Cryptococcus neoformans to resist oxidative stress is one of its most important virulence related traits. To cope with the deleterious effect of cellular damage caused by the oxidative burst inside the macrophages, C. neoformans has developed multilayered redundant molecular responses to neutralize the stress, to repair the damage and to eventually grow inside the hostile environment of the phagosome. We used microarray analysis of cells treated with hydrogen peroxide (H2O2) at multiple time points in a nutrient defined medium to identify a transcriptional signature associated with oxidative stress. We discovered that the composition of the medium in which fungal cells were grown and treated had a profound effect on their capacity to degrade exogenous H2O2. We determined the kinetics of H2O2 breakdown by growing yeast cells under different conditions and accordingly selected an appropriate media composition and range of time points for isolating RNA for hybridization. Microarray analysis revealed a robust transient transcriptional response and the intensity of the global response was consistent with the kinetics of H2O2 breakdown by treated cells. Gene ontology analysis of differentially expressed genes related to oxidation-reduction, metabolic process and protein catabolic processes identified potential roles of mitochondrial function and protein ubiquitination in oxidative stress resistance. Interestingly, the metabolic pathway adaptation of C. neoformans to H2O2 treatment was remarkably distinct from the response of other fungal organisms to oxidative stress. We also identified the induction of an antifungal drug resistance response upon the treatment of C. neoformans with H2O2. These results highlight the complexity of the oxidative stress response and offer possible new avenues for improving our understanding of mechanisms of oxidative stress resistance in C. neoformans.The ability of the opportunistic fungal pathogen  Cryptococcus neoformans is pathogenic basidiomycetous yeast with a ubiquitous worldwide distribution. It exists primarily as an environmental organism associated with soil and is known to have a particular association with bird guano C. neoformans is also an important opportunistic pathogen that causes invasive fungal infections and is responsible for about 1 million cases and 700,000 mortalities annually Aspergillus fumigatus, Candida albicans, and C. neoformans by host immune cells in vitro and the corresponding respective virulence and avirulence phenotypes of these strains in a murine cryptococcosis model In the mammalian host, cell mediated immunity based on phagocytic cells is crucial to counteract fungal infections. Macrophages, neutrophils and other phagocytic cells generate potent reactive oxygen (ROS) and nitrogen (RNS) species that are toxic to most fungal and bacterial pathogens and cause damage to their DNA, protein and lipids. ROS and RNS are implicated in the killing of fungal pathogens such as C. neoformans is its ability to survive inside phagocytic cells. It not only resists killing by macrophages after phagocytosis, but can continue to replicate by budding within this environment and subsequently exit the macrophage without causing host cell lysis C. neoformans to survive and thrive inside this harsh environment suggests it must have mechanisms not only to neutralize the reactive molecular species to which it is exposed within the macrophages but also to repair the cellular damages caused by the oxidative and nitrosative stresses.An important virulence related trait of C. neoformans, a number of genes coding for enzymes of antioxidant defense systems have been shown to be important for both in vitro oxidative stress resistance and also for in vivo pathogenesis C. neoformans resistance to peroxide stress C. neoformans, deletion of these genes renders them sensitive to oxidative stress, albeit with a less severe phenotype than that of a tsa1Δ strain, and decreased survival in macrophage culture conditions C. neoformans also contains two glutathione peroxidases Gpx1 and Gpx2, both of which respond differently to various stressors; with only Gpx2 induced in response to H2O2 generated oxidative stress. Furthermore, both GPX1 and GPX2 deletion mutants were only mildly sensitive to oxidant killing by macrophages and exhibited no effect on virulence in a murine model C. neoformans include a cytosolic copper-zinc superoxide dismutase (Sod1) In C. neoformans experiencing oxidative stress have been reported in which the authors either used fungal cells engulfed by macrophages or grown in the nutrient rich YPD medium and treated with exogenous H2O22O2 in YPD medium, suggesting that the environment has a direct effect on the transcriptional response. This may also be due to the differences in the concentration of ROS released by the exogenous application of stress agent compared to that released inside the macrophages. Gene expression studies of oxidative stress resistance in a number of other fungal organisms such as S. cerevisiae, Schizosaccharomyces pombe and Candida sp have been published 2O2 and the magnitude of the elicited cellular response was highly dependent on the composition of the medium in which the cells were grown and treated. For example, S. pombe triggered different signaling networks mediated either by Pap1 or by Sty1 depending on the concentration of H2O2 used for the treatment 2O2 than the Sty1-mediated pathway and was responsible for inducing an adaptive response. This was shown by an induction of Pap1 with an extracellular concentration of 0.2 mM H2O2, whereas H2O2 concentrations above 0.2 mM failed to trigger this activation.Whole genome microarray studies of S. cerevisiae, the authors reported different degrees of sensitivity of yeast cells to external H2O2 when grown in different media 2O2 for gene induction, even though the same strains grown in the presence of glucose exhibited 70% death when treated with 1.5 mM H2O2. The pathogenic fungi C. glabrata has shown to be resistant to up to 40 mM of H2O2, while C. albicans was found to be sensitive at this concentration of H2O2A. fumigatus, continued to grow during two-hour incubations with 5 mM H2O2 and tolerated up to 30 mM H2O2In a study of oxidative stress and aging in 2O2 inside the phagosome during the oxidative burst is not precisely known, however multiple reports suggest that the effective H2O2 concentration may reach hundreds of micro molar in vivo conditions, we chose to treat C. neoformans cells with H2O2 in a nutrient limited YNB medium at pH 4.0. To stress the cells and avoid the induction of an overwhelming apoptotic death response, cells were treated with a concentration of H2O2 that resulted in the killing of ∼ 15% of initial cell population or lethal dose (LD∼15) which was determined via H2O2-generated oxidative stress death curves. These curves dictated the use of 1 mM H2O2 for treatment. By quantitatively measuring the concentration of H2O2 in the culture during the treatment, we discovered that within 30 minutes of incubation, all of the H2O2 was completely degraded from the medium. Therefore, we subjected RNA samples isolated from the cells at 5, 15, 30, 45 and 60 minutes post H2O2 treatment to microarray hybridization. We used a custom designed C. neoformans serotype A array using the predicted ORFs in the H99 (serotype A) C. neoformans genome identified by the Broad Institute genome sequencing project (http://www.broadinstitute.org/annotation/genome/cryptococcus_ neoformans/FeatureSearch.html). To facilitate the identification of underlying biological processes from the gene expression dataset, we generated a new gene ontology gene association file for C. neoformans genome. Herein, we report a global time-resolved genomic expression pattern of C. neoformans and identified over-represented biological processes which may point to potential mechanisms by which the fungus detects and destroys the oxidative stressor as well as repairs and recovers from the damage caused by the oxidative stress.In the context of pathogenic fungi and phagocytosis, the concentration of HC. neoformans serotype A strain KN99α was used throughout this experiment. Cells were grown on rich medium, YPD , or minimal medium, YNB, pH 4.0 (6.7 g/liter yeast nitrogen base without amino acids plus 20 g/liter dextrose and 25 mM sodium succinate at pH 4.0). Solid media contained 2% Bacto agar. Antimycin-A from Streptomyces sp. (Cat No A8674), Carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (Cat No -C2920), Salicylhydroxamic acid (SHAM) (Cat No -S607) and hydrogen peroxide solution (Cat No H1009) all were purchased from SIGMA-ALDRICH, St Louis, MO, USA. Estimation of H2O2 was performed using OxiSelect H2O2 assay kit from Cell Biolabs, San Diego, CA, USA following the protocol supplied with the reagent. Three independent experiments were carried out to calculate the standard error which is indicated by the error bars in the figures.650 = 1.5) were treated with various concentrations of H2O2 in a shaking 30°C incubator. Aliquots were taken at various time points, cells pelleted by centrifugation at 4°C. Cell pellet was washed two times with cold PBS and finally resuspended in PBS for plating on solid YPD and incubated at 30°C. Colony forming units (CFU) were counted after 2 days.Exponentially growing cells  C. neoformans KN99α were used to inoculate three bioreplicates into YNB pH 4.0 media and incubated at 30°C with shaking (200 rpm) until the cells were in mid-log phase (OD650 ∼1.5–2.0). Each biological replicate culture was split into two cultures and H2O2 was added to one of each of these two cultures to a final concentration of 1 mM. Cultures were incubated for 60 minutes and sampled at 5, 15, 30, 45 and 60-minute intervals. Sodium citrate  was added to control and test samples to rapidly halt the hydrogen peroxide stress. Cells were collected by centrifugation at 1800 g for 5 minutes and washed once with sterile phosphate buffered saline. The washed cell pellets were flash frozen and lyophilized. Lyophilized cells were stored at –80°C.Two-day-old cultures of 8 cells) was vortexed with 0.5 g of 1 mm glass beads  for 5 min., followed by the addition of 600 uL lysis solution (kit supplied) and vortexed for a further 5 min. Disrupted cells were centrifuged at 16 000 g for 3 min. and the supernatant transferred to a pre-filtration column. Rest of the procedures was carried out as described in the Manufacturer’s protocol until the final step. Isolated RNA was treated with RNase free DNase. RNA was quantified using a Nanodrop ND-1000 . The quality of purified RNA was assessed on an Agilent 2100 Bioanalyzer .RNA was isolated using the Agilent Total RNA Isolation (Protocol for Yeast)  according to the manufacturer’s instructions with the following modifications. The lyophilized pellet  according to manufacturer’s instructions with the following modifications. All reaction component volumes were doubled with the exception of RNA concentration resulting in a final reaction mix volume of 40 uL. After the addition of Stop buffer, to remove any unincorporated dye, the reaction mix volume was brought up to 100 uL with nuclease-free water followed by the addition of 3 volumes of 100% nuclease-free ethanol. This 400 uL reaction volume was then applied to an RNeasy Mini Kit column  and processed according to manufacturer’s instructions . Dye incorporation was quantitated using a Nanodrop ND-1000 (Nanodrop Technologies) by measuring emission wavelength at 570 nm (Cy3) and 670 nm (Cy5). Labeled-RNA concentration and dye incorporation were used to determine the RNA labeling specific activity (pmol/ug). Labeled and unlabeled RNA were combined to adjust the sample specific activity to the empirically determined intensities of 40 and 45 pmol/ug for Cy3 and Cy5 labeled samples, respectively. Unstressed Cy3-labeled and 1 mM HMicroarray hybridization and scanning were preformed according to Agilent Microarray processing specifications .rijk = α+Ai+Gk+(AG)ik+Dl+αrijk; where yrijk is the