Asterionella formosa to one genotype of its naturally co-occurring chytrid parasite Zygorhizidium planktonicum across five environmentally relevant temperatures. We found that the thermal tolerance range of the tested parasite genotype was narrower than that of its host, providing the host with a “cold” and “hot” thermal refuge of very low or no infection. Susceptibility to disease was host genotype-specific and varied with temperature level so that no genotype was most or least resistant across all temperatures. This suggests a role of thermal variation in the maintenance of diversity in disease related traits in this phytoplankton host. The duration and intensity of chytrid parasite pressure on host populations is likely to be affected by the projected changes in temperature patterns due to climate warming both through altering temperature dependent disease susceptibility of the host and, potentially, through en- or disabling thermal host refugia. This, in turn may affect the selective strength of the parasite on the genetic architecture of the host population.The cost of parasitism often depends on environmental conditions and host identity. Therefore, variation in the biotic and abiotic environment can have repercussions on both, species-level host-parasite interaction patterns but also on host genotype-specific susceptibility to disease. We exposed seven genetically different but concurrent strains of the diatom  Parasitism is one of the most common consumer strategies Batrachochytriumdendrobatidis is the causative agent of amphibian chytridiomycosis, one of the main drivers of worldwide population declines in amphibians Asterionella formosa Hassall and its two chytrid parasites: Zygorhizidium planktonicum Canter and Rhizophydium planktonicum Canter emend Asterionella often is a prominent contributor to the diatom spring bloom in lakes worldwide. Its blooms are frequently followed by chytrid epidemics with prevalence of infection exceeding 90% in many cases Asterionella spring-blooms depends on water temperatures in early spring as Asterionella already reproduces at temperatures below 3°C, while the parasite is still inactive Temperature is probably the most pervasive environmental variable and influences the metabolic rates of all organisms Asterionella formosa susceptibility to chytrid infection, we performed an infection experiment using seven concurrent genotypes of the diatom host exposed to a single genotype of its chytrid parasite Zygorhizidium planktonicum across a range of environmentally relevant temperatures. We assessed host and parasite thermal tolerance range and optima for activereproduction. Furthermore, we tested for host genotype (G) and temperature (T) main effects and for host genotype-by-temperature (GxT) interactions in net production of host and parasite. As the host genotypes also showed temperature-dependent differences in cell-size, we checked the extent of co-linearity of host cell size and genotype effects on host susceptibility to disease.Species- and genotype-level host-parasite interaction patterns are expected to depend on their environmental conditions, in particular on their temperature environment. In order to test thermal reaction norms of Asterionella formosa is a pennate diatom that forms uniclonal, stellate colonies. It is a characteristic spring-bloom diatom of temperate lakes Asterionella blooms are often followed by chytrid epidemics exceeding 90% prevalence of infection AsterionellaZygorhizidium planktonicum is an obligate and highly virulent parasite of the diatoms Asterionella formosa and Synedraacus Kützing Asterionella spring blooms to a swift end and can therefore affect the phytoplankton succession in lakes Zygorhizidium forms thick-walled resting spores which are inactive and allow the parasite to weather adverse periods The chytrid parasite Asterionella formosa genotypes used in the experiment were isolated from a single water sample taken during the 2008 Asterionella spring-bloom at 5 m depth in Lake Maarsseveen. Host culture establishment was fairly unbiased with a larger than 95% success rate. As all cells of an Asterionella colony are the asexual offspring of a founding cell, isolating single colonies is an easy way to obtain uniclonal cultures of this diatom. Individual isolates were grown in batch culture on CHU-10 medium 4 and FeCl3. For genetic fingerprinting, 50mL of dense culture were centrifuged, and the DNA of the remaining pellet was extracted by a modified QiagenDNeasy Plant Mini Kit  protocol (see Asterionella formosa as well as of one Fragilariacrotonensis genotype (functioning as out-group) was performed by Keygene® , details of the AFLP data analysis are presented in the All Zygorhizidium planktonicum cultures from the same bloom/epidemic occurred by transfer of infected Asterionella colonies carrying only one single sporangium into a uniclonal culture of Asterionella S122 . Establishing parasite cultures was less successful with only 20 infected cultures out of over 400 isolation attempts. This lower success may suggest, that, by baiting the parasite with a uniclonal host culture, we actually screened for parasite genotypes able to infect this specific host genotype. Hence, our collection of parasite isolates may well represent a limited range of the genetic variation present in Lake Maarsseveen. Since we used only one uniclonal isolate of the parasite, we did not assess the genetic diversity of our isolate collection. Host and parasite cultures were maintained in semi-continuous batch cultures in environment test chambers  at 18°C±1°C and 14: 10 h light:dark cycle at 50 µmol quanta s–1 m–2 provided by cool-white fluorescent lamps . All cultures were uniclonal but had slight bacterial contaminations.Isolation of uniclonal Asterionella genotypes  exposed to one Zygorhizidium genotype (F12) at five different temperatures  in five replicates. This resulted in 35 experimental combinations and 175 experimental units. To compare the performance of Asterionella in parasite exposed and non-exposed populations, non-exposed controls of the seven Asterionella genotypes were grown at the same experimental temperatures resulting in 35 control units (one per temperature-host strain combination).The experiment employed a full-factorial design with seven –2 s–1 provided by cool-white fluorescent lamps (TL-D 30W/830) at a 14: 10 light: dark cycle. Photosynthesis-by-irradiance curves  showed that this irradiance level was saturating but not inhibiting.For temperature acclimation, parasite exposed and non-exposed stock cultures of each host genotype were split into five subcultures and stepwise acclimated in semi-continuous batch cultures in temperature-controlled water baths for at least five generations prior to the experiment –1 into a total volume of 60 mL CHU-10 medium –1 from a nearly 100% infected, temperature- and host genotype-matching exposed subculture. Based on results of pre-experiment trials, the host inoculum of ca 15 000 cells mL–1 was small enough to ensure that the culture medium could support several generations of unlimited host growth before light or nutrient availability could become limiting in the batch set up, but also large enough to support the infection. Similarly, the parasite inoculum of ca 5 000 live infection carrying cells mL–1  was large enough to follow both increase and decrease in prevalence over time. All experimental and control units were started on the same day. Each unit was shaken manually twice and their position within the water bath was randomized once each day. Samples for microscopy enumeration were taken every second day for the temperatures 6°C to 21°C and every fifth day for temperature 1°C. All samples were taken at the same moment in the light cycle, fixed with a Glutaraldehyde-Formaldehyde mixture  and stored cool and dark.To start the experiment, each experimental and control unit was inoculated from the corresponding non-exposed subculture to a starting concentration of ca. 15 000 uninfected host cells mLAsterionella cells or 20 fields of view were counted in a 1 mL sample under an inverted microscope  according to the Utermöhl settling method –1 (uninf); (ii) infected host cells carrying one or more living infection(s) mL–1 (inf); (iii) infected host cells carrying only dehisced / dead infection(s) mL–1; (iv) sporangia mL–1, and (v) resting spores mL–1. Infection prevalence, i.e. proportion of cells carrying live infections in the live host population, was calculated as inf/(uninf+inf).Asterionella cells carrying empty or dead sporangia, i.e. class (iii), were excluded from the calculation of infection prevalence as they did not contribute further to population growth of the host or the parasite.A minimum of 200 –1 for uninf, inf, the combined uninf+inf and for parasite sporangia in the experimental units, were averaged over all host genotypes and plotted against temperature. The host genotype-specific rate of change day–1 of each variable was calculated asx is the abundance of either uninfected or infected host cells and parasite sporangia on the last day of the experiment (endx) and the first day of the experiment (startx) respectively. Furthermore, tott is the total number of experimental days.To assess the thermal ranges of hosts and parasites, the rate of change dayP uninf) and infected (P inf) host cells mL–1, net production of parasite sporangia mL–1 (P spor), net increase/decrease in infection prevalence (P prev) and the infection related percentaged reduction in production of uninf cells (% reduction). The net production (P x) variables were calculated asTo work with comparable experimental times and include at least three samples, the first ten days for temperature treatments 1°C to 16°C were used in the statistical analysis. For treatment 21°C only the first six days were included as the infection had cleared and the host population was approaching carrying capacity, hence analysing a longer interval would have resulted in underestimating host production. The response variables were the net production of uninfected . All statistical analyses and plot graphing were carried out in R Within our wide experimental temperature spectrum, all response variables showed non-linear relationships with the explanatory variable temperature. To allow for these non-linear relationships a generalised additive model (GAM) Asterionella formosa and one Fragilariacrotonensis yielded 113 marker bands, of which 69% were polymorph. Each uniclonal culture showed a unique fingerprint pattern and therefore represented a unique genotype. The UPGMA dendrogram (Fragilariacrotonensis as a clear out-group and clustered the Asterionella formosa genotypes in broadly two clusters of three and five genotypes. The dendrogram was a good representation of the Jaccard's similarity matrix as the cophenetic correlation coefficient was r = 0.98 (Mantel test P = 0.001). Most of the nodes were supported well as shown by bootstrap resampling (n = 5000) results  in experimental units) and parasite sporangia abundance  were averaged across host genotypes and plotted against temperature treatment  and host genotype (G), as well as genotype-by-temperature interaction (GxT) effects (Table S1):The most parsimonious GAM model included main effects of temperature (f (based on thin plate regression splines), a Gaussian error distribution εi, and a link function by identity. Main and interaction effects were significant for each of the four response variables: net production of uninfected . The formation of resting spores at 1°C resulted in a loss of prevalence (i.e. negative P prev) over time at 1°C, while the loss of prevalence at 21°C was caused by the death of the parasite population (P prev at intermediate temperatures (11 and 16°C) suggested that parasite production rates surpassed those of the host. This was also reflected in the% reduction patterns (% reduction of uninfected cells) and lowest at the two temperature extremes. Negative values in % reduction indicated that the production of uninfected cells in the experimental units surpassed that in the controls.The highest  at 21°C  and 4B. pulation  and 4C. patterns  and 4D. To exclude that we confounded genotype effects with host cell bio-volume effects on host susceptibility and parasite productivity, we checked for the respective explanatory power of predictors a) host cell bio-volume and b) genotype using ANOVA models , which may have consequences in the potential for host-parasite co-evolution. In any thermal refugium, the host population is freed of parasite mediated selection but experiences abiotic selection pressures. If host genotypes show different performance ranking orders under abiotic stress than under parasite pressure, then selection in the thermal refugia may also favour a different set of host genotypes, disrupt any directional selection for increased parasite resistance in the host population, and cause the host population to lose, to some extent, any pre-adaptation to the parasite. This would increase the infection success of the parasite when re-invading from resting stages. Such examples indicate that the mechanisms behind (temporary) disappearance of disease need to be taken into account in theoretical approaches as well as in the management of infectious diseases.Host and chytrid parasite thermal tolerance ranges do not necessarily overlap fully. If the thermal tolerance range of the parasite is narrower than that of its host, the host can benefit from thermal refugia of low or no disease pressure. This seems to be the case in chytrid-File S1Fingerprinting of Asterionellaformosa isolates. Description of the AFLP fingerprinting and the statistical analysis methods used for the assessment of genetic diversity in the experimental Asterionellaformosa genotypes.(DOCX)Click here for additional data file.File S2ANOVA models testing co-linearity between predictors host genotype and host cell-size. To assess whether genotype or host cell-size is the more appropriate predictor for host and parasite productivity, two ANOVA models were compared including either temperature and host genotype  or temperature and host cell-size . Response variables were net production of uninfected cells (see S2.1 and S2.2) and net production of infected cells in parasite exposed cultures (see S2.4 and S2.5).(DOCX)Click here for additional data file.


