment in plasma iron content. The moderate (spleen) and drastic reduction of ferroportin (duodenum) proteins in the WT treated mice yielded a 75% decrease of plasma iron . In the b, and DMT1, divalent metal transporter 1 , increased , or decreased  by LPS treatment in WT mice followed exactly the same trend in Hepc−/− mice . Indeed, in the BMP6 KO mice presenting, similarly to the iron deficient mice et al. in 2005 who showed, as reported Why hepcidin does not increase in heterozygote Hepc+/− mice after LPS injection is not yet clear. One explanation might be the mild but highly significant 1.5-fold increase in liver iron measured in these heterozygote mice . Indeed, recent evidence suggested that the amount of cellular hepatic iron could influence the response of the hepcidin gene to LPS. Villarroel To conclude, our study shows that the development of hypoferremia during acute inflammation involve both hepcidin-dependent and -independent mechanisms. The rapid and coordinate decrease of ferroportin expression .(TIFF)Click here for additional data file.Figure S2Relative hepcidin1 mRNA levels in the liver of WT and Hepc−/− mice treated with PBS or LPS. Error bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols (*p< 0.05).(TIFF)Click here for additional data file.Figure S3Relative mRNA levels of ferroportin A (FpnA) and ferroportin B (FpnB) in the duodenum and spleen of WT and Hepc−/− mice treated with PBS or LPS. Error bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols .(TIFF)Click here for additional data file.Figure S4Relative lipocalin-2 (Lcn2) mRNA and protein levels in the liver of WT and Hepc−/− mice treated with PBS or LPS. rror bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols . For the Western blot, cytosolic proteins (20 µg/lane) were separated on SDS-PAGE, electro-transferred onto nitrocellulose membrane and analyzed with anti-Lcn2 antibody.(TIFF)Click here for additional data file.Figure S5Relative liver hepcidin1 mRNA levels and plasma iron in Hepc+/− mice treated with PBS or LPS. rror bars represent SD for n = 3 mice in each group. Statistical significance is indicated by * symbols (***p< 0.0005).(TIFF)Click here for additional data file.Table S1Primers sequences used for Real-time PCR.(DOCX)Click here for additional data file.


Tgm4 knockout males show normal sperm count, sperm motility, and reproductive morphology. However, very little of their ejaculate migrates into the female's reproductive tract, suggesting the plug prevents ejaculate leakage. Poor ejaculate migration leads to a reduction in the proportion of oocytes fertilized. However, Tgm4 knockout males fertilized between 3–11 oocytes, which should be adequate for a normal litter. Nevertheless, females mated to Tgm4 knockout males for approximately 14 days were significantly less likely to give birth to a litter compared to females mated to wild-type males. Therefore, it appears that the plug also affects post-fertilization events such as implantation and/or gestation. This study shows that a gene influencing the viscosity of seminal fluid has a major influence on male fertility.Seminal fluid proteins affect fertility at multiple stages in reproduction. In many species, a male's ejaculate coagulates to form a copulatory plug. Although taxonomically widespread, the molecular details of plug formation remain poorly understood, limiting our ability to manipulate the structure and understand its role in reproduction. Here I show that male mice knockouts for  transglutaminase IV cannot form a plug and suffer severe fertility defects. In spite of normal reproductive morphology, less of the ejaculate migrates through the female's reproductive tract and Tgm4 knockout males sire significantly fewer litters than wild type. This study demonstrates that the copulatory plug and/or Tgm4 itself is necessary for normal fertility.Male reproductive fitness is strongly affected by seminal fluid. In many animals, the male's ejaculate coagulates in the female's reproductive tract to form a structure known as the copulatory plug. Here, I show that male mice without a functional copy of the gene  The non-sperm component of an ejaculate can have large effects on male reproductive fitness. In internally fertilizing species, seminal proteins can modify female receptivity in vitro upon contact with extract from the anterior lobe of the prostate  in vitrotransglutaminase IV (Tgm4), was later characterized from humans Since the first published observation of a copulatory plug in a rodent nearly 165 years ago In spite of these early advances, it remains unknown whether Tgm4 is necessary for the formation of the copulatory plug. It has been suggested that some seminal vesicle proteins self-coagulate in the absence of Tgm4 More fully characterizing the biochemical basis of seminal fluid coagulation is critical for understanding the function of the copulatory plug. Early attempts to study copulatory plug function necessarily relied on surgical removal of male accessory glands Tgm4 knockout mice to better understand the molecular basis and functional importance of the copulatory plug, and report two main findings. First, Tgm4 knockout males failed to produce a copulatory plug after mating, demonstrating