logarithm of the rth replicate model-based expression value of gene k at the ith time point with the jth treatment . This model accounts for variance in genes (G), arrays (A), and array plus gene; α term accounts for error.The LOESS balanced data from the Feature Extraction was used to assess replication consistency across biological and process replicates using a linear ANOVA model that considers all 13,864 probes on the array and data for all the replicates, to determine the significance of differential expression Archive RNA extracted for the microarray experiments was used to make first-strand cDNA using the First-Strand cDNA synthesis kit for reverse transcription-PCR (Roche). This cDNA was used as template for real-time PCR analysis using SsoFast SYBR green PCR reagents (Biorad) according to the manufacturer’s recommendations. A BioRad CFX96 thermal cycler was programmed with the following two step PCR cycles: 5 s at 98°C, 5 s at 60°C, and a plate read was repeated in the second step for a total of 40 cycles. A melting curve was performed at the end of the reaction to confirm a single product. Standard curves were performed for each primer set and efficiencies calculated. The data were normalized to glucose-6-phosphate dehydrogenase cDNA expression included with each experiment.http://www.broadinstitute.org/annotation/genome/cryptococcus_neoformans/Info.html) were submitted to the GOAnno program of AgBase −10. The sequences and annotations from the Uniprot database were filtered to remove sequences that had only annotations with an evidence code of IEA (Inferred from Electronic Annotation) or ND . Additional annotation was obtained by submitting those proteins without a match in the UniProt database to InterProScan −20. Gene ontology term enrichment was carried out using a hypergeometic test as implemented in the GOstats program, using a p-value of ≤0.01 as a criteria for significance The predicted protein sequences of H99 serotype A from the Broad assembly 2  for 30 min. At the end of the incubation, cells were rapidly chilled and collected by centrifugation. Cell pellet was lysed in 8 M urea containing buffer and equal amounts of protein per sample were used for immune blot analysis using antibodies specific to ubiquitin proteins . Uniform transfer of proteins across the membrane was verified by staining the membrane with Swift Membrane Stain  before subjecting them to immuno blot analysis.2O2 once it has been added to C. neoformans culture, actively growing yeast cells in YNB, pH 4 medium were treated with 1 mM H2O2. At different time intervals aliquot of the cell culture was removed, cells were separated and the supernatant was used to measure the residual H2O2 in the medium. A concentration of 1 mM H2O2 was rapidly degraded by growing C. neoformans cells and was completely absent in the medium after 30 minutes  were decreased to ∼40–50% of the initial concentration by two hours, clearly indicating that unlike the cells grown in YPD, cells grown in nutrient limited YNB medium are much less efficient in degrading exogenous H2O2. at 30°C . However of H2O2 . Higher 650 = 1–8) in either YPD or YNB medium and treated with various concentrations of H2O2 (2–8 mM), the rate of H2O2 breakdown increased as the culture density increased (data not shown). A cell culture grown in YPD to a density of OD650 = 2.7 was able to degrade 4 mM of H2O2 in just 20 min, while those with a density of OD650 ≥ 4.0 took only 10 minutes . A concentration of 0.5 mM H2O2 had no significant effect on cell viability at any time point  model as previously described 2O2 in C. neoformans  at the five minute time point . At 15 mresponse  is consist cells .2O2 treatment in our experiment (treatment in YNB medium) to a previously published microarray dataset by Ko et al in which authors employed YPD medium for growth and treatment with H2O2. Consistent with our present findings on the influence of media composition and treatment conditions on the oxidative stress response we found considerable differences in the gene sets, but a high level of concordance for those genes identified as differentially expressed in both datasets 2O2 degradation, array probe differences, strains used and the intrinsic variation of experiments done in two different laboratories. We believe the high degree of concordance between the overlapping probes strongly validates both experimental approaches.We compared the sets of genes differentially expressed in response to H2O2 treatment followed similar profile to microarray dataset validating the quality of our array. The linear regression analysis of the microarray and qRT-PCR measurements resulted in a correlation coefficient  of 0.94, suggesting that array dataset correlated positively and closely with qRT quantification (data not shown).Microarray results were validated by performing qRT-PCR analyses of six genes  that encompassed a range of expression levels at 30 min time point. The mRNA abundance of these transcripts at 30 min after H2O2 treatment and recovery of the viable cells at two hour time point  enrichment analysis was performed and several biological processes were significantly enriched in our lists at all time points  and S3. me point  emphasiz2O2 causes substantial perturbation of cellular processes.In addition to the above biological processes, a greater number of unique cellular processes significantly affected only at 30 and 45 min time points were identified . They in2O2 treatment  TRR1 coding for thioredoxin reductase (CNAG_05847) CCP1; CNAG_01138) CAT1 (CNAG_0498) and CAT3 (CNAG_00575) 2O2-induced stress and further validating the quality of our array.As expected, our dataset identified oxidation-reduction process as one of the top ten enriched categories affected by Hreatment . Moreovereatment . Some ofC. neoformans genome using our GO database assigned a total of 514 genes to the oxidation-reduction functional category. We discovered that 205 genes exhibited differential expression at one or more time point with a maximum of 124 genes showing altered mRNA levels at 45 min post H2O2 treatment . The sulfiredoxins are critical for oxidative stress resistance in yeast and higher eukaryotes C. neoformans or in other fungal species. A preliminary bioinformatic analysis of a few select genes supported their potential roles during oxidative stress in C. neoformans and these are discussed below.In addition to the core antioxidant response, our microarray data revealed the contribution of several additional gene products in oxidative stress resistance that may provide clues to novel mechanisms of stress resistance. The strong transcriptional regulation of 50 genes for a significant period of time during He stress . For exa2O2 treatment. Bacterial dioxygenases are reported to have either iron-sulphur center or non-heme mononuclear iron as cofactors. Importantly, these enzymes catalyze oxidation of the substrates at the expense of reduced NADPH C. neoformans after H2O2 treatment suggests that this may be a part of protective mechanism of the yeast cells to inhibit NADPH-requiring reactions while cells are coping with oxidative stress.One of the genes that are under persistent transcriptional regulation in response to peroxide stress is CNAG_03238, predicted to belong to dioxygenase subfamily. It is similar to a putative dioxygenase gene from a saprophytic soil borne filamentous bacteria, and shows significant repression at four of the five time points post HS. cerevisiae provides increased resistance to menadione-induced stress Arabidopsis and other higher eukaryotes Another uncharacterized gene, CNAG_01542, is predicted to have domains belonging to taurine catabolism dioxygenase super-family and exhibits increased expression at 30, 45 and 60 minutes. Proteins containing these domains have been demonstrated to be important for sulfonate metabolism, in the synthesis of Fe-S cluster protein family members and their expression in C. neoformans genome, with 272 (∼45%) showing differential expression at one or more time points during H2O2 treatment  a majority of the metabolic process related genes were still under transcriptional regulation  as well. Sterol analysis of the flucanazole resistant C. albicans showed defects in ERG2 and ERG3S. cerevisiae ERG2 (CNAG_00854) and ERG3 (CNAG_00519) showed altered expression levels in our microarray dataset  and a homolog of MDR1 (CNAG_00796). All three genes show differential transcriptional regulation, with CDR11 showing consistent up regulation at 15, 30 and 45 minutes and MDR1 exhibiting increased levels at 30, 45 and 60 minutes , and AOX1, encoding an alternative oxidase (CNAG_00162). C. neoformans CCP1 is known to protect against external oxidative stress inducing agents and consistent with that role, its transcription was increased at 30 and 45 minutes . By contrast, yeasts such as C. albicans and S. cerevisiae cells were able to grow normally in the presence of 2.5–5 ug/ml of FCCP 2O2 in the presence and absence of FCCP and incubated at 30°C. The capacity to withstand oxidative stress triggered by H2O2 challenge was markedly decreased by FCCP in a concentration dependent manner  gene family have been implicated in a wide variety of cellular functions including those closely connected with oxidative stress resistance such as respiratory growth, glutathione homeostasis, UV and metal ion resistance and double strand DNA repair UBC gene deletion strains exhibit varying degree of sensitivities to diverse external stress conditions indicating the functional overlap between different UBC gene products 2O2 induced oxidative stress involves protein damage by irreversible oxidation and these oxidized proteins are recycled through the ubiquitin dependent proteasomal pathway in other systems C. neoformans, we treated fungal cells with increasing concentration of H2O2 and subjected the cell lysates to immuno-blot analysis. The blots were probed with ubiquitin specific antibody to quantify the amount of ubiquitin tagged proteins in treated cells to their untreated counterpart. We observed an H2O2 concentration dependent increase in the amount of ubiquitin conjugated proteins in the total cell lysate , deubiquitinating enzyme (GO: 0004843), ubiquitin-dependent protein catabolism (GO: 0006511), protein ubiquitinylation (GO: 0016567), and proteosomal ubiquitin-dependent protein catabolism (GO: 0043161) in the s genome . Of the reatment . In S. cl lysate . Densitol lysate .C. neoformans both for establishing an infection in the host and also for surviving extended periods of time in the environment. The kinetics of H2O2 degradation by cells growing in YNB indicates that actively growing C. neoformans cells have an efficient mechanism to degrade exogenous H2O2. This may involve the activity of functional catalase enzymes reported to be present in C. neoformans2O2 degrading activity in the culture supernatant is consistent with an earlier study reporting the absence of secreted catalase family proteins in C. neoformans genome 2O2 breakdown by cells growing in YPD compared to those grown in YNB was rather surprising and clearly suggests that growth media composition may significantly affects cell’s constitutive redox potential. Moreover, we also demonstrated that cells grown to a higher optical density were able to degrade exogenous H2O2 more rapidly compared to the culture at lower optical density, again emphasizing that the cell culture and treatment conditions may significantly influence the magnitude and intensity of the induced cellular transcriptional response to oxidative stress. Therefore these important experimental parameters need to be considered when comparing different datasets generated for H2O2 mediated stress induced either in the same organism or between different organisms.The transcriptional regulation of protective mechanisms against oxidative stress is critical for 2O2 treatment  at the 15 and 30 minute time points consistent with its established role in oxidative stress and virulence C. neoformans