PIWIL2 and TDRD1. The downstream effects mediated by the epigenetic inactivation of the PIWI pathway genes were a defective production of piRNAs and a hypomethylation of the LINE-1 repetitive sequence in the affected patients. Overall, our data suggest that DNA methylation, at least that affecting PIWIL2/TDRD1, has a role in the control of gene expression in spermatogenesis and its imbalance contributes to an unsuccessful germ cell development that might explain a group of male infertility disorders.Epigenetic changes are involved in a wide range of common human diseases. Although DNA methylation defects are known to be associated with male infertility in mice, their impact on human deficiency of sperm production has yet to be determined. We have assessed the global genomic DNA methylation profiles in human infertile male patients with spermatogenic disorders by using the Infinium Human Methylation27 BeadChip. Three populations were studied: conserved spermatogenesis, spermatogenic failure due to germ cell maturation defects, and Sertoli cell-only syndrome samples. A disease-associated DNA methylation profile, characterized by targeting members of the PIWI-associated RNA (piRNA) processing machinery, was obtained. Bisulfite genomic sequencing and pyrosequencing in a large cohort (n = 46) of samples validated the altered DNA methylation patterns observed in piRNA-processing genes. In particular, male infertility was associated with the promoter hypermethylation-associated silencing of  Genetic etiologies have only been able to explain about 15% of cases of male infertility DNA methylation is an epigenetic process that plays a crucial role in determining the time point and magnitude of gene expression. Unlike the genetic code, the epigenetic code is dynamic and tissue-specific Abnormal sperm DNA methylation of imprinted genes is associated with spermatogenic impairment PIWIL1PIWIL2TDRD1TDRD9It is currently unclear whether DNA methylomes of men with impaired sperm production significantly differ from those presenting a complete and efficient spermatogenic process. To address this matter, we analyzed genome-wide DNA methylation in infertile men with spermatogenic failure. Using the Infinium Human Methylation27 BeadChip technology Our study recruited thirty-two infertile patients (aged 30–49 years) due to severe spermatogenic failure (SpF), with a phenotype consistent with non-obstructive (secretory) azoospermia or severe oligozoospermia (<5 million sperm/ml). Only testicular samples with homogeneous phenotypes were selected on the basis of the histological pattern of >20 tubules from the same testicular section; samples with mixed histological patterns were excluded from the study. In addition, five patients with a Sertoli cell-only syndrome (SCO) phenotype were studied as methylation/gene expression controls of somatic cells and nine infertile patients (aged 32–50 years), who were diagnosed with obstructive azoospermia  and showed conserved spermatogenesis (CS) were studied as methylation/gene expression controls of a complete spermatogenic process . InfertiThe clinical procedures for infertile patients included medical history, physical examination, semen analyses  was fixed in Bouin’s solution and reserved for histological analysis, a second aliquot (≈100–200 mg) processed for sperm extraction, and the third (≈10 mg) was immediately transferred to liquid nitrogen and then stored at –80°C until used for molecular analysis.Fixed testicular biopsies were cut into 5-µm sections and stained with hematoxylin–eosin. Germ cells of the different levels of maturation  and Sertoli cells were quantified, and the average number per tubule was calculated after analysis of at least 15–20 cross-sectioned tubules/testis. Assessment of the spermatogenic status and the severity of the alteration is shown by a modified Johnsen score (JS) Using this strategy we confirmed the diagnosis of SCO and CS phenotypes. With respect to SpF patients, eight of them presented maturation failure at the round spermatid level (rsMF), fifteen at the spermatocyte level (scMF) and nine at the spermatogonia level (sgMF), due to the presence of a diminished number of this specific stage and the subsequent germ cell stages in their tubules compared with CS samples .Semen samples obtained from normozoospermic men were collected and allowed to liquefy for 30 min. Before the standard swim-up separation technique, whole semen was centrifuged on a 25% Percoll gradient (20 minutes) to discard somatic cell contamination, further ensuring the purity of the sperm population. The swim-up procedure results in selection of spermatozoa with good motility.Sperm DNA was extracted with an user-developed version of the QIAamp® DNeasy&Tissue Kit purification protocol (Qiagen). Fresh washed (in PBS) sperm was incubated 1∶1 with a lysis buffer containing 20 mM TrisCl (pH 8), 20 mM EDTA, 200 mM NaCl and 4% SDS, supplemented prior to use with 100 mM DTT and 250 ug/ml Proteinase K. Incubation was performed for 4 hours at 55°C with frequent vortexing. Prior to processing in the columns, 200 ul of absolute ethanol and 200 ul of the kit-provided lysis buffer were added to the samples. Then, purification was performed according to kit instructions.Genomic DNA was extracted from testicular biopsies by using the Wizard Genomic DNA Purification kit . DNA methylation profile was assessed using the Infinium Human Methylation27 BeadChip , which assays DNA methylation levels at 27,578 CpG sites. Briefly, DNA was quantified by Quant-iT™ PicoGreen dsDNA Reagent  and the integrity was analyzed in a 1.3% agarose gel. Bisulfite conversion of 600 ng of each sample, which results in unmethylated cytosines being converted to uracils, whereas methylated cytosines are not converted, was performed according to the manufacturer’s recommendation for the Illumina Infinium Assay. Effective bisulfite conversion was checked for three controls that were converted simultaneously with the samples. The intensities of the images were extracted and normalized using GenomeStudio  software. The methylation score of each CpG was represented as a beta (β) value. The threshold for concluding differential methylation of probes was set at an average delta β value >0.1.Genomic DNA was bisulfite-modified using the EZ DNA Methylation-Gold Kit  according to the manufacturer’s protocol. The methylation status of selected regions was analyzed by bisulfite genomic sequencing. Bisulfite-converted DNA was amplified  and subsAmplification primers  and sequTotal RNA was obtained from the testicular biopsy using the Absolutely RNA Miniprep Kit , according to the instructions provided by the manufacturer. Furthermore, small RNA-containing total RNA was additionally obtained with a mirVana miRNA Isolation Kit (Ambion) from an extra portion of the testicular biopsy whenever this was possible. The quality of RNA was assessed using the Agilent 2100 Bioanalyzer . Testicular RNA samples included in the study had a 28S/18S ratio >1.3 and an RIN value >7.5. Single-stranded cDNA was obtained by reverse transcription (RT) of 500 ng of RNA using random hexamer primers and the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems).PIWIL2: Hs00216263_m1; TDRD1: Hs00229805_m1; PGM1: Hs00160062_m1) and custom-designed small RNA TaqMan Assays (Applied Biosystems). Negative controls without template were included in each set of PCR assays as well as a calibrator sample to compare the change in expression of a nucleic acid sequence against the expression in all samples in the same study. PGM1 was previously selected as an appropriate reference gene among ten candidate genes tested (data not shown) for PIWIL2 and TDRD1 data normalization in our study, showing similar Ct values to the ones obtained from target genes, no statistical differences in expression among groups_ and low M-value  reactions were performed on an ABI 7300 real-time PCR system (Applied Biosystems) using gene-specific TaqMan Assays (−ΔΔCt strategy and stored in SDS 2.1 software (Applied Biosystems). Expression levels are shown as relative quantification (RQ) values.Patient and control group samples were always analyzed as paired samples in the same analytical run in order to exclude between-run variations. Real-time qPCR data were pre-processed using the 2Statistical analyses were performed using SPSS 12.0 software . The nonparametric Mann-Whitney U test was used to analyze differences in absolute expression and methylation level in SpF patient groups compared with controls. Pearson product-moment correlation coefficients were calculated to determine the correlation between the methylation status, expression ratios of the target genes and the various histological parameters in patient groups and controls. A value of p<0.05 was considered significant. Gene Ontology (GO), pathways enrichment analysis was performed using the Database for Annotation, Visualization and Integrated Discovery .http://pirnabank.ibab.ac.in) and aligned (BLAT) to the reference genome. Subsequently, the promoter regions (transcription start site +/−2 kb) of the 580 differentially methylated genes were analyzed for the presence of piRNA complementarity. Promoters