for the first time that this gene is necessary for the coagulation of seminal fluid in mice. Tgm4 knockout males therefore provide a powerful model to investigate the function of the copulatory plug. Second, in spite of normal sperm count, sperm motility, and reproductive morphology, Tgm4 knockout males sired significantly fewer litters than their wild type brothers. Analyses presented below suggest Tgm4 knockout males suffer fertility defects at two important stages: 1) less of their ejaculate migrates into the female's reproductive tract, and 2) females mated to Tgm4 knockout males produce significantly fewer litters even though a “normal” absolute number of oocytes were fertilized, suggesting additional defects in implantation and/or gestation. This study demonstrates that a gene influencing the viscosity of semen has major affects on male reproductive success.Here, I use Tgm4.Heterozygous “knockout first” mice were acquired from the Knockout Mouse Project see . HeterozTgm4 knockout males  did not form a copulatory plug , complete dissection of each female's reproductive tract failed to yield a copulatory plug or plug-like structure  .In the absence of a plug, the ejaculates of al fluid . In contssection . The difTgm4 knockout males. Sperm count was not statistically different between wild type (N = 19) vs. Tgm4 knockout (N = 10) males , nor was sperm motility . From these same males, testis and seminal vesicle weight were analyzed in a full factorial ANCOVA to account for the potential covariation with body weight. There was not a significant difference in testis weight between Tgm4 knockout vs. wild type males , nor was there a genotype×body weight interaction effect on testis weight . Similarly, there was no difference in seminal vesicle weight between genotypes , nor was there a genotype×body weight interaction effect on seminal vesicle weight . Furthermore, Tgm4 knockout males successfully copulated at a rate similar to wild type; from the 3-hour pairings, 16/26 to wild type, and 13/28 pairings to Tgm4 knockout males succeeded . Therefore, both genotypes display normal copulatory behavior.The defect in ejaculate migration cannot be explained by defects in reproductive morphology of Tgm4 knockout males fertilized significantly fewer oocytes in 20-hour assays. Among successful 20-hour pairings, Tgm4 knockout males fertilized 45 of 122 oocytes dissected from the female's oviducts (36.9%), compared to wild type males, which fertilized 153 of 231 oocytes (66.2%) , significantly lower than 12 females successfully mated to wild type  . The number of females analyzed (7 mated to Tgm4 knockout and 12 mated to wild type) does not add up to the numbers in Tgm4 knockout and 15 mated to wild type) because scorable oocytes were not always recovered from oviduct dissections. It should be noted that even though Tgm4 knockout males fertilized a lower proportion of oocytes compared to wild type, they always fertilized at least 3 oocytes , suggesting they should be able to impregnate females without difficulty.As might be expected from the reduced number of sperm that make it into the female's uterus,  (66.2%) . FertiliTgm4 knockout males sired significantly fewer litters than wild type  , and 14/106 (13.2%) resulted in 3 or fewer pups . In other words, Tgm4 knockout males sired significantly fewer litters in spite of the fact that they appeared to fertilize enough oocytes for a healthy litter.In contrast to this prediction, after being paired with females for 10–14 days, ild type . Of 30 p= 0.005) . Among aTgm4 knockout males sired significantly fewer litters , compared to 106 of 135 (79%) litters born to a wild type male . Sometimes litters do not reach weaning age because of maternal neglect. Furthermore, the number of offspring weaned per litter was not significantly different among the two male genotypes , nor was weanling weight . The lack of statistical significance may be due to small sample sizes, but suggests that once litters are born, the pups have an equal chance of reaching healthy weaning age regardless of sire genotype.Although  litters , there wTgm4 knockout males failed to produce a copulatory plug, demonstrating for the first time that this gene is necessary for plug formation. In spite of normal sperm count, motility and reproductive morphology, Tgm4 knockout males suffered reduced fertility, most importantly in the significant reduction of litters born compared to wild type. Taking all the data into consideration, a model of the copulatory plug acting at two important stages of reproduction seems to explain the fertility defects of Tgm4 knockout males. First, the plug may facilitate passage of the ejaculate through the cervix and into the uterine horns and oviducts , suggesting that it has a limited biological role. Third, although transglutaminases may alter the sperm surface in vitroTgm4 has accumulated multiple loss-of-function mutations in some species that do not form a plug Four observations suggest that the fertility defects observed in the current study arose from the absence of the copulatory plug rather than from additional pleiotropic functions of Although the present study demonstrates the importance of the copulatory plug in