TSA1 is a typical 2-cys peroxiredoxin and its homolog in S. cerevisiae is located in the cytoplasm C. neoformans TSA3 (CNAG_06917) and DOT5 (CNAG_02854) belong to 1-cys peroxidase family and have shown increased expression at both protein and mRNA levels in response to oxidative stress in a previous study C. neoformans TSA3 and DOT5 in S. cerevisiae are located to mitochondria and nucleus respectively and are known to play a major role in oxidative stress resistance C. neoformans is not known and their deletion in C. neoformans did not affect oxidative stress resistance in either in vitro or in vivo conditions TSA3 and DOT5 shown in our dataset is similar to the pattern of expression of the majority of genes of potential mitochondrial origin suggesting that both TSA3 and DOT5 may contribute additionally to oxidative stress resistance in C. neoformans by unknown mechanisms.In the oxidation-reduction category we found interesting gene regulation patterns of thioredoxin family genes. Among the genes of thioredoxin family, we found increased expression of C. neoformans showed differential expression. Increased expression of CAT1 is consistent it being the only functional catalase as determined by the in-gel activity staining of C. neoformans cell extracts CAT1 and CAT3 suggests these genes may be regulated by similar factors, consistent with both belonging to the same phylogenetic clade 2O2 in the medium had been degraded. This suggests that the CAT1 and CAT3 gene products may have additional novel roles during in vitro oxidative stress induced by H2O2.Three of the four known catalase genes in C. neoformans undergoes major metabolic adaptation during growth inside the host 2O2 involves the genes encoding pentose phosphate pathway (PPP) enzymes. A major product of PPP is the production of NADPH that is critical for the function of proteins required for repairing oxidative protein damage. Accordingly the components and function of PPP have been shown to be important for resistance and adaptation to oxidative stress in yeast and higher eukaryotes. In both S. cerevisiae and C. glabrata, H2O2 treatment increased the expression of genes belonging to the pentose phosphate pathway ZWF1) is increased during exposure of C. albicans to nitrosative stress S. cerevisiae, independent deletion strains of pentose phosphate pathway genes such as 6-phospho gluconate dehydrogenase (GND1), D-ribulose-5-phosphate 3-epimerase (RPE1), transketolase 1 and transketolase 2 (TKL1and TKL2), glucose-6 phosphate dehydrogenase (ZWF1) and transaldolase (TAL1) all exhibit increased sensitivity to oxidative stress C. neoformans. Previous studies demonstrated that neither the ZWF1 mRNA nor protein levels were altered during nitrosative stress in C. neoformans. Consistent with this deletion of ZWF1 gene in C. neoformans did not increase their sensitivity to either oxidative or nitrosative stress C. neoformans.In addition to their initial exposure to oxidative stress inside the macrophages, CCP1-mediated mitochondrial electron transport chain to meet the sudden increase in demand for ATP necessary for repairing damaged proteins. Previous serial analysis of gene expression (SAGE) revealed a high abundance of tags corresponding to phopsphophenol pyruvate carboxykinase (PCK1), a main control enzyme for the regulation of gluconeogenesis in the lung-exposed cryptococcal library suggesting that gluconeogenesis is important for fungal survival in the host lung due to the limited availability of glucose C. neoformans cells to H2O2 in the presence of glucose we also observed significant and consistent up-regulation of PCK1 (CNAG_04217) after H2O2 treatment , malate synthase (MLS1), pyruvate decarboxylase (PDC1) or alcohol dehydrogenase (ADH1) during oxidative stress in contrast to their increased expression during growth conditions in the host lung. The up-regulation of the glyoxylate pathway during pulmonary infection conditions has been attributed to the higher amount of acetate present in lung tissues 2O2.Unlike the response of the TCA cycle and the gluconeogenic pathway, genes of the glyoxylate cycle that converts acetyl-CoA to succinate for the synthesis of carbohydrates responded completely differently in our dataset compared to the previously published lung exposed cryptococcal SAGE library C. neoformans during nitrosative stress Peroxide-induced oxidative stress also caused significant perturbation of genes of amino acid biosynthesis pathway . IncreasS. cerevisiae transcriptional up-regulation of CCP1 after H2O2 treatment and its role in stress signaling has been reported to contribute to oxidative stress resistance C. neoformans CCP1 was initially identified by bioinformatic analysis, the AOX1gene was identified by its significant up-regulation in C. neoformans in response to exposure to 37°C temperature AOX1 as being enriched in the mouse lung library, but not CCP1C. neoformans CCP1 and AOX1are potentially subject to different mechanisms. Moreover, the AOX1 deleted strains exhibited a slight virulence phenotype in a mouse model while CCP1 deleted strains were avirulent. However, both CCP1 and AOX1 independent gene deletion strains exhibited in vitro oxidative stress sensitivity towards H2O2 and tert-butyl hydroperoxide respectively CCP1 and AOX1 observed in our dataset in response to peroxide stress further supports that their transcription is under the control of different signaling mechanisms. The absence of AOX1 up regulation seen in our dataset is similar to the one reported in the previously published microarray analysis AOX1observed in our microarray analysis was in agreement with the results obtained in our laboratory when we subjected RNA samples to RNA seq analysis (unpublished results). The functional consequence of differential expression of CCP1 during H2O2 treatment is dramatic and is reflected in the ability of its specific inhibitor antimycin to completely abolish oxidative stress resistance to H2O2, whereas specific inhibition of AOX1 had little effect on oxidative stress resistance.In C. neoformans in the presence of various stress conditions such as nitrosative stress, heat shock and growth inside the host, all identified genes related to the mitochondria and respiratory chain to be differentially expressed S. cerevisiae and C. albicans2O2 provides evidence that C. neoformans cells undergoes substantial damage by H2O2 and energy dependent repair mechanisms are critical for recovering from oxidative stress. The increased concentration of ubiquitin tagged proteins observed by us during H2O2 treatment . UBI4 dependent ubiquitination has been shown to play a major role in the ubiquitin dependent protein degradation pathway during thermal, cell wall and oxidative stress conditions in S. cerevisiae and C. albicansS. cerevisiae RSP5 (CNAG_05355)  . RSP5 enUBC4 and UBC5 in S. cerevisiae were exceedingly sensitive to stress conditions S. cerevisae upon heat stress S. cerevisiae UBC4 in C. neoformans  and RPT gene  families. Several recent reports in yeast (S. cerevisiae) and mammalian systems indicate that degradation of oxidized proteins may occur either by ubiquitin/ATP-dependent  or ubiquitin/ATP-independent  mechanisms 2O2 treatment indicates that the 19S regulatory particle may play a minor role in maintaining cellular homeostasis during oxidative stress in C. neoformans.Deletion mutants of ubiquitin conjugating enzymes ent time . S. cere2O2 removal, we identified many more genes affected by oxidative stress than were identified in previous studies of oxidative stress in C. neoformans. The pattern of the transcriptional response mirrored the kinetics of peroxide removal and allowed us to infer potential mechanisms for the response as well as the recovery from oxidative stress. We found potential novel mechanisms for the role of mitochondria and expanded our understanding of the role of ubiquitin-dependent proteolysis in recovery from oxidative e stress.By examining gene expression differences over a time course that paralleled the kinetics of HFigure S12O2 breakdown by The affect culture density on HC. neoformans cells. A 4 mM of H2O2 was added to cultures at various densities (OD650) growing in YPD (A) and YNB (B). At various time points samples were withdrawn, cells separated by centrifugation and the supernatant was used for H2O2 estimation. The percentage of residual H2O2 was plotted against H2O2 treatment time. Standard bars reflect standard error calculated from three independent experiments.(TIF)Click here for additional data file.Figure S2Differential expression pattern of C. neoformans genes related to anti-fungal drug resistance upon peroxide stress.(TIF)Click here for additional data file.Table S1Global transcriptional profile of C. neoformans2O2 induced oxidative stress. genome during H(XLSX)Click here for additional data file.Table S22O2 treatment.Gene ontology enrichment analysis of the differentially expressed probes at various time points during H(PDF)Click here for additional data file.Table S3Master list of gene ontology annotation of the differentially expressed probes at various time points.(XLSX)Click here for additional data file.Table S4Differential expression pattern of C. neoformans genes assigned to oxidation-reduction functional category at various time points during exposure to oxidative stress.(XLSX)Click here for additional data file.Table S5List of C. neoformans2O2 induced oxidative stress. differentially expressed genes assigned to oxidation-reduction functional category that exhibited persistent induction or repression for a minimum of three time points during H(PDF)Click here for additional data file.Table S6Differential gene expression profile of the genes belonging to metabolic process functional category with proposed gene names.(XLSX)Click here for additional data file.Table S7Transcript abundance at multiple time points of the genes belonging to C. neoformans2O2 induced oxidative stress. ubiquitin dependent protein catabolic processes during H(XLSX)Click here for additional data file.


To quantify myocardial T2 value in patients with myocarditis and correlate the distribution of abnormal T2 values with the extent of macroscopic late gadolinium enhancement (LGE).25 patients with myocarditis were retrospectively evaluated for the utility of T2 mapping in diagnosing myocarditis. Patients with elevated troponins, negative coronary angiogram, and atypical LGE were diagnosed as acute myocarditis. Patients with normal troponins and macroscopic LGE at the time of cardiac MRI were diagnosed as remote myocarditis. As per our institutional protocol, T2 mapping sequences were performed in all cases with suspected myocarditis in addition to standard LGE images on 1.5 T scanner . T2 mapping was performed on three short axis images , yielding 16 myocardial segments for analysis (AHA segments). Single 4 chamber view image was obtained in addition. Minimum, peak and mean segmental T2 values were calculated by the first reader. Average segmental T2 values were documented along with documentation of the number of segments with elevated T2 values. The presence or absence of LGE was documented by a second reader blinded to the T2 results. Average segmental T2 values were then correlated with troponin levels at the time of the MRI examination.In patients with acute myocarditis, mean T2 values were elevated in segments showing LGE . The T2 values were also elevated in myocardial segments with no macroscopic LGE (avg 60 msec). On an average, there were 6 additional segments that showed elevated T2 values and no macroscopic LGE. In patients with remote myocarditis, the T2 values were normal in areas of LGE.T2 mapping can be utilized as an objective tool for diagnosing acute myocarditis. In addition, T2 values were normal in patients with normal cardiac enzymes and a typical pattern of myocarditis associated LGE. Given the non contrast nature of this technique, normal T2 values may exclude a diagnosis of acute myocarditis in patients with renal insufficiency.None.