with sequence identity of 100% to any piRNA in the data set were regarded as potential regulative target.The complete set of piRNA sequences was obtained from piRNA bank . However, unsupervised hierarchical clustering revealed a distinct methylation profile of the three SpF patient samples that entirely lacked elongated spermatids, unlike the CS controls , reproductive cellular process  and gamete generation   . In partve to CS .PIWIL1/2 and their associated molecules TDRD1/9, were able to cluster normal tissue and patient samples (including sample no.10) separately, suggesting that hypermethylation of these genes is a disease-associated event (It is of particular note that genes involved in the biogenesis of PIWI-associated RNAs (piRNAs), such as the differentially methylated in the array ed event .PIWIL1/2 and TDRD1/9 in three CS samples, six SpF samples (three rsMF and three scMF) and three SCO samples  samples. TDRD9 also exhibited an elevated level of methylation, although we had already detected increased levels in normal tissue. This was even more evident for PIWIL1, for which half of the CpG sites analyzed were found to be methylated in CS samples. As expected, we observed striking differences between CS testis tissue and SCO samples, which is consistent with the germline-specific expression associated with the analyzed genes. Here, hypermethylation was detected in all five genes, probably due to the total absence of germ cells, in SCO specimens.The disruption of genes associated with the piRNA processing machinery has been directly related to spermatogenic failure due to maturation arrest, resulting in male sterility in mouse models. As genome-wide analysis of patient samples with spermatogenic failure identified deregulation of genes involved in piRNA production, we aimed to validate these genes in a larger cohort of samples. To assess the impact of DNA methylation on genes involved in the piRNA processing machinery, we performed bisulfite genomic sequencing of the promoter regions of  samples  and S2. PIWIL2 and TDRD1 in SpF-affected patients, locus-specific pyrosequencing was performed in a larger validation patient cohort. In detail, nine normal CS control, thirty-two SpF  affected patient samples and five SCO specimens were analyzed to detect the CpG site immediately upstream of the transcription start site. To gain a better insight into the tissue specificity of PIWIL2 and TDRD1, we also included five samples from mature swim up-selected spermatozoa and six somatic tissues from colon, breast, blood, skin, lung and brain in the study. As expected, spermatozoa exhibited an absence of DNA methylation in both genes, whereas both promoters were heavily methylated in all somatic tissues  and TDRD1 . We additionally analyzed the germ cell–specific transcript levels per cell in SpF subgroups compared to CS controls in order to exclude the differences in gene expression due to changes in testicular cellularity and to determine whether transcript level per cell is also altered in SpF. Values of transcript amount per cell, in arbitrary units, were obtained for each testicular sample by dividing the PIWIL2/TDRD1 expression values by the proportion of expressing germ cell stages present in a seminiferous tubule of the sample, being spermatogoniae and primary spermatocytes the germ cell stages that predominantly express PIWIL2, whereas spermatocytes predominantly express TDRD1 in the testis   and TDRD1 .Most importantly, the hypermethylation observed in patients was accompanied by reduced transcript levels . Both genih.gov) . SignifiPIWIL2 and TDRD1 expression values, being remarkable the correlation coefficient between the cellular transcript levels of PIWIL2 and the number of elongated spermatids in the tubule , whilst a negative correlation coefficient was obtained with their DNA methylation levels.In order to assess whether there was an association between molecular and histological data and to confirm any physiological relevance, using all the samples in the study, we calculated the correlations between the normalized gene expression values, pyrosequencing data and histological parameters such as the number of each type of cell from the germline, Sertoli cell number and JS count . All thePIWIL2 and TDRD1 repression in patient samples with spermatogenic defects. First, we determined the expression of five primary piRNAs with strictly restricted germline expression PIWIL2/TDRD1 expression  and DNA methylation , respectively, as well as with the severity of spermatogenic impairment measured as the JS value . Remarkably, expression of DQ601291, DQ591415 and DQ601609 was abolished in those samples with complete meiotic arrest (samples no. 26 and 27) suggesting that they were postmeiotic piRNAs and that the absence of expression might be at least partially due to the loss of postmeiotic germ cells. However, this was not the case for DQ589977 and DQ598918: they had a moderate level of expression in samples with meiotic arrest, suggesting a premeiotic expression of these piRNAs. Thus, we additionally analyzed the germ cell–specific levels of both piRNAs (DQ589977 and DQ598918) per cell in SpF samples compared to CS controls in order to obviate the differences in piRNA expression due to changes in tissular germ cell composition. Transcript levels per cell, in arbitrary units, were obtained for each testicular sample by dividing the piRNA expression value by the proportion of spermatogoniae and primary spermatocytes present in a seminiferous tubule of the sample. Interestingly, piRNA expression per cell positively correlated with PIWIL2/TDRD1 expression , PIWIL2/TDRD1 expression per cell  and the severity of spermatogenic impairment measured as the JS value . Negative correlation was observed between piRNA expression per cell and DNA methylation .We examined the potential consequences of PIWIL2 and TDRD1, we observed hypomethylation of LINE-1 sequences using pyrosequencing technology in five out of the thirty-two patients suffering spermatogenetic failure , kallikrein 1 (KLK1), G protein-coupled receptor 156 (GPR156), histone cluster 1 H2aa (HIST1H2AA), RAB24, member RAS oncogene family (RAB24), sphingomyelin phosphodiesterase 3 (SMPD3) and dolichyl-phosphate mannosyltransferase polypeptide 1 (DPM1) showed piRNA sequence identity in their promoter region, suggesting an association of piRNA deregulation with the promoter methylation and gene expression.In addition to repetitive elements, piRNAs also showed the ability to guide DNA methylation and thereby regulate the expression of protein-coding and non-coding genes. In order to identify the DNA methylation changes of potential piRNA target genes associated with spermatogenic failure, we screened the promoter regions (transcription start site +/−2 kb) of the 580 genes differentially methylated in SpF for sequence overlap with piRNAs. We identified seven genes that were differentially methylated between normal and patient samples and also found complementarity to piRNAs sequences within their promoter , which iAlthough, several studies have reported the impact of an aberrant DNA methylation SFN gene and the maternally imprinted genes PLAGL1 and DIRAS3) We screened for global DNA methylation changes in diseased specimens using an array technology capable of profiling the DNA methylation level of 14,495 genes. It has been described that although the vast majority of methylation acquisition in male germ cells is completed in primordial germ cells, before birth, changes of DNA methylation continue to occur at a reduced number of CpGs during spermatogenesis before pachytene PIWIL1/2, TDRD1/9 were able to cluster normal tissue and patient samples separately. These results and the fact that impaired expression of these genes leads to sterility in animal mouse models PIWIL2 and TDRD1 were hypermethylated in SpF specimens. The increase in methylation in SpF patients could be partially explained by the increase proportion of somatic cells in the samples but interestingly, an additional statistically significant increase in the methylation level of germ cells was observed , maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and with Sertoli cell-only syndrome (SCO). Black and white squares indicate CpG methylation and unmethylated sites, respectively. Sample numbers are indicated.(PDF)Click here for additional data file.Figure S2Bisulfite sequencing of TDRD1 and TDRD9 in testis with conserved spermatogenesis (CS), maturation failure at the spermatocyte (scMF) or at the round spermatid (rsMF) stage, and with Sertoli cell-only syndrome (SCO). Black and white squares indicate CpG methylation and unmethylated sites, respectively. Sample numbers are indicated.(PDF)Click here for additional data file.Table S1Primer sequences and locations.(PDF)Click here for additional data file.Table S2633 differentially methylated CpG sites in SpF relative to normal testis tissue.(PDF)Click here for additional data file.Table S3Gene ontology analysis of differentially methylated genes in SpF.(PDF)Click here for additional data file.Table S4Differentially methylated gene promoters overlapping piRNAs.(PDF)Click here for additional data file.