non-competitive matings, it does not reject the hypothesis that the copulatory plug evolved in response to sperm competition Peromyscus polionotus forms a plug Interestingly, copulatory plugs do not always bias fertilizations towards the first male to mate in one-female-two-male mating experiments Tgm4 mRNA, including 120 missense mutations (www.ensembl.org version 69), and Tgm4 was not detected in all ejaculates of five humans Tgm4 associated with differences in human male fertility.Many human seminal fluid proteins have orthologs in mouse ejaculates, including Tgm4 All mouse husbandry techniques, experimental methods, and personnel involved were approved by the University of Southern California's Institute for Animal Care and Use Committee, protocols #11394 and #11777.Tgm4 knockout mouse model was constructed by the multi-institutional Knockout Mouse Project The All experimental males used in this study had 6N parents that were heterozygous for the knockout (KO) and wild type (+) allele. When possible, all three genotypes were taken from the same litter to control for simple maternal effects.Sires and dams were paired for one to two weeks, then separated so the dam gave birth in isolation. Between 21–28 days after birth, males were weaned in groups until genotyping, at which point they were separated into their own cages to avoid dominance interactions between brothers. Sexually mature males show reduced fertility when grouped together, presumably as a result of dominance interactions 5′-AGGTGAAAAACCAAGAAATACCATC-3′ and 5′-CTATTCCAAAACCACCAGACAGTAC-3′) amplified a 704 bp fragment and Reaction 2 primers (GTGGACAGATATTCACTCTGAAGGT and GGAAACACCAATAGAAAAGTGAGTC) amplified a 1,170 bp fragment. Two PCR reactions specifically amplified the knockout first allele: Reaction 3 primers (GCTTTACATGTGTTTAGTCGAGGTT and GTTAAAGTTGTTCTGCTTCATCAGC) amplified a 1,244 bp fragment and Reaction 4 primers (GATTAAATATGATGAAAACGGCAAC and ATTATTTTTGACACCAGACCAACTG) amplified a 1,349 bp fragment. DNA was amplified using 35 cycles of denaturation , annealing  and extension . All PCR reactions used Fermentas 2× PCR premix. Presence/absence of bands was scored on agarose gels. Only genotypes consistent across all four reactions were included in experiments.Shortly after weaning, ear snips were taken for PCR-based genotyping. DNA isolated from ear snips was genotyped with four PCR reactions. Two PCR reactions specifically amplified the wild type allele: Reaction 1 primers , ∼28 day-old females were induced to ovulate using standard techniques −11), mean values were used for subsequent statistical analyses.Twelve hours after administration of hCG, males and females were paired, left together for three hours, and then females were removed and sacrificed. Female reproductive tracts, extending from the vagina through the cervix and uterine horns, were dissected and examined under a dissecting microscope to assess i) the presence/absence of sperm, ii) the presence/absence of a copulatory plug, and iii) the number of oocytes with two pronuclei, a sign of recent fertilization [following 94]. Two different researchers who were blind to genotype measured uterine horns digitally, from a subset of females. Since their measurements were significantly correlated  or customized Python scripts (www.python.org).Testis and seminal vesicle weight were each analyzed in a full factorial ANCOVA using male genotype (knockout vs. wild type) and body weight as factors. An ANCOVA was employed to account for the potential covariation of testis or seminal vesicle weight with body weight. To test for differences in the number of litters born to


A recent meta-analysis suggested an association between exposure to paternal smoking during pregnancy and childhood brain tumor risk, but no studies have evaluated whether this association differs by polymorphisms in genes that metabolize tobacco-smoke chemicals.We assessed 9 functional polymorphisms in 6 genes that affect the metabolism of polycyclic aromatic hydrocarbons (PAH) to evaluate potential interactions with parental smoking during pregnancy in a population-based case-control study of childhood brain tumors. Cases (N = 202) were ≤10 years old, diagnosed from 1984–1991 and identified in three Surveillance, Epidemiology, and End Results (SEER) registries in the western U.S. Controls in the same regions (N = 286) were frequency matched by age, sex, and study center. DNA for genotyping was obtained from archived newborn dried blood spots.EPHX1 H139R, and childhood brain tumors , such that children with the high-risk (greater PAH activation) genotype were at a higher risk of brain tumors relative to children with the low-risk genotype when exposed to tobacco smoke during pregnancy. A dose-response pattern for paternal smoking was observed among children with the EPHX1 H139R high-risk genotype only .We found positive interaction odds ratios (ORs) for both maternal and paternal smoking during pregnancy, Parental smoking during pregnancy may be a risk factor for childhood brain tumors among genetically susceptible children who more rapidly activate PAH in tobacco smoke. The association between parental smoking during pregnancy and risk of childhood brain tumors is inconsistent