The emergence of X4 tropic viral strains throughout the course of HIV infection is associated with poorer prognostic outcomes and faster progressions to AIDS than for patients in whom R5 viral strains predominate. Here we investigate a stochastic model to account for the emergence of X4 virus via mutational intermediates of lower fitness that exhibit dual/mixed (D/M) tropism, and employ the model to investigate whether the administration of CCR5 blockers in-vivo is likely to promote a shift towards X4 tropism. We show that the proposed stochastic model can account for X4 emergence with a median time of approximately 4 years post-infection as a result of: 1.) random stochastic mutations in the V3 region of env during the reverse transcription step of infection; 2.) increasing numbers of CXCR4-expressing activated naive CD4+ T cells with declining total CD4+ T cell counts, thereby providing increased numbers of activated target cells for productive infection by X4 virus. Our model indicates that administration of the CCR5 blocker maraviroc does not promote a shift towards X4 tropism, assuming sufficient efficacy of background therapy (BT). However our modelling also indicates that administration of maraviroc as a monotherapy or with BT of suboptimal efficacy can promote emergence of X4 tropic virus, resulting in accelerated progression to AIDS. Taken together, our results demonstrate that maraviroc is safe and effective if co-administered with sufficiently potent BT, but that suboptimal BT may promote X4 emergence and accelerated progression to AIDS. These results underscore the clinical importance for careful selection of BT when CCR5 blockers are administered in-vivo. CCR5 blockers are a promising new class of anti-HIV drugs that act by binding to the CCR5 coreceptor, thereby reducing the number of CD4-CCR5 complexes available for viral binding by HIV and consequently inhibiting the viral entry stage of the infection cycle A key concern with the administration of CCR5 blockers in-vivo relates to the emergence of CXCR4 (X4) tropic virus Over the course of untreated infection, X4 tropic virus generally emerges at later stages of infection stochastic model to account for X4 emergence in-vivo. While previous deterministic modelling of the X4 switch demonstrated that X4 emergence can in principle be accounted for by increased activation of naive CD4+ T cells at later stages of infection stochastic process. We assume that the tropism shift is also driven by random viral mutations during the reverse transcription step of the infection cycle stochastic event, with a median emergence time of approximately 4 years post-infection with considerable variation around this time deterministic models.In the present study we investigate a Our model is employed to investigate the course of HIV infection when maraviroc is administered at early, intermediate or late stages of infection, and also when co-administered with background therapy (BT) of variable efficacies. Previous modelling has only considered the administration of therapy at a single time-point Three uninfected CD4+ T cell subpopulations are modelled : restingM respectively denote the total number of resting naive, activated and resting memory CD4+ T cells at time MA respectively denote the number of activated naive CD4+ T cells (CD4+HLA-DR+CD45RA+) and activated memory CD4+ T cells (CD4+HLA-DR+CD45RO+) in PB. It is assumed in our model that only activated cells may give rise to productively infected cells upon infection The variables The term Here In our model The term In the present model The term The parameters modulated infectivities” of X4 and of D/M for activated memory CD4+ T cells. Since activated memory CD4+ T cells predominately express CCR5 modulated infectivitiesmodulated infectivitiestive see , but theThe term For any HIV virion that enters a cell, it is assumed that viral mutations may occur during the reverse transcription step of infection (see above). It is assumed that the 3 amino acids at positions 11, 25 and 29 in the V3 region of the env gene determine viral tropism The term The course of infection is simulated from age 30 to age 40  to below 200 cells/µL in 10 years (by age 40) for an individual infected with R5-tropic virus, in whom no D/M and/or X4 tropic virus emerges Viral recrudescence from below detection (50 HIV RNA copies/mL) to approximately 4.5 log10 HIV RNA copies/mL after 4 weeks following cessation of HAART Median time of X4 emergence of approximately 4 years post-PHI not to inhibit D/M strains in order to consider the worst-case scenario in the present analysis, despite previous studies reporting that maraviroc may inhibit some D/M strains not to inhibit X4 viral strains, since these utilize the CXCR4 coreceptor that is not blocked by maraviroc. It is assumed in all simulations that maraviroc has an efficacy of 90%  assuming viral mutations in our model. A single simulation for an individual in whom X4 emerges at approximately 4 years post-PHI is shown in We also performed Monte Carlo sampling over 250 trials for the case of untreated infection  C,D,E,F.Next we investigated whether administration of maraviroc without BT (i.e. BT has efficacy 0%) would promote selection for X4 tropic virus and result in accelerated progression to AIDS. To this aim, we simulated an individual who receives maraviroc as monotherapy at age 32  A,B. Admdeterministic modelling Following selection for X4 tropic virus after administration of maraviroc as monotherapy, total CD4+ T cell exhibit a rapid progression to AIDS within 1 year  A, so thTo further investigate the likelihood of selection for D/M and/or X4 tropic virus following administration of maraviroc, we performed Monte Carlo simulations of maraviroc administration at early, intermediate and late stages of infection respectively corresponding to therapy administration after 1, 3.5 and 6 years post-PHI. We considered scenarios in which maraviroc was applied with BT of moderate efficacy (BT has 90% efficacy) and also with BT of high efficacy (BT has 99% efficacy). Maraviroc was assumed to have an efficacy of 90% in all scenarios.First we investigated whether BT therapy of moderate efficacy (BT has 90% efficacy) would ensure sufficient X4 suppression . When thNext we ran Monte Carlo simulations for the case that maraviroc is administered with BT of high efficacy (BT has 99% efficacy) as shown in stochastic model of X4 emergence to examine whether administration of CCR5 blockers in-vivo is likely to promote X4 emergence, thereby resulting in accelerated progression to AIDS. While previous studies investigated deterministic models of X4 dynamics where X4 emergence was driven by increasing activation of naive CD4+ T cells that express CXCR4 but not CCR5 stochastic model of X4 emergence that explicitly includes CD4+ T cell activation dynamics. In addition to increasing activation of naive CD4+ T cells modelled previously stochastic event, with a median emergence time of approximately 4 years post-infection and a considerable variation in the time when X4 emerges deterministic models.Here we investigated a not result in a single instance of X4 emergence  is safe at early, intermediate and also late stages of infection if administered with sufficiently potent BT (99% efficacy). Monte Carlo simulations of this scenario in the present analysis did ence see . These rThe results of the present analysis are further supported by observations that the virus can find novel ways to bind the CCR5 chemokine coreceptor when CCR5 blockers are administered in-vivo, rather than resorting to a tropism switch Our analysis also indicates that the in-vivo administration of CCR5 blockers as monotherapy  can promote X4 emergence, thereby resulting in accelerated progression to AIDS. These results are in agreement with previous reports from three macaques dually infected with R5 and X4 tropic virus, where administration of the CCR5 blocker CMPD 167 as monotherapy was observed to promote outgrowth of X4 tropic virus not inhibit D/M virus, whereas previous studies reported inhibition of some D/M strains by maraviroc The present conclusion, regarding safety of maraviroc with sufficiently potent BT, is likely to be very robust. We assumed that the infectivities of D/M and of X4 (for activated memory CD4+ T cells) increase following suppression of R5 virus, thereby resulting in increased selection for D/M and X4 viral strains We extend on previous modelling by assuming that mutation from R5 to X4 occurs via intermediates that are of D/M tropism and that are of lower fitness than R5 and X4 The model presented here has a number of limitations. Firstly, our modelling did not include additional cell populations such as macrophages that might contribute to increased selection for X4 virus at later stages of the infection when total CD4+ T cell counts are low, since macrophages provide an additional source of CXCR4-CD4 complexes deterministic models of X4 selection dynamics. The key result of our analysis and modelling is that the administration of maraviroc is safe and that it does not promote X4 emergence if employed with BT of sufficient potency. Our results also indicate that selection for X4 tropic virus may occur if BT of insufficient efficacy is administered with maraviroc. These results highlight the need for careful selection of BT when CCR5 blockers are administered in-vivo, as well as raise the question whether alternative anti-HIV CCR5-targeting treatments like gene therapy In conclusion, we have presented the first stochastic model of in-vivo X4 selection dynamics and investigated whether CCR5 blockers promote X4 emergence resulting in accelerated progression to AIDS. Since X4 emergence is a stochastic event with a median emergence time of approximately 4 years post-infection, and with considerable variation in the time when X4 emerges, the modelling results presented here are more likely to reflect likely clinical outcomes than previously presented Figure S1modulated infectivity” Plot of the “ Here (TIF)Click here for additional data file.Figure S2modulated infectivity” Plot of the “ Here (TIF)Click here for additional data file.Figure S3Plots of the term  Negative values are not shown above. The function values (TIF)Click here for additional data file.


Classical modified Look-Locker sequence (MOLLI) can induce a long breath-hold and is prone to cardiac and respiratory motion. Several shorter sequences based on inversion  and saturation recovery (SASHA) of longitudinal relaxation have been proposed for derivation of T1 values or calculation of extracellular volume fraction or lambdas. Despite the validation in T1 gel phantoms, it has not been determined whether these novel sequences provide equivalent information on T1 when performed within the same individual.Twenty-three subjects underwent T1 mapping in a mid-ventricular equatorial short axis slice using above sequences on 3T clinical scanner prior and after gadolinium contrast (0.2 mmol/kg) administration. The images were analyzed using PRIDE tool with in-built automated motion correction. ROIs were drawn conservatively within the septal and lateral myocardium and the blood pool. Comparison of the native T1 values and lambdas were performed and expressed as percentage of mean difference from the values obtained with gold-standard MOLLI sequence.3'5MOLLI shows the nearest approximation to MOLLI derived values with an underestimation of values in native myocardium by 1.2% and lambdas by 11%. T1 values obtained with SASHA were overestimated by 17%, and lambdas by 29%, whereas shMOLLI led to an underestimation of T1 values by 13% and lambdas by 22%.We demonstrate that 3'5'MOLLI provides near identical T1 values and its derivatives.We propose that 3'5'MOLLI is the optimal 'shortened' sequence for clinical derivation of T1 values.NIHR


IDH1 mutation appeared to be a strong predictor of clinical outcome, overruling histological malignancy grade [IDH1 mutation alters the cellular metabolism and epigenetic phenotype influencing cellular proliferation. IDH1 mutation infers increased levels of D2HGDH leading to the inhibition of DNA and histone demethylating enzymes, resulting in the glioma-CpG island phenotype [IDH1 mutant cells show alterations in glutamine, fatty acid and citrate synthesis pathways, which all may have their influence on cellular proliferation [Recently heterozygous mutations in the active site of the enzyme isocitrate dehydrogenase 1 (IDH1) were discovered in glioblastomas [cy grade . IDH1 iscy grade . IDH1 muhenotype . Alteredhenotype . In addiferation .IDH1 function affect the glucose metabolism, which may explain the different biological behaviour of tumors with and without the IDH1 mutation [IDH1 mutated samples  and in 39 IDH1 wild-type glioma samples  and in four samples of normal brain (Table IDH1 mutated cells seem to compensate for the low production of α-ketoglutarate by overexpressing D2HGDH and L2HGDH in the cytoplasm. They also overexpress GLUD1, which converts glutamate to α-ketoglutarate inside the mitochondria. In addition, we found that IDH1 mutated cells overexpress HIF1AN. The HIF1AN gene inhibits HIF1α. Since HIF1α acts as an oxygen sensor that promotes angiogenesis, the formation of dysfunctional tumor vasculature is counteracted in IDH1 mutated cells. Furthermore, the IDH1 mutated cells overexpress the LDHB gene while cells without IDH1 mutation overexpress the LDHA gene. The present results illustrate that tumor cells without IDH1 mutation switch their energy production from a low rate of glycolysis followed by the TCA cycle to a high rate of glycolysis followed by aerobic glycolysis (LDHA up-regulation; Figure LDHA and LDHB influences the progression of cancer cells [IDH1 mutation tend to correct their energy production through the TCA cycle by overexpressing LDHB (Figure IDH1 mutated gliomas.The changes in mutation . In ordeer cells ,9. It maIDH1 mutation normalize their glucose metabolism, which appears to result in a slower tumor progression. Depending on the IDH1 status of the tumor, specific interference with the glucose metabolism and aerobic glycolysis should therefore be considered for future therapeutic strategies.We conclude that gliomas with All authors declare that they have no competing interests.DM and LB carried out the molecular genetic analyses; SS and PvdS carried out the data analysis and DM and JMK conceptualized and designed the study and wrote the manuscript. All authors read and approved the final manuscript.