Microorganisms play a critical role in the decomposition of organic matter, which contributes to energy and nutrient transformation in every ecosystem. Yet, little is known about the functional activity of epinecrotic microbial communities associated with carrion. The objective of this study was to provide a description of the carrion associated microbial community functional activity using differential carbon source use throughout decomposition over seasons, between years and when microbial communities were isolated from eukaryotic colonizers . Additionally, microbial communities were identified at the phyletic level using high throughput sequencing during a single study. We hypothesized that carrion microbial community functional profiles would change over the duration of decomposition, and that this change would depend on season, year and presence of necrophagous insect colonization. Biolog EcoPlates™ were used to measure the variation in epinecrotic microbial community function by the differential use of 29 carbon sources throughout vertebrate carrion decomposition. Pyrosequencing was used to describe the bacterial community composition in one experiment to identify key phyla associated with community functional changes. Overall, microbial functional activity increased throughout decomposition in spring, summer and winter while it decreased in autumn. Additionally, microbial functional activity was higher in 2011 when necrophagous arthropod colonizer effects were tested. There were inconsistent trends in the microbial function of communities isolated from remains colonized by necrophagous insects between 2010 and 2011, suggesting a greater need for a mechanistic understanding of the process. These data indicate that functional analyses can be implemented in carrion studies and will be important in understanding the influence of microbial communities on an essential ecosystem process, carrion decomposition. Decomposition is an important component of nutrient and organic matter cycling in ecosystems sensu Benbow et al. Microbial communities associated with carrion are part of the necrobiome, Prokaryotes can compete with eukaryotes by producing toxic compounds that affect resource “appeal” to consumers and thus reduce competition from eukaryotes There are several factors influencing microbial community assembly and function during vertebrate carrion decomposition: temperature There are several emerging areas of research focused on a better understanding of microbial community functional ecology importance in carrion decomposition: 1) how does the functional activity of microbial communities change during decomposition; 2) the role of temperature driving this functional succession; 3) how microbial function is mediated by necrophagous insect colonization and succession; and 4) intra- and inter-seasonal variation in these processes Gleditsia triacanthos) and a variety of maples (Acer spp.), while the most common sub-canopy cover was Amur honeysuckle (Lonicera maackii). Sus scrofa (swine) carcasses weighing 14–18 kg were used as models of vertebrate carrion decomposition Seasonal variation in MMCPs was studied within Morris Bean Reserve of Greene County, Ohio, USA , a Midwest temperate forest of 12.2 hectares surrounded by agricultural fields with a small tributary stream running adjacent to the reserve that empties into the Little Beavercreek River. The predominant trees were honey locust  for a total of 30 plots within 10–80 m from each other as previously described Carcasses purchased from a local farm were double bagged during transport and prior to field placement to prevent insect colonization. Using the same experimental design of Benbow et al. Quercus spp.) and maple. Carcasses ranging from 5–30 kg were sampled from 5 August 2010 until 14 August 2010 and 26 July 2011 until 2 August 2011. Carcasses purchased from the same farm as described above were double bagged during transport and prior to field placement to prevent insect colonization. In 2010, six male carcasses were randomly placed at a minimum of 20 m apart along three transects. In 2011, using the same methods as described in 2010, six carcasses  were purchased from the same farm, double bagged during transport to the field and randomly placed along three new transects.Annual variation of microbial community activity and effects of necrophagous insect colonization was studied in a Midwestern temperate forest habitat surrounded by agricultural fields in Xenia, Ohio, USA . The dominant tree fauna consisted of oak (3 Lumite® screen (18×14 mesh size) portable field cages  to exclude insect colonization. These carcasses were considered the necrophagous insect exclusion treatment (EXC), while the three remaining carcasses were the insect access treatment (ACC) with insects immediately allowed access to the carcasses. As with the previous seasonal study, all carcasses were covered with anti-scavenging cages with NexSens DS1923 micro-T temperatures loggers measuring local ambient temperature every 0.25 h. Temperature data were converted into ADH.During each year, three random carcasses were enclosed in individual 1.8 mPermission was granted by the Green County Park District for the seasonal trials at Morris Bean Reserve, while no permits were necessary for the annual and necrophagous insect colonization studies, as the landowners granted permission to use the privately owned property. The field studies did not involve endangered or protected species.During the seasonal trials, sterile cotton applicators were used to sample the microbial communities of the buccal cavity (the top area of the mouth and under the tongue), skin and the interior anal cavity. Epinecrotic microbial communities were sampled from the carcasses every three days, weather and field conditions permitting, until the dry stage of decomposition as defined by Payne In both the annual and necrophagous insect effect studies, sterile cotton applicators were used to sample microbial communities from two regions on each carcass for 60s: the buccal cavity and the skin, which consisted of combining three areas (approximately 2.54×15.24 cm) along a single transect of a carcass. Care was taken to assure that new areas were swabbed at subsequent samplings. Duplicate samples were collected from the carcasses for 16S rRNA analysis of the bacterial communities using 454-pyrosequencing during the first necrophagous insect effect study (2010). Carcasses were sampled until the dry stage of decomposition as described by Payne 2™ with Wallac 1420 Workstation software version 2.0  during 2009 and 2010, and a Tecan Sunrise™ with Magellan™ software version 7.0  during 2011. A two-tailed paired t-test was used to test for any differences between the plate reader models, and there was not a significant difference in data between plate readers .Microbial community function was evaluated using phenotype Biolog EcoPlates™  to provide MMCPs in all experiments After all seasonal samples were collected, three dates representing three different phases of decomposition  were chosen for analysis. The spring and summer trials followed the same sampling regime, with the first sampling date corresponding with the initial oviposition of Calliphoridae (Insecta: Diptera) signifying the early phase of decomposition . The second sampling date took place when the carcasses reached the middle phase of decomposition , while the last sampling date represented the late phase of decomposition . For the autumn trial, the first sampling date corresponded with initial Calliphoridae oviposition; however, the second sampling date occurred prior to the first snow of the season. It was decided at the beginning of the study that carcasses would not be disturbed once they were covered with snow. The third sampling date took place in January of 2011, which corresponded with increased temperatures and snowmelt. For the winter trial, there were four sampling dates instead of three because of little insect activity during the beginning of the trial. The first sampling date took place immediately after carcass exposure since a delay in initial oviposition was expected during winter temperature conditions. The second date coincided with the next snowmelt. Both a third and fourth sampling date were chosen to represent different decomposition time points, even though there appeared to be negligible visible change to the carcasses and little adult or larval blow fly activity on the carcasses during the entire trial. Sampling for the annual and necrophagous insect colonizer effects studies occurred at initial field placement and on days 1, 3 and 5 during 2010. While in 2011, sampling occurred at initial field placement and then daily until 5 days of decomposition had occurred.Analyses were performed according to Stefanowicz Samples collected in 2010 were used to identify microbial communities through 454-pyrosequencing using methods described in Pechal et al. Bray-Curtis distance with nonmetric multidimensional scaling (NMDS) and 999 permutations using vegan 2.0–7 library in R Differences in microbial function and variation over decomposition were tested using Bray-Curtis dissimilarity values with permutational multivariate analysis of variance (PERMANOVA) with the adonis function using the vegan 2.0–7 library in the R statistical package Spring overall microbial functional use of the carbon resources was highest of all seasons  and incr2 = 0.88) was sufficient to describe normalized carcass MMCPs among seasons (P = 0.001) in MMCPs among all seasons (P = 0.001), but no differences during the autumn (PERMANOVA: P = 0.226) or winter studies (PERMANOVA: P = 0.101).Using PERMANOVA we found no significant differences in normalized MMCPs among the sample locations  of each carcass; therefore, data were pooled for the remainder of analyses. A two-dimensional NMDS ordination  in 2011 (2 = 0.87) described the variation in carcass MMCPs between years  and a significant interaction effect . Overall2 = 0.91) was sufficient to describe the variation in carcass MMCPs between ACC and EXC carcasses  and ACC (70%) carcasses with Firmicutes being the next predominant phyla at 33% and 20% of EXC and ACC communities, respectively. Proteobacteria remained predominant on the first (35%), third (63%), and fifth (82%) sampling days for EXC carcasses. While Firmicutes decreased as decomposition progressed on the first (43%), third (28%), and fifth (16%) day. However, ACC displayed an inverse trend as Proteobacteria decreased from the first (42%) and third (44%) days to only 3% total abundance on the fifth day, but with some inter-carcass variation as discussed in detail in Pechal et al. Over decomposition, mean microbial functional activity of ACC carcasses decreased by 46.3% while there was a 45.2% increase on EXC carcasses . There w2 = 0. 