in the literature. Most studies have reported positive associations between paternal smoking during pregnancy and childhood brain tumor risk, although the findings from only three studies were statistically significant Studies examining the association between maternal smoking during pregnancy and childhood brain tumors generally suggest little to no increased risk. Ten studies reported no association EPHX1, detoxifies selected substances , and activates others, including PAH EPHX1 alter enzyme activity through amino acid changes EPHX1 Y113H results in decreased mEH activity, whereas a variant leading to an arginine (R) substitution of a histidine (H) at H139R results in increased mEH activity Several genes are associated with the activation (transformation to more carcinogenic intermediates) or detoxification of PAH. We focused on 6 genes of potential importance to our analysis of parental smoking (PAH exposure) and childhood brain tumors . MicrosoMPO) and sulfotransferase (SULT1A1) also activate carcinogens found in tobacco smoke, including PAHs. Variations in genotype at MPO G-463A SULT1A1 R213H NQO1), and glutathione S-transferases (including GSTM1 and GSTP1) detoxify PAHs. Variant alleles at NQO1 (P187S) GSTP1 I105V and GSTP1 A114V GSTM1Myeloperoxidase   codes 191.0–192.1] between 1984–1991. Controls living in the same regions were identified using random digit dialing, and were frequency matched to cases by age, sex, and study center. This analysis includes children born in Washington State in 1978 or later, or in California in 1982 or later, the birth years for which a specimen could still remain in the state archives. Children meeting these criteria were ≤10 years old. Specimens were obtained for 93% of eligible cases and 83% of eligible controls, as detailed elsewhere Participants were cases and controls enrolled in the West Coast Childhood Brain Tumor study Parental smoking was assessed by in-person interview with the subjects’ mothers. Mothers were asked if they ever smoked tobacco during their pregnancy with the enrolled child (yes/no), and the number of cigarettes smoked per day or week. They also were asked whether there was regular tobacco smoke exposure during pregnancy  from the child’s father in the home, from any other household resident, or at work. Maternal exposure to tobacco smoke from the child’s father during pregnancy will be hereafter referred to as “paternal smoking.” Mothers and fathers also were asked if they ever smoked at least once a day for 3 months or longer prior to the pregnancy with the participating child (yes/no).Maternal smoking during pregnancy was categorized by the typical number of cigarettes smoked per day: never smoked, 1–10, or 11+ cigarettes. Paternal smoking during pregnancy was categorized by the median number of hours per day the mother was exposed to tobacco smoke from the father .EPHX1 H139R (rs2234922), EPHX1 Y113H (rs1051740), and EPHX1 C-613T (rs2854448), SULT1A1 R213H (rs9282861), NQO1 P187S (rs1800566), GSTP1 I105V (rs1695), GSTP1 A114V (rs1138272), and rs2243828 (in complete linkage disequilibrium with MPO G-463A (rs2333227)). Microsomal epoxide hydrolase (mEH) activity was computed using EPHX1 H139R and Y113H polymorphisms: low activity–0,1, or 2 stable alleles at H139R/Y113H , or high activity–3 or 4 stable alleles . One multiplex PCR-based assay P>0.01) for all genotype frequencies for controls when stratified by race/ethnicity, with the exception of EPHX1 Y113H for Los Angeles non-Hispanic Whites (P<0.0001), and for NQ01 P187S for the heterogeneous ‘Other’ ethnicity (P = 0.0003).Subjects’ DNA was extracted from dried blood spot specimens from neonatal screening archives in California and Washington using the QIAamp DNA Mini Kit  at the Center for Ecogenetics and Environmental Health Functional Genomics Laboratory at the University of Washington . Custom TaqMan Detection System-based assays-by-Design Service  were used to assess a priori with the expectation that maternal education is associated both with maternal or paternal smoking and childhood brain tumors. A parallel set of models were additionally adjusted for spousal smoking. Formal tests of interaction were conducted using a product term in each model. Case-only analyses were conducted after confirming independence of each gene-smoking association among controls. Consistencies of all associations were further evaluated by race/ethnicity (non-Hispanic White or Hispanic). Polytomous logistic regression was used to evaluate whether gene-environment interactions differed by histological tumor type ; formal tests of heterogeneity were conducted. Tests for trend in dose analyses were evaluated using a 1df test for the categorized dose variable. Due to a priori hypotheses regarding the suspected functionality of the tested polymorphisms in the metabolism of tobacco smoke, no corrections for multiple comparisons were made. All reported P-values are two-sided.We used unconditional logistic regression to evaluate the primary associations and potential interaction of genotype at each locus with maternal and/or paternal smoking during pregnancy. Odds ratios (ORs) and 95% confidence intervals (CIs) were computed to estimate relative risks. For