Pknox2 was mainly expressed in the zeugopod domain of the murine limb at E10.5 and E11.5. Misexpression of Pknox2 in the limb bud mesenchyme of transgenic mice led to deformities in the zeugopod and forelimb stylopod deltoid crest, but left the autopod and other stylopod skeletons largely intact. These malformations in zeugopod skeletons were recapitulated in mice overexpressing Pknox2 in osteochondroprogenitor cells. Molecular and cellular analyses indicated that the misexpression of Pknox2 in limb bud mesenchyme perturbed the Hox10-11 gene expression profiles, decreased Col2 expression and Bmp/Smad signaling activity in the limb. These results indicated that Pknox2 misexpression affected mesenchymal condensation and early chondrogenic differentiation in the zeugopod skeletons of transgenic embryos, suggesting Pknox2 as a potential regulator of zeugopod and deltoid crest formation.The TALE (Three Amino acid Loop Extension) family consisting of Meis, Pbx and Pknox proteins is a group of transcriptional co-factors with atypical homeodomains that play pivotal roles in limb development. Compared to the in-depth investigations of Meis and Pbx protein functions, the role of Pknox2 in limb development remains unclear. Here, we showed that  Hox genes  are expressed in restricted domains along the axes of the limb buds in spatial and temporal colinearity, and provide positional information for limb patterning, skeletal condensation and differentiation. Expression of the Hoxd 9-13 genes in the limb bud is activated sequentially from Hoxd9 to Hoxd13 at the posterior border of the limb bud. The Hoxa9-13 genes are activated similarly to the Hoxd9-13 genes. The sequential activation of these genes correlate with the malformations in the limb skeletons of specific Hox mutants. For instance, compound mutants of Hoxa9/d9 have a shorter humerus and a loss of deltoid crest formation in the forelimb stylopod Hox9 (Hox9aabbccdd) mutant mice exhibit severe forelimb defects with a loss of posterior skeletal elements including complete autopod loss and partial zeugopod loss, suggesting a role of Hox9 in forelimb anterior-posterior patterning Hox10 (Hox10aaccdd) results in severe agenesis in the hindlimb styplopod, and defects in the forelimb zeugopod and deltoid crest to a lesser degree Hoxa11/d11 selectively display deformities in the forelimb zeugopod Hox11aaccdd) demonstrate dramatic malformations of the fore- and hindlimb Hoxa13/d13 exhibit malformations in autopod development Hoxa and Hoxd genes results in early arrest of limb outgrowth, with severe truncations in distal elements Hox genes in limb development: Hox9 participates in stylopod specification, Hox10/11 contributes to zeugopod formation and Hox12/13 regulates autopod development.The vertebrate limb bud arises from the lateral plate mesoderm and then develops into three segments  along the proximal-distal (PD) axis through an endochondral ossification mechanism. Limb patterning and morphogenesis are established by diffusible signals originating from different domains of the limb bud, including the Fgf, Wnt and RA signaling pathways Hox genes have a universal role in a variety of developmental processes, the specificities of Hox function are achieved by interactions with co-factors such as the members of the TALE (Three Amino acid Loop Extension) superfamily. The TALE superfamily consists of transcription factors with atypical homeodomains including the Pbx, Meis and Pknox proteins. Similar to the Hox genes, these TALE genes are dynamically expressed in the limb bud. Genetic studies reveal that TALE genes regulate limb patterning and development of the skeletal elements. For instance, Pbx1 is exclusively expressed in the proximal limb bud, whereas Pbx2 is expressed throughout the limb mesenchyme Pbx1-deficient mice have malformations in the proximal limb elements, while double Pbx1/2 mutants exhibit distal limb deformities in addition to the proximal limb defects Meis1 is a specific marker for the proximal domain, especially for the presumptive stylopod skeleton in early limb bud Meis1 gene in the limb bud shifts limb PD patterning and promotes the formation of proximal limb segments Shh expression as well as Hox expression in the regulation of limb development Pknox1/2 are also dynamically expressed in the avian limb bud Pknox1-null mutation is lethal in mice at E7.5 and hypomorphic Pknox1 mutant mice exhibit no obvious alterations in limb development Pknox2 protein in limb development remains unclear.While the Pknox2 genes in limb mesenchyme or osteochondroprogenitor cells in the early limb bud in transgenic mice. Misexpression of Pknox2 in the limb bud mesenchyme resulted in deformities in zeugopod elements and a loss of deltoid crest formation. The malformations in these transgenic mice were correlated with the perturbations of Hoxd10-11 gene expression profiles in the zeugopod elements. Therefore, Hox-dependent patterning alterations underlie, at least in part, the limb zeugopod defect in mice of Pknox2 misexpression.Here, we overexpressed the Pknox2 gene  was cloned into a vector harboring the Prx1 promoter Col2a1 promoter Col1a1-3.6 kb promoter Prx1-Pknox2 transgenic mice: 5′- TCTGGTGGCAGCGAAAGTC-3′; forward oligo for Col2a1-Pknox2 transgenic mice: 5′-AGGGTGTTGTTTAGAATGGGA-3′; forward oligo for Col1a1-Pknox2 transgenic mice: 5′-CACTCCAGTGACAGCACCTCT-3′; reverse oligo for transgenic mice: 5′-ATGGAGGATAGTTCAGGGCTT-3′.The 1.4 kb coding sequence CDS of All the mouse embryonic manipulations comply with the guidelines of the Bioethics Committee of Bio-X Institutes of Shanghai Jiao Tong University (SYSK-SH-2011–0112).Mouse embryos at P0 were eviscerated, and their skins were removed. Mice were fixed overnight in 95% ethanol followed by staining overnight in Alcian Blue and Alizarin Red solution (SIGMA) as previously reported in situ hybridization, embryos were sacrificed at various ages, dissected, and fixed in 4% paraformaldehyde (PFA)/PBS at 4°C overnight. After fixation, the tissues were dehydrated in 100% ethanol and embedded in paraffin. The embedded tissues were cut to generate 8 µm-thick sections and mounted onto slides. HE staining and Safranin O staining were performed following standard protocols. Whole mount in situ hybridization were performed as described For histology and Pknox2 expression has been detected throughout the limb mesenchyme and is particularly strong in the mesenchyme underlying the ectoderm in the chick limb bud Pknox2 expression during mouse limb bud development. Meis1, Hoxa11 and Hoxd13 are used as markers for the stylopod, zeugopod and autopod, respectively Pknox2 expression was first detected as a stripe in the central region of the hindlimb at E10.5, which was between the Meis1-expression domain and the progress zone  controls, the expression domains of Hoxa10, Hoxd10 and Hoxd11 in Prx1-Pknox2 transgenic mice were anteriorly shifted . Safranin O staining on the sections at E15.5 showed that chondrocyte hypertrophy and bone ossification were delayed in the radius and ulna . Collectively, these results suggest that misexpression of Pknox2 disrupts zeugopod formation at the condensation stage and during early chondrogenic differentiation.To gain insight into the timing and appearance of the defects in and ulna . The choand ColX , which aand ColX . In factBmp7 and BmpR1b double knockout mice have a nearly absent ulna and a shortened and bent radius Pknox2 affects Bmp signaling activity, the protein expression of p-Smad1/5/8 was examined in the ulna and radius of Col2-Pknox2 transgenic embryos at E12.5 via IHC. Interestingly, p-Smad1/5/8 expression levels were markedly decreased in the developing ulna and radius of transgenic mice, whereas p-Smad1/5/8 expression levels were relatively normal in the humerus of transgenic mice and WT controls , which was active as early as the osteoblast progenitor stage. Interestingly, there were no apparent defects in other skeletal elements except for the missing deltoid crest in the forelimbs of the Col1-Pknox2 transgenic mice . Therefore, it is likely that perturbations in Hoxa10/d10 expression profiles could not fully account for the forelimb zeugopod deformities caused by misexpression of Pknox2. Double mutants of Hoxa11/Hoxd11 exhibit deformities in the forelimb Prx1-Pknox2 transgenic mice. Several lines of evidence have demonstrated that Pknox2 could form a heterotrimeric complex with the Hox and PBX proteins in vitroPknox2 forms complex with Hox11 and Pbx1/2 in controlling Hox gene expression. Ectopic expression of Meis1 in the distal limb bud also results in abnormalities in forelimb zeugopod formation Pbx1-independent manner Prx1-Pknox2 mice may result from the combination of Hox10/11 gene activity in zeugopod elements.In our study, misexpression of Prx1-Pknox2 mice were recapitulated to a lesser extent in mice ectopically expressing Pknox2 in osteochondroprogenitor cells. On the other hand, early limb patterning was largely intact in both the Prx1-Pknox2 and Col2-Pknox2 mice, which had relatively normal skeletal segmentation in the proximal-distal axis as well as normal anterior-posterior polarity in the autopod skeletal elements. Only chondrogenic differentiation and ossification were strikingly perturbed in the zeugopod elements of transgenic mice at E12.5. Misexpression of Pknox2 in the limb bud mesenchyme altered the expression of Hoxa10/d10 and Hoxa11/d11 in the zeugopod at E11.5. In fact, 5′ Hoxd (Hoxd 9-13) genes have been shown to function at the mesenchymal condensation stage of skeletal formation Hox11 functions from the condensation stage onwards and programs chondrogenic and endochondral ossification Hoxa10/d10 and Hoxd11 domains and a diminished Hoxa11-expression domain in the posterior side might lead to fewer mesenchymal progenitor cells available for ulna condensation and differentiation. These findings indicate that overexpression of Pknox2 perturbs zeugopod development at the condensation and chondrogenic differentiation stages as a co-factor of Hox proteins.The defects in the Pknox2 misexpression was the decrease of Bmp/Smad signaling in the zeugopod elements. Loss of Bmp7 and Bmpr1b in the mouse limb leads to malformations in the ulna, radius and the deltoid crest, similar to the phenotype observed in Prx1-Pknox2 transgenic mice TGFb2 also results in a slightly shortened zeugopod lacking a deltoid crest and olecranon Prx1-Pknox2 mice could be recapitulated in the Col1-Pknox2 mice but not in the Col2-Pknox2 mice, indicating that Pknox2 misexpression affects deltoid crest formation at the osteoblast differentiation stage. In fact, the deltoid crest is induced by the Bmp4 signal from neighboring tendon cells at E13.5 and then develops through endochondral ossification Pknox2 overexpression. For instance, it has been reported that Hoxa13 directly binds to the enhancer elements of Bmp2 and Bmp7 to regulate their expression Pknox2 misexpression may suppress Bmp/Smad signaling activity during zeugopod development and deltoid crest formation as a co-factor for Hox genes.Another molecular event in mice with Figure S1qRT-PCR analysis of Hox10-11 gene expression in the limbs from the Prx1-Pknox2 embryos. qRT-PCR is performed for Hoxa10, Hoxa11, Hoxd10 and Hoxd11 genes in the limbs from Prx1-Pknox2 embryos at E12.5. No obvious alteration is detected in the expression levels of these genes.(TIF)Click here for additional data file.