89) was sufficient to describe the variation in carcass MMCPs between ACC and EXC carcasses  by 8–18% than 2010 (data not shown) throughout decomposition, except at initial placement in the field when ADH for all carcasses was zero.Mean daily ambient temperature among carcasses was 23.2±2.1°C during 2010 and 25.1±1.0°C in 2011. Mean ADH associated with the carcasses in 2011 was significantly higher  seasonal variation of microbial community function, with overall higher and more variable activity during spring when compared to the other seasons; 2) annual differences in metabolic profiles; and 3) inconsistent effects of necrophagous insect activity  on microbial functional profiles and overall activity.Fungi and bacteria have been documented as facilitators of the initial decomposition processes of carrion Seasonal variance in abiotic conditions was hypothesized to significantly impact microbial community function. For the spring and winter trials, microbial functional activity decreased during early decomposition followed by an increase in later phases of decomposition. The increase in functional activity during the winter trial most likely results from a lack of competition with invertebrates  because of the colder temperatures . Summer had an increase in microbial activity throughout decomposition. Autumn was the only season that demonstrated a decreasing trend in overall activity throughout decomposition, which can be due to competition with invertebrates, the depletion of the carrion resource, or both. Competition between microbes and insects for a resource occurs by microbes producing toxins that deter eukaryotic consumers We found significantly different metabolic profiles over decomposition for carcasses in spring and summer but not autumn and winter. However, why did microbial functional activity increase in the winter trial as temperatures decreased? Carter and Tibbett Qualitative phases of decomposition could be differentiated for carcasses based on microbial functional activity, and insects appeared to be reducing the microbial community throughout the decomposition process in 2010. However, these trends were not consistent between years. The lack of similarities between years may result from abiotic  and biotic factors , as was demonstrated in our seasonal study. Abiotic factors including temperature may account for microbial community function variation between years and throughout decomposition. Ambient temperatures were up to 18% higher in 2011. Previous studies, however, have reported conflicting microbial species response to changing temperature. For instance, soil bacteria abundance increased with temperature (+3°C) in the presence of elevated carbon dioxide, but decreased under conditions of similar temperature conditions without elevated carbon dioxide Musca domestica (Diptera: Muscidae), which carries over 100 pathogenic microbes Proteus mirabilis, which is commonly associated with blow flies Primary insect colonizers utilize the carrion resource as nutrition, mating or an oviposition site. Subsequent larval development may disrupt established microbial communities through direct or indirect competitive interactions on the carcass Additionally, we documented four major phyla associated with the carcasses throughout decomposition using 454-pyrosequencing that included Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. These phyla have been reported in one other study where epinecrotic communities were described using 454-pyrosequencing . The MMCPs were significantly different (PERMANOVA: P = 0.001) among seasons. The circles indicate 95% standard error of each season.(TIFF)Click here for additional data file.Figure S2Decomposition phase within season Bray-Curtis dissimilarity based non-metric multidimensional scaling. Non-metric multidimensional scaling ordinations of MMCPs were plotted on two dimensions for carrion communities of decomposition phases  during A) spring , B) summer , C) autumn , and D) winter  seasons. There were significant differences among decomposition phases in spring and summer (PERMANOVA: P = 0.001), but no significant difference among decomposition phases in autumn (PERMANOVA: P = 0.226) or winter (PERMANOVA: P = 0.011). The circles indicate 95% standard error of each decomposition phases.(TIFF)Click here for additional data file.Figure S3Mean functional activity over decomposition between years. The mean (SEM) microbial functional activity between the 2010 (gray circle) and 2011 (black square) field trials at initial field placement (Day 0) and subsequent sampling on days 1, 3, and 5.(TIFF)Click here for additional data file.Figure S4Annual Bray-Curtis dissimilarity based non-metric multidimensional scaling. Using Bray-Curtis dissimilarity values, MMCPs from both 2010 and 2011 field seasons were plotted using a two dimensional non-metric multidimensional scaling model . There were significantly different MMCPs (PERMANOVA: P = 0.001) between years. The circles indicate 95% standard error of each year.(TIFF)Click here for additional data file.Figure S5Mean functional activity over decomposition between sampling regions in both field seasons. The mean microbial functional activity for the buccal (orange circle) and skin (yellow square) sampling region in the A) 2010 and B) 2011 field trials at initial field placement (Day 0) and subsequent sampling on days 1, 3, and 5.(TIFF)Click here for additional data file.Figure S6Treatment within field season Bray-Curtis dissimilarity based non-metric multidimensional scaling. Non-metric multidimensional scaling ordinations of MMCPs were plotted on two dimensions for ACC and EXC carrion communities during A) 2010 , and B) 2011  field seasons.(TIFF)Click here for additional data file.Materials S1Supplementary Tables S1–S2. Table S1, Carbon sources and their respective groupings  in a Biolog EcoPlate™. Table S2, PERMANOVA results testing MMCPs between sampling region  communities and among carcass decomposition days including the interaction for both 2010 and 2011 field seasons with significant results indicated by an asterisk.(DOCX)Click here for additional data file.


Fetal chromosomal abnormalities are the most common reasons for invasive prenatal testing. Currently, G-band karyotyping and several molecular genetic methods have been established for diagnosis of chromosomal abnormalities. Although these testing methods are highly reliable, the major limitation remains restricted resolutions or can only achieve limited coverage on the human genome at one time. The massively parallel sequencing (MPS) technologies which can reach single base pair resolution allows detection of genome-wide intragenic deletions and duplication challenging karyotyping and microarrays as the tool for prenatal diagnosis. Here we reported a novel and robust MPS-based method to detect aneuploidy and imbalanced chromosomal arrangements in amniotic fluid (AF) samples. We sequenced 62 AF samples on Illumina GAIIx platform and with averagely 0.01× whole genome sequencing data we detected 13 samples with numerical chromosomal abnormalities by z-test. With up to 2× whole genome sequencing data we were able to detect microdeletion/microduplication (ranged from 1.4 Mb to 37.3 Mb of 5 samples from chorionic villus sampling (CVS) using SeqSeq algorithm. Our work demonstrated MPS is a robust and accurate approach to detect aneuploidy and imbalanced chromosomal arrangements in prenatal samples. Chromosomal abnormalities occur in 1 of 160 live births To overcome these limitations, in this study we have developed a new method based on massively parallel sequencing (MPS) platform to detect fetal chromosomal abnormalities, which is independent of particular genetic markers and cell culture in medical practice. We directly sequenced 62 DNA samples extracted from uncultured amniotic fluid (AF) of pregnant women. After statistical analysis, we can clearly detect numerical chromosomal abnormalities among 46 chromosomes. The whole process only takes 7 days and the results were validated by full karyotyping analysis. Subsequently, we investigated the presence of copy number variations among 5 prenatal samples previously identified by arrayCGH. We demonstrated that with different whole genome sequencing coverage MPS platform could be applied for identification of aneuploidy and imbalanced chromosomal arrangements, and those approaches are more sensitive and effective compared with conventional methods.z-scores of each of the chromosomes, except the Y chromosome, for each of the normal controls and test cases were calculated according to the formula described in In this study, the 32 test cases were used for sequencing analysis under double-blind conditions. For all 62 AF samples , we obtained 0.55∼3.28 million 35-bp single-end reads (or 19.25 Mb∼114.80 Mb sequencing data) which corresponds to 0.006∼0.038× human genome depth. Consequently, 0.20∼1.15 million unique reads (UR) were obtained for each sample . For eacNormalized UR% values for all 22 autosomes and chromosome X were calculated . 5 casesTo further validated our developed and optimized analysis method among cases with imbalanced chromosomal arrangements, we obtained 44∼70 M sequence tags for the 5 “known” samples with 1.4 to 2.3× whole genome depth. To detect segmental copy number variations, a sequence-based Matlab CNV detection package, SegSeq, with YH reference genome as comparative genome were used. By applying the SegSeq analysis, we detected microdeletions and microduplications in all 5 samples . All theTwo samples  were selected to further ascertain the results on the HumanOmni2.5 M chip (Illumina). CNV partition Algorithm plug-in, was used to detect copy number variations in these two samples. A duplication at 2q36∼q37.3 (230369496∼242444380) and a deletion at 13q32.3q33.3 (97091318∼106462788) were correctly detected, totally in concordance with the arrayCGH and sequencing analysis results.Massively parallel sequencing has been reported only to apply in noninvasive prenatal diagnosis of trisomy 21, 18 and 13 based on cell-free fetal DNA, due to it is limited amount of fragmented fetal DNA By establishing a normal control sequencing tag data set, we have been able to demonstrate this new approach only requires a minimum among of DNA materials (100 ng) to achieve the identification of aneuploidies with a ultra low sequencing coverage (0.01×). Comparing to the golden standard (G-Band karyotyping) in clinical practice, MPS has no time limitation. Also as long as 100 ng genomic DNA can be extracted from tissues at any gestational weeks, MPS can be performed and report all fetal aneuploidies in 7 days. If necessary, more sequence reads can be performed to detect whether microdeletion or microduplication exists in the fetal genome which may result in severe developmental retardation. In our study, tissues obtained from fetus, such as amniotic fluid, CVS and placenta can be analyzed without cell culture since 100 ng genomic DNA is sufficient for library preparation and sequencing. So it can be used for the research into the molecular mechanism of miscarriage, stillbirth and fetal death when tissues are difficult to culture. Furthermore, our validation study on the 5 arrayCGH samples show that when more sophisticated sequencing protocols and bioinformatics algorithms are applied to the analysis, it is possible to detect smaller size chromosomal copy number variations as well as complex rearrangements across the whole genome of the fetus, such as balanced chromosomal