main associations and interaction analyses, genotypes were dichotomized and classified as low- or high-risk based on the ability of each variant to increase or decrease the activation or detoxification of PAHs . All modInstitutional Review Board approvals were obtained in California from the University of Southern California Institutional Review Board and the Committee for the Protection of Human Subjects at the Health and Human Services Agency of the State of California, and in Washington from the Fred Hutchinson Cancer Research Center and the Washington State Department of Health. Written informed consent for all participants was obtained prior to interview. Before release from neonatal archives in both California and Washington, all dried blood-spot specimens were anonymized by the assignment of a random specimen identification number that could not be linked to identifying information.P = 0.02), and control mothers were more likely to have a college or graduate degree .Cases and controls were similar with regard to frequency-matched variables . A higheThe ORs for childhood brain tumors in relation to maternal smoking during pregnancy were less than one, but not statistically significant . One excWe observed a statistically non-significant increased OR associated with paternal smoking during pregnancy . Exposure to paternal smoking for >3 hours per day, vs. no exposure, was positively associated with childhood brain tumors . The OR for smoking by both parents during pregnancy was consistent with no association (data not shown). Results were similar when examined by histology (data not shown). No association was observed for maternal exposure to tobacco smoke from other household residents. However, the number of mothers reporting exposure from other household members during pregnancy was small .We modeled the direct genotype-childhood brain tumor association using ‘low-risk’ or ‘high-risk’ genotypes see . No polyEPHX1 H139R , whereas there was little observed association in children with a low-risk genotype (HH) . The case-only analysis showed a similar association . Results were comparable when log-additive models were evaluated (data not shown). Other potential interactions were either statistically non-significant  or did not manifest in a biologically plausible manner (e.g. GSTP1 A114V) . Results were similar when examined by histology (data not shown).When we examined the association between maternal and paternal smoking (never/ever during pregnancy) and childhood brain tumor risk, by ‘low-risk’ or ‘high-risk’ genotype, we found a positive interaction OR for paternal smoking and 14V) see . We obseEPHX1 H139R . A similar interaction was observed for mEH activity . The findings were supported by case-only analyses .As with paternal smoking, we observed an interaction between maternal smoking and EPHX1 H139R . A similar association was seen for SULT1A1 R213H, although the interaction did not reach statistical significance. Among children with a high-risk genotype (RR), children exposed to >3 hours per day of smoke from the father were 2.57 times as likely as unexposed children to develop a childhood brain tumor . This association was greater after adjusting for maternal smoking during pregnancy . No increased risk was observed among children with a low-risk genotype . Adjustment for maternal smoking had minimal effects on remaining polymorphisms. A suggestion of increasing ORs among carriers of high-risk genotypes also was observed by duration of exposure for EPHX1 Y113H and mEH activity .A positive association between hours per day of exposure to paternal smoking during pregnancy and childhood brain tumor risk was observed only among children with a high-risk genotype (HR or RR) for vity see , and forEPHX1 H139R genotype . Among children with a high-risk variant (RR or HR) for EPHX1 H139R, children whose mothers smoked 11 or more cigarettes per day were twice as likely to develop a childhood brain tumor as children of mothers who did not smoke , however, the number of children exposed to high levels of maternal smoking was low. Among children with a low-risk genotype, there was no increase in childhood brain tumor risk observed in relation to smoking. Results were similar for high mEH activity. Effect estimates decreased slightly after adjustment for paternal smoking.Similar to the paternal smoking data, a statistically significant interaction was observed for level of maternal smoking during pregnancy and EPHX1 H139R genotype for children exposed to paternal or maternal tobacco smoke during pregnancy. The pattern of increased risk associated with exposure to tobacco smoke for children with a high-risk genotype, in contrast to those with a low-risk genotype, persists for children exposed to either maternal or paternal smoking, as evidenced by the parallel interactions presented. Similar patterns were observed for mEH activity.EPHX1 H139R and both maternal and paternal smoking overall (never/ever) and by level of exposure. Our results suggest that childhood brain tumor risk may be associated with exposure to parental smoking during pregnancy for children with genetic susceptibility to carcinogenic PAHs present in tobacco smoke.Our study expands on