Imprinting is an epigenetic mechanism that restrains the expression of about 100 genes to one allele depending on its parental origin. Several imprinted genes are implicated in neurodevelopmental brain disorders, such as autism, Angelman, and Prader-Willi syndromes. However, how expression of these imprinted genes is regulated during neural development is poorly understood. Here, using single and double KO animals for the transcription factors Neurogenin2 (Ngn2) and Achaete-scute homolog 1 (Ascl1), we found that the expression of a specific subset of imprinted genes is controlled by these proneural genes. Using in situ hybridization and quantitative PCR, we determined that five imprinted transcripts situated at the Dlk1-Gtl2 locus  are upregulated in the dorsal telencephalon of Ngn2 KO mice. This suggests that Ngn2 influences the expression of the entire Dlk1-Gtl2 locus, independently of the parental origin of the transcripts. Interestingly 14 other imprinted genes situated at other imprinted loci were not affected by the loss of Ngn2. Finally, using Ngn2/Ascl1 double KO mice, we show that the upregulation of genes at the Dlk1-Gtl2 locus in Ngn2 KO animals requires a functional copy of Ascl1. Our data suggest a complex interplay between proneural genes in the developing forebrain that control the level of expression at the imprinted Dlk1-Gtl2 locus (but not of other imprinted genes). This raises the possibility that the transcripts of this selective locus participate in the biological effects of proneural genes in the developing telencephalon. The cerebral cortex is populated by two main neuronal classes, pyramidal neurons and interneurons that represent respectively 85% and 15% of cortical neurons.Two distinct, though tightly linked processes, control the development and placement of these neurons within the cortex. On one hand, ‘spatial patterning’ by which the telencephalon is regionally subdivided into defined morphological and molecular progenitor territories, thereby underlying the generation of future interneurons and pyramidal neurons The proneural genes Ngn2 and Ascl1 are respectively expressed in the dorsal and ventral telencephalon, where they determine the regional identity of neural progenitors During recent years a series of transcriptional targets of Ngn2 and Ascl1 that could mediate their functions have been reported H19 gene is transcribed from the maternal allele only , whereas its imprinted neighbor Igf2 is a paternally expressed gene (PEG) de novo DNA methyltransferases (Dnmt) Parental genomic imprinting is an epigenetic mechanism that restrains the expression of about 100 mammalian genes to one parental allele Ube3a) is linked to Angelman disease and several imprinted loci are linked to Autism Spectrum Disorders (ASD) and to Prader-Willi, two syndromes in which cerebral functions are altered Both MEGs and PEGs are required during mouse embryonic development as uniparental embryos  that express only PEGs or only MEGs, die during early development Here we identify 5 imprinted genes, 2 PEGs  and 3 MEGs  situated at an imprinted locus at 12qF1 Gtl2  was highly upregulated in the dorsal telencephalon of Ngn2 KO mice. First, to shed light on Gtl2 localization and infer about its function in the normal developing telencephalon, we performed in situ hybridizations (ISH) on WT animals. At E12.5, Gtl2 is mostly expressed in postmitotic neurons of the ventral telencephalon where it remains strongly expressed up to E17.5 . From E13.5 onward, Gtl2 starts to be expressed in the nascent cortical plate in the dorsal telencephalon , we observed that the non-coding RNA encoded by the imprinted gene cephalon Fig.1A−CM. m. domesticus and M. m. molossinus, we were able to distinguish between parental alleles and to determine that Gtl2 is maternally expressed in the developing dorsal telencephalon , confirming that Gtl2 is a MEG at this developmental stage in this structure. We then confirmed by ISH . We didn’t detect any changes in Gtl2 expression in the ventral telencephalon at any developmental stages in Ngn2 KO mice (data not shown). This data thus shows that in the developing telencephalon, the imprinted gene Gtl2 encodes for a nuclear RNA expressed mostly in post-mitotic neurons of the ventral and dorsal telencephalon and thalamus .In addition, by using reciprocal hybrid cortex issued from crosses between d by ISH Fig.1C ain Dlk1 gene in Ngn2 KO cortex , Mirg and Rtl1  Fig.2A [To determine whether the absence of Ngn2 selectively affects the Dlk1-Gtl2 locus or whether it also impacts on IGs located at other genomic regions, we measured RNA levels of 14 additional IGs present on 4 imprinted loci. These loci are situated at chromosomes 6 Fig.3A a. To further investigate the link between Ascl1 and IGs at the Dlk1-Gtl2 locus, we performed ISH on Ascl1 KO mice. We confirmed that Ascl1 is directly linked to the expression of at least two imprinted transcripts (Gtl2 and Dlk1) in the ventral telencephalon. As shown in We next thought to identify the molecular mechanism responsible for the overexpression of Dlk1, Gtl2, Rian, Mirg and Rtl1 in the Ngn2 KO dorsal telencephalon. One obvious candidate to test is Ascl1 that is known to be highly upregulated in the cortex of Ngn2 KO animals . FurtherAltogether, this suggests a model in which Ascl1 plays a central role in regulating the expression of IGs of the Dlk1-Gtl2 locus in the developing brain. First, Ascl1 would be necessary for their expression in the normal developing ventral telencephalon. Secondly, the upregulation of the RNAs from the Dlk1-Gtl2 locus in Ngn2 KO animals would be a consequence of the ectopic expression of Ascl1 in the respecified dorsal telencephalon.This study has uncovered a link between neurogenic transcription factors and a set of imprinted transcripts. First, we have observed that the imprinted gene Gtl2 is upregulated in the telencephalon of Ngn2 KO animals Fig.1. GGtl2 is part of the Dlk1-Gtl2 imprinted locus at chromosomal position 12qF1 that contains several additional MEGs and PEGs , Fig.S1); on the other hand their upregulation in Ngn2 KO animals is reduced in Ngn2/Ascl1 double KO animals . For analysis, WT and heterozygous animal were pooled as loss on one allele does not markedly alter phenotype The GFP+/−Ngn2ki or GFP+/− Ascl1+/−Ngn2ki heterozygous intercrosses. RNA was isolated using RNeasy minikit according to the manufacturer’s instructions (Qiagen). Alternatively, RNA was extracted using RNA now (Ozyme). All RNA samples were treated with DNase (Qiagen or Ambion for RNeasy and RNA now methods respectively). Reverse transcription was performed using N6 primers and MMLV-RT (Promega). Quantitative PCR (qPCR) was performed in duplicate in 384 well plates using 2× SybrGreen Mix and a LC480 Real-Time PCR System (Roche). Results are presented as linearized Cp-values normalized to housekeeping genes TBP, Gus2 and Gapdh and the indicated reference value (2-ΔΔCt). The sequence of primers is available upon request.E13.5 dorsal telencephalons were dissected out from embryos obtained from All steps and solutions for RNA in situ hybridization (ISH) were achieved in RNase free environment. Embryos were perfused intracardiacally with a solution of 4% paraformaldehyde (in PBS) and the brains were post-fixed in the same fixative over night. Brains were then cryoprotected in 30% sucrose (in PBS). ISH was carried out on 10 µm cryostat sections  using digoxygenin-labeled (Roche) RNA probes as previously described Immunostaining was performed on 20 µm thick cryosections. Blocking solution consisted of PBS supplemented with 5% horse serum (Invitrogen), 0.3% Triton X-100 (Sigma) and 3% Bovine Serum Albumin . Antibody solution consisted of PBS supplemented with 1% horse serum, 0.1% Triton X-100 and 3% BSA. Dlk1 or Tbr1 antibodies (a kind gift of Charlotte Harken Jensen and Robert Hevner respectively) were incubated overnight at 4°C and secondary during 2 hours at RT. Nuclei were stained with bisbenzimide . Sections were mounted with glycergel (DAKO).in-situ RNA hybridization and immunofluorescence staining were acquired with an Axioplan2 Zeiss microscope and a Spot RT camera, converted in false colors and overlayed using Adobe Photoshop software.Pictures of the Data are presented as mean + standard error of the mean of at least three biologically independent experiments or of WT and KO embryos. A Mann-Whitney’s test was used for comparing the distribution of data of measurements of RT-qPCR experiments.Figure S1Expression of Gtl2 RNA (ISH) at E17.5 in Ngn2 WT and Ngn2 KO mice. (A-E) Co-labeling with Tbr1 protein (immunohistochemistry) was used to show specific localization of Gtl2 positive neurons in the dorsal telencephalon . Cells that show ectopic expression of Gtl2 mRNA in Ngn2 KO mice are localized in the IZ (red arrows in B and D). Scale bars: 150 µm.(TIF)Click here for additional data file.