arrangements,structure variations or even single-gene disorders. With the application of third-generation sequencing system in clinical laboratories, such as Miseq/Illumina and Ion Torrent PGM/Life Technologies, the whole process will take less time and acceptable price. Thus, it is likely that MPS will play an increasingly important role in the future development of prenatal screening and diagnosis.A potential weakness of the study was that in the figure of describing the ratio of unique reads in each chromosome, chromosome19 and 22 have a differently huge coefficient of variation because of their extremely high GC content and made the detection of trisomy19 and trisomy22 difficult. Further study will be set up to deal with these problems, for example the computational correction of GC content among chromosomes. Other chromosomal abnormalities, such as balanced translocation or incomplete aneuploidy caused by mosaics or partial duplication or deletion of a chromosome should, in principle, also be detectable. Further studies are required to determine the effectiveness of massively parallel genomic sequencing in detecting these rare aberrations. Another weakness for the new methodology was the starting materials for library construction; with the conventional Illumina library construction approach100 ng genomic DNA of fetus was required. To further reduce the risk to pregnancies it would be important to reduce the amount for AF or CVS samples, other library construction methods such as using in vitro transposition may be an alternative solution.In conclusion, we have demonstrated the usefulness of massively parallel sequencing to detect fetal aneuploidy and imbalanced chromosomal abnormalities of genomic DNA in prenatal samples. In principle, massively parallel sequencing can also reveal other features of the genomic material from amniotic fluid such as histone modifications as well as epigenetic DNA methylation. With the rapid reduction of sequencing cost, we expect that the strategy described in this article will become a powerful tool in the detection of all kinds of chromosomal abnormalities in clinical settings.The study was approved by the Institutional Review Board of Beijing Obstetrics and Gynecology hospital of the Capital Medical University. Informed consent was obtained from each participant.A total of 32 pregnant women at a high risk of Down's syndrome were recruited as test cases from the Beijing Obstetrics and Gynecology hospital during the period of January to May 2010. Amniocentesis was applied at 19∼22th gestational week and standard G-band karyotyping analysis was performed. Another 30 euploid AF samples  were included as normal control at the same hospital. To validate the sensitivity of analysis method for detection of microdeletion/microduplication, 5 chorionic villus sampling (CVS) samples validated by karyotyping and arrayCGH were recruited from the Prenatal Genetic Diagnosis Centre (PGDC) at Department of Obstetrics & Gynecology, Chinese University of Hong Kong.Genomic DNA was extracted from uncultured AF samples with Micro DNA Kit (Tiangen) and quantified with the Quant-iT dsDNA HS Assay Kit (Invitrogen). 100 ng genomic DNA from each sample was sheared into small fragments ranged from 100 to 400 bp with Bioruptor (Diagenode). After end-repair, “A”- overhanging and adapter-ligation, DNA fragments of 300 bp (±25 bp) in length were selected by 2% agarose gel electrophoresis and underwent 12 cycles of PCR with multiplex primers. PCR products were purified by Agencourt AMPure Kit (Beckman). Size distribution of the library was detected by Agilent Bioanalyzer DNA 1000 kit (Agilent Technologies) and the concentration was measured by quantitative PCR (qPCR). Libraries with different index tags were mixed in equal moles into a pool and sequenced with single-end 36 cycle multiplex sequencing on Illumina GAIIx platform.For the 5 DNA samples from CVS tissues, sequencing libraries with the insert size of 500 bp (±25 bp) were prepared. Paired-end 100 cycle multiplex sequencing was performed on Illumina HiSeq 2000.z-score of chromosome N (N stands for chromosome numbers) is calculated as,35-bp single-end reads from 62 AF samples  were aligned against repeat-masked human genome build 36 (hg18) by ELAND. Unique reads (UR), which can be mapped to reference genome sequence without any mismatches or alternative positions, were used for further analysis. The UR percentage (UR%) of chromosome N  can be used as an indicator of fetal trisomy for autosomes. To satisfy central-limit theorem, 30 euploid samples were used as normal controls. The http://soap.genomics.org.cn/) We mapped the Illumina reads to the reference sequence of human genome  by Short Oligonucleotide Analysis Package aligner (SOAP2) (http://www.broadinstitute.org/cgi-bin/cancer/publications/pub_paper.cgi?mode=view&paper_id=182) For the CNV detection, we employed a MATLAB packet, SegSeq  http://www.fetalmedicine.hk/en/Fetal_DNA_chip/Appendix_I.pdf) with most of the known common non-pathogenic CNVs regions removed. This chip provides a means to detect chromosomal aberrations with resolution of <100 Kb across the genome. The quality of the array was analysed using Agilent DNA analytics software and cases where the Derivative Log Ratio spread of the array was >0.25 were excluded from further data analysis. Data reporting variations in copy number were released after excluding known non-pathogenic chromosome copy number variants that have been listed at the Database of Genomic Variants.Genomic chromosomal copy number variants (CNVs) were detected using a targeted high resolution 44 K oligonucleotide array specifically constructed for prenatal screening with the intention of targeting common trisomic aneuploidies and most known microdeletion and microduplication syndromes. This Fetal DNA chip included telomeric and pericentromeric regions, examining the genome to a resolution of 100 kb Click here for additional data file.Table S2The z-scores of each chromosome (except chr Y) for 62 AF samples.(XLS)Click here for additional data file.Table S3Outcome and clinical data of CUHK cases.(XLS)Click here for additional data file.


HTLV-1 is the etiologic agent for ATL. The virally encoded regulatory protein Tax is critical to the oncogenic process and a variety of biological activities are attributed to the protein. Tax localizes to the nucleus where it has been reported to impair the cellular DNA damage response and activate viral and cellular gene transcription. Tax also exhibits nucleocytoplasmic shuttling and in response to DNA damage translocates to the cytoplasm where it initiates activation of anti-apoptotic NFκB signaling. Thus, subcellular compartmentalization drives Tax function and likely plays an important role in HTLV-1 biology and host disease development. Although it seems clear that post-translational modification of Tax by ubiquitin/SUMO dictates subcellular localization, it is not clear how this process is regulated. We present evidence that RNF4, a SUMO-targeted ubiquitin ligase (STUbL), mediates nuclear-cytoplasmic translocation of Tax. Over-expression of RNF4 but not an RNF4 RING mutant resulted in a dramatic cytoplasmic enrichment of Tax. Concomitant with the RNF4-induced cytoplasmic accumulation of Tax was an increase in “cytoplasmic” (NFkB activation) and decrease in “nuclear” (HTLV-LTR activation) Tax functions. We demonstrated that RNF4 binds adjacent to the putative Tax ubiquitin/SUMO modification sites K280/K284 suggesting that Tax may be a substrate for RNF4. Indeed, we show that RNF4, but not RNF4 RING mutant, ubiquitylates Tax both in vitro and in vivo. Finally, depletion of RNF4 by RNAi prevents the nuclear-cytoplasmic translocation of Tax following DNA damage. These results provide important new insights into STUbL mediated pathways that regulate the functional dynamics of viral oncogenes.


Saccharomyces cerevisiae as a model organism to study the pathways targeted by NSNM. Our screening with yeast mutants revealed that it affects chromatin, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair processes. We also show that NSNM acts as an epigenetic modifier as its treatment causes reduction in global histone acetylation and formation of histone adducts. Cells treated with NSNM exhibited increase in mitochondrial membrane potential as well as intracellular ROS levels and the effects were rescued by addition of reduced glutathione to the medium. We also report that deletion of SOD1 and SOD2, the superoxide dismutase in Saccharomyces cerevisiae displayed hypersensitivity to NSNM. Furthermore, NSNM treatment causes rapid depletion of total glutathione and reduced glutathione. We also demonstrated that NSNM induces degradation of Sml1, a ribonucleotide reductase inhibitor involved in regulating dNTPs production. In summary, we define the various biological pathways targeted by isocyanates.Isocyanates, a group of low molecular weight aromatic and aliphatic compounds possesses the functional isocyanate group. They are highly toxic in nature hence; we used N-succinimidyl N-methylcarbamate (NSNM), a surrogate chemical containing a functional isocyanate group to understand the mode of action of this class of compounds. We employed budding yeast  In recent decades, a key center of attention has been on understanding exposure to hazardous agents in the natural environment including water, soil and air. Elucidation of modes of action through studying effects of occupational exposure to environmental contaminants on human health is of foremost concern. Such exposures are often associated with a number of diseases, including cancer, diabetes, and infertility, etc. Isocyanates Saccharomyces cerevisiae. The budding yeast is considered as an attractive candidate for our studies because of its pliability to genetic manipulation and completely known genomic sequence N-succinimidyl N-methylcarbamate (NSNM) is one of the carbamate esters which possess functional isocyanate group To understand the mode of action of isocyanate on yeast cells we screened the sensitivity of yeast mutants for genes involved in key cellular pathways including epigenetics, DNA damage checkpoint kinase, DNA repair and TOR pathways. Our results demonstrated that NSNM acts as an epigenetic modifier. Mitochondrial membrane potential and ROS levels were increased considerably following exposure to NSNM. Rapid depletion of intracellular glutathione was also observed upon treatment with NSNM. Taken together, our result shows that isocyanates target multiple biological pathways which contribute to its toxicity.Saccharomyces cerevisiae strains used in this study are listed in The 600) was measured at regular intervals for 8 h. Clonogenic assay was carried as described earlier To investigate the biological effect of NSNM on the growth of yeast cells, growth assay was carried out by spot testing (as described earlier 2) to make 0, 50, 100, 200, 300, 500 and 1000 U/ml concentrations. MNase digestion was performed for 5 min at 37 °C and immediately reaction was stopped by adding stop solution . Phenol chloroform extraction was carried out, pellets were washed with 70% ethanol, air-dried, and