previous studies by evaluating the modifying effect of selected genetic polymorphisms involved in the metabolism of carcinogens present in tobacco smoke. We identified biologically plausible interactions between EPHX1 results in increased mEH activity EPHX1 is expressed in the brain and during the fetal period mEH is considered a detoxification enzyme for many substrates. However, in the process of PAH detoxification, carcinogenic highly-activated intermediates may be generated. mEH metabolizes PAHs to bay region diol-epoxides EPHX1 H139R and mEH activity. In our primary associations analysis, exposure to maternal smoking during pregnancy resulted in an OR <1, whereas exposure to paternal smoking was positively associated with childhood brain tumors. However, interaction ORs for childhood brain tumors, EPHX1 H139R and parental smoking were above null for both maternal smoking and paternal smoking  and childhood brain tumors are likely related to one of two different explanations. First, the data on maternal smoking during pregnancy may be subject to maternal reporting bias. If mothers of cases were more likely than mothers of controls to underreport smoking, an artificially low association could result. Second, a similar bias could have occurred if among smokers we contacted, mothers of cases were less likely than mothers of controls to participate in the study. The occurrence of ORs <1 for maternal smoking during pregnancy, especially more recently when smoking has become less socially acceptable, is consistent with either possible source of bias. Although these factors may have biased the maternal smoking-childhood brain tumor association downward, they are unlikely to account for the observed interactions. Gene-environment interactions are largely unaffected by selection bias EPHX1 H139R genotype. Our data suggest that children with a high-risk genotype are at a greater risk of childhood brain tumors if exposed to either maternal or paternal smoking during pregnancy, relative to children with a low-risk genotype and similar exposures. This may indicate that PAH activation increases risk regardless of the source of parental exposure.The differences in maternal vs. paternal smoking ORs may be due to true biological differences in these associations with childhood brain tumor risk. However, if this were the case, we might have expected to observe dissimilar interaction ORs for maternal and paternal smoking with respect to 8dG (an oxidative product of DNA damage) The carcinogenic process may be initiated through maternal exposure to environmental tobacco smoke from the father, or through the sperm, as a result of paternal smoking shortly before the child’s conception. Although our primary results focused on paternal smoking during pregnancy, we observed similar interaction ORs for paternal smoking prior to pregnancy. Paternal smoking may induce genotoxic effects on sperm; studies of male smokers have demonstrated greater levels of oxoBoth strengths and limitations of this analysis need to be considered in the interpretation of the data. Although this is a relatively large population-based study of childhood brain tumors with comprehensive ascertainment of cases and highly comparable population-based controls, our sample is small for gene-environment interaction analyses. Therefore, these findings could be explained by chance. We also focused on polymorphisms from a small number of candidate genes relevant to PAH specifically. We did not explore other genes associated with metabolism of other potential carcinogens in tobacco smoke and therefore may have missed some important interactions. We did not have DNA or genotype data for mothers, which during the pregnancy could influence PAH metabolism in combination with the child’s genotype. However, to our knowledge this is the first assessment of these interactions. Moreover, use of archival dried blood spots allowed inclusion of all cases regardless of survival status, therefore minimizing survival bias that may be problematic in case-control studies of highly fatal diseases.Our study supports previous findings that parental smoking may be a risk factor for childhood brain tumors, and provides new information that risk may vary by genetic susceptibility. Studies that have reported no association may have been limited by inaccurate self-report of maternal smoking, and a lack of data on the genetic susceptibility of children in the study. Future studies of childhood brain tumors and parental smoking should include biological markers of smoking, in addition to data on the genetic susceptibility of children to tobacco smoke, to confirm and extend the results reported here.File S1Table S1. Risk of childhood brain tumors in relation to polycyclic aromatic hydrocarbon (PAH) metabolism polymorphisms, West Coast Childhood Brain Tumor Study, N = 479. Table S2. Association between exposure to prenatal parental smoking and selected polymorphisms in a case-only analysis, West Coast Childhood Brain Tumor Study, N = 196. Table S3. Risk of childhood brain tumors in relation to maternal smoking level during pregnancy by polymorphisms in selected genes, West Coast Childhood Brain Tumor Study.(DOCX)Click here for additional data file.