Diabetic ketoacidosis (DKA) is a potentially life threatening complication of initial presentation with type 1 diabetes mellitus (T1DM). Annual DKA incidence data has been reported in selected populations in many studies, however few studies have reported the trend in DKA incidence over time in an unselected representative childhood population.To determine the annual incidence of DKA at initial presentation with type 1 diabetes mellitus in all children <15 years of age between 2000 and 2009 in the Auckland region.Data from Auckland children with newly diagnosed T1DM between 1 January 2000 and 31 December 2009 were collected from Starbase, the Starship Children’s Hospital diabetes database. T1DM was confirmed by the presence of glutamic acid decarboxylase and/or tyrosime phosphatise-like protein (IA2) antibodies. DKA was defined by international criteria as venous or capillary pH and bicarbonate as mild DKA with pH <7.30 and bicarbonate <15 mmol/l, moderate DKA pH <7.20 and bicarbonate <10 mmol/l and severe DKA ph <7.10and bicarbonate <5 mmol/l.There were 481 children diagnosed with T1DM in the Auckland region  between 2000 and 2009. Over the study period the DKA incidence was highly variable (32 to 63% without a discernable change in incidence over the 10 year period [p=0.11]), thus data are expressed as a means over the study period 2000-2009. There were 47.4% of children in DKA at initial presentation which is very similar to the DKA incidence we reported for 1995-96 of 42%[The incidence of DKA at initial presentation of type 1 diabetes mellitus is much higher in Auckland children compared to other published studies. DKA incidence has plateaued over the past 14 years and occurs far more frequently in young children. We speculate that improved community awareness of the symptoms of T1DM will lead to earlier diagnosis of T1DM and avoidance of DKA and associated sequelae.


We showed there are specific ALDH1 autoantibodies in ovarian autoimmune disease and ovarian cancer, suggesting a role for ALDH1 in ovarian pathology. However, there is little information on the ovarian expression of ALDH1. Therefore, we compared ALDH1 expression in normal ovary and benign and malignant ovarian tumors to determine if ALDH1 expression is altered in ovarian cancer. Since there is also recent interest in ALDH1 as a cancer stem cell (CSC) marker, we assessed co-expression of ALDH1 with CSC markers in order to determine if ALDH1 is a potential CSC marker in ovarian cancer.mRNA and protein expression were compared in normal human ovary and serous ovarian tumors using quantitative Reverse-Transcriptase PCR, Western blot (WB) and semi-quantitative immunohistochemistry (IHC). ALDH1 enzyme activity was confirmed in primary ovarian cells by flow cytometry (FC) using ALDEFLUOR assay.ALDH1 mRNA expression was significantly reduced  in malignant tumors compared to normal ovaries and benign tumors. The proportion of ALDH1+ cells was significantly lower in malignant tumors  compared to normal ovaries  and benign tumors . ALDH1+ cells occurred in the stroma and surface epithelium in normal ovary and benign tumors, although surface epithelial expression varied more in benign tumors. Localization of ALDH1 was heterogeneous in malignant tumor cells and little ALDH1 expression occurred in poorly differentiated malignant tumors. In benign tumors the distribution of ALDH1 had features of both normal ovary and malignant tumors. ALDH1 protein expression assessed by IHC, WB and FC was positively correlated (p < 0.01). ALDH1 did not appear to be co-expressed with the CSC markers CD44, CD117 and CD133 by IHC.Total ALDH1 expression is significantly reduced in malignant ovarian tumors while it is relatively unchanged in benign tumors compared to normal ovary. Thus, ALDH1 expression in the ovary does not appear to be similar to breast, lung or colon cancer suggesting possible functional differences in these cancers.These observations suggest that reduced ALDH1 expression is associated with malignant transformation in ovarian cancer and provides a basis for further study of the mechanism of ALDH1 in this process. In previous studies we identified aldehyde dehydrogenase 1A1 (ALDH1) as a novel antigen in ovarian autoimmunity associated with unexplained infertility and premature menopause . This prALDH1A1 gene at chromosome 9q21 . The Primer sequences were: ALDH1A1 Forward (5'- TTGGAATTTCCCGTTGGTTA-3') and Reverse (5'- CTGTAGGCCCATAACCAGGA-3'); Actin Forward (5'-CTGTGGCATCCACGAAACTA-3') and Reverse (5'- ACATCTGCTGGAAGGTGGAC -3'). The PCR amplifications were carried out in a 25 μl reaction volume containing 25 ng of cDNA using Platinum Taq DNA Polymerase (Invitrogen) according to manufacturer's recommendation. The mixture was denatured at 94°C (3 minutes) followed by 35 cycles at 94°C (30 seconds) and 54°C (30 seconds) to anneal and 72°C (1 minute) for extension followed by a final extension at 72°C (10 minutes) in a programmable Peltier Thermo Cycler . The PCR products were separated by electrophoresis in a 3% (W/V) agarose gel (Invitrogen) and visualized using ethidium bromide stain . Amplicon from one positive sample each from normal ovary and ovarian serous carcinoma was purified using a QIAquick PCR purification kit  and sequenced at DNA sequencing facility (University of Illinois at Chicago) using an ABI 3100 Genetic analyzer (Applied Biosystems). The amplicon sequences were blasted against the NCBI RefSeq human mRNA database and confirmed with a perfect match for ALDH1A1 gene [GenBank: NM_000689.3]. Quantitative Reverse Transcriptase-PCR (qRT-PCR) was carried out using SYBR green master mix in an ABI 7500 RT-PCR system and analyzed using the ΔCt method with human Actin as an internal control according to the manufacturer's recommendation (Applied Biosystems). The ΔΔCt was determined by subtracting ΔCt of each sample from the average ΔCt of normal ovary. The differences in ALDH1 mRNA expression levels were calculated as the fold change using the formula 2-ΔΔCt as previously described  (data not shown).ALDH1 mRNA expression was significantly lower in malignant ovarian tumors (n = 5) compared to normal ovary  and benign ovarian tumors   compared to normal ovaries  and benign ovarian tumors  Figure . There wALDH1 protein was detected as a single band at 55 kDa in all of the ovarian tissues tested by Western blot Figure . DensitoALDH1 immunostaining was observed in various cell types in normal ovary and serous ovarian tumors. In normal ovary, a diffuse ALDH1 staining pattern was observed in the stroma in fibroblasts-like cells and fibrous tissue. In addition, the surface epithelial cells stained intensely although there were occasional cells without stain Figure  and 3B. The staining pattern of ALDH1 in uninvolved areas adjacent to benign serous ovarian tumors was similar to that of normal ovary Figure . In contIn malignant serous ovarian tumors ALDH1 staining varied  compared to normal ovary (22.8 ± 6.4%) and benign tumors (16.3 ± 5.6%). The ALDEFLUOR assay was positively correlated with the proportion of cells expressing ALDH1 by semi-quantitative immunohistochemistry . Overall, the estimation of enzyme activity in ovarian cells was consistent with ALDH1 mRNA and protein expression levels.The mean fluorescence intensity (MFI) was significantly decreased in malignant ovarian tumors  compared to normal ovary  and benign ovarian tumors   was smaller than the proportion of ALDH1 immunostained cells suggesting that not all ALDH1 may be active. This was also observed by Deng et al. [The ALDH1 protein expression and enzyme activity were correlated. However, the proportion of ALDEFLUOR positive cells  malignant ovarian tumors have a higher survival rate than patients with less differentiated (high-grade) tumors . ALDH1 sPrevious studies showed that higher ALDH1 expression in tumor cells is associated with poor clinical outcomes in breast, ,47 lung,Recent observations suggest ALDH1 is a marker for CSCs in various malignancies and that ALDH1 in CSCs is associated with chemoresistance and increased malignant potential ,37,47,48We found that the total ALDH1 expression is significantly reduced in malignant serous ovarian tumors compared to normal ovaries and that expression in benign serous ovarian tumors is similar to normal ovary. ALDH1 was expressed in malignant tumor cells but at a low level and was absent in the more aggressive poorly differentiated malignant tumor cells. The heterogeneity of ALDH1 expression pattern suggests ALDH1 could be used as a novel indicator of prognosis and possibly as an indicator of responses to chemotherapy. Further investigation could facilitate understanding the role of ALDH1 in the ovary and ovarian tumors.The authors declare that they have no competing interests.KP, JL and SE worked to develop the experimental design. KP performed the experiments, statistical calculations and wrote the manuscript. SE facilitated the PCR and gene sequencing, and assisted KP in data analysis. AB developed the immunohistochemistry (IHC) protocols, facilitated tissue collection and assisted in writing the manuscript. MB assisted with tissue collection and IHC tissue processing. JL conceived the study, mentored KP in scientific methods and data analysis and assisted in drafting and finalizing the manuscript. All authors approved the final version of the manuscript.