re-suspended in 30 μl TE containing 100 μg/ml RNase. Samples were loaded on a 1.2% agarose gel and visualized by EtBr (Ethidium bromide) staining.MNase assay was performed as described previously Core histones were purified from white leghorn chicken brain tissue as described earlier 7 yeast cells were harvested and washed with ice-cold PBS. Cells were resuspended in 100 μl of PBS followed by staining with MitoTracker. After staining, cells were visualized under fluorescence microscope (AXIOVERT 4.0) using Rhodamine filter.To measure ROS production, we used 2, 7-dichlorodihydrofluorescein diacetate (DCF-DA) . ROS and mitochondrial membrane potential were measured following protocol described previously Glutathione levels were measured using the method described earlier Yeast cells in exponential phase were treated with alpha factor to synchronize cells in G1 phase as described earlier For ROS and mitochondrial membrane potential measurement, wild type yeast strain was grown till exponential phase in presence or absence of antioxidant agent GSH (10 mM) followed by treatment with indicated concentration of NSNM for three hours. Cells were stained with either MitoTracker or DCF-DA as described above before being analyzed by FACS.To determine the effect of NSNM on Sml1 and Rad52, we analyzed Sml1-YFP or Rad52-YFP yeast strains by confocal microscopy as described previously Exponentially growing yeast cultures were treated with DMSO or NSNM for 3 h and harvested. Whole cell extracts were obtained from the yeast cell pellets by 20% trichloro acetic acid (TCA) precipitation following standard protocol In a pilot experiment, we tested the effect of NSNM on the growth of yeast cells. In liquid medium, significant growth inhibition was observed at very low dose of 5.0 μM NSNM and the growth was completely inhibited at 100 μM concentration . On soliBased on our growth curve experiments, we observed significant reduction in the growth rate of yeast cells upon NSNM treatment, suggesting that the NSNM is possibly causing defects in cell cycle. For confirmation, we performed FACS analysis to monitor cell cycle progression in the presence or absence of this chemical. Exponentially growing yeast cells were synchronized by alpha factor treatment for 2 h. After synchronization G1 arrested cells were released in 50 μM NSNM or DMSO (control) containing media. The DMSO treated cells moved to G2 phase within 60 min of release from alpha factor arrest  while NSgcn5Δ, hat1Δ, rtt109Δ, nlp3Δ, atf2Δ, hpa2Δ, sas2Δ and nut1Δ) and histone deacetylases . Deletion of GCN5 and RTT109 exhibited enhanced growth inhibition  were found to be hypersensitive for NSNM , which are generated during different stress conditions. To investigate the possible role of Sod1 or Sod2 for conferring resistance to NSNM, we compared the growth of sod1Δ/sod2Δ and wild type cells in the presence of increasing dose of NSNM. As shown in sod1Δ/sod2Δ mutants were hypersensitive to NSNM. Because Sod1 or Sod2 are primarily antioxidant enzymes, we sought to investigate whether the increased sensitivity of sod1Δ/sod2Δ for NSNM is due to increased ROS levels. We analyzed the effect of the antioxidants, such as reduced glutathione (GSH), on the growth phenotype of sod1Δ/sod2Δ. As shown in sod1Δ/sod2Δ in the presence of NSNM suggesting the role of ROS behind the toxicity exhibited by NSNM. Furthermore, to determine whether the ROS accumulation in NSNM treated cells is facilitated by increase in free radicals, we investigated the effect of NSNM on ROS levels in presence of GSH by FACS. Interestingly, the increase in ROS levels (Superoxide dismutases (SODs) are a class of enzymes that catalyze the dismutation of superoxide into hydrogen peroxide and oxygen. In budding yeast SODs have two forms S levels  and mitoS levels  due to NTaken together, these results indicate that NSNM causes generation of reactive oxygen species leading to growth inhibition/death of yeast cells.mec1Δ, rad53Δ, and dun1Δ deletion mutants in presence of NSNM , NER and BER  pathways did not show any significant sensitivity to NSNM , NER (Nucleotide Excision Repair) and BER (Base Excision Repair). Mutants defective in DNA single or double-strand break repair pathways  to study the response in yeast cells. We screened some of important pathways including epigenetics, histone modifications, DNA damage response, protein-ubiquitylation and chaperones, oxidative stress, TOR pathway and DNA repair pathways. Through screening we identified few molecules including Gcn5, Rtt109, Ssq1 and Slx5 which are required to provide tolerance to NSNM.In the present study we used budding yeast GCN5 and RTT109 showed enhanced growth inhibition in response to NSNM treatment suggesting that it might target epigenetic machinery of the cells. Additionally, we observed a decrease in histone acetylation marks upon treatment with this compound, suggesting that the adduct formation might be the mechanism responsible for the decrease in histone acetylation. Based on our present study we can propose that NSNM reacts with histones to make adducts, therefore compromising downstream processes that may represent a vital mechanism by which NSNM influences its biological functions.One of the important findings of the current study is that NSNM acts as an epigenetic modifier. It makes adduct with histone and its treatment causes decrease in the global histone acetylation. Change in epigenetics or alteration in histone modifications occurs during regulation of various biological processes 2− to H2O2 are the superoxide dismutases (SODs) 2O2 to H2O+O2 occurs through the action of catalase, a heme-based enzyme that is normally localized in the peroxisome 2O2 is converted to O2 through coupled reactions, with the conversion of reduced glutathione (GSH) to oxidized glutathione (GSSG), catalyzed by glutathione peroxidase (GPX) NSNM is a reactive compound and based on our sensitivity assays we observed that it is targeting multiple cellular pathways in yeast. Next, we measured the cellular redox status upon treatment with NSNM. Interestingly, we detected increase in mitochondrial membrane potential as revealed by MitoTracker staining. Increase in mitochondrial membrane potential is known to increase intracellular ROS levels in vivo cellular dNTPs pool. Although, degradation of Sml1 upon exposure to NSNM partly supports our hypothesis, its effect on cellular dNTPs levels needs to be further tested.Oxidative stress is often associated with induction of DNA damage response (DDR) Altogether, we have illustrated the key pathways affected by isocyanates. Our findings suggest that the toxic effects of isocyanate are largely contributed by cumulative effect of alteration in redox status and epigenetic modifications. Thus, it is likely that many lethal challenges by isocyanates are the result of hijacking natural stress-response machineries to induce ROS production above threshold levels leading to cell death. Our studies undoubtedly provide important insights into the physiological pathways affected by isocyanates. Further analysis on global and gene-specific changes in response to isocyanates will provide more insights into the mechanisms of toxicity exhibited by them. We anticipate that these facts would assist to devise improved approaches in assessment of toxicity caused by isocyanates.Table S1List of yeast strains used in this study.(DOCX)Click here for additional data file.


Cell type-specific patterns of gene expression reflect epigenetic changes imposed through a particular developmental lineage as well as those triggered by environmental cues within adult tissues. There is great interest in elucidating the molecular basis and functional importance of epigenetic mechanisms in both normal physiology and disease – particularly in cancer, where abnormal ‘-omic’ states are often observed. In this article we review recent progress in studies of epigenetic mechanisms in the most common primary adult brain cancer, glioblastoma multiforme. Three distinct areas are discussed. First, the evidence in support of ongoing ‘normal’ epigenetic processes associated with differentiation – as predicted by ‘cancer stem cell’ models of the disease. Second, identification of site-specific and global epigenetic abnormalities. Third, genetic disruptions directly within the core epigenetic machinery, exemplified by the recently identified mutations within isocitrate dehydrogenase genes IDH1/2 and variant histone genes H3.3/H3F3A. These constitute the ‘good, the bad and the ugly’ of epigenetic mechanisms in cancer. Nowaday1.1clonal genetic model of cancer    and moreia (AML) . ProgramThe specific focus of this article is the high grade glioma, glioblastoma multiforme (GBM). We review three major areas: (1) The ‘good’ epigenetic mechanisms that operate during differentiation to generate the cellular hierarchy. (2) The ‘bad’ epigenetic mechanisms, such as silencing of tumour suppressors, which are cancer-specific alterations that are nevertheless potentially reversible. (3) The ‘ugly’ mechanisms, disrupted in a cancer irreversibly through genetic alterations to the core epigenetic machinery .2IDH1/2 as a hallmark of secondary GBM. We will discuss the significance of this oncogene and its potential relevance to epigenetics later in this review.GBM is the most common and aggressive form of primary brain cancer. Current treatments for patients with these cancers are largely ineffective and the median survival is only fourteen months . GBM is TP53 and IDH1 mutations as well as PDGFRA mutations and overexpression. It includes significantly younger patients and also correlated with increased survival compared to other subtypes. The ‘classical’ subtype is mainly characterised by a high frequency of EGFR aberrations and the absence of TP53 mutations, while the ‘mesenchymal’ subtype typically displays loss of the tumour suppressor gene NF1. The ‘neural’ group was less defined and did not show a unique deregulation of specific genes or pathways. This knowledge of the diversity of GBM will need to be incorporated into laboratory studies for a comprehensive understanding and modelling of the disease. Inevitably these differences need to be considered in design of new clinical trials – particularly targeted molecular therapies.Simultaneous genetic disruptions to the canonical P53, RTK/PI3K and RB/CDK pathways are common in primary GBMs . HoweverPDGFRA is the most frequently occurring target of focal amplification in paediatric cases. Even those tumours that lack genetic amplification of PDGFRA or PDGFRB still show strong expression of the genes associated with PDGFRA amplification, suggesting a central role for this pathway. IDH1/2 mutations, commonly found in adult secondary gliomas, are also not present.GBM is primarily a disease of adults. However, paediatric high grade glioma (HGG) has histological features of adult GBM and accounts for 15–20% of all childhood CNS tumours. Sadly only 10–30% of these patients survive more than 2 years after diagnosis. Recently our knowledge of the genetics of paediatric HGG has advanced considerably. Despite the similarities to GBM, studies have identified substantial differences in the molecular features underlying paediatric and adult high-grade gliomas and no correspondence to specific adult GBM subtype has been identified . Common Noteworthy from these genetic analyses was the observation that paediatric GBM has a much more limited spectrum of genetic alterations than adult, with many of the tumours displaying a remarkably normal karyotype. This suggested the involvement of epigenetic disruptions, perhaps related to a specific spatial or temporal developmental context. In fact, in a highly significant advance, it has recently been reported by two groups that mutations within histone H3 and the associated chromatin modifying apparatus are frequent in paediatric brain cancers (discussed below)  Schwart.2.1Alongside the inter- and intra-tumoural genetic heterogeneity, there is also cellular heterogeneity within the GBM tumour population with regard to differentiation status, with some cells displaying immature stem/progenitor cell markers while others have more mature differentiated features. The cancer stem cell hypothesis proposes that this variety of cellular developmental states is reflective of the normal cellular hierarchy that would exist within that tissue. Consequently the tumour might be viewed as a complex tissue/organ, or a ‘caricature’ of normal development . Thus, t+ and nestin. Isolation and characterisation of this subpopulation using cell sorting for CD133 expression, or using other markers, has shown this subpopulationc tumour initiating , which use S-adenosyl methionine (SAM) as the methyl donor. The majority of CpGs reside within repetitive elements that are methylated. Another site of variable methylation is within the CpG islands which are often associated with promoter regions of genes, and are usually unmethylated. DNA hypermethylation of CpG islands is associated with gene silencing and is also typically present at imprinted genes, repetitive elements and in genes on the inactivated X-chromosome in females .RB gene in retinoblastoma .RASSF1A and HIC1 were subjected to targeted methylation in human mesenchymal stem cells (MSCs), which resulted in the formation of cancer stem cell-like cells and initiated transformation , which has antigenic and immunotherapeutic value in GBM , cell cycle regulators (p16INK4a and p15INK4b), DNA repair genes , and genes involved in tumour invasion and apoptosis   .MGMT, a DNA repair enzyme that is methylated in 68% of glioblastomas  were found hypermethylated in more than 50% of cases . 5hmC was originally identified in human and mouse brain and was proposed to have a role in epigenetic control of neuronal function  Tahilia. This moWhether 5hmC has additional functions in addition to being an intermediate in DNA demethylation is unclear and remains controversial. According to one study, the distribution of 5hmC cannot be explained by the distribution of 5mC . Genome-In a study analysing the tissue distribution of 5hmC, the highest 5hmC levels were observed in terminally differentiated cells, while less differentiated tissue stem/progenitor cells had very low levels . Also, t4DNA is folded and compacted by histone and non-histone proteins into chromatin in eukaryotic cells. Chromatin is an important gene regulatory structure and variations are achieved through specialized histone proteins and by covalent modifications. Aberrations of histone modifications, the usage of histone variants as well as altered expression of chromatin-associated regulators are often disrupted in cancer. Histones can be modified post-translationally, which alters their interaction with DNA and nuclear proteins. Modifications of the histone tails  include methylation, acetylation, phosphorylation, ubiquitination, sumoylation, citrullination and ADP-ribosylation . It has It has become increasingly clear that chromatin modifications are at least as widespread and important as alterations in DNA methylation in cancers. Modifications such as the acetylation of lysine residues alter the charge and thus change the bulk of the nucleosome. This changes interactions with other nuclear components. Methylation, on the other hand, provides specific binding platforms for chromatin-associated proteins. H3K4 methylation positively regulates transcription by recruiting nucleosome remodelling enzymes and histone acetylases, while H3K27 regulates transcription negatively by promoting a compact chromatin structure . MoreoveThe polycomb group (PcG) proteins form large complexes that are involved in gene silencing through modifications to chromatin. They are subdivided into two groups, the polycomb repressive complex 1 and 2 (PRC1 and PRC2). The histone modifications induced by the PRC1 and PRC2 complexes allow stable silencing of gene expression . The triThe PcG proteins reversibly repress genes encoding transcription factors required for lineage choice and differentiation in stem cells . InteresEZH2-dependent epigenetic silencing of the BMP receptor 1B (BMPR1B) in a subset of glioblastoma tumour-initiating cells (TICs) has also been noted, thereby limiting the extent to which the TICs can respond to differentiation signals  is another example of histone modifier that is silenced by DNA hypermethylation in glioma and neuroblastoma,   . Restoraene body .5IDH1 and IDH2 were uncovered in secondary GBM following genome-wide sequencing   , chondroia (AML) . Initialia (AML) . Thus, aetiology .IDH1/2 mutated gliomas show distinct epigenetic profiles, characterised by a high frequency of methylated CpG islands, the “CpG island methylator phenotype” (CIMP) (” (CIMP) . The CIM” (CIMP) . CIMPs h” (CIMP) . DespiteIDH1 mutations and a role in the establishment of the glioma-CIMP (G-CIMP) has been explored using immortalised primary human astrocytes and isogenic cells expressing either mutant or wild-type IDH1 , or in the related HIST1H3B (encoding histone H3.1). These were observed in 78% of DIPGs and in 22% of non-BS-PGs respectively   Schwart. The Wu ectively .H3F3A gene, which encodes for H3.3, were highly specific to GBM cases and were observed in 31% of tumours, leading to amino acid substitutions at two critical positions within the histone tail (K27M and G34R/G4V). Mutations in the genes DAXX and ATRX, which encodes for two subunits of a chromatin remodelling complex required for H3.3 incorporation at pericentric heterochromatin and telomeres, were identified in 31% of samples overall and in 100% of the tumours with a H3.3 mutation. The mutations in H3.3, ATRX and DAXX were shown to be largely specific to paediatric cases, further highlighting the distinct molecular pathology of these cancers.The Schwartzentruber et al., study also identified mutations in H3.3, and in chromatin remodelling genes . SomaticH3F3A causing a p.Gly34Arg alteration. The alterations are both located in key regulatory sites within the histone tails that are normally associated with gene repression or gene activation depending on the histone tail modification present. Trimethylation of Lys27 in histone H3 (H3K27me3) is associated with the silencing of genes and monomethylation (H3K27me) is associated with gene activation. Acetylated Lys27 has also been shown to be associated with transcriptionally active regions. The replacement of Lys27 with a methionine suggests a loss of function, as it removes the ability to methylate or acetylate this position. However, the mutations are present in the heterozygous state and always encode the same amino acid substitutions, which suggest a gain-of-function phenotype. Thus, the functional consequences of these alterations still remain to be elucidated. Whether these genetically-driven direct disruptions to epigenetic mechanisms in paediatric glioma are mirroring changes in adult GBM that are driven by alternative mechanisms remains to be determined.These mutations lead to a p.Lys27Met amino acid substitution. In addition, 14% of the non-BS-PGs had somatic mutations in 6Epigenetic pathways are important therapeutic targets. The altered ‘bad’ epigenetic defects that accumulate in cancer are potentially reversible, and the ‘good’ epigenetic mechanisms which may still operate in cancer stem cell driven contexts could be promoted through inductive differentiation promoting signals. However, the ‘ugly’ epigenetics driven directly through altered genetics within key regulatory molecules may be more challenging to suppress.Treating tumours with demethylating agents or histone deacetylases could activate silenced tumour suppressor genes and have clinical value. More than 40 years ago, the cytidine ribose nucleoside analogue 5-azacytidine was discovered as a potent agent for cancer treatment . It was The fact that the nucleoside analogues need to be incorporated into DNA during DNA synthesis limits the activity of the drugs in slowly proliferating cells, potentially limiting their effectiveness against quiescent cancer stem cell populations. Non-nucleoside DNMT inhibitors are therefore under development, also with a second aim of avoiding the toxicity associated with the incorporation of nucleoside analogues into DNA. The future also holds great promise for targeted epigenetic reprogramming through the use of technologies such as engineered zinc finger nucleases (ZFNs), artificial transcription factors (ATFs) and transcription activator-like effector nucleases (TALENs) . SuccessHistone deacetylases (HDACs) are a family of 18 deacetylating enzymes that remove acetyl groups from lysine residues of histone proteins, as well as on other proteins including transcription factors . HDACs ain vitro, which might be due to that HDACi promote a more open chromatin conformation and therefore permit better access of DNA damaging agents to the chromatin, with an increased efficacy of these agents. The HDACi SAHA (Vorinostat) has been successfully utilised in clinical trials of patients with cutaneous T cell lymphoma  and Cancer Genome Atlas (cancergenome.nih.gov), we should soon have comprehensive reference data defining the spectrum of all major ‘omic’ changes for a range of gliomas and related malignancies. These will provide a foundation for exploring the functional consequence and cell type specific effects of various altered pathways.Exploration of epigenetic processes within GBMs is clearly of great importance for a full understanding of the biology of this disease, and for the management of patients . As descH3.3, ATRX and DAXX) highlight the importance epigenetic alterations have in the development of glioma. Efforts to understand the biological significance of these various disruptions, particularly the elucidation of which mechanisms are key functional players either early during tumour initiation, during disease progression or in response to therapies, will help in design of rational therapeutic strategies and improve currently dismal patient outcomes.Intra-tumoural heterogeneity of cell populations also presents a major obstacle to our understanding of GBM. It is therefore now important to make use of single cell analysis and try to reconcile cancer stem cell models of the disease with clonal genetic evolution models. The recent findings of somatic mutations in key regulatory genes (