ZNF133. However, direct sequence analysis of coding and untranslated exons did not reveal a potential pathogenic mutation. Microdeletion or duplication was also excluded by comparative genomic hybridization using a dense chromosome 20 specific array. Geographical origin, haplotype and statistical analysis suggest that in 14 unrelated families an as yet undiscovered mutation on 20p11.23 was inherited from a common ancestor. Prevalence of PPCD in the Czech Republic appears to be the highest worldwide and our data suggests that at least one other novel locus for PPCD also exists.Posterior polymorphous corneal dystrophy (PPCD) is a rare autosomal dominant genetically heterogeneous disorder. Nineteen Czech PPCD pedigrees with 113 affected family members were identified, and 17 of these kindreds were genotyped for markers on chromosome 20p12.1- 20q12. Comparison of haplotypes in 81 affected members, 20 unaffected first degree relatives and 13 spouses, as well as 55 unrelated controls, supported the hypothesis of a shared ancestor in 12 families originating from one geographic location. In 38 affected individuals from nine of these pedigrees, a common haplotype was observed between D20S48 and D20S107 spanning approximately 23 Mb, demonstrating segregation of disease with the PPCD1 locus. This haplotype was not detected in 110 ethnically matched control chromosomes. Within the common founder haplotype, a core mini-haplotype was detected for D20S605, D20S182 and M189K2 in all 67 affected members from families 1–12, however alleles representing the core mini-haplotype were also detected in population matched controls. The most likely location of the responsible gene within the disease interval, and estimated mutational age, were inferred by linkage disequilibrium mapping (DMLE+2.3). The appearance of a disease-causing mutation was dated between 64–133 generations. The inferred ancestral locus carrying a PPCD1 disease-causing variant within the disease interval spans 60 Kb on 20p11.23, which contains a single known protein coding gene,  Posterior polymorphous corneal dystrophy (PPCD) is a rare bilateral disorder transmitted as an autosomal dominant trait. Clinically PPCD is characterized by vesicles, bands and polymorphous opacities with pathology at the level of Descemet membrane and the corneal endothelium. Peripheral anterior iris adhesions, iris atrophy, pupillary ectropion and corectopia may also develop. Occasional severe visual disability results from secondary glaucoma or corneal edema VSX1; MIM ID #605020) within this locus was reported as disease-causing in a few PPCD cases COL8A2; MIM ID #120252) located on 1p34.3-p32.3, however only one disease-causing mutation in one family has been described to date ZEB1; MIM ID #189909) mapping to chromosome 10p11.2 were identified as disease-causing in PPCD3 (MIM ID #609141) and it has been estimated that pathogenic changes within this gene account for approximately 25% of all PPCD cases, with a range from 9%–45% depending on population studied VSX1 from this genetic interval by a linkage study, and lack of disease-causing changes, implies that an as yet undiscovered gene is causative for PPCD1 The genetic heterogeneity of PPCD is currently known to be represented by three loci on chromosomes 20, 1, and 10 Families affected by rare dominantly inherited disorders are often unrelated, however occasionally they share a chromosomal genomic region implying that the pathogenic mutation arose in a common ancestor In this study we observed that PPCD in the Czech Republic appears to have a remarkably high prevalence. A total of 19 Czech PPCD families, including two previously linked pedigrees The study was approved by the Ethics Committee of General University Hospital in Prague, Czech Republic and conformed to the tenets of the Declaration of Helsinki. All participants signed an informed consent prior to inclusion into the study.Subjects from 19 Czech pedigrees with familial PPCD were examined between the years 1995–2010 in the Department of Ophthalmology of the First Faculty of Medicine, Charles University in Prague. Ophthalmologic assessment included visual acuity, slit lamp examination, intraocular pressure measurements and specular microscopy using Noncon ROBO Pachy SP-9000 . Diagnosis of PPCD was based on positive family history and the presence of vesicles and polymorphic opacities at the level of Descemet membrane and the corneal endothelium. Pedigrees were drawn and residency within the Czech Republic of the eldest family member known to suffer from PPCD was noted. Geographic origin of the families was plotted on a map.DNA was isolated from venous blood samples using the Nucleon III BACC3 genomic DNA extraction kit according to manufacturer’s instructions . Genotyping was performed using 11 polymorphic microsatellite markers on chromosome 20 which were fluorescently labeled and amplified by polymerase chain reaction (PCR). Ten microsatellites were commercially available: D20S98, D20S118, D20S114, D20S48, D20S605, D20S182, D20S139, D20S190, D20S106 and D20S107 . A dinucleotide marker used in this study, M189K21, was reported previously To investigate the possibility of a common lineage, haplotypes of affected individuals were constructed based on segregation within the families, and then compared between families. In order to calculate allele frequencies and haplotype frequencies in the population, 55 unrelated Czech population matched controls (110 chromosomes) were also genotyped for each marker.www.dmle.org) was used. The program DMLE+ uses Bayesian estimates of the location of a gene with a mutation affecting a discrete (disease) trait based on the observed linkage disequilibrium at multiple genetic markers. Other parameters are also estimated, such as mutation age To infer the location of a gene responsible for PPCD1 in the population studied and to estimate the age of the mutation (i.e. the time elapsed since the appearance of the common ancestor in the population) DMLE+ (Disease Mapping using Linkage disequilibrium) version 2.3 (http://research.marshfieldclinic.org/genetics/GeneticResearch/compMaps.asp). For markers which do not appear on this genetic map  the distance in cM was estimated empirically by interpolation using the standard curve of physical distance. Since DMLE+2.3 does not accept identical distances for more than one marker, where the position of two markers obtained from the Marshfield genetic map was identical  position was recalculated using the standard curve as above  reference sequence (NM_003434 and NM_001083330). Both coding and untranslated regions as well as intron-exon boundaries were screened.Two probands from families 1 and 2, previously shown to be linked to 20p11.2 Array comparative genomic analysis (CGH) was used to evaluate DNA copy number variation (CNV) on chromosome 20  with a median probe spacing of 134 bp. Patient DNA from family 1, previously shown to be linked to 20p11.2 Clinical ascertainment demonstrated bilateral corneal changes consistent with the diagnosis of PPCD in 113 subjects  from 19 families. None of the PPCD families were known to be related to one another. In all families PPCD was consistent with autosomal dominant inheritance pattern, with incomplete penetrance noted for family 18 and 19 Analysis of genetic markers on 20p12.1- 20q12 revealed a common haplotype spanning a region of at least 23 Mb surrounding the centromere and encompassing both the short and long arm of chromosome 20. This full common haplotype was detected in 16 affected members of the two previously linked PPCD1 families (1 and 2) and also in 22 affected members from families 3–8 and 12 , however no potential pathogenic changes in the annotated coding and untranslated regions were identified. Since a deletion or duplication of the ZNF133 gene would not be detected by PCR amplification and sequencing, we also performed dense chromosome 20 CGH analysis (Nimblegen) to detect any CNVs in an affected individual from family 1. No microdeletions or duplications on 20p12.1-20p11.23 were detected in this patient.The Identification of 113 affected individuals from 19 Czech PPCD families demonstrates, to the best of our knowledge, the highest reported prevalence of PPCD worldwide. Correlated to the population, at least 1 in 100,000 inhabitants in the Czech Republic are affected with PPCD. Because of the relative rarity of this disorder, a founder effect was suspected as the most likely explanation.In this study we show that 38 affected individuals from nine families, including two previously reported families Affected individuals within Czech PPCD1 pedigrees were observed in all subsequent generations implying that the disorder is highly if not fully penetrant, consistent with other studies Although the fully shared haplotype among members of nine families comprised about 1/3 of chromosome 20 including the centromere and pericentromeric regions, we have calculated that the original mutational event did not occur recently. Since the families originate from one particular geographical area, we assume that they represent a relatively isolated population, which are known to have more extensive linkage disequilibrium than outbred populations The mutation age was estimated to be 1800 years assuming a 20-year generation time. It remains to be elucidated if the disease-causing variant arose in the Czech Republic and is specific for that particular geographical region or if it is an ancient mutation introduced from elsewhere. Once the PPCD1 causing gene is discovered it will be possible to further explore these hypotheses.et al. narrowed the proximal boundary of the PPCD1 genetic interval to D20S182 defining the critical disease physical interval to a 1.8 Mb region, under the assumption that there is no micro-heterogeneity The minimal shared region in families 1–12 was observed between D20S48 and D20S139, which corresponds to the locus delineation by linkage analysis we described previously in Czech families ZNF133 which is a transcriptional repressor containing KRAB box and zinc finger domains http://www.corneanet.net/) ZNF133 to be the best positional candidate in the refined PPCD1 region, however we did not detect a pathogenic change within currently annotated coding or untranslated regions of this gene. Similarly, no pathogenic variants were observed for ZNF133 in two other studies using probands from different PPCD families linked to chromosome 20 ZNF133 is disease-causing, the change might be located in regulatory sequences, as yet unidentified exons or deep within the introns of this gene.The strongest candidate disease gene based on our current analysis is ZNF133, is very limited such that splice variants and important elements controlling transcription remain undefined. The causative mutations may lie in these uncharacterised upstream regions, exons, and introns or within as yet undiscovered genes on 20p12.1-20p11.23. Independent analysis of each family, by targeted next generation sequencing of the disease interval defined by recombinations within a single large linked family, would circumvent any assumption of locus homogeneity, and may represent the most comprehensive approach, if complemented with Sanger sequencing of gaps and gene expression studies.The data presented here, taken together with previous linkage studies, candidate gene screening, targeted genomic next-generation sequencing ZEB1 were detected in families 18 and 19 Families 15–19 apparently originate from different parts of the country than families 1–14 and these families provided an excellent internal control for our analyses. None of these kindreds had the full consensus haplotype and, with the exception of the proband from family 19, they did not share the minimal core haplotype segment found in all 64 affected individuals from pedigrees 1–12. Based on these observations, it is likely that PPCD in these families is caused by a different mutation or a different gene. In support of this, disease-causing mutations in ZEB1 changes account for approximately 4% cases, whereas a disease-causing gene at the 20p12.1-20p11.23 locus is likely to be responsible for more than 80% of all PPCD cases. A recent study by Vincent et al. highlights the fact that in some cohorts ZEB1 accounts for a smaller proportion of cases than originally thought We conclude that in the Czech population Clinical implications of our study lie in the fact that PPCD1 seems to be more severe showing a higher percentage of secondary glaucoma and necessity for keratoplasty than PPCD3, which has a direct impact on patient counseling ZEB1 mutations were previously identified in two families , and lack of the PPCD1 haplotype in two families  suggests they may not be associated with the PPCD1 locus. In family 15, the PPCD1 locus is excluded and no mutation was detected in VSX1, COL8A2 or ZEB1 suggesting a novel locus may exist.In summary we have demonstrated that the high frequency of PPCD in the Czech Republic is attributable to a founder mutation located on 20p12.1-20p11.23. The disease gene and causative variant have yet to be identified. We anticipate that approximately 80% of PPCD in the Czech population will be attributed to the mutant allele at the locus on chromosome 20p, however it is also clear that other PPCD genes or alleles are implicated as not all families described in this study share a common founder haplotype. Consistent with this finding Table S1Microsatellite markers used to construct haplotypes. Microsatellite markers on chromosome 20 and their extrapolated genetic positions used for genotyping in the current study. Physical distances were from Ensembl release 57 and sex-averaged genetic distances were from The Marshfield Comprehensive Human Genetic Map.(DOC)Click here for additional data file.Table S2Analysis of the ZNF133 gene. Primers used for amplification and sequencing ZNF133, their melting temperature (Tm), length of each amplified fragment and variations identified in the proband from family 1 (A), proband from family 2 (B) and first degree relative from family 2 (C) are shown. Exon number corresponds to reference sequence NM_003434.(DOC)Click here for additional data file.Table S3Identification of a founder haplotype in Czech PPCD families. Each affected individual is represented by a column, presence of the same allele as the consensus haplotype is indicated by x, and presence of the full common haplotype spanning a region of at least 23 Mb is indicated by o. All 67 affected members from Families 1–12 originating from the same geographic area within the Czech Republic shared a conserved chromosomal region between D20S48 and D20S139 (highlighted in bold). In families 15–19 originating from other parts of the Czech Republic the core haplotype segment between D20S48 and D20S139 was not shared among affected individuals. Affected members from families 13–14 were not available for genotyping.(DOC)Click here for additional data file.